Nat Genet, 1997 May, 16(1), 28 - 36 Functional screening of 2 Mb of human chromosome 21q22.2 in transgenic mice implicates minibrain in learning defects associated with Down syndrome; Smith DJ et al.; Using Down syndrome as a model for complex trait analysis, we sought to identify loci from chromosome 21q22.2 which, when present in an extra dose, contribute to learning abnormalities . We generated low-copy-number transgenic mice, containing four different yeast artificial chromosomes (YACs) that together cover approximately 2 megabases (Mb) of contiguous DNA from 21q22.2 . We subjected independent lines derived from each of these YAC transgenes to a series of behavioural and learning assays . Two of the four YACs caused defects in learning and memory in the transgenic animals, while the other two YACs had no effect . The most severe defects were caused by a 570-kb YAC; the interval responsible for these defects was narrowed to a 180-kb critical region as a consequence of YAC fragmentation . This region contains the human homologue of a Drosophila gene, minibrain, and strongly implicates it in learning defects associated with Down syndrome. Genetics, 1997 May, 146(1), 227 - 38 Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene; Kelly WG et al.; In screening for embryonic-lethal mutations in Caenorhabditis elegans, we defined an essential gene (let-858) that encodes a nuclear protein rich in acidic and basic residues . We have named this product nucampholin . Closely homologous sequences in yeast, plants, and mammals demonstrate strong evolutionary conservation in eukaryotes . Nucampholin resides in all nuclei of C . elegans and is essential in early development and in differentiating tissue . Antisense-mediated depletion of LET-858 activity in early embryos causes a lethal phenotype similar to characterized treatments blocking embryonic gene expression . Using transgene-rescue, we demonstrated the additional requirement for let-858 in the larval germline . The broad requirements allowed investigation of soma-germline differences in gene expression . When introduced into standard transgene arrays, let-858 (like many other C . elegans genes) functions well in soma but poorly in germline . We observed incremental silencing of simple let-858 arrays in the first few generations following transformation and hypothesized that silencing might reflect recognition of arrays as repetitive or heterochromatin-like . To give the transgene a more physiological context, we included an excess of random genomic fragments with the injected DNA . The resulting transgenes show robust expression in both germline and soma . Our results suggest the possibility of concerted mechanisms for silencing unwanted germiline expression of repetitive sequences. Cancer Res, 1997 May 1, 57(9), 1630 - 3 Identification of a 790-kilobase region of common allelic loss in chromosome 10q25-q26 in human endometrial cancer; Nagase S et al.; We previously identified two commonly deleted regions on chromosome bands 10q25-q26 in endometrial cancer: an 8-cM region and a 12-cM region . In the present study, we further studied the 8-cM region with 83 endometrial cancer specimens and 14 microsatellite markers and narrowed it down to a 1-cM region between D10S587 and D10S1723 . This result was confirmed by two-color fluorescence in situ hybridization analysis . An association between histopathologically lower grade tumor and allelic loss (P = 0.03 by Fisher's exact test) was also observed . We also constructed a yeast artificial chromosome (YAC) contig and found that one YAC clone, which was 790 kb in size, harbored the whole 1-cM region of common allelic loss. Diabetes, 1997 May, 46(5), 900 - 6 Cloning and characterization of an uncoupling protein homolog: a potential molecular mediator of human thermogenesis; Gimeno RE et al.; We have identified a novel cDNA encoding a protein highly homologous to the mammalian brown fat uncoupling protein (UCP) . Unlike the known UCP, which is expressed specifically in brown adipose tissue, the UCP homolog (UCPH) mRNA is expressed in a variety of tissues, with predominant expression in human white adipose tissue and skeletal muscle . In the white adipose tissue of ob/ob and db/db mice, the UCPH transcript is induced approximately fivefold relative to lean littermate controls . Expression of murine UCPH in yeast results in growth inhibition under conditions that require aerobic respiration, but does not affect growth under anaerobic conditions . Furthermore, UCPH expression in yeast causes a decrease in the mitochondrial membrane potential, as judged by staining with the potential-sensitive dye DiOC6 . These observations suggest that UCPH, like UCP, uncouples oxidative phosphorylation . The possibility that the UCPH protein is an important mediator of human thermogenesis is discussed. Curr Biol, 1997 May 1, 7(5), 301 - 7 Phosphatidic acid formation by phospholipase D is required for transport from the endoplasmic reticulum to the Golgi complex; Bi K et al.; BACKGROUND: Lipid molecules may play a regulatory role in the secretory pathway of mammals and yeast . The lipid hydrolase phospholipase D (PLD) is one candidate for mediating regulation of secretion, based on the location of this enzyme and its requirements for activation . RESULTS: We found that primary alcohols, which block formation of phosphatidic acid (PA) by PLD, inhibited the transport of two different viral glycoproteins from the endoplasmic reticulum to the Golgi complex in Chinese hamster ovary cells . Corresponding secondary alcohols, which are much less potent in blocking PA formation, were also less effective in blocking transport of the glycoproteins . The block in glycoprotein transport imposed by primary alcohols was reversed when PA, in the form of liposomes, was exogenously supplied to the culture medium . CONCLUSIONS: We suggest that the earliest site of regulation of membrane transport by PLD is within the intermediate compartment between the endoplasmic reticulum and the Golgi complex. Infect Immun, 1997 May, 65(5), 1849 - 55 Activation, binding, and processing of complement component 3 (C3) by Blastomyces dermatitidis; Zhang MX et al.; Complement plays a key role in phagocyte recognition and killing of Blastomyces dermatitidis, but little is known about how complement components interact with the yeast . We report the characteristics of activation, binding, and processing of C3 by B . dermatitidis . In pooled normal human serum (NHS), deposition of C3 on the yeast was detectable within 2 min, whereas in NHS containing MgEGTA, deposition was delayed by 6 min, indicating that the yeast activates C3 by both classical and alternative pathways . When both pathways were operative, maximal binding of 4.5 x 10(6) C3 molecules/cell was achieved in less than 30 min . In the absence of the classical pathway, yeast cells bound 80% of the maximum C3, indicating that the yeast intrinsically activates the alternative pathway . Delayed deposition of C3 in NHS-MgEGTA was similar to that in NHS preabsorbed by the yeast or by immobilized protein A/G to remove serum immunoglobulin . Purified immunoglobulin restored C3 binding to NHS preabsorbed by the yeast, suggesting that antibody in nonimmune serum initiates the classical pathway . beta-Glucan absorption of NHS abolished the classical pathway, suggesting that cell wall beta-glucan is a target of initiating antibodies . Hydroxylamine treatment of NHS-opsonized yeast cells showed that 76% of C3 was bound to yeast cells by ester linkage, supporting a role for hydroxyl groups on cell wall polysaccharides . Hydroxylamine-cleaved fragments were chiefly C3b and iC3b; 70% of hydroxylamine-sensitive C3b was converted to iC3b within 1 min of opsonization, and the ratio was stable over 1 h . Our data predict that C3b and iC3b on opsonized yeast cells direct binding to CR1 and CR3 receptors on human phagocytes, which, in turn, may influence the fate of this host-pathogen interaction. J Mol Evol, 1997 May, 44(5), 477 - 91 Molecular evolution of cytochrome c oxidase subunit IV: evidence for positive selection in simian primates; Wu W et al.; Cytochrome c oxidase (COX) is a multi-subunit enzyme complex that catalyzes the final step of electron transfer through the respiratory chain on the mitochondrial inner membrane . Up to 13 subunits encoded by both the mitochondrial (subunits I, II, and III) and nuclear genomes occur in eukaryotic organisms ranging from yeast to human . Previously, we observed a high number of amino acid replacements in the human COX IV subunit compared to mouse, rat, and cow orthologues . Here we examined COX IV evolution in the two groups of anthropoid primates, the catarrhines (hominoids, cercopithecoids) and platyrrhines (ceboids), as well as one prosimian primate (lorisiform), by sequencing PCR-amplified portions of functional COX4 genes from genomic DNAs . Phylogenetic analysis of the COX4 sequence data revealed that accelerated nonsynonymous substitution rates were evident in the early evolution of both catarrhines and, to a lesser extent, platyrrhines . These accelerated rates were followed later by decelerated rates, suggesting that positive selection for adaptive amino acid replacement became purifying selection, preserving replacements that had occurred . The evidence for positive selection was especially pronounced along the catarrhine lineage to hominoids in which the nonsynonymous rate was first faster than the synonymous rate, then later much slower . The rates of three types of "neutral DNA" nucleotide substitutions (synonymous substitutions, pseudogene nucleotide substitutions, and intron nucleotide substitutions) are similar and are consistent with previous observations of a slower rate of such substitutions in the nuclear genomes of hominoids than in the nuclear genomes of other primate and mammalian lineages. Mol Cell Biol, 1997 May, 17(5), 2521 - 8 Heteromeric and homomeric interactions correlate with signaling activity and functional cooperativity of Smad3 and Smad4/DPC4; Wu RY et al.; Homologs of Drosophila Mad function as downstream mediators of the receptors for transforming growth factor beta (TGF-beta)-related factors . Two homologs, the receptor-associated Smad3 and the tumor suppressor Smad4/DPC4, synergize to induce ligand-independent TGF-beta activities and are essential mediators of the natural TGF-beta response . We now show that Smad3 and Smad4 associate in homomeric and heteromeric interactions, as assessed by yeast two-hybrid and coimmunoprecipitation analyses . Heteromeric interactions are mediated through the conserved C-terminal domains of Smad3 and Smad4 . In Smad3, the homomeric interaction is mediated by the same domain . In contrast, the homomeric association of Smad4 requires both the N-terminal domain and the C-terminal domain, which by itself does not homomerize . Mutations that have been associated with impaired Mad activity in Drosophila or decreased tumor suppressor activity of Smad4/DPC4 in pancreas cancer, including a short C-terminal truncation and two point mutations in the conserved C-terminal domains, impair the ability of Smad3 and Smad4 to undergo homo- and heteromeric associations . Analyses of the biological activity of Smad3 and Smad4 and their mutants show that full signaling activity correlates with their ability to undergo efficient homo- and heteromeric interactions . Mutations that interfere with these interactions result in decreased signaling activity . Finally, we evaluated the ability of Smad3 or Smad4 to induce transcriptional activation in yeast . These results correlate the ability of individual Smads to homomerize with transcriptional activation and additionally with their biological activity in mammalian cells. Nucleic Acids Res, 1997 May 1, 25(9), 1665 - 77 Extracting protein alignment models from the sequence database; Neuwald AF et al.; Biologists often gain structural and functional insights into a protein sequence by constructing a multiple alignment model of the family . Here a program called Probe fully automates this process of model construction starting from a single sequence . Central to this program is a powerful new method to locate and align only those, often subtly, conserved patterns essential to the family as a whole . When applied to randomly chosen proteins, Probe found on average about four times as many relationships as a pairwise search and yielded many new discoveries . These include: an obscure subfamily of globins in the roundworm Caenorhabditis elegans ; two new superfamilies of metallohydrolases; a lipoyl/biotin swinging arm domain in bacterial membrane fusion proteins; and a DH domain in the yeast Bud3 and Fus2 proteins . By identifying distant relationships and merging families into superfamilies in this way, this analysis further confirms the notion that proteins evolved from relatively few ancient sequences . Moreover, this method automatically generates models of these ancient conserved regions for rapid and sensitive screening of sequences. J Virol, 1997 May, 71(5), 4005 - 15 Retrovirus-like end processing of the tobacco Tnt1 retrotransposon linear intermediates of replication; Feuerbach F et al.; The tobacco retrotransposon Tnt1 can transpose through an RNA intermediate in the heterologous host Arabidopsis thaliana . We report here the identification and characterization of extrachromosomal linear and circular DNA forms of Tnt1 in this heterologous host . Our results demonstrate that Tnt1 linear intermediates possess two extra base pairs at each end compared with Tnt1's integrated forms . Prior to integration into the host genome, the two terminal nucleotides at the 3' end of these linear intermediates are removed, as in the case of the yeast Ty3 retrotransposon and of retroviruses . Our data, together with those from recent studies of Ty3, reinforce the idea that 3' dinucleotide cleavage is not restricted to retroviral integrases and is probably a feature shared by many different retrotransposons' enzymes. J Virol, 1997 May, 71(5), 3574 - 9 The baculovirus single-stranded DNA binding protein, LEF-3, forms a homotrimer in solution; Evans JT et al.; LEF-3 is one of six proteins from Autographa californica multinucleocapsid polyhedrosis virus required for transient DNA replication and has the properties of a single-stranded DNA binding protein . In this report we demonstrate that LEF-3 interacts with itself in both yeast two-hybrid assays and glutathione S-transferase fusion affinity assays . LEF-3 deletion clones which were unable to interact with full-length LEF-3 also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3 . LEF-3 was purified to homogeneity and characterized by analytical ultracentrifugation and native polyacrylamide gel electrophoresis . These studies revealed that LEF-3 was present as a 132-kDa complex, indicating that its native conformation is that of a homotrimer . This result was confirmed by cross-linking with glutaraldehyde followed by matrix-assisted laser desorption/ionization mass spectrometry. Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4739 - 44 The regulator of early gliogenesis glial cells missing is a transcription factor with a novel type of DNA-binding domain; Schreiber J et al.; Absence or presence of glial cells missing (GCM) in cells of the developing nervous system of Drosophila decides over their future fate as neurons or glia with only those cells turning into glia that express GCM . To understand how GCM exerts its function we performed a detailed structure-function analysis . Using fusions between the DNA binding domain of the yeast GAL4 protein and GCM, we detected a transactivation function within the C-terminal part of GCM . In addition to this transactivation domain we mapped a sequence-specific DNA-binding domain within the N-terminal part of the GCM protein in close proximity to a bipartite nuclear localization signal . Binding site selection assays determined the motif 5'-AT(G/A)CGGGT-3' as the preferred binding site for GCM . Both the lack of homology to known proteins and the novel DNA binding specificity indicate that GCM contained a new type of DNA-binding domain . In transiently transfected cells, GCM also activated transcription from promoters consisting of the newly identified GCM-binding site and a TATA box . Thus, GCM is a novel type of transcription factor involved in early gliogenesis. Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4246 - 9 Effects of folding on metalloprotein active sites; Winkler JR et al.; Experimental data for the unfolding of cytochrome c and azurin by guanidinium chloride (GuHCl) are used to construct free-energy diagrams for the folding of the oxidized and reduced proteins . With cytochrome c, the driving force for folding the reduced protein is larger than that for the oxidized form . Both the oxidized and the reduced folded forms of yeast cytochrome c are less stable than the corresponding states of the horse protein . Due to the covalent attachment of the heme and its fixed tetragonal coordination geometry, cytochrome c folding can be described by a two-state model . A thermodynamic cycle leads to an expression for the difference in self-exchange reorganization energies for the folded and unfolded proteins . The reorganization energy for electron exchange in the folded protein is approximately 0.5 eV smaller than that for a heme in aqueous solution . The finding that reduced azurin unfolds at lower GuHCl concentrations than the oxidized protein suggests that the coordination structure of copper is different in oxidized and reduced unfolded states: it is likely that the geometry of CuI in the unfolded protein is linear or trigonal, whereas CuII prefers to be tetragonal . The evidence indicates that protein folding lowers the azurin reorganization energy by roughly 1.7 eV relative to an aqueous Cu(1, 10-phenanthroline)22+/+ reference system. DNA Res, 1997 Apr 28, 4(2), 133 - 40 Ordered YAC clone contigs assigned to rice chromosomes 3 and 11; Tanoue H et al.; Yeast artificial chromosome (YAC) clones were arranged on the positions of restriction fragment length polymorphism (RFLP) and sequence-tagged site (STS) markers already mapped on the high-resolution genetic maps of rice chromosomes 3 and 11 . From a total of 416 and 242 YAC clones selected by colony/Southern hybridization and polymerase chain reaction (PCR) analysis, 238 and 135 YAC clones were located on chromosomes 3 and 11, respectively . For chromosomes 3 and 11, 24 YAC contigs and islands with total coverage of about 46% and 12 contigs and islands with coverage of about 40%, respectively, were assigned . Although many DNA fragments of multiple copy marker sequences could not be mapped to their original locations on the genetic map by Southern hybridization because of a lack of RFLP, the physical mapping of YAC clones could often assign specific locations of such multiple copy sequences on the genome . The information provided here on contig formation and similar sequence distribution revealed by ordering YAC clones will help to unravel the genome organization of rice as well as being useful in isolation of genes by map-based cloning. Biochem Biophys Res Commun, 1997 Apr 28, 233(3), 723 - 8 Two N-terminal self-association domains are required for the dominant negative transcriptional activity of WT1 Denys-Drash mutant proteins; Holmes G et al.; Patients with Denys-Drash syndrome (DDS) have been shown to be constitutionally heterozygous for mutations of the WT1 gene . Almost all DDS mutations inactivate or remove the DNA-binding zinc finger region of WT1 and the resulting mutant proteins appear to act in a dominant negative manner . This may occur via WT1 self-association, which has been shown to involve the first 180 amino acids . By creating a series of N-terminal deletions, we have further investigated WT1 self-association using a yeast di-hybrid system and an in vitro protein binding assay . Our results suggest that there are two distinct domains within the N-terminal region facilitating self-association, residing from amino acids 1-45 and 157-253 . Co-transfection of WT1 with progressively shorter N-terminal constructs demonstrates that both of these sites are required for a dominant negative activity as assessed by activation of a reporter construct. Biochem Biophys Res Commun, 1997 Apr 28, 233(3), 592 - 600 Binding sites of cytoplasmic effectors TRAF1, 2, and 3 on CD30 and other members of the TNF receptor superfamily; Boucher LM et al.; CD30 is present on the surfaces of malignant cells from patients with Hodgkin's lymphoma, anaplastic large cell lymphoma, and other lymphomas . The yeast two hybrid genetic screen method was used to identify molecular effectors which mediate CD30 signalling events . Clones corresponding to genes coding for TRAF1, TRAF2, and TRAF3 molecules, postulated to be involved in signalling via the TNF and CD40 receptors, were isolated . In this report, we show that the CD30 intracellular tail contains two motifs that bind TRAFs . The more amino terminal motif, 558PHYPEQET565, binds TRAF2 and 3, while the more carboxyl terminal motif, 576MLSVEEEG583, binds TRAF1 and 2 . We show that these amino acid motifs are conserved in TNFRp75 and CD40 and that sequences in these receptors homologous to TRAF-binding sequences found in CD30 can selectively bind the TRAFs in a predictable manner. J Biol Chem, 1997 Apr 25, 272(17), 11452 - 6 Ligand-dependent interaction of the aryl hydrocarbon receptor with a novel immunophilin homolog in vivo; Carver LA et al.; In an effort to identify regulators of aryl hydrocarbon receptor (AHR) signaling, we have employed the yeast two-hybrid system to screen for human proteins that interact in a ligand-dependent manner with the AHR . After screening 1.4 x 10(6) clones from a human B cell library, two distinct clones were identified that associated specifically with the liganded receptor . No clones were identified that interacted preferentially with the unliganded AHR . One of the ligand-dependent clones, ARA9, encodes a novel 330-amino acid protein with regions of amino acid sequence similarity to the 52-kDa FK506-binding protein known to be associated with the glucocorticoid receptor . Yeast two-hybrid experiments with ARA9 demonstrated a strong interaction with the AHR that is enhanced 11-fold in the presence of the ligand beta-naphthoflavone . In vitro experiments using proteins generated in reticulocyte lysates confirmed this interaction and indicated that ARA9 can be co-immunoprecipitated with the AHR using antisera raised specifically for either the AHR or the 90-kDa heat shock protein . The observation that ARA9 has a high affinity for both the 90-kDa heat shock protein-associated and ligand-activated forms of the AHR suggests that ARA9 is a component of the AHR-signaling pathway in vivo. J Biol Chem, 1997 Apr 25, 272(17), 11350 - 5 A common binding site mediates heterodimerization and homodimerization of Bcl-2 family members; Diaz JL et al.; Bcl-2 inhibits apoptosis induced by a wide variety of stimuli . In contrast, the Bcl-2 homologue, Bax, antagonizes Bcl-2's death protecting function . Bcl-2 forms protein-protein homodimers with itself and heterodimers with Bax, and previous experiments have shown that point mutations in Bcl-2 can abrogate Bax binding while leaving homodimerization intact . These mutagenesis results can be interpreted to suggest that Bcl-2 has separate binding sites that are responsible for homodimer and heterodimer formation . Results from yeast two-hybrid studies have also suggested that homodimerization and heterodimerization reflect distinct modes of interaction . However, using quantitative plate binding assays, we now show that Bax as well as peptides derived from the BH3 domains of Bax and Bak block both Bcl-2/Bax binding and Bcl-2/Bcl-2 binding . Similar assays demonstrate that Bcl-xL can form both homodimers and heterodimers and that these interactions are also inhibited by Bax and the BH3-derived peptides . These results demonstrate that the same binding motifs are responsible for both homodimerization and heterodimerization of Bcl-2 family members. J Biol Chem, 1997 Apr 25, 272(17), 10990 - 3 Modulation of RNA polymerase II elongation efficiency by C-terminal heptapeptide repeat domain kinase I; Lee JM et al.; Hyperphosphorylation of the C-terminal heptapeptide repeat domain (CTD) of the RNA polymerase II largest subunit has been suggested to play a key role in regulating transcription initiation and elongation . To facilitate investigating functional consequences of CTD phosphorylation we developed new templates, the double G-less cassettes, which make it possible to assay simultaneously the level of initiation and the efficiency of elongation . Using these templates, we examined the effects of yeast CTD kinase I or CTD kinase inhibitors on transcription and CTD phosphorylation in HeLa nuclear extracts . Our results showed that polymerase II elongation efficiency and CTD phosphorylation are greatly reduced by CTD kinase inhibitors, whereas both are greatly increased by CTD kinase I; in contrast, transcription initiation is much less affected . These results demonstrate that CTD kinase I modulates the elongation efficiency of RNA polymerase II and are consistent with the idea that one function of CTD phosphorylation is to promote effective production of long transcripts by stimulating the elongation efficiency of RNA polymerase II. Gene, 1997 Apr 21, 189(2), 213 - 9 Drosophila melanogaster NADPH-cytochrome P450 oxidoreductase: pronounced expression in antennae may be related to odorant clearance; Hovemann BT et al.; Insects perceive a large number of airborne chemicals as olfactory components mainly through the antenna . It is thought that detection of the odorants by specific receptors is followed by a degradative pathway that clears the olfactory organ from accumulating chemicals . In Drosophila, a number of P450 monooxygenases are involved in the metabolism of foreign chemicals {Dunkov et al . (1996) Cytochrome P450 gene cluster in Drosophila melanogaster, Mol . Gen . Genet . 251, 290-297} . NADPH-cytochrome P450 oxidoreductases serve to transfer reducing equivalents to P450 monooxygenases . We isolated cDNA and genomic clones coding for a Drosophila NADPH cytochrome P450 oxidoreductase (CPR) . The largest cDNA of 2471 nucleotides in length contained an open reading frame of 693 amino acids that includes the putative CPR sequence . CPR is a single copy gene as shown by genomic Southern hybridisation and maps to the cytogenetic map position 26C on the second chromosome . Comparison of genomic and cDNA CPR sequences revealed a gene structure that is split into at least six exons . The CPR protein sequence is almost identical with that of house fly and remarkably conserved when compared to vertebrates and yeast . RNA expression is high in embryos and antennae as compared to adult heads, adult bodies and larvae . High expression in antennae may reflect the putative function in olfactory clearance. J Mol Biol, 1997 Apr 18, 267(5), 1149 - 56 Crystal structure of an alternating octamer r(GUAUGUA)dC with adjacent G x U wobble pairs; Biswas R et al.; The crystal structure of the RNA duplex, r(GUAUGUA)dC, with a 3'-terminal deoxy C residue, has been determined at 1.38 A resolution . The r(GUAUGU) hexameric consensus sequence is present at the exon-intron junction in pre-mRNAs of yeast and higher eukaryotic organisms . The crystal belongs to the rhombohedral space group R3 . The hexagonal unit cell dimensions are a = b = 39.71 A, c = 68.15 A and gamma = 120 degrees with one duplex in the asymmetric unit . The structure was solved using the molecular replacement method . The final model contains 332 atoms of the duplex and 67 solvent molecules . The R-factor is 17.6% (Rfree of 23.1%) for 4035 reflections with F > or = 1.5sigma(F) in the resolution range 10.0 to 1.38 A . The duplex is of the A-type with a pseudodyad relating the two strands . The RNA helix is slightly distorted, in spite of the presence of two adjacent G x U wobble base-pairs located at the center of the helix . The twist angle between the wobble pairs, 38.1 degrees, is above the average value and those between the wobble base-pairs and the flanking Watson-Crick base-pairs, 26.7 degrees and 26.3 degrees, respectively, are lower than the average values . The twist between the junction base-pairs are about 24 degrees . The G x U wobble pairs are bridged by water molecules and solvated in the grooves . G x U base-pairs are as stable as the Watson-Crick A x U pairs and only slightly less stable than the G x C pairs accounting for their frequent occurrence in RNA. Cell, 1997 Apr 18, 89(2), 297 - 308 MARK, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption; Drewes G et al.; MARK phosphorylates the microtubule-associated proteins tau, MAP2, and MAP4 on their microtubule-binding domain, causing their dissociation from microtubules and increased microtubule dynamics . We describe the molecular cloning, distribution, activation mechanism, and overexpression of two MARK proteins from rat that arise from distinct genes . They encode Ser/Thr kinases of 88 and 81 kDa, respectively, and show similarity to the yeast kin1+ and C . elegans par-1 genes that are involved in the establishment of cell polarity . Expression of both isoforms is ubiquitous, and homologous genes are present in humans . Catalytic activity depends on phosphorylation of two residues in subdomain VIII . Overexpression of MARK in cells leads to hyperphosphorylation of MAPs on KXGS motifs and to disruption of the microtubule array, resulting in morphological changes and cell death. Oncogene, 1997 Apr 17, 14(15), 1869 - 74 Two functional assays employed to detect an unusual mutation in the oligomerisation domain of p53 in a Li-Fraumeni like family; Lomax ME et al.; Previous investigations of a Li - Fraumeni like family (Barnes et al., 1992) demonstrated that both the proband and her mother had elevated p53 protein levels in both tumour tissue and normal tissue at sites distant from the tumour, although no mutation was found in the p53 gene . In the present study two recently described functional assays for p53, an apoptotic assay and the functional assay for the separation of alleles in yeast (FASAY), have been employed to study the functional activity of p53 from this patient . The results of the apoptotic assay demonstrated that this patient had a p53 functional defect and the FASAY result suggested that this defect was in fact a germline mutation of the p53 gene . A point mutation of codon 337, which results in an amino acid substitution of a cysteine for an arginine, was demonstrated initially in cDNA and was confirmed by sequencing of genomic DNA . This is an unusual mutation as it is in the oligomerisation domain of p53, in contrast to the majority of p53 mutations which are in the core DNA binding domain . This mutation results in a protein which still retains partial transactivational activity in the FASAY . The mutation of codon 337 is only the second reported case of a germline missense mutation occurring in the oligomerisation domain of p53. Genomics, 1997 Apr 15, 41(2), 275 - 8 Isolation and chromosomal mapping of a novel ATP-binding cassette transporter conserved in mouse and human; Savary S et al.; We report here on the identification and genomic mapping of a novel member of the family of the ATP-binding cassette (ABC) transporters, ABC7, conserved in mouse and in humans . The ABC7 gene encodes a protein with the typical features of half-transporters, such as those involved in translocation of antigenic peptides or in peroxisomal disorders . ABC7 shows a ubiquitous expression pattern and maps to the X chromosome both in mouse and in humans . The high sequence similarity to those of two yeast half-transporters supports once again the extreme evolutionary conservation of this family of proteins. Genomics, 1997 Apr 15, 41(2), 218 - 26 BAC and PAC contigs covering 3.5 Mb of the Down syndrome congenital heart disease region between D21S55 and MX1 on chromosome 21; Hubert RS et al.; Chromosome 21 is a model for the study of human chromosomal aneuploidy, and the construction of its physical and transcriptional maps is a necessary step in understanding the molecular basis of aneuploidy-dependent phenotypes . To identify the gene(s) responsible for Down syndrome congenital heart disease (DS-CHD), we constructed a physical map of the D21S55 to MX1 region . A bacterial artificial chromosome (BAC) library was screened using several YACs spanning the interval, and a P1-derived artificial chromosome (PAC) library was screened using radiolabeled STS PCR products and whole BACs in gap-filling initiatives . FISH confirmed the location of all BAC and PAC clones to 21q22.2-q22.3 . Overlaps were established using clone-to-clone Southerns and 24 new STSs, generated from the direct sequencing of BAC and PAC ends, along with 35 preexisting STSs . Approximately 3.5 Mb of the 4- to 5-Mb D21S55 to MX1 interval is covered in 85 BACs and 24 PACs, representing fourfold coverage within the contigs . These BAC and PAC contigs are valuable reagents for isolating the genes for DS-CHD. Genomics, 1997 Apr 15, 41(2), 185 - 92 A sequence-ready high-resolution physical map of the best macular dystrophy gene region in 11q12-q13; Cooper PR et al.; Best disease, an autosomal dominant inherited macular degenerative disorder, was previously localized between D11S1765 and UGB (uteroglobin) in 11q13 by genetic linkage analysis . Since this region was found to be refractory to cloning in YAC (yeast artificial chromosome)-based vectors, a P1 artificial chromosome (PAC) contig was assembled . Gridded PAC libraries representing a 16-fold genome equivalent were screened by hybridization using PCR products representing STSs derived from YAC end sequences, markers binned to 11q13, and PAC-derived insert ends . A highly marker dense approximately 1.7-Mb PAC contig that encompassed the disease gene region was constructed, allowing us to order accurately the markers throughout the region and to provide the most precise estimate of its physical size . Using this contig, thus far we have mapped seven anonymous ESTs and five known genes into this region . This high-resolution physical map will facilitate the isolation of polymorphic markers for refinement of the disease gene region, as well as the identification of candidate genes by exon trapping, cDNA selection, and gene prediction from PAC-derived genomic sequence. Genomics, 1997 Apr 15, 41(2), 141 - 54 Binning clones by hybridization with complex probes: statistical refinement of an inner product mapping method; Andrews C et al.; Molecular methods that use long-range information to solve genomics problems (i.e., top-down strategies) efficiently have become increasingly prominent in the genomics literature . One such method, an implementation of inner product mapping (IPM), uses noisy, long-range radiation hybrid (RH)/YAC overlap data and relatively noise-free RH/STS overlap data to localize clones to specific chromosomal regions . Because the molecular data are rarely noise-free, statistical models tailored to the top-down molecular methods make the methods far more effective . We develop two statistical models for IPM (or any other top-down strategy of similar form), a parametric logit model and a nonparametric order-restricted model, and show how these models can be implemented within a hierarchical Bayes framework . Using these models, we refine the chromosome 11 map reported in M . Perlin et al . (1995, Genomics 28: 315-327) . Our analyses improve the IPM map, both in terms of successful localization of clones and in terms of the confidence with which they are localized. Cancer Res, 1997 Apr 15, 57(8), 1516 - 22 Identification of mutations at DNA topoisomerase I responsible for camptothecin resistance; Wang LF et al.; A camptothecin-resistant cell line that exhibits more than 600-fold resistance to camptothecin, designated CPT(R)-2000, was established from mutagen-treated A2780 ovarian cancer cells . CPT(R)-2000 cells also exhibit 3-fold resistance to a DNA minor groove-binding ligand Ho33342, a different class of mammalian DNA topoisomerase I inhibitors . However, CPT(R)-2000 cells exhibit no cross-resistance toward drugs such as Adriamycin, amsacrine, vinblastine, and 4'-dimethyl-epipodophyllotoxin . The mRNA, protein levels, and enzyme-specific activity of DNA topoisomerase I are relatively the same in parental and CPT(R)-2000 cells . However, unlike the DNA topoisomerase I activity of parental cells, which can be inhibited by camptothecin, that of CPT(R)-2000 cells cannot . In addition, parental cells after camptothecin treatment results in a decrease in the level of DNA topoisomerase I, whereas CPT(R)-2000 cells are insensitive to camptothecin treatment . These results suggested that the mechanism of camptothecin resistance is most likely due to a DNA topoisomerase I structural mutation . This notion is supported by DNA sequencing results confirming that DNA topoisomerase I of CPT(R)-2000 is mutated at amino acid residues Gly717 to Val and Thr729 to Ile . We also used the yeast system to examine the mutation(s) responsible for camptothecin resistance . Our results show that each single amino acid change results in partial resistance, and the double mutation gives a synergetic effect on camptothecin resistance . Because both mutation sites are near the catalytic active center, this observation raises the possibility that camptothecin may act at the vicinity of the catalytic active site of the enzyme-camptothecin-DNA complex. Proc Natl Acad Sci U S A, 1997 Apr 15, 94(8), 3616 - 20 Design of TATA box-binding protein/zinc finger fusions for targeted regulation of gene expression; Kim JS et al.; Fusing the TATA box-binding protein (TBP) to other DNA-binding domains may provide a powerful way of targeting TBP to particular promoters . To explore this possibility, a structure-based design strategy was used to construct a fusion protein, TBP/ZF, in which the three zinc fingers of Zif268 were linked to the COOH terminus of yeast TBP . Gel shift experiments revealed that this fusion protein formed an extraordinarily stable complex when bound to the appropriate composite DNA site (half-life up to 630 h) . In vitro transcription experiments and transient cotransfection assays revealed that TBP/ZF could act as a site-specific repressor . Because the DNA-binding specificities of zinc finger domains can be systematically altered by phage display, it may be possible to target such TBP/zinc finger fusions to desired promoters and thus specifically regulate expression of endogenous genes. Virology, 1997 Apr 14, 230(2), 217 - 27 Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein; Gale MJ Jr et al.; Hepatitis C virus (HCV) is the major cause of non-A non-B hepatitis and a leading cause of liver dysfunction worldwide . While the current therapy for chronic HCV infection is parenteral administration of type 1 interferon (IFN), only a fraction of HCV-infected individuals completely respond to treatment . Previous studies have correlated the IFN sensitivity of strain HCV-1b with mutations within a discrete region of the viral nonstructural 5A protein (NS5A), termed the interferon sensitivity determining region (ISDR), suggesting that NS5A may contribute to the IFN-resistant phenotype of HCV . To determine the importance of HCV NS5A and the NS5A ISDR in mediating HCV IFN resistance, we tested whether the NS5A protein could regulate the IFN-induced protein kinase, PKR, a mediator of IFN-induced antiviral resistance and a target of viral and cellular inhibitors . Using multiple approaches, including biochemical, transfection, and yeast genetics analyses, we can now report that NS5A represses PKR through a direct interaction with the protein kinase catalytic domain and that both PKR repression and interaction requires the ISDR . Thus, inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects of IFN . Finally the inhibition of the PKR protein kinase, by NS5A is the first described function for this HCV protein. Gene, 1997 Apr 11, 189(1), 25 - 9 Isolation of a full-length human WNT7A gene implicated in limb development and cell transformation, and mapping to chromosome 3p25; Bui TD et al.; The Wnt gene family has a role in development as well as tumourigenesis . One mouse member, Wnt7a, is vital for limb development in vivo and also possesses transforming ability in vitro . This study reports the isolation of a full length of human homologue of mouse Wnt7a gene by library screening . Yeast artificial chromosome-fluorescence in situ hybridisation (YAC-FISH) mapped the WNT7A gene to chromosome 3p25 . Human WNT7A had an ORF encoding a deduced protein of 349 aa that exhibited 97% and 92% identity to mouse Wnt7a at the aa and nucleic acid levels, respectively . It possessed the 22 conserved cysteine residues and 3 more at the amino terminus, and a putative poly A tail . This is the fifth human WNT gene in which a complete cDNA sequence had been determined. J Mol Biol, 1997 Apr 11, 267(4), 1002 - 11 Modeling of protein conformational fluctuations in pKa predictions; Zhou HX et al.; A method is presented to account for conformational fluctuations of a protein in predicting the pK(a) values of its titrating groups . Conformations of the protein are generated by conventional molecular dynamics or Monte Carlo simulations, in which the protonations of the titrating groups are fixed . For each protein conformation, the electrostatic free energies required to add a proton charge to a titrating group while other groups are either unprotonated or protonated are calculated within a dielectric continuum model . These are used to determine the mean protonations of the titrating groups in the conformation at a series of pH values . The mean protonations are then used to determine the relative weight of the particular conformation with the titrating groups having all possible protonations . A conformationally averaged mean protonation for each titrating group is finally obtained by the weighted sum of the group's mean protonations in all the conformations . This method is applied to yeast iso-1-ferricytochrome c . The predicted pK(a) values are in general agreement with experimental results. J Biol Chem, 1997 Apr 11, 272(15), 10035 - 40 Interaction of MAD2 with the carboxyl terminus of the insulin receptor but not with the IGFIR . Evidence for release from the insulin receptor after activation; O'Neill TJ et al.; We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor (IR) . We identified a human cDNA encoding a protein that appears to be the human homolog of the yeast MAD2 protein, which we term hMAD2 . The yeast MAD2 protein was first identified in a genetic screen to identify cell cycle checkpoint regulatory proteins, yet the mechanism by which MAD2 functions in cell cycle control is currently unclear . Here we show that hMAD2 requires the COOH-terminal 30 amino acids of the IR for interaction and that hMAD2 does not interact with the related insulin-like growth factor I receptor . Interestingly, hMAD2 does not require IR tyrosine autophosphorylation for interaction because it interacts with a kinase-dead IR in the yeast two-hybrid system . In support of this finding, hMAD2-GST fusions were found to interact strongly in vitro with receptors derived from noninsulin-stimulated cells . Furthermore, using two independent in vitro assays, IR activation was found to significantly reduce the interaction of hMAD2 with the IR . Lastly, we show that hMAD2 can be coimmunoprecipitated with the IR from Chinese hamster ovary IR cell lysates, suggesting that this interaction occurs in vivo in cells of mammalian origin . Our results suggest that hMAD2 represents a novel class of proteins that is specific for interaction with the IR as compared with the insulin-like growth factor I receptor and that interacts best with the inactive IR and is released upon receptor autophosphorylation . The function of hMAD2 and its potential role in insulin signaling remain to be elucidated. J Biol Chem, 1997 Apr 4, 272(14), 9141 - 6 p40(phox) down-regulates NADPH oxidase activity through interactions with its SH3 domain; Sathyamoorthy M et al.; The NADPH oxidase of phagocytes generates microbicidal oxidants in response to a variety of stimuli . Its activation and assembly involve multiple SH3 domain interactions among several oxidase components . Here we present evidence that the cytosolic oxidase-associated protein, p40(phox), mediates down-regulation of NADPH oxidase through interactions with its SH3 domain . Recombinant p40(phox) was produced in several eukaryotic expression systems (insect, mammalian, and yeast) to explore its role in oxidase function in relation to domains involved in interactions with other factors, p47(phox) and p67(phox) . p40(phox) inhibited oxidase activity in vitro when added to neutrophil membranes and recombinant p47(phox), p67(phox), and p21rac . Co-transfection of p40(phox) into K562 cells resulted in significant decreases ( approximately 40%) in whole cell oxidase activity . Furthermore, the isolated SH3 domain of p40(phox) was even more effective in inhibiting whole cell oxidase activity, consistent with experiments showing that this domain binds to the same proline-rich target in p47(phox) (residues 358-390) that interacts with p67(phox) . In contrast, deletion of the carboxyl-terminal domain of p40(phox) that binds to p67(phox) did not relieve its oxidase inhibitory effects . Thus, p40(phox) appears to down-regulate oxidase function by competing with an SH3 domain interaction between other essential oxidase components. J Biol Chem, 1997 Apr 4, 272(14), 9086 - 92 Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance; Bush KT et al.; The accumulation of misfolded proteins in the cytosol leads to increased expression of heat-shock proteins, while accumulation of such proteins in the endoplasmic reticulum (ER) stimulates the expression of many ER resident proteins, most of which function as molecular chaperones . Recently, inhibitors of the proteasome have been identified that can block the rapid degradation of abnormal cytosolic and ER-associated proteins . We therefore tested whether these agents, by causing the accumulation of abnormal proteins, might stimulate the expression of cytosolic heat-shock proteins and/or ER molecular chaperones and thereby induce thermotolerance . Exposure of Madin-Darby canine kidney cells to various proteasome inhibitors, including the peptide aldehydes (MG132, MG115, N-acetyl-leucyl-leucyl-norleucinal) and lactacystin, inhibited the degradation of short-lived proteins and increased markedly the levels of mRNAs encoding cytosolic heat-shock proteins (Hsp70, polyubiquitin) and ER chaperones (BiP, Grp94, ERp72), as shown by Northern blot analysis . However, inhibitors of cysteine proteases (E64), serine proteases (leupeptin), or metalloproteases (1, 10-phenanthroline) had no effect on the levels of these mRNAs . The relative efficacies of the peptide aldehyde inhibitors in inducing these mRNAs correlated with their potencies against the proteasome . Furthermore, reduction of the aldehyde group of MG132 decreased its inhibitory effect on proteolysis and largely prevented the induction of these mRNAs . Although treatment with the proteasome inhibitors caused rapid increases in mRNA levels (as early as 2 h after treatment with MG132), the inhibitors did not detectably affect total protein synthesis, total protein secretion, ER morphology, or the retention of ER-lumenal proteins, even after 18 h of treatment . Together, the findings suggest that inhibition of proteasome function induces heat-shock proteins and ER chaperones due to the accumulation of sufficient amounts of abnormal proteins and/or the inhibition of degradation of a key regulatory factor (e.g . heat-shock factor) . Since expression of heat-shock proteins can protect cells from thermal injury, we tested whether the proteasome inhibitors might also confer thermotolerance . Treatment of cells with MG132 for as little as 2 h, markedly increased the survival of cells subjected to high temperatures (up to 46 degrees C) . Thus, these agents may have applications in protecting against cell injury. J Biol Chem, 1997 Apr 4, 272(14), 8918 - 23 An 18-kDa domain of a glycosylphosphatidylinositol-linked NAD:arginine ADP-ribosyltransferase possesses NAD glycohydrolase activity; Kim HJ et al.; Transfection of NMU (rat mammary adenocarcinoma) cells with NAD:arginine ADP-ribosyltransferase cDNAs from Yac-1 murine lymphoma cells or rabbit muscle increased NAD glycohydrolase and ADP-ribosyltransferase activities . The ADP-ribosyltransferase activity was released from transformed NMU cells by phosphatidylinositol-specific phospholipase C (PI-PLC) and hence glycosylphosphatidylinositol (GPI)-anchored, whereas the NAD glycohydrolase (NADase) activity remained cell-associated . By gel permeation chromatography, the size of the PI-PLC-released transferase was approximately 40 kDa and that of the detergent-solubilized NADase was approximately 100 kDa . Using polyclonal antibodies against rabbit muscle transferase on Western blots, approximately 18- and approximately 30-kDa band were visualized among proteins from the NADase fractions and 38-40-kDa bands with protein from the transferase fractions . Incubation of blots with {32P}NAD led to the incorporation of radioactivity into the immunoreactive transferase bands of 38 kDa and the immunoreactive NADase band of approximately 18 kDa . These data suggest that proteolysis of ADP-ribosyltransferase synthesized in transformed NMU cells might result in the formation of aggregates of an 18-kDa NAD glycohydrolase . A fusion protein with glutathione S-transferase linked to the amino terminus of Yac-1 transferase, from which the amino-terminal 121 amino acids had been deleted (GST-Yac-1-delta121), exhibited NADase, but not transferase, activity . The size of the recombinant fusion protein was similar to that of the proteolytic fragment seen in NMU cells transformed with transferase cDNA . These results are compatible with the conclusion that the NAD glycohydrolase activity was generated in NMU cells by proteolysis of ADP-ribosyltransferase, with release of a carboxyl-terminal fragment that possesses glycohydrolase but not transferase activity, i.e . the carboxyl-terminal portion of the transferase can exist as a catalytically active NADase. J Biol Chem, 1997 Apr 4, 272(14), 8901 - 4 RNA-diethylstilbestrol interaction studied by Fourier transform infrared difference spectroscopy; Neault JF et al.; Diethylstilbestrol (DES), a synthetic estrogen, is known to be a carcinogen in human and in animals . This study was designed to examine the interaction of DES with yeast RNA in aqueous solution at physiological pH with drug/RNA-phosphate (P) molar ratios of 1/80, 1/40, 1/20, 1/10, 1/4, and 1/2 . Fourier transform infrared (FTIR) difference spectroscopy was used to determine the drug binding mode, the binding constant, the sequence selectivity, and RNA secondary structure in the RNA.DES complexes . Spectroscopic evidence showed that at low drug concentration (1/80 and 1/40), DES is intercalating through both Gua-Cyt and Ade-Urd base pairs with minor interaction with the backbone PO2 group (external binding) . The calculated binding constant of K approximately 8.5 x 10(4) M-1 at a drug concentration of 3.12 x 10(-4) M shows that DES is a weaker intercalator than those of the methylene blue, acridine orange, and ethidium bromide . At high drug content (r > 1/40, where r represents the DES/RNA-phosphate molar ratio), a partial helix destabilization occurs with no alteration of RNA conformation upon drug complexation . However, a comparison with DNA.DES complexes showed that drug intercalation causes major reduction of the B-DNA structure in favor of A-DNA with no participation of the backbone PO2 group in the DES . DNA complexation. J Biol Chem, 1997 Apr 4, 272(14), 8878 - 84 A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin; Ma Q et al.; To identify new proteins involved in dioxin-dependent signal transduction and transcriptional regulation, we used a yeast two-hybrid system to identify proteins that interact with the Ah receptor (AhR) . We cloned a mouse cDNA, which encodes a novel approximately 37-kDa protein that binds to AhR; we have designated the protein as Ah receptor-interacting protein (AIP) . The amino acid sequence of mouse AIP exhibits homology with members of the FK506-binding protein family . AIP also contains three tetratricopeptide repeat (TPR) motifs; the TPR sequence is present in proteins required for cell cycle control and RNA synthesis and in steroid receptor-binding immunophilins . Coimmunoprecipitation experiments in mouse hepatoma cells reveal that AIP is cytoplasmic and associates with unliganded Ah receptor and with hsp90; 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment disrupts the AhR-AIP-hsp90 interaction . Overexpression of AIP augments the response of the CYP1A1 gene to 2,3,7,8-tetrachlorodibenzo-p-dioxin . Our data suggest that AIP influences ligand receptivity and/or nuclear targeting of AhR. Science, 1997 Apr 4, 276(5309), 126 - 31 Flexibility in DNA recombination: structure of the lambda integrase catalytic core; Kwon HJ et al.; Lambda integrase is archetypic of site-specific recombinases that catalyze intermolecular DNA rearrangements without energetic input . DNA cleavage, strand exchange, and religation steps are linked by a covalent phosphotyrosine intermediate in which Tyr342 is attached to the 3'-phosphate of the DNA cut site . The 1.9 angstrom crystal structure of the integrase catalytic domain reveals a protein fold that is conserved in organisms ranging from archaebacteria to yeast and that suggests a model for interaction with target DNA . The attacking Tyr342 nucleophile is located on a flexible loop about 20 angstroms from a basic groove that contains all the other catalytically essential residues . This bipartite active site can account for several apparently paradoxical features of integrase family recombinases, including the capacity for both cis and trans cleavage of DNA. Biochim Biophys Acta, 1997 Apr 3, 1325(1), 117 - 25 A protein disulfide-thiol interchange activity of HeLa plasma membranes inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl) urea (LY181984) Morre DJ, Jacobs E, Sweeting M, de Cabo R, Morre DM. Plasma membrane vesicles isolated from HeLa cells grown in suspension culture contain a protein disulfide-thiol interchange (protein disulfide-like) activity . The activity was estimated from the restoration of activity to inactive (scrambled) pancreatic RNAase . RNAase activity was measured either by hydrolysis of cCMP or by a decrease in acid precipitable yeast RNA . The ability of plasma membrane vesicles to restore activity to inactive (scrambled) pancreatic ribonuclease was inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984) . The activity correlated with that of a cyanide-resistant NADH oxidase also associated with the plasma membrane vesicles that exhibited a similar pattern of drug response . The activity was stimulated by reduced glutathione and inhibited by oxidized glutathione but did not depend on either for activity . The antitumor sulfonylurea-inhibited activity was greatest in the presence of reduced glutathione and least in the presence of oxidized glutathione . The antitumor sulfonylurea-inhibited activity was unaffected by a monoclonal antibody to protein disulfide isomerase . Also the antitumor sulfonylurea-inhibited activity was unaffected by peptide antisera to the consensus active site sequence of protein disulfide isomerase . Thus the antitumor sulfonylurea-inhibited activity appeared to reside with a novel cell surface protein capable of oxidation of both NADH and protein thiols and of carrying out a protein disulfide isomerase-like protein disulfide-thiol interchange activity in the absence of NADH or other external reductants. Yi Chuan Xue Bao, 1997 Apr, 24(2), 99 - 108 {Some YAC contig construction and long range physical mapping at human X chromosome Xp11.3-21.3}; Miao WM et al.; Human X chromosome short arm Xp11.3-p21.3 is an area, where several genetic disease gene loci are located . In this work, the YAC conting construction, long range physical mapping were done for this region . Some DNA probes and STS markers were used for YAC screening . Totally 77 YACs were obtained from the YAC libraries of CEPH, ICRF and ours . The size determination, 26 pairs of microsatelite STS analysis, single copy probe hybridization, Alu-PCR finger printing and long range physical mapping were conducted with these YACs . These results allowed us to map these YACs, and finally 6 YAC contigs were obtained in Xp11.3-21.3, covering about 15.3 Mb . This work will greatly facilitate the positional cloning of disease genes or the genome sequencing in this important region. J Bioenerg Biomembr, 1997 Apr, 29(2), 151 - 63 Human cytochrome c oxidase: structure, function, and deficiency; Taanman JW; As the terminal component of the mitochondrial respiratory chain, cytochrome c oxidase plays a vital role in cellular energy transformation . Human cytochrome c oxidase is composed of 13 subunits . The three major subunits form the catalytic core and are encoded by mitochondrial DNA (mtDNA) . The remaining subunits are nuclear-encoded . The primary sequence is known for all human subunits and the crystal structure of bovine heart cytochrome c oxidase has recently been reported . However, despite this wealth of structural information, the role of the nuclear encoded subunits is still poorly understood . Yeast cytochrome c oxidase is a close model of its human counterpart and provides a means of studying the effects of mutations on the assembly, structure, stability and function of the enzyme complex . Defects in cytochrome c oxidase function are found in a clinically heterogeneous group of disorders . The molecular defects that underlie these diseases may arise from mutations of either mitochondrial or the nuclear genomes or both . A significant number of cytochrome c oxidase deficiencies, often associated with other respiratory chain enzyme defects, are attributed to mutations of mtDNA . Mutations of mtDNA appear, nonetheless, uncommon in early childhood . Pedigree analysis and cell fusion experiments have demonstrated a nuclear involvement in some infantile cases but a specific genomic lesion has not yet been reported . Detailed analyses of the many steps involved in the biogenesis of cytochrome c oxidase, often pioneered in yeast, offer several starting points for further molecular characterizations of cytochrome c oxidase deficiencies observed in clinical practice. Mech Dev, 1997 Apr, 63(1), 29 - 38 Cloning and expression analysis of a novel mouse gene with sequence similarity to the Drosophila fat facets gene; Wood SA et al.; The Drosophila fat facets (faf) gene is a ubiquitin-specific protease necessary for the normal development of the eye and of the syncytial stage embryo in the fly . Using a gene trap approach in embryonic stem cells we have isolated a murine gene with extensive sequence similarity to the Drosophila faf gene and called it Fam (fat facets in mouse) . The putative mouse protein shows colinearity and a high degree of sequence identity to the Drosophila protein over almost its entire length of 2554 amino acids . The two enzymatic sites characteristic of ubiquitin-specific proteases are very highly conserved between mice and Drosophila and this conservation extends to yeast . Fam is expressed in a complex pattern during postimplantation development . In situ hybridisation detected Fam transcripts in the rapidly expanding cell populations of gastrulating and neurulating embryos, in post-mitotic cells of the CNS as well as in the apoptotic regions between the digits, indicating that it is not associated with a single developmental or cellular event . The strong sequence similarity to faf and the developmentally regulated expression pattern suggest that Fam and the ubiquitin pathway may play a role in determining cell fate in mammals, as has been established for Drosophila. Biochem J, 1997 Apr 1, 323 ( Pt 1), 197 - 206 An apparent association between glycosylphosphatidylinositol-anchored proteins and a sphingolipid in Tetrahymena mimbres; Zhang X et al.; Sphingolipids are thought to stabilize glycosylphosphatidylinositol (GPI)-anchored protein-rich membrane domains of yeast and polarized higher animal cells during the processing and targeting of these proteins to the plasma membrane . A widely used criterion for identifying the stable sphingolipid- and GPI-anchored protein-enriched membrane domains is the resistance of these lipid-modified proteins to solubilization by the detergent Triton X-100 (TX-100) at low temperature . Surprisingly, there have been no reports of sphingolipid/GPI-anchored protein association in protozoans, despite the fact that these cells contain considerably higher levels of GPI-anchored proteins than does any other organism . We report here the presence in Tetrahymena mimbres of a significant pool of GPI-anchored proteins which resisted extraction by 1% TX-100 at 4 degrees C but not at 37 degrees C . Of the total cellular complement of GPI-anchored proteins, which together accounted for more than 2% of whole-cell protein and were especially enriched in surface membranes, 10% of the major 63kDa component (gpi63) and 23% of a somewhat less abundant component (gpi23) were insoluble in TX-100 at 4 degrees C . A substantial proportion of the cell's only abundant sphingolipid, ceramideaminoethylphosphonate (CAEP), was also insoluble in 1% TX-100 at 4 degrees C . Radiolabelling studies involving {3H}leucine incorporation into proteins and {3H}palmitic acid incorporation into lipids revealed that the TX-100-resistant gpi63, gpi23 and CAEP molecules were all metabolically distinct from their TX-100-soluble counterparts in other compartments of the cell . The presence of detergent-resistant sphingolipid/GPI-anchored protein domains in non-polarized ciliate and trypanosomatid cells was probably obscured in previous studies by the profusion of accompanying detergent-soluble molecules. Biochem Mol Med, 1997 Apr, 60(2), 169 - 73 Identification of a ferritin light chain pseudogene near the glycerol kinase locus in Xp21 by cDNA amplification for identification of genomic expressed sequences; Guo W et al.; We used cDNA amplification for identification of genomic expressed sequences (CAIGES) to identify genes in the glycerol kinase region of the human X chromosome . During these investigations we identified the sequence for a ferritin light chain (FTL) pseudogene in this portion of Xp21 . A human liver cDNA library was amplified by vector primers, labeled, and hybridized to Southern blots of EcoRI-digested human genomic DNA from cosmids isolated from yeast artificial chromosomes in the glycerol kinase region of Xp21 . A 3.1-kb restriction fragment hybridized with the cDNA library, was subcloned and sequenced, and a 440-bp intronless sequence was found with strong similarity to the FTL coding sequence . Therefore, the FTL pseudogene that had been mapped previously to Xp22.3-21.2 was localized specifically to the glycerol kinase region . The CAIGES method permits rapid screening of genomic material and will identify genomic sequences with similarities to genes expressed in the cDNA library used to probe the cloned genomic DNA, including pseudogenes. Br J Dermatol, 1997 Apr, 136(4), 490 - 3 Non-dermatophytes in onychomycosis of the toenails; Ellis DH et al.; A multicentre trial for the treatment of dermatophyte onychomycosis of the toenails with terbinafine was carried out in Australia and New Zealand . Between eight and 12 nail samples were obtained from each of the 118 patients in the 48-week trial, and each sample was investigated by direct microscopy and culture for dermatophyte and non-dermatophyte fungi . Patients were randomized to treatment with terbinafine at 250 mg/day or placebo for the first 12 weeks of the study, then non-responders were offered a 12-week course of terbinafine from week 28 . All patients had a dermatophyte infection . In 42 patients (36%) microscopy and mycological culture identified dermatophytes alone . In the remaining 76 patients (64%), a non-dermatophyte mould or yeast was also isolated at some stage during the trial, but in only three patients did the same non-dermatophyte persist in two or more successive nail specimens . The presence of a fungal contaminant in addition to a dermatophyte had no apparent effect on the efficacy of treatment with terbinafine . We conclude that non-dermatophyte moulds and yeasts are generally found as contaminating organisms in dermatophyte onychomycosis, secondary to the dermatophytes, and that they do not influence the outcome of treatment. J Cell Sci, 1997 Apr, 110 ( Pt 8), 1013 - 22 Characterization of the interactions of alpha-catenin with alpha-actinin and beta-catenin/plakoglobin; Nieset JE et al.; Cadherins are calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion . To function in cell-cell adhesion, the transmembrane cadherin molecule must be associated with the cytoskeleton via cytoplasmic proteins known as catenins . Three catenins, alpha-catenin, beta-catenin and gamma-catenin (also known as plakoglobin), have been identified . beta-catenin or plakoglobin is associated directly with the cadherin; alpha-catenin binds to beta-catenin/plakoglobin and serves to link the cadherin/catenin complex to the actin cytoskeleton . The domains on the cadherin and betacatenin/plakoglobin that are responsible for protein-protein interactions have been mapped . However, little is known about the molecular interactions between alpha-catenin and beta-catenin/plakoglobin or about the interactions between alpha-catenin and the cytoskeleton . In this study we have used the yeast two-hybrid system to map the domains on alpha-catenin that allow it to associate with beta-catenin/plakoglobin and with alpha-actinin . We also identify a region on alpha-actinin that is responsible for its interaction with alpha-catenin . The yeast two-hybrid data were confirmed with biochemical studies. Glycoconj J, 1997 Apr, 14(3), 345 - 56 Optimizing lectin-carbohydrate interactions: improved binding of divalent alpha-mannosylated ligands towards Concanavalin A; Page D et al.; The synthesis and binding properties to Jack bean phytohaemagglutinin in (Concanavalin A, Con A) of a new family of divalent alpha-D-mannopyranoside ligands are described . The synthesis of these ligands is based on the coupling of commercially available diamines to p-isothiocyanatophenyl 2,3,4,6 tetra-O-acetyl-alpha-D-mannopyranoside (4) . The resulting dimers 6, 15 to 22 and 30 were tested for their relative inhibitory potency by solid-phase enzyme-linked lectin assays (ELLA) using methyl alpha-D-mannopyranoside as standard . Divalent mannosylated ligand 35 bearing a non-aromatic aglycon was also tested for comparison purposes . Concentrations necessary for 50% inhibition (IC50s) of binding of yeast mannan to Jack bean phytohaemagglutinin (Con A) were determined . The inhibitions showed dimers to be approximately 10- to 90-fold more potent than methyl alpha-D-mannopyranoside . Variations in the intra-mannosyl distance proved to be an important factor for optimum binding. Comput Appl Biosci, 1997 Apr, 13(2), 137 - 43 LASSAP, a LArge Scale Sequence compArison Package; Glemet E et al.; MOTIVATION: This paper presents LASSAP, a new software package for sequence comparison . LASSAP is a programmable, high-performance system designed to raise current limitations of sequence comparison programs in order to fit the needs of large-scale analysis . LASSAP provides an API (Application Programming Interface) allowing the integration of any generic pairwise-based algorithm . RESULTS: Whatever pairwise algorithm is used in LASSAP, it shares with all other algorithms numerous enhancements such as: (i) intra- and inter-databank comparisons; (ii) computational requests (selections and computations are achieved on the fly); (iii) frame translations on queries and databanks; (iv) structured results allowing easy and powerful post-analysis; (v) performance improvements by parallelization and the driving of specialized hardware . LASSAP currently implements all major sequence comparison algorithms (Fasta, Blast, Smith/Waterman), and other string matching and pattern matching algorithms . LASSAP is both an integrated software for end-users and a framework allowing the integration and the combination of new algorithms . LASSAP is used in different projects such as the building of PRODOM, the exhaustive comparison of yeast sequences, and the subfragments matching problem of TREMBL. Chromosome Res, 1997 Apr, 5(2), 118 - 24 Analysis of chromosome 6 deletions in lymphoid malignancies provides evidence for a region of minimal deletion within a 2-megabase segment of 6q21; Sherratt T et al.; Fluorescence in situ hybridization has been used to define deletion breakpoints within chromosome bands 6q16-21 in cases of lymphoid malignancy . Previous evidence suggested that the region might contain a tumour-suppressor gene . Six yeast artificial chromosome probes, each selected using a single marker, were localized to 6q16-21 and the following order was confirmed; D6S330-D6S283-D6S301-D6S447-D6S246-FYN+ ++ . Of 32 cases of lymphoid malignancy, 30 showed deletion of D6S246 and, in the two cases in which D6S246 was retained, the adjacent marker, D6S447, was deleted . These observations imply that a region of minimal deletion is located within a 2-megabase segment of 6q21, between D6S447 and D6S246, providing a candidate region for the location of a tumour-suppressor gene. FASEB J, 1997 Apr, 11(5), 322 - 30 Aging processes, DNA damage, and repair; Gilchrest BA et al.; The second triennial FASEB Summer Research Conference on "Clonal Senescence and Differentiation" (August 17-22, 1996) focused on the interrelationships between aging processes and DNA damage and repair . The attendees represented a cross section of senior and junior investigators working in fields ranging from classic cellular gerontology to yeast and nematode models of aging to basic mechanisms of DNA damage and repair . The meeting opened with a keynote address by Dr . Bruce Ames that emphasized the documented relationships between oxidative damage, cancer, and aging . This was followed by eight platform sessions, one poster discussion, one featured presentation, and an after-dinner address . The following sections highlight the key points discussed. J Med Genet, 1997 Apr, 34(4), 343 - 5 Physical localisation of the breakpoints of a constitutional translocation t(5;6)(q21;q21) in a child with bilateral Wilms' tumour; Hoban PR et al.; A 6 month old boy presented with bilateral Wilms' tumour . Cytogenetic analysis of the lymphocytes from the patient showed a de novo balanced translocation t(5;6)(q21;q21), which was also present in the tumour material as the sole cytogenetic abnormality . To facilitate the identification of the translocation breakpoints, we have established a lymphoblastoid cell line (MA214L) from the patient which maintains the translocation in culture . We have used Genethon microsatellite markers as sequence tagged sites (STSs) to isolate yeast artificial chromosome (YAC) clones to 5q and 6q from human genomic libraries . Using fluorescence in situ hybridisation (FISH) on metaphase preparations of MA214L, we have physically defined the translocation breakpoints between YAC clones on each chromosome arm . The genetic distance separating the flanking YACs on 6q21 is 3 cM, while that on 5q21 is 4 cM . To date this is the first report of these chromosomal regions being implicated in Wilms' tumourigenesis. J Med Genet, 1997 Apr, 34(4), 335 - 9 A 4 Mb cryptic deletion associated with inv(8)(q13.1q24.11) in a patient with trichorhinophalangeal syndrome type I; Sasaki T et al.; We report on an 11 year old girl with trichorhinophalangeal syndrome type I (TRPS1), postaxial polydactyly of the fingers, and a de novo paracentric inversion on the long arm of chromosome 8 involving bands q13.1 and q24.11 . Molecular analysis using FISH and polymorphic DNA markers detected an approximately 4 Mb, cytogenetically unidentified deletion occurring between two STSs markers, AFMB331YA9 and D8S1200, around the region of the distal inversion breakpoint . Although the deletion is large, mental retardation was not present in the patient . This is the first report of a cryptic deletion in a TRPS1 patient, both ends of which were analysed at the molecular level . The data obtained are useful for defining the location of the putative mental retardation gene(s) in TRPS1 and Langer-Giedion syndrome (TRPS2), as well as a locus for postaxial polydactyly. J Wildl Dis, 1997 Apr, 33(2), 285 - 9 Lung parasites of shrews from Pennsylvania; Laakkonen J et al.; We examined lung parasites of three species of soricids, Sorex cinereus (n = 58), Sorex fumeus (n = 23) and Blarina brevicauda (n = 45) collected from Pennsylvania (USA), from 1990 to 1995 . Yeast-like cells of Hisfoplasma capsulatum var . capsulatum were found in lung sections stained with Grocott's modification of Gomori's methenamine silver, periodic acid-Schiff, Giemsa, and hematoxylin-eosin in two (3%) S . cinereus, eight (35%) S . fumeus and two (4%) B . brevicauda . The number of spores of H . capsulatum in the lungs was low and no inflammatory reaction was evident . The infection was not disseminated to other organs . This is the first report of H . capsulatum infection in any species of shrews of the genus Sorex and the prevalence in S . fumeus was remarkably high compared to those reported for other wild mammals . A nematode, possibly Angiostrongylus michiganensis, was found in the lungs of one S . fumeus on necropsy and in a stained lung section of one S . cinereus . In both cases the host was also infected with the fungus . Pneumocystis carinii, which is the most common lung parasite in Sorex araneus (the numerically dominant Eurasian species of shrew), was not found in any of the North American species of shrew examined in this study. EMBO J, 1997 Apr 1, 16(7), 1610 - 9 Lyn tyrosine kinase is essential for erythropoietin-induced differentiation of J2E erythroid cells; Tilbrook PA et al.; Erythropoietin stimulates the immature erythroid J2E cell line to terminally differentiate and maintains the viability of the cells in the absence of serum . In contrast, a mutant J2E clone (J2E-NR) fails to mature in response to erythropoietin; however, it remains viable in the presence of the hormone . We have shown previously that intracellular signalling is disrupted in the J2E-NR cell line and that tyrosine phosphorylation is dramatically reduced after erythropoietin stimulation . In this study we investigated the defect in J2E-NR cells that is responsible for their inability to differentiate . Screening of numerous signalling molecules revealed that the lyn tyrosine kinase appeared to be absent from J2E-NR cells . On closer examination, both lyn mRNA and protein content were reduced >500-fold . Consistent with a defect in lyn, amphotropic retroviral infection of J2E-NR cells with lyn restored the ability of the cells to synthesize haemoglobin and enabled the cells to mature morphologically . Conversely, the ability of J2E cells to differentiate in response to epo was severely curtailed when antisense lyn oligonucleotides or a dominant negative lyn were introduced into the cells . However, erythropoietin-supported viability was unaffected by reducing lyn activity . The ability of two other erythropoietin-responsive cell lines (R11 and R24) to differentiate in response to the hormone was also reduced by dominant negative lyn . Finally, co-immunoprecipitation and yeast two-hybrid analyses indicated that lyn directly associated with the erythropoietin receptor complex . These data indicate for the first time an essential role for lyn in erythropoietin-initiated differentiation of J2E cells but not in the maintenance of cell viability. Eur J Biochem, 1997 Apr 1, 245(1), 156 - 63 Differential expression of three Arabidopsis genes encoding the B' regulatory subunit of protein phosphatase 2A; Latorre KA et al.; Numerous plant processes ranging from signal transduction to metabolism appear to be mediated, in part, by type 2A protein serine/threonine phosphatases (PP2A) . In an effort to identify factors that control the activity of this enzyme in plants, we have isolated and characterized DNA sequences encoding the B' regulatory subunit of PP2A from Arabidopsis thaliana . Specifically, we used PCR to amplify a segment of Arabidopsis cDNA that encodes a conserved section of the B' polypeptide . This PCR fragment was subsequently used as a probe to screen an Arabidopsis cDNA library and cDNA clones derived from three distinct genes were identified . The AtB' alpha and AtB' beta genes encode highly similar 57-kDa B' regulatory subunits while the third gene, AtB' gamma, encodes a more divergent 59-kDa B' protein . A comparison of the three Arabidopsis B' polypeptides to those of yeast and animals shows the core region of this protein to be the most conserved while the amino and carboxy termini vary both in length and sequence . Genomic Southern blots indicate that at most the Arabidopsis genome contains five genes encoding the B' regulatory subunit . The three genes identified in this study are expressed in all Arabidopsis organs, albeit at varying levels . In addition, mRNAs derived from the three genes accumulate differentially in response to heat shock . Our results indicate that the activity of plant PP2A might be regulated by a B' type regulatory subunit similar to those found in animals and yeast, and suggest possible roles for B'-containing PP2A complexes within plant cells. J Med Microbiol, 1997 Apr, 46(4), 321 - 5 GM-CSF-modulated phagocytosis of Trichosporon beigelii by human neutrophils; Richardson MD et al.; Trichosporon beigelii has emerged as a lethal opportunist pathogen in granulocytopenic and corticosteroid-treated patients . Little is known of the host defence mechanisms against this yeast . The interaction between human neutrophils and serum-opsonised T . beigelii and the effect of GM-CSF on binding and ingestion of the yeast by neutrophils were investigated by a microscopic analysis of neutrophil monolayers stained with FITC-Concanavalin A . Positive staining with FITC-Concanavalin A distinguished between intracellular and extracellular yeast cells . Binding of T . beigelii to neutrophils was an energy- and complement-dependent process involving movement of actin in the neutrophil cytoskeleton . The mean percentage binding of T . beigelii was 37.5% and the mean binding index (BI) was 1.30 whereas the mean percentage ingestion was 3.5% and the mean phagocytic index (PI) was 1.34 . GM-CSF increased percentage ingestion of T . beigelli from 2.8% to 30.5% and the PI was increased from 1.3 to 1.86 . The percentage binding was 36.8% and the mean BI was 1.3 in control experiments compared with 49.3% and 1.6, respectively, in the presence of GM-CSF . In conclusion, GM-CSF significantly increased percentage ingestion of opsonised T . beigelii by neutrophils, but its effect on percentage binding of the yeast was not statistically significant. Genomics, 1997 Apr 1, 41(1), 84 - 92 The XRCC2 DNA repair gene: identification of a positional candidate; Tambini CE et al.; The human XRCC2 gene, complementing a hamster cell line (irs1) hypersensitive to DNA-damaging agents, was previously mapped to chromosome 7q36.1 . Following radiation reduction of human/hamster hybrids, the gene was found to be associated with the marker D7S483 . Yeast artificial chromosomes (YACs) carrying D7S483 were fused to the irs1 cell line to identify a YAC that complemented the sensitivity defect . Transcribed sequences were isolated by direct cDNA selection using the complementing YAC, and these were mapped back to the YAC and hybrids to define a 400-kb region carrying XRCC2 . Sequencing of cDNAs led to the identification of both known and novel gene sequences, including a candidate for XRCC2 with homology to the yeast RAD51 gene involved in the recombinational repair of DNA damage . Strong support for the candidacy of this gene was obtained from its refined map position and by the full complementation of irs1 sensitivity with a 40-kb cosmid carrying the gene. Genomics, 1997 Apr 1, 41(1), 49 - 55 A high-density STS map based on a single contig of YAC and P1 clones in the chromosome 8p12-p21 region; Mitsuda N et al.; We have constructed a yeast artificial chromosome (YAC) and P1 contig in the 8p12-p21 region . The contig comprises 16 overlapping YAC clones and 44 overlapping P1 clones . Twelve dinucleotide-repeat polymorphic sequence-tagged site (STS)-markers that were previously isolated mainly from these YAC and P1 clones were genetically mapped . A total of 46 nonpolymorphic STS markers were newly established mainly from the YAC and P1 clone end fragments, and 28 of the 46 nonpolymorphic STSs, as well as the 12 polymorphic STSs, were also mapped physically onto the contig based on STS content analysis of YAC pools and of the P1 and YAC clones . As a result, the YAC and P1 clones were assembled into a single contig covering a minimum of 1.5 Mb physically and 2.8 cM genetically with 12 polymorphic and 28 nonpolymorphic STSs within the 8p12-p21 region . Average STS spacing in the contig was estimated to be 40 kb/STS . In addition, further characterization of the contig suggested that this contig includes a region where genetic recombination occurs frequently . Thus, the resulting cloned region, together with densely mapped STS markers on the contig, should help to promote our understanding of this region. Blood, 1997 Apr 1, 89(7), 2516 - 22 11q deletions identify a new subset of B-cell chronic lymphocytic leukemia characterized by extensive nodal involvement and inferior prognosis; Dohner H et al.; Deletions of the long arm of chromosome 11 (11q) are one of the most frequent structural chromosome aberrations in various types of lymphoproliferative disorders . However, in most conventional chromosome banding studies of B-cell chronic lymphocytic leukemia (B-CLL), 11q deletions were not identified as a frequent aberration . The objective of this study was to analyze the frequency and clinical impact of 11q deletions in B-CLL by interphase cytogenetics using fluorescence in situ hybridization (FISH) . Mononuclear cells from 214 patients with B-CLL were studied by FISH using the yeast artificial chromosome (YAC) clone 755b11 from chromosome region 11q22.3-q23.1; we previously showed that this clone was contained within a 2- to 3-Mb sized segment of 11q commonly deleted in lymphoproliferative disorders . Forty-three of the 214 (20%) tumors exhibited 11q deletions; 11q deletions were the second most frequent chromosome aberration following 13q14 (RB1 and/or D13S25) deletions (45%); they were more frequent than trisomy 12 (15%) or deletion of 17p (TP53 gene) (10%) . Patients with 11q deletions were younger (P = .01) and had more advanced clinical stages (P = .01) . 11q deletions were associated with extensive peripheral, abdominal, and mediastinal lymphadenopathy (P < .001) . Patients with 11q deletions had a more rapid disease progression as shown by a shorter treatment-free interval (9 months v 43 months; P < .001) . The prognostic effect of 11q deletion on survival strongly depended on the age: in patients less than 55 years old, the median survival time was significantly shorter in the deletion group (64 months v 209 months; P < .001), whereas in patients > or = 55 years old there was no significant difference (94 months v 111 months; P = .82) . 11q deletions identify a new clinical subset of B-CLL characterized by extensive lymph node involvement . In younger B-CLL patients, this aberration is an important predictor of survival. Curr Opin Genet Dev, 1997 Apr, 7(2), 264 - 73 Centromeres, checkpoints and chromatid cohesion; Allshire RC; An emerging view is that the formation of active centromeres is modulated in an epigenetic manner reflecting the association of centromeres with heterochromatin . Support for this comes from studies on fission yeast centromeres, the properties of human neocentromeres and dicentric chromosomes, and analyses of Drosophila minichromosome deletion derivatives . A link has been established between tension across kinetochores and the phosphorylation status of kinetochore components . Vertebrate homologues of yeast MAD2 have recently been isolated and localized to kinetochores, indicating that components of the spindle integrity checkpoint are conserved . The linkage between sister chromatids is only dissolved at anaphase during mitotic and meiotic divisions . Phenotypic and localization data combined with their pattern of rapid degradation at anaphase have implicated several yeast and Drosophila proteins in aspects of sister chromatid cohesion. Curr Opin Genet Dev, 1997 Apr, 7(2), 249 - 58 PcG complexes and chromatin silencing; Pirrotta V; Silencing complexes in yeast and in the fly have many similarities . This repressive complex is assembled by a chain of recruitment; its extent and stability depend on the concentration of components and affect an extended chromatin region, probably through interactions with nucleosomes . Recent results show that assembly of the complex is antagonized by transcriptional activity in the region but is favored by interactions with other complexes nearby or in other regions that associate in the same nuclear environment . How such a complex interferes with transcriptional activity is not entirely clear but current evidence suggests that they compete with the chromatin structure required for the binding of activators. Carcinogenesis, 1997 Apr, 18(4), 605 - 10 Which DNA polymerases are used for DNA-repair in eukaryotes? Wood RD, Shivji MK. There are five well-characterized nuclear DNA polymerases in eukaryotes (DNA polymerases alpha, beta, delta, epsilon and zeta) and this short review summarizes our current knowledge concerning the participation of each in DNA-repair . The three major DNA excision-repair pathways involve a DNA synthesis step that replaces altered bases or nucleotides removed during repair . Base excision-repair removes many modified bases and abasic sites, and in mammalian cells this mainly involves DNA polymerase beta . An alternative means for completion of base excision-repair, involving DNA polymerases delta or epsilon, may also operate and be even more important in yeast . Nucleotide excision-repair uses DNA polymerases delta or epsilon to resynthesize the bases removed during repair of pyrimidine dimers and other bulky adducts in DNA . Similarly, mismatch-repair of replication errors appears to involve DNA polymerases delta or epsilon . DNA polymerase alpha is required for semi-conservative replication of DNA but not for repair of DNA . A more recently discovered enzyme, DNA polymerase zeta, appears to be involved in the bypass of damage, without excision, and occurs during DNA replication of a damaged template. Genome Res, 1997 Apr, 7(4), 368 - 77 The E6-Ap ubiquitin-protein ligase (UBE3A) gene is localized within a narrowed Angelman syndrome critical region; Sutcliffe JS et al.; Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are distinct clinical phenotypes resulting from maternal and paternal deficiencies, respectively, in human chromosome 15qll-q13 . Although several imprinted, paternally expressed transcripts have been identified within the PWS candidate region, no maternally expressed gene has yet been identified within the AS candidate region . We have developed an integrated physical map spanning the PWS and AS candidate regions and localized two breakpoints, including a cryptic t(14;15) translocation associated with AS and a non-AS 15q deletion, which substantially narrow the AS candidate region to approximately 250 kb . Mapping data indicate that the entire transcriptional unit of the E6-AP ubiquitin-protein ligase (UBE3A) gene lies within the AS region . The UBE3A locus expresses a transcript of approximately 5 kb at low to moderate levels in all tissues tested . The mouse homolog of UBE3A was cloned and sequenced revealing a high degree of conservation at nucleotide and protein levels . Northern and RT-PCR analysis of Ube3a expression in mouse tissues from animals with segmental, paternal uniparental disomy failed to detect substantially reduced or absent expression compared to control animals, failing to provide any evidence for maternal-specific expression from this locus . Recent identification of de novo truncating mutations in UBE3A taken with these observations indicates that mutations in UBE3A can lead to AS and suggests that this locus may encode both imprinted and biallelically expressed products. Genome Res, 1997 Apr, 7(4), 339 - 52 Cloning, characterization, and copy number of the murine survival motor neuron gene: homolog of the spinal muscular atrophy-determining gene; DiDonato CJ et al.; Because of a 500-kb inverted duplication, there are two copies of the survival motor neuron (SMN) gene in humans, cenSMN and telSMN . Both genes produce identical ubiquitously expressed transcripts; however, only mutations in telSMN are responsible for spinal muscular atrophy (SMA), the second most common autosomal recessive childhood disease . We have cloned the murine homolog Smn and mapped the gene to Chromosome 13 within the conserved syntenic region of human chromosome 5q13 . We show that the Smn transcript (1.4 kb) is expressed as early as embryonic day 7 . In contrast to humans, we found no evidence of alternative splicing . The predicted amino acid sequence between mouse and human SMN is 82% identical, and a putative nuclear localization signal is conserved . FISH data indicate that the duplication of the SMA region observed in humans is not present in the mouse . We also found no evidence of multiple Smn genes using Southern blot hybridization and single-strand conformation analysis . Using these methods, we detected at least four copies of Naip exon 5 clustering distal to Smn . Finally, three biallelic markers were identified within the Smn coding region; two are silent polymorphisms, whereas the third changes a cysteine residue to a tyrosine residue in exon 7 . Overall, our results indicate that Smn is single copy within the mouse genome, which should facilitate gene disruption experiments to create an animal model of SMA. Genome Res, 1997 Apr, 7(4), 307 - 14 Evolutionary features of the 4-Mb Xq21.3 XY homology region revealed by a map at 60-kb resolution; Mumm S et al.; Forty-three yeast artificial chromosomes (YACs) from the X chromosome have been overlapped across the 4-Mb Xq21.3 region, which is homologous to a segment in Yp11.1 . The region is formatted to 60-kb resolution with 57 STSs and is merged at its edges with contigs specific for X . This allows a direct comparison of marker orders and distances on X and Y . In addition to some sequence variation and possible differences in marker order, two larger evolutionary divergencies between the X and Y homologs were revealed: (1) The X homolog is interrupted by a small X-specific region detected by a 3-kb plasmid probe for locus DXS214 . An STS was developed from one end of the probe, but the sequence at the other end was highly homologous to an L1 repetitive element . This suggests that the interpolation of the X-specific segment may have involved an L1-mediated event . (2) A 250-kb portion containing DXYS1 is several megabases away from the rest of the homologous DNA on the Y but is contiguous with the remainder of the homologous region on X . Marker orders are consistent with the origin of the Y-specific 250-kb region in a paracentric inversion after the initial transfer of X DNA to the Y chromosome. Am J Hum Genet, 1997 Apr, 60(4), 860 - 8 Molecular characterization of patients with 18q23 deletions; Strathdee G et al.; The 18q- syndrome is a deletion syndrome that is characterized by mental retardation, hearing loss, midfacial hypoplasia, growth deficiency, and limb anomalies . Most patients with this syndrome have deletions from 18q21-qter . We report on three patients with deletions of 18q23 . A mother and daughter with identical deletions of 18q23 have many of the typical features of the 18q- syndrome, including midfacial hypoplasia and hearing loss . In contrast, the third patient has few of the symptoms of the 18q- syndrome . A contig of the 18q23 region was generated to aid in the mapping of the breakpoints . FISH was used to map both breakpoints to the same YAC clone . Furthermore, somatic-cell hybrids from the daughter and the third patient were isolated . The mapping results of sequence-tagged sites relative to the two breakpoints were identical, suggesting that the two deletion breakpoints map very close to one another . The analyses of these patients demonstrate that the critical region for the 18q- syndrome maps to 18q23 but that a deletion of 18q23 does not always lead to the clinical features associated with the syndrome . These patients demonstrate the wide phenotypic variability associated with deletions of 18q. FEMS Microbiol Lett, 1997 Apr 1, 149(1), 31 - 7 Induction of aldose reductase and xylitol dehydrogenase activities in Candida tenuis CBS 4435; Kern M et al.; In this study the ability of various sugars and sugar alcohols to induce aldose reductase (xylose reductase) and xylitol dehydrogenase (xylulose reductase) activities in the yeast Candida tenuis was investigated . Both enzyme activities were induced when the organism was grown on D-xylose or L-arabinose as well as on the structurally related sugars D-arabinose or D-lyxose . Mixtures of D-xylose with the more rapidly metabolizable sugar D-glucose resulted in a decrease in the levels of both enzymes formed . These results show that the utilization of D-xylose by C . tenuis is regulated by induction and catabolite repression . Furthermore, the different patterns of induction on distinct sugars suggest that the synthesis of both enzymes is not under coordinate control. Ann Pharmacother, 1997 Apr, 31(4), 445 - 56 Oral terbinafine: a new antifungal agent; Abdel-Rahman SM et al.; OBJECTIVE: To review the pharmacology, pharmacokinetics, efficacy, adverse effects, drug interactions, and dosage guidelines of terbinafine . Available comparative data of terbinafine and other antimycotic agents are described for understanding the potential role of terbinafine in patient care . DATA SOURCES: A MEDLINE search restricted to English language during 1966-1996 and extensive review of journals was conducted to prepare this article . MeSH headings included allylamines, terbinafine, SF 86-327, dermatophytosis, dermatomycosis . DATA EXTRACTION: The data on pharmacokinetics, adverse effects, and drug interactions were obtained from open-label and controlled studies and case reports . Controlled single- or double-blind studies were evaluated to describe the efficacy of terbinafine in the treatment of various fungal infections . DATA SYNTHESIS: Terbinafine is the first oral antimycotic in the allylamines class: a fungicidal agent that inhibits ergosterol synthesis at the stage of squalene epoxidation . Terbinafine demonstrates excellent in vitro activity against the majority of dermatophyte species including Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton floccosum; less activity is seen against Dematiaceae and the filamentous fungi . It is least active against the pathogenic yeast and this correlates with the relatively poor efficacy against these organisms in vivo . High concentrations of terbinafine are achieved in keratinous tissues, the site of superficial infections, and these concentrations are maintained for up to 3 months . The clinical efficacy of terbinafine against a number of dermatophyte infections exceeds that of the current standard of therapy, griseofulvin . The efficacy of terbinafine may be as good or better than that of the azole antifungals . Additional studies are required to confirm these observations . Terbinafine demonstrates a good safety profile, and relatively few drug interactions have been identified . CONCLUSIONS: Terbinafine is more effective than the gold standard, griseofulvin, in the treatment of tinea pedis and tinea unguinum, with considerably shorter treatment duration in the latter . It has been proven as effective as griseofulvin in the treatment of tinea capitis, tinea corporis, and tinea cruris . Terbinafine does not appear to offer any advantage in the treatment of nondermatophyte infections; its utility in the treatment of systemic infections has yet to be established . Depending on individual institutional costs, terbinafine may be a front-line drug for some superficial infections responding poorly to the current standard of therapy. Hum Genet, 1997 Apr, 99(4), 450 - 3 Molecular mapping of a translocation breakpoint at 14q13 in a patient with mirror-image polydactyly of hands and feet; Matsumoto N et al.; Mirror hands and feet (MIM, 135750) is a rare congenital anomaly, and mirror-image polydactyly is considered to be a variant of mirror hands and feet . To our knowledge, seven patients with the disorder have been reported in the literature . Parent-to-child transmission was reported in two families, which may indicate a single-gene defect inherited in an autosomal dominant fashion . We had previously encountered a boy with mirror-image polydactyly whose karyotype showed 46,XY,t(2;14) (p23.3;q13) de novo . We hypothesized that at least one of the putative genes responsible for the determination of an anterior-posterior limb pattern is disrupted by a translocation breakpoint . In this study, we identified a yeast artificial chromosome clone spanning a translocation breakpoint at 14q13, and the breakpoint was confirmed to be located between two loci, AFM200ZH4 and D14S306, within a genetic distance of 0.6 cM. Protein Sci, 1997 Apr, 6(4), 761 - 70 Structural comparison of cupredoxin domains: domain recycling to construct proteins with novel functions; Murphy ME et al.; The three-dimensional structures of the copper-containing enzymes ascorbate oxidase, ceruloplasmin, and nitrite reductase, comprised of multiple domains with a cupredoxin fold, are consistent with having evolved from a common ancestor . The presence or absence of copper sites has complicated ascertaining the structural and evolutionary relationship among these and related proteins . Simultaneous structural superposition of the enzyme domains and their known cupredoxin relatives shows clearly that there are at least six cupredoxin classes, and that the evolution of the conserved core of these domains is independent of the presence or absence of copper sites . Relationships among the variable loops in these structures show that the two-domain ancestor of the blue oxidases contained a trinuclear-copper interface but could not have functioned in a monomeric state . Comparison of the sequence of the copper-containing, iron-regulating protein . Ferrous transport (Fet3) from yeast to the structurally defined core and loop residues of the cupredoxins suggests specific residues that could be involved in the ferroxidase activity of Fet3. Hum Mol Genet, 1997 Apr, 6(4), 513 - 8 Identification of a self-association region within the SCA1 gene product, ataxin-1; Burright EN et al.; Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract within the SCA1 gene product, ataxin-1 . Expansion of this tract is believed to result in a gain of function by the mutant protein, perhaps through altered self-associations or interactions with other cellular proteins . We have used the yeast two hybrid system to determine if ataxin-1 is capable of multimerization . This analysis revealed that ataxin-1 does have the ability to self-associate, however, this association does not appear to be influenced by expansion of the polyglutamine tract . Consistent with this finding, deletion analysis excluded the involvement of the polyglutamine tract in ataxin-1 self-association, and instead localized the multimerization region to amino acids 495-605 of the wild type protein . These results, while identifying an ataxin-1 self-interaction region, fail to support a proposed model of polar-zipper mediated multimerization involving the ataxin-1 polyglutamine tract. Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 3145 - 50 Functional antagonism between RNA polymerase II holoenzyme and global negative regulator NC2 in vivo; Gadbois EL et al.; Activation of eukaryotic class II gene expression involves the formation of a transcription initiation complex that includes RNA polymerase II, general transcription factors, and SRB components of the holoenzyme . Negative regulators of transcription have been described, but it is not clear whether any are general repressors of class II genes in vivo . We reasoned that defects in truly global negative regulators should compensate for deficiencies in SRB4 because SRB4 plays a positive role in holoenzyme function . Genetic experiments reveal that this is indeed the case: a defect in the yeast homologue of the human negative regulator NC2 (Dr1 x DRAP1) suppresses a mutation in SRB4 . Global defects in mRNA synthesis caused by the defective yeast holoenzyme are alleviated by the NC2 suppressing mutation in vivo, indicating that yeast NC2 is a global negative regulator of class II transcription . These results imply that relief from repression at class II promoters is a general feature of gene activation in vivo. Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 3046 - 51 A conserved domain is present in different families of vesicular fusion proteins: a new superfamily; Weimbs T et al.; We have analyzed conserved domains in t-SNAREs {soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors in the target membrane}, proteins that are believed to be involved in the fusion of transport vesicles with their target membrane . By using a sensitive computer method, the generalized profile method, we were able to identify a new homology domain that is common in the two protein families previously identified to act as t-SNAREs, the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) families, which therefore constitute a new superfamily . This homology domain of approximately 60 amino acids is predicted to form a coiled-coil structure . The significance of this homology domain could be demonstrated by a partial suppression of the coiled-coil properties of the domain profile . In proteins belonging to the syntaxin family, a single homology domain is located near the transmembrane domain, whereas the members of the SNAP-25 family possess two homology domains . This domain was also identified in several proteins that have been implicated in vesicular transport but do not belong to any of the t-SNARE protein families . Several new yeast, nematode, and mammalian proteins were identified that belong to the new superfamily . The evolutionary conservation of the SNARE coiled-coil homology domain suggests that this domain has a similar function in different membrane fusion proteins. Nat Genet, 1997 Apr, 15(4), 385 - 8 Isolation of the human PEX12 gene, mutated in group 3 of the peroxisome biogenesis disorders; Chang CC et al.; The peroxisome biogenesis disorders (PBDs) are a group of genetically heterogeneous diseases lethal in early infancy . Although the clinical features of PBD patients may vary, cells from all PBD patients exhibit a defect in the import of one or more classes of peroxisomal matrix proteins . This cellular phenotype is shared by yeast pex mutants, and human orthologues of yeast PEX genes have been shown to be defective in some groups of PBD patients . We identified a putative human orthologue of ScPEX12 by screening the database of expressed sequence tags for cDNAs capable of encoding a protein similar to yeast Pex12p . Although its sequence similarity to yeast Pex12 proteins was limited, PEX12 shared the same subcellular distribution as yeast Pex12p and localized to the peroxisome membrane . PEX12 expression restored peroxisomal protein import in fibroblasts from PBD patients of complement group 3 (CG3) and frameshift mutations in PEX12 were detected in two unrelated CG3 patients . These data demonstrate that mutations in PEX12 are responsible for CG3 of the PBD and that PEX12 plays an essential role in peroxisomal matrix protein import. Nat Genet, 1997 Apr, 15(4), 369 - 76 Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic chondrodysplasia punctata; Braverman N et al.; Rhizomelic chondrodysplasia punctata (RCDP) is a rare autosomal recessive phenotype that comprises complementation group 11 of the peroxisome biogenesis disorders (PBD) . PEX7, a candidate gene for RCDP identified in yeast, encodes the receptor for peroxisomal matrix proteins with the type-2 peroxisome targeting signal (PTS2) . By homology probing we identified human and murine PEX7 genes and found that expression of either corrects the PTS2-import defect characteristic of RCDP cells . In a collection of 36 RCDP probands, we found two inactivating PEX7 mutations: one, L292ter, was present in 26 of the probands, all with a severe phenotype; the second, A218V, was present in three probands, including two with a milder phenotype . A third mutation, G217R, whose functional significance is yet to be determined, was present in five probands, all compound heterozygotes with L292ter . We conclude that PEX7 is responsible for RCDP (PBD CG11) and suggest a founder effect may explain the high frequency of L292ter. Genes Chromosomes Cancer, 1997 Apr, 18(4), 305 - 9 Physical mapping of the 5 Mb D9S196-D9S180 interval harboring the basal cell nevus syndrome gene and localization of six genes in this region; Xie J et al.; The basal cell nevus syndrome (Gorlin syndrome) is characterized by multiple basal cell carcinomas and diverse developmental defects . The gene responsible for this syndrome has been mapped previously to a 2 cM interval between D9S196 and D9S 180 at 9q22.3, and very recently mutations of a candidate gene in this region--the human homolog of the Drosophila patched gene have been identified . We report here on physical mapping studies integrating a contig of yeast artificial chromosomes and bacterial artificial chromosomes with a long-range map spanning approximately 5 Mb between the recombination-determined flanking markers . Six genes have been mapped to this interval. Genes Chromosomes Cancer, 1997 Apr, 18(4), 279 - 85 Malignant astrocytoma-derived region of common amplification in chromosomal band 17p12 is frequently amplified in high-grade osteosarcomas; Hulsebos TJ et al.; Recently, we reported a new amplification event that involves marker D17S67 in 17p12 in three malignant astrocytomas of patients with a very short survival . The amplified region may contain an oncogene implicated in astrocytoma tumorigenesis . To determine the extent of the amplified regions, we constructed a yeast artificial chromosome contig spanning the D17S67 region and tested the amplification status of markers that map to the contig . We determined a commonly amplified region between markers D17S1311 and D17S1875 with a maximal length of 1,630 kb . By using marker 745R, from within the commonly amplified region, we screened 60 high-grade astrocytomas but could not detect additional tumors with the amplification event . This suggests that the incidence of the amplification event in high-grade astrocytoma is low (5%) . It has recently been shown by comparative genomic hybridization that amplification of 17p11-p12 is a frequent event in high-grade osteosarcomas, occurring in 20-30% of cases . Since the commonly amplified region is within 17p12, we tested 745R in 20 osteosarcomas, including 6 lung metastases, and detected amplification in 9 cases (45%) . Marker 745R was found to be amplified in 4 of the 6 lung metastases (66%) . From this frequent involvement and the association with clinically aggressive astrocytomas we conclude that for both tumor types presence of the amplification event seems to correlate with aggressive clinical behaviour. Genes Chromosomes Cancer, 1997 Apr, 18(4), 254 - 68 A t(6;12)(q23;p13) results in the fusion of ETV6 to a novel gene, STL, in a B-cell ALL cell line; Suto Y et al.; ETV6 (TEL) is rearranged in various types of hematologic malignancies . The B-cell precursor acute lymphoblastic leukemia (ALL) cell line SUP-B2 has a t(6;12)(q23;p13) involving ETV6 at 12p13 and a submicroscopic deletion of the other ETV6 allele . The reciprocal translocation results in the fusion of ETV6 to a previously unknown gene at 6q23, which we named STL (six-twelve leukemia gene) . Both reciprocal fusion transcripts can be detected: On the der(6) chromosome, the ETV6/STL mRNA shows an apparently out of frame fusion of ETV6 at nucleotide 187 to STL, which would result in the addition of 14 amino acids to the first 54 amino acids of ETV6 . On the der(12) chromosome three different variants of the STL/ETV6 fusion mRNA could be detected; variable size segments were inserted at the breakpoint between STL and ETV6 exon 3 . One of these variants could give rise to a protein in which the first 54 amino acids of ETV6 are replaced by 12 amino acids from one of the STL short open reading frames . Sequence analysis of a 1.4 kb STL cDNA clone from a skeletal muscle library revealed no long open reading frames . This cell line will be very useful in studying the different mechanisms by which alterations of ETV6 contribute to leukemogenesis and in testing the hypothesis that ETV6 might act as a tumor suppressor gene. Curr Opin Biotechnol, 1997 Apr 1, 8(2), 189 - 94 Translational regulation of gene expression in plants; Cohen A et al.; Translational regulation plays a major role in plant gene expression . Recent advances have been made in understanding how this mechanism of control is directed in the chloroplast as well as in the cytoplasm . Our knowledge of translational regulation in plant mitochondria, however, is limited . Translational control of gene expression in yeast mitochondria suggests that similar mechanisms will also play a significant role in the expression of plant mitochondrial genes. Curr Opin Cell Biol, 1997 Apr, 9(2), 193 - 204 Protein tyrosine phosphatases in signal transduction; Neel BG et al.; Protein-tyrosyl phosphorylation, regulated by protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is a key cellular control mechanism . Until recently, little was known about PTPs . However, the past two years have witnessed an explosion of information about PTP structure, regulation and function . Crystal structures of several PTPs have provided insights into enzymatic mechanisms and regulation and suggested the design of 'substrate-trapping' mutants . Candidate homophilic and heterophilic ligands for transmembrane PTPs have been identified, and roles for transmembrane PTPs in regulating cell-cell interactions have been suggested . Finally, progress has been made in understanding signaling by Src homology 2 domain containing PTPs and PTPs controlling yeast osmoregulatory pathways. J Virol, 1997 Apr, 71(4), 3188 - 96 Nucleolar and nuclear localization properties of a herpesvirus bZIP oncoprote