Yeasts

J Biol Chem, 1997 Jan 10, 272(2), 1180 - 7
Protease footprinting analysis of ternary complex formation by human TFIIA; Hori R et al.; Transcription factor (TF) IIA performs two important regulatory functions during RNA polymerase II transcription: it is required for efficient binding of TFIID to a core promoter and it mediates the effects of upstream activators, both through direct interaction with the TATA box binding protein (TBP) . To begin studying how TFIIA mediates these effects, we used a highly sensitive protease footprinting methodology to identify surfaces of human TFIIA participating in TFIIA x TBP x TATA ternary complex formation . Chymotrypsin and proteinase K cleavage patterns of TFIIA bearing a 32P-end-labeled gamma subunit revealed that amino acids 59-73 were protected from cleavage both in the context of an immobilized ternary complex and in a binary complex with TBP alone . In contrast, amino acids 341-367 in the beta portion of a 32P-labeled alpha-beta subunit were protected in the ternary but not in the binary complex, implying that those residues interact with promoter DNA . The regions of human TFIIA identified by protease footprinting are homologous to and encompass the yeast TFIIA residues that contact TBP and DNA in the recently solved crystal structure of the yeast ternary complex . The conservation of the regions and residues mediating complex formation implies that yeast and human TFIIA employ the same mechanism to stabilize the binding of TFIID to a core promoter.

J Biol Chem, 1997 Jan 10, 272(2), 1171 - 9
Mutational analysis of a plant defensin from radish (Raphanus sativus L.) reveals two adjacent sites important for antifungal activity; De Samblanx GW et al.; Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression . The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties . Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified . In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine . Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength . Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI beta-turn connecting beta-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting beta-strand 1 and the alpha-helix and contiguous residues on the alpha-helix and beta-strand 3, on the other hand . When added to F . culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca2+ uptake by up to 20-fold . An arginine substitution variant with enhanced antifungal activity caused increased Ca2+ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca2+ uptake.

J Biol Chem, 1997 Jan 10, 272(2), 1076 - 81
Complementary DNA cloning and sequencing of the chicken muscle ecto-ATPase . Homology with the lymphoid cell activation antigen CD39; Kirley TL; The ecto-ATPase from chicken gizzard (smooth muscle) was solubilized, and the 66-kDa cell membrane ecto-ATPase protein was purified . The protein was then subjected to both enzymatic and chemical cleavage, and the resultant peptides were purified by reverse phase high pressure liquid chromatography and sequenced . Several of these internal peptide sequences were used to design oligonucleotides to screen a chicken muscle library to identify the cDNA encoding the ecto-ATPase . Two overlapping partial clones were sequenced, yielding the complete coding region and a long 3'-untranslated sequence . The deduced amino acid sequence is in agreement with the N-terminal and peptide sequences obtained from the purified protein . The chicken muscle ecto-ATPase is a slightly basic (predicted pI = 7.93) 494-amino acid protein (54.4 kDa), containing a single transmembrane domain at each end of the protein . The majority of the protein is predicted to be extracellular, making it a Type Ia plasma membrane protein . There are four putative N-glycosylation sites, a single potential cAMP/cGMP-dependent protein kinase phosphorylation site, as well as a single putative tyrosine kinase phosphorylation site . Analysis of the sequence using the BLAST programs demonstrated homology with other ecto-ATPases and ecto-apyrases, including those from the parasitic protozoan Toxoplasma gondii, potato tubers, and garden pea, as well as a guanosine diphosphohydrolase from yeast . However, the most striking homology observed was to the human and mouse lymphoid cell activation antigen 39 (CD39), a molecule now known to have apyrase activity . The chicken ecto-ATPase showed considerable amino acid sequence homology with CD39 over the entire length of the sequence, excluding about 30-40 amino acids at the extreme ends of the protein (which include the two membrane-spanning helices) . The sequence homology between the gizzard ecto-ATPase and CD39 was confirmed by Western blots demonstrating immunocross-reactivity between mono- and polyclonal antibodies raised against the chicken ecto-ATPase and two commercially available monoclonal antibodies against the human CD39 protein . The results suggest that the muscle ecto-ATPase may be involved in cell adhesion, since the highly homologous CD39 protein is involved in homotypic adhesion of activated B lymphocytes.

J Biol Chem, 1997 Jan 10, 272(2), 1046 - 53
The phosphorylation state of translation initiation factors is regulated developmentally and following heat shock in wheat; Gallie DR et al.; Several translation initiation factors in mammals and yeast are regulated by phosphorylation . The phosphorylation state of these factors is subject to alteration during development, environmental stress (heat shock, starvation, or heme deprivation), or viral infection . The phosphorylation state and the effect of changes in phosphorylation of the translation initiation factors of higher plants have not been previously investigated . We have determined the isoelectric states for the wheat translation initiation factors eIF-4A, eIF-4B, eIF-4F, eIF-iso4F, and eIF-2 and the poly(A)-binding protein in the seed, during germination, and following heat shock of wheat seedlings using two-dimensional gel electrophoresis and Western analysis . We found that the developmentally induced changes in isoelectric state observed during germination or the stress-induced changes were consistent with changes in phosphorylation . Treatment of the phosphorylated forms of the factors with phosphatases confirmed that the nature of the modification was due to phosphorylation . The isoelectric states of eIF-4B, eIF-4F (eIF-4E, p26), eIF-iso4F (eIF-iso4E, p28), and eIF-2alpha (p42) were altered during germination, suggesting that phosphorylation of these factors is developmentally regulated and correlates with the resumption of protein synthesis that occurs during germination . The phosphorylation of eIF-2beta (p38) or poly(A)-binding protein did not change either during germination or following a thermal stress . Only the phosphorylation state of two factors, eIF-4A and eIF-4B, changed following a heat shock, suggesting that plants may differ significantly from animals in the way in which their translational machinery is modified in response to a thermal stress.

FEBS Lett, 1997 Jan 6, 400(3), 341 - 4
Inhibition of epithelial Na+ currents by intracellular domains of the cystic fibrosis transmembrane conductance regulator; Kunzelmann K et al.; Cystic fibrosis is characterized by an impaired cyclic adenosine 3,5-monophosphate (cAMP) activated Cl- conductance in parallel with an enhanced amiloride sensitive Na+ conductance (ENaC) of the respiratory epithelium . Very recently, acute downregulation of ENaC by the cystic fibrosis transmembrane conductance regulator (CFTR) was demonstrated in several studies . The mechanism, however, by which CFTR exerts its inhibitory effect on ENaC remains obscure . We demonstrate that cytosolic domains of human CFTR are sufficient to induce inhibition of rat epithelial Na+ currents (rENaC) when coexpressed in Xenopus oocytes and stimulated with 3-isobutyl-1-methylxanthine (IBMX) . Moreover, mutations of CFTR, which occur in cystic fibrosis, abolish CFTR-dependent downregulation of rENaC . Yeast two hybrid analysis of CFTR domains and rENaC subunits suggest direct interaction between the proteins . Enhanced Na+ transport as found in the airways of cystic fibrosis patients is probably due to a lack of CFTR dependent downregulation of ENaC.

Biochem Biophys Res Commun, 1997 Jan 3, 230(1), 100 - 4
Identification of a human cDNA homologue to the Drosophila translocation protein 1 (Dtrp1); Daimon M et al.; In yeast, several integral membrane proteins such as Sec61p, Sec62p and Sec63p have been reported as the components involved in protein translocation across and into the endoplasmic reticulum (ER) membrane . Among them, the homologues of Sec61p have been found both in bacterias and mammals, whereas those of Sec62p or Sec63p have not . So, Sec61p seem to be the evolutionary conserved component, while Sec62p and Sec63p may not . To date, no homologues of Sec62p have been found in mammals yet . Here, we report a novel human cDNA, HTP1 (for human translocation protein 1), that encodes a protein of 399 amino acids that is 36.3% identical (64.6% similar) to Drosophila homologue of Sec62p, Drosophila translocation protein 1 (Dtrpl) . Northern blot analysis showed two HTP1 transcripts of about 2.8 and 5.5 kb, which were expressed concomitantly in various human tissues such as heart, brain, placenta, liver and pancreas.

Biochim Biophys Acta, 1997 Jan 3, 1350(1), 27 - 32
An orphan nuclear receptor lacking a zinc-finger DNA-binding domain: interaction with several nuclear receptors; Masuda N et al.; The yeast two-hybrid screening was applied to cloning cDNAs of proteins that interact with peroxisome proliferator-activated receptor alpha (PPAR alpha) . We obtained from a rat liver cDNA library a clone encoding a protein related to the ligand-binding domain of the members of nuclear hormone receptor superfamily, whereas apparently lacking the zinc-finger DNA-binding domain . This protein interacted with the activated forms of several nuclear receptors, and thus is a novel type of heterodimer-forming nuclear receptor.

FEBS Lett, 1997 Jan 3, 400(2), 177 - 82
Co-elevation of brain natriuretic peptide and proprotein-processing endoprotease furin after myocardial infarction in rats; Sawada Y et al.; We investigated the expression of the yeast Kex2 family endoproteases furin and PACE4, and brain natriuretic peptide (BNP) in the atrium and ventricle after infarction as well as the conversion of the BNP precursor gammaBNP to BNP-45 . In a rat heart failure model, plasma BNP rose in two phases--first at day 3, and again at day 14 . BNP mRNA, as measured by Northern blot analysis, increased strongly at day 3, then at days 14 and 28 less strongly in the atrium, and in the ventricle it increased weakly at day 3, then strongly at days 14 and 28 . Furin mRNA showed the same pattern of expression as that of BNP message, whereas PACE4 message stayed unchanged after the infarction . Both furin and BNP were immunostained in the myocardium adjacent to the infarcted tissue . We suggest that after myocardial infarction, furin is co-expressed with BNP in both the atrium and ventricle, and that furin may be responsible for the conversion of gammaBNP to BNP-45.

FEBS Lett, 1997 Jan 2, 400(1), 25 - 30
From BRCA1 to RAP1: a widespread BRCT module closely associated with DNA repair; Callebaut I et al.; Inherited mutations in BRCA1 predispose to breast and ovarian cancer, but the biological function of the BRCA1 protein has remained largely elusive . The recent correspondence of Koonin et al . {Koonin, E.V., Altschul, S.F . and Bork, P . (1996) Nature Genet . 13, 266-267} has emphasized the potential importance of the BRCA1 C-terminal region for BRCA1-mediated breast cancer suppression, as this domain shows similarities with the C-terminal regions of a p53-binding protein (53BP1), the yeast RAD9 protein involved in DNA repair, and two uncharacterized, hypothetical proteins (KIAA0170 and SPAC19G10.7) . The highlighted domain has been suggested to be the result of an internal duplication, each of the tandem domains being designated as a 'BRCT domain' (for BRCA1 C-terminus) . Sequence analysis using hydrophobic cluster analysis reveals here the presence of 50 copies of the BRCT domain in 23 different proteins, including, in addition to BRCA1, 53BP1 and RAD9, XRCC1, RAD4, Ect2, REV1, Crb2, RAP1, terminal deoxynucleotidyltransferases (TdT) and three eukaryotic DNA ligases . Most of these proteins are known to be involved in DNA repair . The BRCT domain is not limited to the C-termini of protein sequences and can be found in multiple copies or in a single copy as in RAP1 and TdT, suggesting that it could well constitute an autonomous folding unit of approx . 90-100 amino acids.

Cytogenet Cell Genet, 1997, 77(3-4), 290 - 5
Mapping AAC1, AAC2 and AACP, the genes for arylamine N-acetyltransferases, carcinogen metabolising enzymes on human chromosome 8p22, a region frequently deleted in tumours; Matas N et al.; Arylamine N-acetyltransferases (NATs) are encoded at two loci on 8p22, a region subject to deletions in bladder tumours . The two functional genes (AAC1 and AAC2 alias NAT1 and NAT2) without introns in the coding region, encode enzymes which metabolise carcinogens, including bladder carcinogens . They are both multi-allelic and certain alleles have been implicated as susceptibility factors in bladder cancer . There is a third N-acetyltransferase gene, a pseudogene, AACP alias NATP, which we show is also located on chromosome 8 at the p22 region . We have mapped a series of YAC clones (ICI and CEPH) containing the NAT genes and the markers D8S21, an RFLP marker, and D8S261, a microsatellite marker . We show that D8S21 is a portion of the coding region of AAC2 . The order of genes in this region, covering some 2 Mb, is TEL-D8S261-AAC1-AACP-AAC2 (D8S21)-CEN . The restriction map also illustrates that there are likely to be other expressed genes in the region through the identification of CpG islands.

Cytogenet Cell Genet, 1997, 77(3-4), 246 - 51
Development of diagnostic tools for the analysis of 5p deletions using interphase FISH; Gersh M et al.; Cri-du-chat syndrome is associated with a deletion of the short arm of chromosome 5 . Through the phenotypic and molecular analyses of individuals with a subset of the features associated with the syndrome, the genes involved in the syndrome have been mapped to two distinct critical regions . Deletion of a critical region in 5p15.2 results in the distinct facial features associated with the syndrome as well as the severe mental and developmental delay, while a deletion of 5p15.3 is associated only with the characteristic cat-like cry, the key diagnostic feature of the syndrome . Therefore, subtle differences in the extent of the 5p deletion can have a profound affect on the prognosis of the patient . In order to more easily differentiate between deletions that lead to the cri-du-chat syndrome phenotype and deletions that lead only to the isolated cat-like cry, we have constructed YAC contigs that span both critical regions . The YAC clones have been used to isolate cosmids mapping to each critical region and cosmids that lie just within the two critical region boundaries have been identified . We report here on the use of these cosmids as probes for fluorescent in situ hybridization experiments on interphase nuclei as a means of more accurately differentiating between small 5p deletions that coincide with a complete cri-du-chat syndrome phenotype and the severe mental and developmental delay that is associated with it and deletions that only delete the distal critical region that coincide with the isolated cat-like cry and a much improved prognosis.

Cytogenet Cell Genet, 1997, 77(3-4), 232 - 7
A panel of radiation hybrids and YAC clones specific for human chromosome 5; Marzella R et al.; We report the characterization, by reverse fluorescence in situ hybridization (FISH), of 59 hybrids retaining fragments of human chromosome 5 . Most of these hybrids are radiation hybrids generated by gamma irradiating, at low dosage, a monochromosomal hybrid retaining chromosome 5 as its only human contribution . The partial chromosome paints generated from these hybrids will make powerful tools for cytogenetic investigations, especially on the cytogenetic evolution of primates, and examples are reported . The molecular characterization of these hybrids was refined using 74 sequence-tagged sites (STSs), which allowed the physical dissection of chromosome 5 into 71 distinct regions with an average length of 2.7 Mb . The panel, therefore, is also suitable for high-precision subregional mapping of new genes or sequences located on chromosome 5 . As an additional resource for cytogenetic studies involving chromosome 5, we report the characterization, by FISH, of 73 YACs from CEPH . The vast majority of these YACs are recognized by at least one of the STSs used for hybrid characterization, thus enabling the integrated use of YACs and partial chromosome paints derived from the hybrids.

Cytogenet Cell Genet, 1997, 77(3-4), 169 - 74
Human type I cytokeratin genes are a compact cluster; Ceratto N et al.; A YAC clone (211F11) containing approximately 0.5 Mb of human DNA was isolated from a human genomic library by PCR-based screening with cytokeratin (KRT) 13-specific primers . The YAC clone was mapped by FISH to the long arm of chromosome 17 (17q12-->q21), a region to which several other type I KRT genes had been mapped previously . We now show by Southern blot hybridization and PFGE analyses that KRT13, 14, 15, and 16 are all contained within YAC clone 211F11 . Long-range restriction mapping analysis of clone 211F11 and of two smaller YAC clones that were also isolated with KRT13-specific primers, suggests that KRT13, 14, 15, 16 and their linked type I genes KRT17 and 19, are contained in less than 150 kb of genomic DNA . According to our reconstruction it then appears that at least six type I KRT genes are arranged in a highly compact cluster . The three YACs reported in this study represent a new tool to dissect the molecular structure of the locus of the human type I KRT genes.

SAAS Bull Biochem Biotechnol, 1997, 10, 43 - 8
Genetic approaches to biochemical questions: insights into the functional requirements of proline 185 in the active site of human galactose-1-phosphate uridylyltransferase; Quimby BB et al.; Saturating random mutagenesis at a given position within a polypeptide sequence can provide powerful insights into the functional requirements of the position . By coupling this genetic methodology with expression of human proteins in yeast, we and others have begun to ask pointed and important questions about the structure-function relationships of proteins associated with human genetic disease.

SAAS Bull Biochem Biotechnol, 1997, 10, 7 - 11
Molecular approaches to identify novel genes expressed in Arabidopsis thaliana; Nuccio ML et al.; Our laboratory is engaged in an effort to identify genes expressed primarily during plant embryogenesis . Genes which exhibit unique expression profiles in the plant are also being sought . To this end, several methods to identify and clone novel genes based on specific expression patterns have been developed . These methods include virtual subtraction, differential display and other PCR based technologies . In addition to this, a yeast one-hybrid approach has been established to identify transcription factors which regulate these genes . To date, this work has identified several novel genes.

Arch Tierernahr, 1997, 50(3), 213 - 25
Formation and disappearance of mycophenolic acid, patulin, penicillic acid and PR toxin in maize silage inoculated with Penicillium roqueforti; Muller HM et al.; Maize silage was inoculated with Penicillium roqueforti strains which are able to form the mycotoxins mycophenolic acid (MPA), patulin (PAT), penicillic acid (PA), or PR toxin (PRT) . The silage was incubated for 160 d without agitation under aerobic conditions at 15 degrees C in the dark . The mycotoxins were quantified by HPLC and identified by HPLC combined with diode array detection and by two-dimensional thin layer chromatography . MPA, PAT, PA, and PRT above the detection limit were measured for the first time at 36, 22-27, 13, and 49 days of incubation, maximum toxin contents (mg/kg) were 3.56 MPA, 15.10 PAT, 3.06 PA, and 2.17 PRT . With increasing storage time toxin contents decreased to a low or non detectable level . The production of MPA, PAT and PRT was preceded by an increase in pH from 4 to 8-9 . Along with the initial pH increase the content of ergosterol as well as of P . roqueforti and yeast propagules increased whereas the levels of total soluble sugar and of water extractable NH3 decreased . It is concluded that the probability to detect MPA, PAT, PA, and PRT in maize silage moulded by P . roqueforti under practical conditions of agriculture is low during the growth of this fungus and again after prolonged storage.

Microbios, 1997, 89(358), 47 - 54
Regulation of rubratoxin-B biosynthesis: assessment of role of gamma-irradiation, pH and carbohydrates; Aziz NH et al.; The maximum rubratoxin-B yield was obtained at pH 5.5,, and by increasing the initial pH to near neutrality the yield decreased for both yeast extract sucrose (YES) and Sabouraud dextrose yeast extract (SDYE) media, but the concentration of mycotoxin was higher in YES medium . The rubratoxin-B yield from Penicillium purpurogenium decreased with increasing gamma-irradiation, and at 1.0 kGy no mycotoxin was detected at any pH values . In both the unirradiated and irradiated P . purpurogeniium cultures, as the rubratoxin-B synthesis increased from 46 to 72 h, the lipid content decreased . The concentration (mmoles/g dry wt mycelium) of puridine nucleotides in the mycelium of P . purpurogenium during growth in YES and SDYE media may be a factor in rubratoxin-B synthesis . An elevated NADPH/ NADP ratio favours fatty acid synthesis whereas a depressed NADPH/NADP ratio favours mycotoxin formation . The gamma-irradiation played a role in the regulation of rubratoxin-B biosynthesis.

DNA Seq, 1997, 7(3-4), 127 - 39
Organization and sequence of the human gene for the mitochondrial citrate transport protein; Iacobazzi V et al.; The citrate (tricarboxylate) carrier transports citrate (or other tricarboxylates) across the inner membranes of mitochondria in an electroneutral exchange for malate (or other dicarboxylic acids) . We have determined the sequence of the human citrate transporter gene from overlapping genomic clones generated by polymerase chain reactions by use of primers and probes based on the rat cDNA sequence and on emerging sequences . The gene is spread over 2.8 kb of human DNA and is divided into eight exons . All the introns are located at the level of the sequences coding for the extramembranous loops (and not for the transmembrane segments) of the mature protein . The open reading frame of the human gene encodes the mature protein consisting of 298 amino acids, preceded by a presequence of 13 amino acids to help to target it into mitochondria . 84 identities and 106 highly conservative substitutions are present in CTPs from man to yeast . In addition, we have determined the sequences of two human pseudogenes related to the citrate carrier gene encompassing the coding sequence of the gene between nucleotides 260 and 720.

Annu Rev Biophys Biomol Struct, 1997, 26, 139 - 56
Flexibility of RNA; Hagerman PJ; One of the fundamental properties of the RNA helix is its intrinsic resistance to bend- or twist-deformations . Results of a variety of physical measurements point to a persistence length of 700-800 A for double-stranded RNA in the presence of magnesium cations, approximately 1.5-2.0-fold larger than the corresponding value for DNA . Although helix flexibility represents an important, quantifiable measure of the forces of interaction within the helix, it must also be considered in describing conformational variation of nonhelix elements (e.g . internal loops, branches), since the latter always reflect the properties of the flanking helices; that is, such elements are never completely rigid . For one important element of tertiary structure, namely, the core of yeast tRNAPhe, the above consideration has led to the conclusion that the core is not substantially more flexible than an equivalent length of pure helix.

Annu Rev Nutr, 1997, 17, 501 - 26
Comparative nutrition of iron and copper; Winzerling JJ et al.; The suggestion from nutritional studies with mammals of a link between iron and copper metabolism has been reinforced by recent investigations with yeast cells . Iron must be in the reduced ferrous (FeII) state for uptake by yeast cells, and reoxidation to ferric (FeIII) by a copper oxidase is part of the transport process . Thus, yeast cells deficient in copper are unable to absorb iron . In an analogous way, animals deficient in copper appear to be unable to move FeII out of cells, probably because it cannot be oxidized to FeIII . Invertebrate animals use copper and iron in ways very similar to vertebrates, with some notable exceptions . In the cases where vertebrates and invertebrates are similar, the latter may be useful models for vertebrate metabolism . In cases where they differ (e.g . predominance of serum ferritin in insects, oxygen transport by a copper protein in many arthropods, central importance of phenoloxidase, a copper enzyme in arthropods), the differences may represent processes that are exaggerated in invertebrates and thus more amenable to study in these organisms . On the other hand, they may represent processes unique to invertebrates, thus providing novel information on species diversity.

Recent Prog Horm Res, 1997, 52, 141 - 64; discussion 164-5
Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor action; Shibata H et al.; Steroid/thyroid hormone receptors are ligand-dependent transcription factors that regulate diverse aspects of growth, development, and homeostasis by binding as homodimers or heterodimers to their cognate DNA response elements to modulate transcription of target genes . Transactivation by steroid/ thyroid hormone receptors involves a conserved AF-2 domain located in the distal carboxy-terminus of the receptors . The existence of co-factors, termed co-activators or adapters, was first suggested by transcriptional squelching between progesterone receptors and estrogen receptors . Co-repressors were also postulated to contribute to the silencing function of unliganded thyroid hormone receptor (TR) . The yeast two-hybrid system and Far-Western blotting have been used to identify several proteins that interact with members of the steroid/thyroid hormone receptor superfamily in a ligand-sensitive manner . Our laboratory cloned the first functional co-activator, termed steroid receptor co-activator-one (SRC-1), that appears to be a general co-activator for all steroid receptors tested and enhances transactivation of steroid hormone-dependent target genes . Subsequently, many more putative co-activators have been reported, including the SRC-1 related proteins, TIF2 and GRIP1, and other putative and unrelated co-activators such as ARA70, Trip1, RIP140, and TIF1 . In addition, another co-activator, CREB-binding protein (CBP), has been shown to enhance steroid receptor-dependent target gene transcription . CBP and SRC-1 interact and synergistically enhance transcriptional activation by the ER and PR . Therefore, a ternary complex-consisting of CBP, SRC-1, and liganded steroid receptors-may form to increase the rate of hormone-responsive gene transcription . Similarly, co-repressors, such as SMRT and N-CoR, for TR and retinoic acid receptors (RAR) have been identified . The unliganded TR and RAR have been shown to inhibit basal promoter activity; this silencing of target gene transcription by unliganded receptors is mediated by these co-repressors . Collectively, available evidence supports the following model of steroid-responsive gene transcription . Upon binding of agonist the receptor changes its conformation in the ligand-binding domain that enables recruitment of co-activators, which allows the receptor to interact with the basal transcriptional machinery more efficiently and to activate transcription . In contrast, binding of antagonists induces a different conformational change in the receptor . Although some antagonist-bound receptor can dimerize and bind to its cognate DNA element, it fails to dislodge the associated co-repressors, which results in a nonproductive interaction with the basal transcriptional machinery . Similarly, the TR and RAR associate with co-repressors in the absence of ligand, thereby resulting in a negative interaction with the transcriptional machinery that silences target gene expression . In the case of mixed agonist/antagonists, such as 4-hydroxytamoxifen, activation of gene transcription may depend on the relative ratio of co-activators and co-repressors in the cell or cell-specific factors that determine the relative agonistic or antagonistic potential of different compounds . These co-activators and co-repressors appear to act as an accelerator and/or a brake that modulates transcriptional regulation of hormone-responsive target gene expression . Thus, the recent discovery of co-activators and co-repressors expands our knowledge of the mechanisms of steroid receptor action.

Biosystems, 1997, 43(1), 1 - 24
Dynamics of two-component biochemical systems in interacting cells; synchronization and desynchronization of oscillations and multiple steady states; Wolf J et al.; Systems of interacting cells containing a metabolic pathway with an autocatalytic reaction are investigated . The individual cells are considered to be identical and are described by differential equations proposed for the description of glycolytic oscillations . The coupling is realized by exchange of metabolites across the cell membranes . No constraints are introduced concerning the number of interacting systems, that is, the analysis applies also to populations with a high number of cells . Two versions of the model are considered where either the product or the substrate of the autocatalytic reaction represents the coupling metabolite (Model I and II, respectively) . Model I exhibits a unique steady state while model II shows multistationary behaviour where the number of steady states increases strongly with the number of cells . The characteristic polynomials used for a local stability analysis are factorized into polynomials of lower degrees . From the various factors different Hopf bifurcations may result in leading for model I, either to asynchronous oscillations with regular phase shifts or to synchronous oscillations of the cells depending on the strength of the coupling and on the cell density . The multitude of steady states obtained for model II may be grouped into one class of states which are always unstable and another class of states which may undergo bifurcations leading to synchronous oscillations within subgroups of cells . From these bifurcations numerous different oscillatory regimes may emerge . Leaving the near neighbourhood of the boundary of stability, secondary bifurcations of the limit cycles occur in both models . By symmetry breaking the resulting oscillations for the individual cells lose their regular phase shifts . These complex dynamic phenomena are studied in more detail for a low number of interacting cells . The theoretical results are discussed in the light of recent experimental data on the synchronization of oscillations in populations of yeast cells.

Immunogenetics, 1997, 46(4), 307 - 11
The human natural killer gene complex is located on chromosome 12p12-p13; Renedo M et al.; Natural killer (NK) cells preferentially express several type II glycoproteins of the calcium-dependent lectin superfamily . The genes coding for these molecules are clustered on the distal mouse chromosome 6 and on the rat chromosome 4 in a region designated the NK gene complex . To date, no definite evidence of the presence of a NK gene complex has been found in humans . Here we report the assignment by fluorescence in situ hybridization of the CD94 gene to human chromosome 12p12-p13, in the same region where the CD69 and NKG2A genes had been previously mapped . In addition, using a yeast artificial chromosome contig spanning this region we determined that the human CD94, NKG2A, NKG2C, NKG2E, and NKR-P1A (NKR) genes map to the short arm of chromosome 12 . The distal to proximal position of these loci are: NKR- CD69 - CD94/NKG2A/NKG2C/NKG2E . These data demonstrate the existence of a human NK gene complex located within a 5.6 cM interval flanked by the genetic markers D12S397 and D12S89 . The physical distance spanned by the NK gene complex in humans ranges between 0.7 and 2.4 megabases.

Immunogenetics, 1997, 46(3), 206 - 12
Identification of new HLA class I region genes by sample sequencing; Goldsworthy M et al.; Although many human major histocompatibility genes have been identified, relatively few have been localized to the class I region . We searched for new class I region genes by sample sequencing, a process in which short stretches of random genomic sequence are generated from cosmids and then compared with sequences deposited in nucleotide databases . Four class I region cosmids were isolated for sample sequencing by screening a chromosome 6 specific cosmid library with probes derived from specific class I region genes or with overlapping class I region yeast artificial chromosomes . Cosmids were sonnicated to produce fragments of 0.5 - 1 kilobases, subcloned, and sequenced using an automated sequencer . Sequences were then compared with nucleotide sequences deposited in the GenBank databases using the BLASTN algorithm . A number of potential new class I region genes were identified, including a cDNA with similarity to the tre oncogene, the trans-activating factor SC1 (TCF19), and a member of the interferon inducible 1 - 8 gene family . These observations suggest that sample sequencing is an efficient method for identifying new class I region genes, which can be applied to other regions of the genome and to other species, and support previous observations that the class I region contains a variety of genes other than those encoding HLA antigens.

Soc Gen Physiol Ser, 1997, 52, 227 - 39
Dissection of protein kinase and phosphatase targeting interactions; Scott JD; Protein phosphorylation is a primary means of mediating signal transduction events that control cellular processes . Accordingly, the activities of protein kinases and phosphatases are highly regulated . One level of regulation is that the subcellular distribution of several kinases and phosphatases is restricted by association with targeting proteins or subunits . This mechanism promotes rapid and preferential modulation of specific targets within a defined microenvironment in response to diffusible second messengers . The type II cAMP-dependent protein kinase (PKA) is targeted by association of its regulatory subunit (RII) with A-kinase anchoring proteins (AKAPs) . To date, 36 unique AKAPs have been identified . Each of these proteins contains a conserved amphipathic helix responsible for AKAP association with cellular structures . Disruption of PKA/AKAP interaction with peptides patterned after the amphipathic helix region blocks certain cAMP responses, including the modulation of glutamate receptor ion-channel activity in neurons and transcription of cAMP-responsive genes . Yeast two-hybrid screening methods have identified neuronal specific AKAP79-binding proteins including the beta isoform of the phosphatase 2B, calcineurin . Biochemical and immunological studies have confirmed the two-hybrid results and identified additional members of this multienzyme signaling complex, including certain protein kinase C isoforms . These findings are consistent with colocalization of CaN, PKC, and type II PKA by AKAP79 and suggest a novel model for reversible phosphorylation in which the opposing kinase and phosphatase actions are colocalized in a signal transduction complex by association with a common anchor protein.

Rapid Commun Mass Spectrom, 1997, 11(9), 1015 - 24
Rapid 'de novo' peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer; Shevchenko A et al.; Protein microanalysis usually involves the sequencing of gel-separated proteins available in very small amounts . While mass spectrometry has become the method of choice for identifying proteins in databases, in almost all laboratories 'de novo' protein sequencing is still performed by Edman degradation . Here we show that a combination of the nanoelectrospray ion source, isotopic end labeling of peptides and a quadrupole/ time-of-flight instrument allows facile read-out of the sequences of tryptic peptides . Isotopic labeling was performed by enzymatic digestion of proteins in 1:1 16O/18O water, eliminating the need for peptide derivatization . A quadrupole/time-of-flight mass spectrometer was constructed from a triple quadrupole and an electrospray time-of-flight instrument . Tandem mass spectra of peptides were obtained with better than 50 ppm mass accuracy and resolution routinely in excess of 5000 . Unique and error tolerant identification of yeast proteins as well as the sequencing of a novel protein illustrate the potential of the approach . The high data quality in tandem mass spectra and the additional information provided by the isotopic end labeling of peptides enabled automated interpretation of the spectra via simple software algorithms . The technique demonstrated here removes one of the last obstacles to routine and high throughput protein sequencing by mass spectrometry.

Genet Eng (N Y), 1997, 19, 183 - 99
Switching of gene expression: analysis of the factors that spatially and temporally regulate plant gene expression; Meisel L et al.; In this chapter, we have reviewed the present research and understanding of several families of transcription factors in plants . From this information, it appears there is good conservation between the types of transcription factors in plants and animals . However, there are several types of factors which have been isolated in plants that remain to be documented in animals (e.g., HD-Zip and GT) . These as well as the presence of two types of TATA-binding proteins (TBPs) in plants suggest that although transcription in eukaryotes is highly conserved, fundamental differences may exist . Despite the differences, the modes of regulating transcription are well conserved . Figure 3 summarizes these modes of regulation . In recent years, the role of chromatin structure as well as subcellular localization have been the focus of a vast amount of research in mammals, Drosophila and yeast . However, very little research in these areas has been done in plants . Isolation of genes such as Curly leaf suggest a conservation of genes that influence the formation of heterochromatin-like structures . Whether or not this gene influences chromatin/heterochromatin structure in plants, however, remains to be tested . The study of nuclear localization of factors such as COP1 and KN1 is now leading to models for regulating nuclear transport as well as intercellular transport of transcription factors . Further study of the inter- and intracellular movement of these and other transcription factors may provide information on new modes of regulating transcription . In addition to understanding the role chromatin structure and subcellular localization of transcription factors may have on transcription initiation, the biological role of many plant transcription factors remains to be identified . Several approaches may be taken to understand the mechanisms by which transcription factors influence biochemical and physiological processes in the plant . These steps include 1) identification of the DNA-binding sites of the factors as well as the promoter regions which contain these sites . Presently, this approach is limiting in that not many non-coding regions have been sequenced and characterized in detail . Furthermore, the presence of a putative binding site within a promoter does not necessarily indicate that the factor will bind to the site in vivo . 2) Analysis of the binding affinity for a particular factor to a binding site in comparison to other related factors, via in vitro competition assays and quantitative titrations . This will provide information on how strongly these factors are binding to the sites, but without knowledge of all the factors present in a single cell it is difficult to recreate the in vivo conditions . 3) Generation of transgenic plants or microinjection of DNA/RNA to express a particular factor ectopically, reduce expression of the factor via antisense expression, and creation of dominant negative mutants by overexpression of key dimerization domains may provide information concerning what biological pathways these factors influence . 4) Isolation of mutations in particular transcription factors has been extremely informative in floral development . However, this approach usually entails isolation of a mutant due to a phenotype and eventual mutated locus . The cloning of the locus may or may not involve a transcription factor . 5) Many plant transcription factors have been isolated via sequence similarity to other previously identified and/or characterized transcription factors . However, the biological role of may of these factors is not known . In addition to ectopic expression of these factors by creating transgenic plants, isolation of a loss-of-function mutation may provide valuable information concerning the role of this factor in vivo . Many loss-of-function mutations in MADS box genes have led to a better understanding of how the MADS domain proteins interact with one another as well as how they influence floral development . (ABSTRACT TRUNCATED)

Cytogenet Cell Genet, 1997, 76(3-4), 139 - 43
Genomic organization and mapping of the human HEP-COP gene (COPA) to 1q; Quek HH et al.; In eukaryotic cells, protein transport between the endoplasmic reticulum and Golgi compartments is mediated in part by non-clathrin-coated vesicular coat proteins (COP) . Seven COP subunits have been recognized, and represent components of a complex known as coatomer . We have previously isolated the cDNA of the human homolog of alpha-COP, designated HEP-COP and given the official gene symbol COPA . Here we report the genomic organization of COPA, which contains 33 exons ranging in size from 67 to 611 bp . Mapped by PCR and cycle sequencing, all the exon-intron junctions conformed with the GT-AG rule, the 32 introns ranging from about 80 bp to 4 kbp, with the genomic DNA of COPA estimated to span approximately 37 kb . Southern blot analysis of genomic DNAs of nine eukaryotic species, from human to yeast, revealed identical signals totaling 36 kb each for man and monkey only . Using 5' RACE and primer extension analysis, the putative transcriptional start site was localized to 466 nucleotides upstream of the translation initiation codon . Comprising a 126-nucleotide 5' untranscribed genomic sequence and a 466-nucleotide 5' noncoding cDNA sequence, the 592-nucleotide 5' CpG island lacked TATA and CAAT boxes but displayed a high G+C content, was enriched for CpG dinucleotides, and contained a potential Sp1-binding site, i.e., features compatible with a housekeeping gene . COPA was mapped by fluorescence in situ hybridization to chromosome region 1q23-->q25.

J Steroid Biochem Mol Biol, 1997 Jan, 60(1-2), 147 - 52
Pregnenolone-7beta-hydroxylating activity of human cytochrome P450-1A1; Doostzadeh J et al.; In many human and murine tissues, both pregnenolone and dehydroepiandrosterone are hydroxylated at the 7alpha and 7beta positions by a cytochrome P450-containing microsomal complex . The 7alpha- and 7beta-hydroxysteroids produced were shown to activate an immune response in mice . Based upon identification by crystallization to constant specific activity and gas chromatography-mass spectrometry analysis, we ascertained that a yeast-expressed human cytochrome P450-1A1 was able to 7beta-hydroxylate pregnenolone (K(M) from 3.2 +/- 0.5 to 4.1 +/- 0.4 microM, turnover number from 117 +/- 15 to 135 +/- 13 pmol/min/nmol of cytochrome P450-1A1) . The other human cytochromes P450 tested did not produce identifiable quantities of 7alpha- or 7beta-hydroxylated derivatives of pregnenolone or dehydroepiandrosterone . These findings indicate that cytochrome P450-1A1 involvement in the 7beta-hydroxylation of pregnenolone may contribute to the production of the 7-hydroxylated steroids necessary for activation of the immune defences.

Res Commun Inst Ferment, 1997, (18), 20 - 5
Detection of heterogeneity of 18S rRNA inter-genes and mutation arising during PCR amplification; Ueda K et al.; Direct sequencing revealed sequence heterogeneity among ribosomal RNA gene (rDNA) operons, consisting of 8 base heterogeneous sites on the 18S rDNA of Galactomyces citri-aurantii IFO 10822, and 6 base heterogeneous sites in the same region on the 18S rDNA of G . citri-aurantii IFO 10821 . Sequence analysis of the cloned 18S rRNA genes of 14 species (19 strains) of ascomycetous yeast-like fungi detected a total of 32 substitutions between two cloned sequences from each of 10 strains . Eight substitutions came from heterogeneity of G . citri-aurantii IFO 10822, and 24 substitutions were predicted to be due to misincorporation by the Taq DNA polymerase . A low frequency of random substitution, estimated to occur in PCR at approximately 1 in 2690 nucleotides, was detected; and transitions occurred 7 times more frequently than transversions.

Cancer Invest, 1997, 15(3), 227 - 36
Economic analyses of phase III cooperative cancer group clinical trials: are they feasible?
Bennett CL, Golub R, Waters TM, Tallman MS, Rowe JM.
Both economic and clinical evaluations of new pharmaceutical agents are important to physicians who practice in the current health care environment . While cooperative cancer groups carry out large-scale phase III clinical evaluations of these agents, few cooperative group studies incorporate economic analyses because of concerns over overburdening of data management, investigators, and statistical center personnel . In this study, we describe the results and operational considerations of one of the first completed economic analyses of a phase III cooperative group trial of the Eastern Cooperative Oncology Group (ECOG) . We developed an economic model estimating economic benefits of yeast-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) as adjunct therapy for adult patients (56-70 years) with acute myelogenous leukemia . Clinical data were based on prospectively collected information from a recently reported double-blind phase III multi-institutional study carried out by ECOG . Retrospective economic data were obtained from financial information systems at our hospital, one of the study sites . The cost-minimization analyses were based on the perspective of a third-party payer . Indirect costs related to loss of earnings by patients and caregivers as well as quality-of-life adjustments were not incorporated into the model . Clinical trial results indicated that patients treated with GM-CSF had shorter times to recovery of absolute neutrophil count of 500 cells/mm3 and 1000 cells/mm3 and fewer serious infections than patients who received placebo following induction chemotherapy, while no significant differences were noted in red blood cell and platelet transfusion dependency, toxicities, and duration of hospitalization . The economic model estimated that the group treated with GM-CSF was estimated to have lower costs of care, associated with lower frequencies of serious infections and lower overall infection-related costs . Sensitivity analyses indicated that these results held true over a wide range of estimates of costs and infection rates . Prospective economic analyses of phase III cooperative cancer group clinical trials have not been completed to date . Strategies that are not likely to overburden data managers and statistical center personnel are possible to devise . However, these studies require careful planning and coordination between clinical trialists, economists, and health services researchers.

Res Vet Sci, 1997 Jan-Feb, 62(1), 22 - 5
Epidemiological analysis of Malassezia pachydermatis isolates by partial sequencing of the large subunit ribosomal RNA; Guillot J et al.; The opportunistic yeast Malassezia pachydermatis is commonly recovered from both normal and diseased skin of warm-blooded animals . The diversity of M pachydermatis isolates obtained from a wide range of hosts was investigated by the partial sequencing of the large subunit (LSU) ribosomal RNA . Among 100 isolates examined, seven types (Ia-Ig) were discriminated on the basis of nucleotide sequence diversity . The seven types differed by one to five mutations, all of them corresponding to transitions . The predominant sequence, type Ia, appeared to be ubiquitous since it was observed in isolates recovered from domestic and wild carnivora, from a monkey and from man . In contrast the sequence types Ic, Id and Ig seemed to be more host-specific; they included isolates recovered exclusively from rhinoceros, dogs and ferrets, respectively . None of the seven sequence types correlated with isolation from healthy skin or a particular lesion (otitis externa or other dermatitis) . The study indicated that the skin of an animal may be colonised by more than one type of M pachydermatis.

Eur J Hum Genet, 1997 Jan-Feb, 5(1), 9 - 14
Nephropathic cystinosis (CTNS-LSB): construction of a YAC contig comprising the refined critical region on chromosome 17p13; Peters U et al.; A yeast artificial chromosome (YAC) contig was constructed encompassing the entire region on chromosome 17p13 where the autosomal recessive disorder infantile nephropathic cystinosis (MIM 21980, CTNS-LSB) has been genetically mapped . It comprises seven clones ordered by their content of a series of six sequence-tagged sites (STSs) . Fluorescence in situ hybridisation (FISH) revealed two chimaeric clones . The order of four polymorphic STSs mapped with the contig was consistent with that of the known genetic map with the exception of markers D17S1583 (AFMb307zg5) and D17S1798 (AFMa202xf5) where a telomeric location of D17S1583 was inferred from the contig; two non-polymorphic STSs were localised within the marker frame-work . From the analysis of recombination events in an unaffected individual as defined by leucocyte cystine levels we support the high-resolution mapping of this region to a small genetic interval and show that it is entirely represented on a single, non-chimaeric YAC clone in the contig.

Dermatology, 1997, 194 Suppl 1, 32 - 6
Epidemiology and ecology of onychomycosis; Summerbell RC; The epidemiology and ecology of onychomycosis are complex and little understood . Most is known about tinea unguium, dermatophytic nail infection, and its causative agents . This is often categorised according to the precise locus on the nail of the infection . The principal infectious propagules are thought to be the arthroconidia or chlamydospores which form within the solid substratum of invaded nail tissue . The process of infecting new hosts appears to be facilitated by abrasion, moistening and scratching . The role of the non-dermatophyte yeast Candida as an agent of onychomycosis per se may have been overestimated . The range of interactions between dermatophytes and non-dermatophytes in nails is complex and poorly understood . There may be at least six distinct ecological categories of non-dermatophyte isolations from nails . It would be of clinical interest to know which species found in mixed infections were never able to advance beyond 'secondary colonisation', as they would not require specific treatment.

Cytogenet Cell Genet, 1997, 76(1-2), 23 - 6
Mapping of the 8q12 translocation breakpoint to a 40-kb region in a pleomorphic adenoma with an ins(8;3)(q12;p21.3p14.1); Roijer E et al.; The translocation t(3;8)(p21;q12) is the most common chromosome abnormality observed in pleomorphic adenomas of the salivary glands . In this paper we describe the physical mapping of the breakpoints in an adenoma with a variant t(3;8), viz., an ins(8;3)(q12;p21.3p14.1) . Using sequence-tagged sites (STSs) corresponding to landmarks within a previously identified yeast artificial chromosome (YAC) spanning the breakpoint in adenomas with t(3;8), cosmids isolated from a chromosome 8-specific cosmid library . The 8q12 insertion breakpoint was mapped by FISH to a 300-kb region flanked by MOS and a new STS, CH129 . A cosmid within this region was shown to span the breakpoint . To test whether the recently identified FHIT gene, which maps to 3p14.2, was disrupted by the 3p rearrangement, we also isolated an FHIT YAC and mapped this YAC by FISH distal to the most proximal 3p breakpoint . In addition, RT-PCR analysis revealed only a normal-sized FHIT transcript, suggesting that FHIT is not affected by the 3;8-rearrangement.

Biol Trace Elem Res, 1997 Jan, 56(1), 117 - 24
Protective role of selenium against hepatitis B virus and primary liver cancer in Qidong; Yu SY et al.; High rates of hepatitis B virus (HBV) infection and primary liver cancer (PLC) are present in Qidong county . Epidemiological surveys demonstrated an inverse association between selenium (Se) level and regional cancer incidence, as well as HBV infection . Four-year animal studies showed that dietary supplement of Se reduced the HBV infection by 77.2% and liver precancerous lesion by 75.8% of ducks, caused by exposure to natural environmental etiologic factors . An intervention trial was undertaken among the general population of 130,471 . Individuals in five townships were involved for observation of the preventive effect of Se . The 8-yr follow-up data showed reduced PLC incidence by 35.1% in selenized table salt supplemented vs the nonsupplemented population . On withdrawal of Se from the treated group, PLC incidence rate began to increase . However, the inhibitory response to HBV was sustained during the 3-yr cessation of treatment . The clinical study among 226 Hepatitis B Surface Antigen (HBsAg)-positive persons provided either 200 micrograms of Se in the form of selenized yeast tablet or an identical placebo of yeast tablet daily for 4 yr showed that 7 of 113 subjects were diagnosed as having PLC in the placebo group, whereas no incidence of PLC was found in 113 subjects supplemented with Se . Again on cessation of treatment, PLC developed at a rate comparable to that in the control group, demonstrating that a continuous intake of Se is essential to sustain the chemopreventive effect.

Planta, 1997, 201(4), 487 - 95
NopA64, a novel nucleolar phosphoprotein from proliferating onion cells, sharing immunological determinants with mammalian nucleolin; de Carcer G et al.; Five major soluble nuclear proteins associated with cell proliferation were identified in Allium cepa L . root cells . One of them, of 64 kDa, was revealed by Western blotting with anti-mammalian nucleolin antibodies . A polyclonal antibody raised against this protein, which we have named NopA64, localised it in the nucleolus as well as in nuclear coiled bodies . Together with NopA64, the antibody also revealed a smaller form, called NopA61 . Both proteins were present in the soluble ribonucleoprotein fraction and in the nuclear matrix of proliferating cells, but NopA61 was the only form revealed in differentiated cells . NopA64 contained epitopes also present in other plants, in mammalian nucleolin and in its yeast homologue, gar2 . In mammals, the highest homology was with 50-kDa nucleolin fragments containing the RNA-binding motifs and the glycine-arginine-rich (GAR) domain . NopA64 was moderately phosphorylated in vitro by exogenous casein kinase II and cdc2 kinase, whereas NopA61 was highly phosphorylated by casein kinase II . Furthermore, NopA61 was the only band detected after dephosphorylation as well as after endoproteolysis of NopA64 . This protein could be one of the various functional homologues of mammalian nucleolin in plant cells.

J Basic Microbiol, 1997, 37(2), 85 - 91
Optimization of catalase biosynthesis in submerged cultures of Aspergillus niger mutant; Gromada A et al.; The effect of some medium components, viscous substances and metabolic inhibitors, on catalase production by mutant Aspergillus niger has been studied in shake culture . Altering the composition of the basal medium, particularly substituting NaNO3 for KNO3, and peptone for yeast extract brought an increase in extra- and intracellular catalase activity by 1.5- and 3-fold, respectively . The addition of 2.0-6.0 mg sodium alginate or pectin/ml as viscous additive to the medium, containing glucose as carbon source, increased the medium viscosity and catalase production in shake culture by about 2.8- to 3.0-fold . The highest yield of extracellular catalase activity of A . niger was obtained in the presence of sodium orthovanadate and Triton X-100, which improved the activity of this enzyme by about 1.5-2.2-fold . A significant increase in intracellular catalase activity was observed in the presence of hematin, Tween 80 and sodium orthovanadate (1.7-, 1.6- and 1.4-fold respectively) . The time course of growth and enzyme production by A . niger in the optimized medium is also reported.

Annu Rev Pharmacol Toxicol, 1997, 37, 297 - 326
The role of the hsp90-based chaperone system in signal transduction by nuclear receptors and receptors signaling via MAP kinase; Pratt WB; The multicomponent heat-shock protein (hsp) 90-based chaperone system is an ubiquitous protein-folding system in the cytoplasm of eukaryotes . Several signal transduction systems utilize an interaction with hsp90 as an essential component of the signaling pathway . The steroid and dioxin receptors are bound to hsp90 through their hormone-binding domains, and several of them must be bound to hsp90 in order to have a ligand-binding site . The binding of ligands to these receptors promotes their dissociation from hsp90, an event that is the first step in their signaling pathways . Several protein kinases, including the Src and Raf components of the MAP kinase system, are also bound to hsp90 . Genetic studies in yeast have demonstrated that hsp90 is required for normal signaling via steroid and dioxin receptors and for the activity of Src in vivo . The hsp90-based chaperone system has been reconstituted from purified components, permitting detailed analysis of the molecular basis of the chaperone's role in signal transduction.

Prog Clin Biol Res, 1997, 396, 101 - 13
Molecular perspectives on cancer, the cell cycle and the inherited disorder ataxia-telangiectasia; Brown KD et al.; Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder which presents a wide array of clinical symptoms including enhanced cancer predisposition and progressive cerebellar degeneration leading to general neuromotor dysfunction . The A-T cellular phenotype consists of higher levels of chromosome breakage, increased sensitivity to ionizing radiation and radiomimetic drugs, and defective cell cycle checkpoints in response to genome damage . Positional-cloning of the gene mutated in A-T, designated ATM, identified a 13 kb transcript encoding a 3056 amino acid protein which possesses a carboxy-terminal domain with distinct homology to phosphatidylinositol-3 kinase . Furthermore, ATM related proteins have been identified in yeast, Drosophila and other mammalian species which are involved in cell cycle control and cellular responses to DNA damage . Development of cellular and animal models for A-T can serve to better dissect the role and involvement of ATM in cell cycle regulation, cancer development, neuronal cell death and other hallmark symptoms of this disorder.

Cell Motil Cytoskeleton, 1997, 36(4), 363 - 76
Monoclonal antibodies recognizing the N- and C-terminal regions of talin disrupt actin stress fibers when microinjected into human fibroblasts; Bolton SJ et al.; We have characterized a panel of 6 monoclonal antibodies raised against human platelet talin by Western blotting, immune precipitation, and immunofluorescence, and shown that antibodies TA205 and TD77 disrupt actin stress fibers and focal adhesions, and inhibit cell motility when microinjected into human fibroblasts . Using a series of chick talin fusion proteins spanning the entire length of the molecule, we have mapped the epitopes recognized by these antibodies to the conserved N- and C-terminal regions of the protein . TA205 bound to an epitope contained within residues 139-433, a region which overlaps an F-actin binding site, and which shows homology with the ezrin/radixin/moesin family of cytoskeletal proteins . The epitope recognized by TD77 was located within the C-terminal region of the protein (residues 2269-2541) which also contains an F-actin binding site homologous to that in the yeast actin-binding protein SIa2p . To investigate the possibility that TD77 disrupts actin stress fibers by binding directly to the C-terminal actin binding site, additional talin fusion proteins were generated and analyzed for TD77 and actin binding . Fusion proteins containing residues 2269-2541, 2304-2541, and 2304-2463 all cosedimented with F-actin, whereas TD77 did not recognize the latter fusion protein . These results show that the C-terminal actin-binding site is distinct from the region recognized by the anti-functional antibody TD77, raising the possibility that it binds to a novel functionally important ligand-binding site in the talin molecule.

Cell Motil Cytoskeleton, 1997, 36(3), 253 - 65
Local accumulation of alpha-spectrin-related protein under plasma membrane during capping and phagocytosis in Acanthamoeba; Kwiatkowska K et al.; During capping and phagocytosis the interaction between cluster cell surface receptors and the submembraneous actin-based skeleton may be mediated by spectrin-like proteins . To test this possibility we examined the localization of an alpha-spectrin immunoanalogue, that had been previously identified in whole extracts of Acanthamoeba, during capping of Con A receptors and during phagocytosis of Con A-coated yeast . During capping alpha-spectrin and filamentous actin co-migrated with the Con A receptors and accumulated in the region of cap formation, as demonstrated by double immunofluorescence studies . Immunoelectron microscopy revealed submembraneous location of alpha-spectrin in cells exposed to Con A, both at the time of initial cross-linking and during accumulation of alpha-spectrin in the region of the cap . Phagocytosis studies showed that alpha-spectrin and actin filaments were concentrated around phagocytic cups that enclosed ConA-coated yeast upon internalization . The proteins also surrounded nascent phagosomes present in the vicinity of the plasma membrane but were absent at the later time point of phagosome maturation . These data demonstrate a correlation between clustering of cell surface receptors and submembraneous localization of alpha-spectrin, suggesting an involvement of spectrin-like proteins in mediating the interaction of receptor clusters with the actin cytoskeleton.

Ann Hum Genet, 1997 Jan, 61 ( Pt 1), 15 - 24
Fine structure physical mapping of a 1.9 Mb region of chromosome 13q12; Still IH et al.; Through linkage analysis and the identification of structural chromosome rearrangements, a number of disease genes have been mapped to the pericentromeric region of the long arm of chromosome 13 . Structural rearrangements, or deletions, of the 13q12 region have been implicated in a range of myeloproliferative neoplasms, and other haematopoietic malignancies . In particular, seven cases of a t(8;13)(p11;q12.1) rearrangement have been noted in patients with an atypical myeloproliferative disorder associated with T-cell leukemia and eosinophilia . We have previously identified a CEPH mega YAC, 943E4, which crosses the translocation breakpoint in archival tumour samples from two patients with this t(8;13) translocation . As an initial step in the characterisation of this translocation breakpoint, we have generated a fine structure physical map of this 1.9 Mb YAC . We have used the method of YAC fragmentation to generate a series of deletion constructs of known size, which provide discreet physical landmarks convenient for mapping genetic markers along the 943E4 YAC . Analysis of these deletion constructs defined the order of ESTs and microsatellite markers in 943E4 as: cen-NIB1257-(ATP1AL1/D13S283)-D13S179E-(D13S5 04E/D13S505E)-D13S824E-D13S182E -D13S221-tel . These markers have also been assigned to physically defined regions relative to the fragmented YAC endpoints and a derived NotI restriction map.

J Exp Biol, 1997 Jan, 200(Pt 2), 237 - 45
Molecular genetic analysis of V-ATPase function in Drosophila melanogaster; Dow JA et al.; V-ATPases are phylogenetically widespread, highly conserved, multisubunit proton pumps . Originally characterised in endomembranes, they have been found to energise transport across plasma membranes in a range of animal cells and particularly in certain epithelia . While yeast is the model of choice for the rapid generation and identification of V-ATPase mutants, it does not allow their analysis in a plasma membrane context . For such purposes, Drosophila melanogaster is a uniquely suitable model . Accordingly, we have cloned and characterised genes encoding several V-ATPase subunits in D . melanogaster and, using P-element technology, we have succeeded in generating multiple new alleles . Reporter gene constructs reveal ubiquitous expression, but at particularly high levels in those epithelial thought to be energised by V-ATPases, and several of the alleles have lethal recessive phenotypes characterised by epithelial dysfunction . These results, while providing the first gene knockouts of V-ATPases in animals, also illustrate the general utility of D . melanogaster as a model for the genetic analysis of ion transport and its control in epithelia.

Jpn J Cancer Res, 1997 Jan, 88(1), 39 - 43
Induction of p53-independent apoptosis associated with G2M arrest following DNA damage in human colon cancer cell lines; Arita D et al.; The tumor suppressor p53 protein induces apoptosis in response to various kinds of DNA damage in normal cells, but it is still unclear whether or not apoptosis induced by DNA damage correlates with the p53 status in tumor cells . We determined the status of p53 by functional analysis of separated alleles in yeast in five human colon cancer cell lines, SW-480, SW-620, DLD-1, COLO320 and LS174T and investigated whether p53 is necessary for apoptosis and cell cycle arrest after treatment of the cells with a DNA-damaging agent, etoposide (VP-16), or gamma-irradiation . Of these cell lines, only LS174T expresses a functional p53 . Apoptosis was detected in SW-480 and COLO320 cell lines, but not in the other cell lines, including LS174T cell line with a normal p53 function . Furthermore, cell cycle analysis revealed accumulation in the G2M phase preceding induction of apoptosis in SW-480 and COLO320 cells, but not in the other cells . These results suggest that apoptotic induction by DNA damage is not necessarily related to p53 status and that induction of p53-independent apoptosis following DNA damage may correlate with G2M arrest in the cell cycle, at least in the colon cancer cell lines used in this study.

Arch Environ Health, 1997 Jan-Feb, 52(1), 72 - 9
Airborne fungus allergen in association with residential characteristics in atopic and control children in a subtropical region; Li CS et al.; Airborne fungi were collected during the summer and winter seasons . A N6 Andersen sampler was used inside and outside the homes of 46 asthmatic children, 20 atopic children, and 26 nonatopic control children in the Taipei area . In addition, host and house characteristics were obtained by questionnaire . The indoor fungus concentrations of asthmatic and control groups were higher than those in atopic groups in summer, but there were no differences in total fungus concentrations among three groups in winter . Concentration differences among these three groups also occurred for Cladosporium and Penicillium in summer and for Aspergillus, Cladosporium, Penicillium, and yeast in the winter . Moreover, it was demonstrated that no differences in fungus concentration were observed between damp and dry homes . Penicillium concentrations appeared to be related to home dampness . Home dampness was associated with allergic symptoms in children with asthma and rhinitis . An association was also observed between the occurrence of Cladosporium and history of asthma.

Genome Res, 1997 Jan, 7(1), 59 - 64
A collection of 1814 human chromosome 7-specific STSs; Bouffard GG et al.; An established goal of the ongoing Human Genome Project is the development and mapping of sequence-tagged sites (STSs) every 100 kb, on average, across all human chromosomes . En route to constructing such a physical map of human chromosome 7, we have generated 1814 chromosome 7-specific STSs . The corresponding PCR assays were designed by the use of DNA sequence determined in our laboratory (79%) or generated elsewhere (21%) and were demonstrated to be suitable for screening yeast artificial chromosome (YAC) libraries . This collection provides the requisite landmarks for constructing a physical map of chromosome 7 at < 100-kb average spacing of STSs.

Genome Res, 1997 Jan, 7(1), 27 - 36
4.5-Mb YAC STS contig at 50-kb resolution, spanning Xq25 deletions in two patients with lymphoproliferative syndrome; Porta G et al.; Sequence-tagged site (STS) content mapping in yeast artificial chromosomes (YACs) was used to cover the region deleted in two patients affected with X-linked lymphoproliferative disorder . The order of markers includes, centromere to telomere, DXS8009-DXS1206-DXS8078-DXS8044-DXS982- DXS6811-DXS8093-AFM240xblO- DXS75-DXS737-DXS100-DXS6-DXS1046-DXS803 8 . The order of six major markers is confirmed by fluorescent in situ hybridization, and all the markers assigned by linkage mapping fall within a 1.6-cM interval . The contig comprises 90 clones containing 89 STSs, yielding a resolution of 50 kb; DNA in a gap just telomeric to DXS8044 has not been found in > 20 equivalents of YACs or bacterial clones . The two deletions were found to have centromeric breakpoints that lie close to DXS1206 and may be identical; the telomeric breakpoints are -150 kb apart, one falling between DXS737 and DXS100, the other between DXS100 and DXS1046 . Several STSs near the breakpoints show weak amplification from more than one site; one gives products from three groups of YACs, and lie, respectively, within 50 kb of the centromeric and the two telomeric deletion borders . Such partially duplicated segments of DNA are candidates for involvement in the formation of the deletions.

Genome Res, 1997 Jan, 7(1), 10 - 6
Identification and localization of the gene for EXTL, a third member of the multiple exostoses gene family; Wise CA et al.; Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by multiple bony outgrowths from the juxtaepiphyseal region of long bones . In a small proportion of cases, these exostoses progress to malignant chondrosarcomas . Genetic linkage of this disorder has been described to three independent loci on chromosomes 8q24.1 (EXT1), 11p11-13 (EXT2), and 19p (EXT-3) . The EXT1 and EXT2 genes were isolated recently and show extensive sequence homology to each other . These genes are deleted in exostoses-derived tumors, supporting the hypothesis that they encode tumor suppressors . We have identified a third gene that shows striking sequence similarity to both EXT1 and EXT2 at the nucleotide and amino acid sequence levels, and have derived its entire coding sequence . Although the mRNA transcribed from this gene is similar in size to that from EXT1 and EXT2, its pattern of expression is quite different . We have localized this gene by fluorescence in situ hybridization to metaphase chromosomes and by whole genome radiation hybrid mapping to chromosome 1p36.1 between DIS458 and DIS511, region that frequently shows loss of heterozygosity in a variety of tumor types . This gene, EXTL (for EXT-like), is therefore a new member of the EXT gene family and is a potential candidate for several disease phenotypes.

Steroids, 1997 Jan, 62(1), 169 - 75
The multiple murine 3 beta-hydroxysteroid dehydrogenase isoforms: structure, function, and tissue- and developmentally specific expression; Payne AH et al.; The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) is essential for the biosynthesis of all active steroid hormones . To date five distinct isoforms have been identified in the mouse . The different isoforms are indicated by roman numerals (I-V) in the chronological order in which they have been isolated . The different isoforms are expressed in a tissue- and developmentally specific manner and fall into two functionally distinct groups . 3 beta-HSD I, II, and III function as NAD(+)-dependent dehydrogenaselisomerases, and IV and V function as NADPH-dependent 3-keto steroid reductases . These latter two isoforms, therefore, are not involved in the biosynthesis of steroid hormones, but most likely in the inactivation of steroid hormones . In the adult mouse 3 beta-HSD I is expressed in the classical steroidogenic tissues, the adrenal glands and the gonads . 3 beta-HSD II and III are expressed in the liver and kidney, with III being the major isoform expressed in the adult liver . 3 beta-HSD IV is expressed almost exclusively in the kidney of both sexes, and expression of 3 beta-HSD V is observed only in the male liver starting late in puberty . In the fetal liver of both sexes, 3 beta-HSD I is the major or only isoform expressed at 13.5 days postconception and remains the major isoform until the day of birth, after which 3 beta-HSD III becomes the major isoform . Expression of 3 beta-HSD I in the liver decreases after birth and ceases by day 20 postnatally . Thus the liver expresses four distinct isoforms of 3 beta-HSD, I, II, III, and V, at different times during development . The mouse 3 beta-HSD genes, Hsd3b, have been mapped to a small region on mouse chromosome 3 . Analysis of two yeast artificial chromosome (YAC) libraries identified one clone that contains the entire Hsd3b locus within a 1400-kb insert . Hybridization by Southern blot analysis of restriction-enzyme-digested YAC DNA using an 18-base oligonucleotide that hybridizes without mismatch to all known Hsd3b sequences indicates that there are a total of seven Hsd3b genes or pseudogenes in the mouse genome . Further analysis of mouse genomic DNA by pulse field gel electrophoresis suggests that all of the Hsd3b gene family is found within a 400-kb fragment.

Biosci Biotechnol Biochem, 1997 Jan, 61(1), 177 - 8
Isolation and identification of alpha-glucosidase inhibitors from tochu-cha (Eucommia ulmoides); Watanabe J et al.; alpha-Glucosidase inhibitory activity was found in aqueous methanol extracts of tochu-cha, dried leaves of Eucommia ulmoides (Eucommiaceae) . Five active principles against yeast enzyme were isolated and characterized . Among them, quercetin (1, Ki: 8.5 x 10(-6) M) was considered to contribute mostly to the activity of the tochu leaves . In regard to an animal alpha-glucosidase, rat intestinal sucrase activity was also inhibited by 1.

Genomics, 1997 Jan 1, 39(1), 47 - 54
Comparative linkage mapping of human chromosome 13 and bovine chromosome 12; Sun HS et al.; A comparative linkage map of human chromosome 13 and bovine chromosome 12 was constructed using eight polymorphic microsatellite markers associated with six specific genes . Linkage of these was also examined relative to five previously mapped anonymous microsatellite markers . Seven gene-linked markers were developed from bovine large-insert genomic clones containing one of five genes of interest (serotonin receptor subtype 2, fms-related tyrosine kinase, coagulation factor 10, retinoblastoma susceptibility gene, collagen type IV alpha 1), and one additional marker was developed from a microsatellite resident within an intron of the bovine dopachrome tautomerase gene . Four of these loci were previously assigned to bovine chromosome 12 by analysis of a somatic cell hybrid panel . This study provides linkage information for examining gene order in this conserved synteny group . The comparative linkage mapping results indicate that the q arm of human chromosome 13 is almost entirely conserved in bovine chromosome 12 . One intrachromosomal rearrangement was detected in this linkage group relative to human, and this rearrangement was confirmed by fluorescence in situ hybridization results.

Genomics, 1997 Jan 1, 39(1), 19 - 29
Characterization of a Mus spretus YAC that maps to the pseudoautosomal region; Yen CH et al.; A library, containing M . spretus DNA in a half-YAC vector, was made and screened for clones hybridizing with an oligomer of the telomere hexamer TTAGGG . FISH to metaphase spreads of spleen cells showed hybridization of clone YTY3 to the distal ends of both X and Y chromosomes, consistent with localization to the pseudoautosomal region (PAR) . Recombinational mapping in the BXD RI strains and an interspecies backcross, using a plasmid subclone and PCR primers from YTY3, showed linkage to distal Chr X . The restriction map of the YAC contains three NotI sites . Sequences similar to Mov15 lie close to the vector end of the clone, while the other end contains a telomere array that appears to be interstitial within the PAR, suggesting that the insert represents a proximal portion of the PAR . Sequences homologous to the clone are also present at subtelomeric regions of autosomes 4, 9, and 13.

Plant J, 1997 Jan, 11(1), 93 - 103
Characterization of proteins that interact with the GTP-bound form of the regulatory GTPase Ran in Arabidopsis; Haizel T et al.; Ran, a small soluble GTP-binding protein, has been shown to be essential for the nuclear translocation of proteins and it is also thought to be involved in regulating cell cycle progression in mammalian and yeast cells . Genes encoding Ran-like proteins have been isolated from different higher plant species . Overexpression of plant Ran cDNAs, similarly to their mammalian/yeast homologues, suppresses the phenotype of the pim46-1 cell cycle mutant in yeast cells . The mammalian/yeast Ran proteins have been shown to interact with a battery of Ran-binding proteins, including the guanidine nucleotide exchange factor RCC1, the GTPase-activating Ran-GAP, nucleoporins and other Ran-binding proteins (RanBPs) specific for Ran-GTP . Here, the characterization of the first Ran-binding proteins from higher plants is reported . The yeast two-hybrid system was used to isolate cDNA clones encoding proteins of approximately 28 kDa (At-RanBP1a, At-RanBP1b) that interact with the GTP-bound forms of the Ran1, Ran2 and Ran3 proteins of Arabidopsis thaliana . The deduced amino acid sequences of the At-RanBP1s display high similarity (60%) to mammalian/yeast RanBP1 proteins and contain the characteristic Ran-binding domains . Furthermore, interaction of the plant Ran and RanBP1 proteins, is shown to require the acidic C-terminal domain (-DEDDDL) of Ran proteins in addition to the presence of an intact Ran-binding domain . In whole cell extracts, the GST-RanBP1a fusion protein binds specifically to GTP-Ran and will not interact with Rab/Ypt-type small GTP-binding proteins . Finally, in good agreement with their proposed biological function, the At-Ran and the At-RanBP genes are expressed coordinately and show the highest level of expression in meristematic tissues.

Am J Obstet Gynecol, 1997 Jan, 176(1 Pt 1), 138 - 41
Prevalence of and risk factors for fungal vaginitis caused by non-albicans species; Spinillo A et al.; OBJECTIVE: Our purpose was to evaluate the prevalence of symptomatic yeast vaginitis caused by non-albicans species among patients attending a vaginitis clinic over an 8-year period . STUDY DESIGN: A retrospective study of 1263 patients with symptomatic yeast vaginitis confirmed by culture techniques was performed . RESULTS: The prevalence of symptomatic fungal vaginitis caused by non-albicans species increased from 9.9% (10/101) in 1988 to 17.2% (36/209) in 1995 (chi 2 for trend = 9.33, p = 0.002) . Non-albicans species were found more frequently in known human immunodeficiency virus-seropositive patients (23/102 vs 143/1161, odds ratio 2.07, 95% confidence interval 1.2 to 3.46) than in seronegative subjects or subjects of unknown status for the virus . Recurrent vaginal candidiasis was an additional risk factor for vaginitis caused by non-albicans species (odds ratio 2.47, 95% confidence interval 1.72 to 3.52) . The increase in non-albicans isolates during the study period was confirmed in stratified analysis and in the subgroup of self-referred patients with no history of either human immunodeficiency virus infection or recurrent vaginal candidiasis . CONCLUSION: The prevalence of fungal vaginitis caused by non-albicans species has increased sharply in the setting of a vaginitis clinic . The characteristics of risk factors suggest that fungal cultures should be done routinely in human immunodeficiency virus-seropositive subjects with suspected vaginal candidiasis and in patients with recurrent vaginal infection.

DNA Cell Biol, 1997 Jan, 16(1), 9 - 16
The murine L-plastin gene promoter: identification and comparison with the human L-plastin gene promoter; Lin CS et al.; Plastins (or fimbrins) are a family of actin-binding proteins that are conserved from yeast to humans . In mammals, three tissue-specific plastin isoforms have been identified . The L isoform (L-plastin) is normally expressed only in leukocytes but is also found in >90% of neoplastic nonleukocyte human cells . Because L-plastin expression in tissue-specifically regulated in both humans and rodents, it is likely that similar mechanisms regulate L-plastin gene expression in human and rodent cells and that they could be identified by comparing the function and nucleotide sequences of the human and murine L-plastin gene promoters . Previously, we reported the isolation and characterization of the human L-plastin gene promoter . In this study, we isolated a murine L-plastin 5' end cDNA and used it as a probe to isolate several murine genomic clones . A representative clone contained 7 kb of the flanking region, 0.1 kb of the first exon, and 9.9 kb of the first intron . A continuous 1,354-bp sequence was identified around the first exon . Five transcription initiation sites were found 40 to 73 bp downstream from a perfect TATA box . Alignment of the sequence with its human counterpart revealed approximately 60% homology in a 1-kb region spanning the first exon and the flanking region . The TATA box, one ER binding site, and two ETS binding sites were completely conserved . An Sp1 binding sequence in the human promoter was partially conserved in the murine promoter but could still bind to Sp1 . A second ER binding sequence, lying 5' adjacent to the TATA box in the human promoter, was conserved only at the 3' half-site in the murine promoter; the 5' half-site was changed into a potential AP1 binding site . This AP1/ER hybrid sequence was incapable of binding to ER . However, both human and murine promoters were found to function equally well in either human or murine leukocytes.

Eur J Immunol, 1997 Jan, 27(1), 111 - 5
Regulated production of the interferon-gamma-inducible protein-10 (IP-10) chemokine by human neutrophils; Cassatella MA et al.; Interferon-gamma (IFN-gamma)-inducible protein-10 (IP-10), a member of the C-X-C sub-family of chemokines, is known to be produced by monocytes, lymphocytes, keratinocytes and endothelial cells in response to IFN-gamma . Here, we show that human polymorphonuclear neutrophils (PMN) also have the ability to produce IP-10 . IFN-gamma alone had a modest effect on IP-10 mRNA accumulation, whereas tumor necrosis factor-alpha (TNF-alpha), yeast particles opsonized with IgG (Y-IgG), lipopolysaccharide (LPS), and formyl-methionyl-leucyl-phenylalanine (fMLP) all failed to up-regulate IP-10 gene expression . However, stimulation of neutrophils with IFN-gamma in combination with either TNF-alpha or LPS (but not with Y-IgG or fMLP) resulted in a considerable induction of IP-10 mRNA transcripts, as well as in the extracellular release of the protein . In contrast, the best inducer of IP-10 release from peripheral blood mononuclear cells was IFN-gamma alone . Furthermore, mRNA stabilization analyses demonstrated that IP-10 mRNA isolated from PMN stimulated with IFN-gamma only, or with IFN-gamma plus either TNF-alpha or LPS, had similar half-lives . Finally, we found that interleukin-10, a known inhibitor of chemokine production in PMN, moderately suppressed the extracellular production of IP-10 in neutrophils stimulated with IFN-gamma plus either LPS or TNF-alpha . Since IP-10 is a potent chemoattractant for activated T lymphocytes, the ability of neutrophils to produce IP-10 might contribute to the evolution and progression of the inflammatory response.

Eur J Immunol, 1997 Jan, 27(1), 72 - 7
The cytoplasmic domain of rat NKR-P1 receptor interacts with the N-terminal domain of p56(lck) via cysteine residues; Campbell KS et al.; NKR-P1 is a type II transmembrane protein which acts as an activation receptor on natural killer (NK) cells . The cytoplasmic domains of the CD4, CD8 and 4-1BB receptors contain the sequence Cys-X-Cys-Pro which is directly involved in coupling to another pair of cysteines in the N-terminal domain of the src family tyrosine kinase p56(lck) . The cytoplasmic domain of NKR-P1 in rodents also contains the Cys-X-Cys-Pro sequence, but the capacity of the receptor to bind p56(lck) is presently unknown . We tested for direct coupling between these proteins using both protein biochemistry and the yeast two-hybrid technique . Immunoprecipitation studies showed that p56(lck) can be co-immunoprecipitated with NKR-P1 from a rat NK tumor cell line . In addition, the cytoplasmic domain of NKR-P1 interacted with the N-terminal domain of p56(lck) in yeast as assessed by reporter gene activation . Integrity of the cysteine pairs in both proteins was critical in mediating the interaction . The experiments suggest that the association of p56(lck) with NKR-P1 is somewhat weaker than the p56(lck) association with CD8alpha, but of much lower avidity than between CD4 and p56(lck) . This could reflect a higher activation threshold for the NKR-P1 and CD8 receptors, which are involved in cytolytic responses, compared to CD4 which is involved in T cell helper function.

Mamm Genome, 1997 Jan, 8(1), 21 - 8
A medium-density genetic linkage map of the bovine genome; Barendse W et al.; A cattle genetic linkage map was constructed which covers more than 95 percent of the bovine genome at medium density . Seven hundred and forty six DNA polymorphisms were genotyped in cattle families which comprise 347 individuals in full sibling pedigrees . Seven hundred and three of the loci are linked to at least one other locus . All linkage groups are assigned to chromosomes, and all are orientated with regards to the centromere . There is little overall difference in the lengths of the bull and cow linkage maps although there are individual differences between maps of chromosomes . One hundred and sixty polymorphisms are in or near genes, and the resultant genome-wide comparative analyses indicate that while there is greater conservation of synteny between cattle and humans compared with mice, the conservation of gene order between cattle and humans is much less than would be expected from the conservation of synteny . This map provides a basis for high-resolution mapping of the bovine genome with physical resources such as Yeast and Bacterial Artificial Chromosomes as well as providing the underpinning for the interpolation of information from the Human Genome Project.

Mamm Genome, 1997 Jan, 8(1), 9 - 15
Structure and expression of the mouse casein gene locus; Rijnkels M et al.; The analysis of yeast artificial chromosomes (YACs) containing the complete mouse casein gene locus revealed the presence of five casein genes, alpha-, beta-, gamma-, delta-, and kappa-casein, in this order, in the locus . The alpha- and beta-casein genes are only 10 kb apart and have convergent transcriptional orientations . The distance between the beta-casein gene and the alpha s2-like gamma-casein gene is about 70 kb, and these genes have divergent transcriptional orientations . The gamma- and delta-casein genes, both encoding a alpha s2-like casein, are linked within 60 kb and convergently transcribed . The kappa-casein gene is located about 100 kb from the delta-gene . Except for the presence of the delta-casein gene, the organization of the mouse casein locus resembles that of the bovine locus, including the transcriptional orientation of the genes . In contrast to the other casein genes, which are strongly induced at mid-lactation, expression of the delta-casein gene is abruptly induced upon parturition . Comparative analysis of alpha s2-like sequences from various species suggests that the ancestral alpha s2-like gene duplicated around the time of radiation of the rodent and artiodactylid ancestors.

Cutis, 1997 Jan, 59(1), 21 - 4
Malassezia furfur: a fungus belonging to the physiological skin flora and its relevance in skin disorders; Schmidt A; Malassezia furfur is an anthropophilic fungus that belongs to the physiological skin flora . The fungus can grow in a yeast phase as well as in a mycelial phase; on nonaffected skin the fungus is mainly prevalent in the yeast phase . The organism has complex lipid requirements for growth, which also explains its occurrence on the skin . This also leads to the requirement for specially supplemented media for in vitro cultivation . Malassezia furfur is the causative agent of pityriasis versicolor . It also seems to be associated with seborrheic dermatitis and dandruff formation, folliculitis, confluent and reticulate papillomatosis, and the provocation of psoriatic lesions . Many substances for topical application, such as azole antimycotics, ciclopirox olamine, piroctone-olamine, zinc pyrithione, or sulfur-containing substances are effective in the treatment of these diseases . In recent years rare cases of systemic infections and fungemias caused by Malassezia have been reported.

Trends Genet, 1997 Jan, 13(1), 21 - 6
TRF1, a mammalian telomeric protein; Smith S et al.; Telomerase adds TTAGGG repeats onto mammalian chromosome ends, replenishing the terminal sequence loss incurred during DNA replication . This maintenance of telomeric DNA preserves binding sites for telomeric proteins, which form a protective nucleoprotein complex at chromosome ends . The recent isolation of TRF1, the mammalian telomeric-repeat binding factor, should now allow the structure and function of the telomeric complex to be examined in detail.

Hum Genet, 1997 Jan, 99(1), 115 - 20
Refinement of the dominant optic atrophy locus (OPA1) to a 1.4-cM interval on chromosome 3q28-3q29, within a 3-Mb YAC contig; Jonasdottir A et al.; Dominant optic atrophy, type Kjer, is an autosomal dominant eye disease that is characterized by progressive optic atrophy with onset in early childhood, decrease of visual acuity, colour vision defects and centrocecal scotoma . By examination of 5 Danish families and the use of polymorphic markers, we have refined the localization of the OPA1 locus and assigned it to a 1.4-cM interval on chromosome 3q28-3q29, between markers D3S3669 and D3S3562 . This localizes the gene on a 3-Mb YAC contig covering the disease locus . We have also located a possible candidate gene HRY to this contig.

Hum Genet, 1997 Jan, 99(1), 11 - 7
Refined molecular characterization of the breakpoints in small inv dup(15) chromosomes; Huang B et al.; Inv dup(15) is the most common supernumerary marker chromosome in humans . To investigate the mechanism responsible for this frequent chromosome rearrangement, we characterized the breakpoints in 18 individuals with small inv dup(15) chromosomes {i.e., negative for the Prader-Willi (PWS)/Angelman syndrome (AS) critical region} . Since two proximal breakpoint regions ("hotspots") for PWS/AS deletions have been previously identified with the most proximal 15q markers D15S541/S542 and S543, we hypothesized that formation of the small inv dup(15) chromosomes may involve one or both of these breakpoint hotspots . By analysis with S542, both breakpoint regions were found to be involved in approximately equal frequencies . In ten cases, the inv dup(15) was negative for S542 (Class I), indicating the breakpoint is between the centromere and the most proximal marker on chromosome 15 . For the other eight cases, S542 was positive by fluorescence in situ hybridization (5/5) and/or microsatellite analysis (7/7), but S543 was negative (Class II) . These two breakpoint regions appear to be the same as the two proximal breakpoints reported in the common PWS/AS deletions . To initiate cloning and sequencing of the Class II breakpoint, the gap in the yeast artificial chromosome (YAC) contig between S541/S542 and S543 was filled by screening the CEPH YAC and mega-YAC libraries . YACs 705C2 and 368H3 were found to bridge this gap, and therefore contain the more distal breakpoint region . The finding of consistent breakpoints in small inv dup(15), like that found in PWS/AS deletions, provides strong evidence for hotspots for chromosome breakage in this region . In addition, our results show that two extra copies (tetrasomy) of the region from 15cen to the euchromatic region containing S542 are present in individuals with Class II breakpoints . Since most individuals carrying a small inv dup(15) are phenotypically normal, the euchromatin region included in the small inv dup(15) chromosomes does not appear to contain genes with clinically significant dosage effects.

Int Rev Cytol, 1997, 170, 1 - 38
Tropomyosin isoforms in nonmuscle cells; Lin JJ et al.; Vertebrate nonmuscle cells, such as human and rat fibroblasts, express multiple isoforms of tropomyosin, which are generated from four different genes and a combination of alternative promoter activities and alternative splicing . The amino acid variability among these isoforms is primarily restricted to three alternatively spliced exon regions; an amino-terminal region, an internal exon, and a carboxyl-terminal exon . Recent evidence reveals that these variable exon regions encode amino acid sequences that may dictate isoform-specific functions . The differential expression of tropomyosin isoforms found in cell transformation and cell differentiation, as well as the differential localization of tropomyosin isoforms in some types of culture cells and developing neurons suggest a differential isoform function in vivo . Tropomyosin in striated muscle works together with the troponin complex to regulate muscle contraction in a Ca(2+)-dependent fashion . Both in vitro and in vivo evidence suggest that multiple isoforms of tropomyosin in nonmuscle cells may be required for regulating actin filament stability, intracellular granule movement, cell shape determination, and cytokinesis . Tropomyosin-binding proteins such as caldesmon, tropomodulin, and other unidentified proteins may be required for some of these functions . Strong evidence for the distinct functions carried out by different tropomyosin isoforms has been generated from genetic analysis of yeast and Drosophila tropomyosin mutants.

J Rheumatol, 1997 Jan, 24(1), 202 - 5
Identification of polygenic disease genes; Cox RD; I discuss the identification and cloning of genes involved in determining susceptibility to diseases under polygenic control . The process of cloning a susceptibility gene is as follows: identification of new genetic markers in the region by database analysis, isolation of DNA clones in the region and the generation of new genetic markers, refinement of the map position using these markers for linkage disequilibrium analysis, construction of a physical and disequilibrium map, construction of a clone contig across the critical region in yeast artificial chromosomes, PAC, bacterial artificial chromosomes and cosmids, and finally gene identification and etiological mutation detection.

Genes Chromosomes Cancer, 1997 Jan, 18(1), 50 - 8
High-resolution deletion mapping of chromosome arm 17p in childhood primitive neuroectodermal tumors reveals a common chromosomal disruption within the Smith-Magenis region, an unstable region in chromosome band 17p11.2; Scheurlen WG et al.; Loss of heterozygosity (LOH) on chromosome arm 17p is the most common genetic aberration in childhood primitive neuroectodermal tumors (PNETs) . To determine the frequency and extent of 17p deletions, 29 loci on 17p were investigated in 24 tumors by using restriction fragment length polymorphism (RFLP) and microsatellite analysis . LOH on 17p was found in 9 of 24 tumors . In all tumors with LOH, a continuous stretch from the telomere to chromosome band 17p11.2 was completely deleted, and no interstitial or terminal small-scale deletions were detected in the remaining 15 tumors . In four tumors with LOH on 17p, the chromosomal breakpoint was located between D17S953 and D17S805 . To identify this deletion breakpoint on the cytogenetic map of chromosome 17 and to exclude uniparental disomy, we verified our data by using fluorescence in situ hybridization (FISH) analyses . By using two yeast artificial chromosome (YAC) clones that were positive for D17S689 and D17S953, the same breakpoint was confirmed in two specimens of cerebrospinal fluid (CSF) metastases by using FISH on interphase preparations . We demonstrate that, in most childhood PNETs with LOH on 17p, the breakpoint is close to, but not within, the centromere . It varies, and it occurs predominantly between the two markers D17S689 and D17S953, which is an unstable chromosomal region that is deleted or duplicated in the Smith-Magenis syndrome . Because LOH of 17p is associated with the formation of isochromosome 17q in the majority of PNETs, this study provides entry points to determine the molecular nature of this phenomenon.

RNA, 1997 Jan, 3(1), 17 - 26
Identification of specific nucleotide sequences and structural elements required for intronic U14 snoRNA processing; Xia L et al.; Vertebrate U14 snoRNAs are encoded within hsc70 pre-mRNA introns and U14 biosynthesis occurs via an intron-processing pathway . We have shown previously that essential processing signals are located in the termini of the mature U14 molecule and replacement of included boxes C or D with oligo C disrupts snoRNA synthesis . The experiments detailed here now define the specific nucleotide sequences and structures of the U14 termini that are essential for intronic snoRNA processing . Mutagenesis studies demonstrated that a 5', 3'-terminal stem of at least three contiguous base pairs is required . A specific helix sequence is not necessary and this stem may be extended to as many as 15 base pairs without affecting U14 processing . The spatial positioning of boxes C and D with respect to the terminal stem is also important . Detailed analysis of boxes C and D revealed that both consensus sequences possess essential nucleotides . Some, but not all, of these critical nucleotides correspond to those required for the stable accumulation of nonintronic yeast U14 snoRNA . The presence of box C and D consensus sequences flanking a terminal stem in many snoRNA species indicates the importance of this "terminal core motif" for snoRNA processing.

Nat Genet, 1997 Jan, 15(1), 36 - 41
Mutations in TWIST, a basic helix-loop-helix transcription factor, in Saethre-Chotzen syndrome; Howard TD et al.; Saethre-Chotzen syndrome is one of the most common autosomal dominant disorders of craniosynostosis in humans and is characterized by craniofacial and limb anomalies . The locus for Saethre-Chotzen syndrome maps to chromosome 7p21-p22 . We have evaluated TWIST, a basic helix-loop-helix transcription factor, as a candidate gene for this condition because its expression pattern and mutant phenotypes in Drosophila and mouse are consistent with the Saethre-Chotzen phenotype . We mapped TWIST to human chromosome 7p21-p22 and mutational analysis reveals nonsense, missense, insertion and deletion mutations in patients . These mutations occur within the basic DNA binding, helix I and loop domains, or result in premature termination of the protein . Studies in Drosophila indicate that twist may affect the transcription of fibroblast growth factor receptors (FGFRs), another gene family implicated in human craniosynostosis . The emerging cascade of molecular components involved in craniofacial and limb development now includes TWIST, which may function as an upstream regulator of FGFRs.

Nat Genet, 1997 Jan, 15(1), 21 - 9
Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family; Li QY et al.; Holt-Oram syndrome is a developmental disorder affecting the heart and upper limb, the gene for which was mapped to chromosome 12 two years ago . We have now identified a gene for this disorder (HOS1) . The gene (TBX5) is a member of the Brachyury (T) family corresponding to the mouse Tbx5 gene . We have identified six mutations, three in HOS families and three in sporadic HOS cases . Each of the mutations introduces a premature stop codon in the TBX5 gene product . Tissue in situ hybridization studies on human embryos from days 26 to 52 of gestation reveal expression of TBX5 in heart and limb, consistent with a role in human embryonic development.

Cancer Res, 1997 Jan 1, 57(1), 24 - 7
ATM gene product phosphorylates I kappa B-alpha; Jung M et al.; The recently cloned ATM gene is mutated in patients with ataxia telangiectasia, but its biological functions remain to be experimentally determined . Structural analysis has revealed ATM sequence similarities to the catalytic domains of phosphatidyl-3 kinase and other members of this family of yeast and mammalian proteins . Rabbit polyclonal antibodies raised against polypeptide regions unique to the COOH terminus and to the NH2 terminus of the published ATM sequence confirm ATM as M(r) approximately 350,000 protein in normal cells, which is missing in AT cells . Immunoprecipitated protein(s) is capable of phosphorylating I kappa B-alpha in an in vitro kinase assay . However, we did not observe a phosphatidyl-3 kinase or a DNA-dependent protein kinase function by ATM immunoprecipitates . These data support a protein kinase activity for ATM and suggest a role in NF-kappa B activation.

J Neurosci, 1997 Jan 1, 17(1), 152 - 9
Essential role for dlg in synaptic clustering of Shaker K+ channels in vivo; Tejedor FJ et al.; The assemblage of specific ion channels and receptors at synaptic sites is crucial for signaling between pre- and postsynaptic cells . However, the mechanisms by which proteins are targeted to and clustered at synapses are poorly understood . Here we show that the product of the Drosophila discs-large gene, DLG, is colocalized with Shaker K+ channels, which are clustered at glutamatergic synapses at the larval neuromuscular junction . In heterologous cells, DLG can cluster Shaker-type K+ channels, and, in the yeast two-hybrid system, the DLG PDZ1-2 domains bind directly to the C-terminal tail of Shaker proteins . We also demonstrate that DLG-Shaker interactions are required in vivo for Shaker clustering at the neuromuscular junction . Synaptic clustering of Shaker channels is abolished not only by mutations in dlg but also by a mutation in Shaker that deletes its C-terminal DLG binding motif . Analyses of various dlg mutant alleles suggest that channel clustering and synaptic targeting functions depend on distinct DLG domains . These studies demonstrate for the first time that DLG plays an important role in synaptic organization in vivo that correlates with its ability to bind directly to specific membrane proteins of the synapse.

Nat Med, 1997 Jan, 3(1), 89 - 93
Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs; Griffiths M et al.; In most mammalian cells nucleoside uptake occurs primarily via broad-specificity, es (e, equilibrative; 5, sensitive to NBMPR inhibition) transporters that are potently inhibited by nitrobenzylthioinosine (NBMPR) . These transporters are essential for nucleotide synthesis by salvage pathways in hemopoietic and other cells that lack de novo pathways and are the route of cellular uptake for many cytotoxic nucleosides used in cancer and viral chemotherapy . They play an important role in adenosine-mediated regulation of many physiological processes, including neurotransmission and platelet aggregation, and are a target for coronary vasodilator drugs . We have previously reported the purification of the prototypic es transporter from human erythrocytes and have shown that this glycoprotein of apparent M, 55,000 is immunologically related to nucleoside transporters from several other species and tissues, including human placenta . Here we report the isolation of a human placental cDNA encoding a 456-residue glycoprotein with functional characteristics typical of an es-type transporter . It is predicted to possess 11 membrane-spanning regions and is homologous to several proteins of unknown function in yeast, nematodes, plants and mammals . Because of its central role in the uptake both of adenosine and of chemotherapeutic nucleosides, study of this protein should not only provide insights into the physiological roles of nucleoside transport but also open the way to improved therapies.

J Virol, 1997 Jan, 71(1), 179 - 90
Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid protein (pUL86) through two different domains; Wood LJ et al.; We have used the yeast GAL4 two-hybrid system to examine interactions between the human cytomegalovirus (HCMV) major capsid protein (MCP, encoded by UL86) and the precursor assembly protein (pAP, encoded by UL80.5 and cleaved at its carboxyl end to yield AP) and found that (i) the pAP interacts with the MCP through residues located within the carboxy-terminal 21 amino acids of the pAP, called the carboxyl conserved domain (CCD); (ii) the pAP interacts with itself through a separate region, called the amino conserved domain (ACD), located between amino acids His34 and Arg52 near the amino end of the molecule; (iii) the simian CMV (SCMV) pAP and AP can interact with or replace their HCMV counterparts in these interactions, whereas the herpes simplex virus pAP and AP homologs cannot; and (iv) the HCMV and SCMV maturational proteinase precursors (ACpra, encoded by UL80a and APNG1, respectively) can interact with the pAP and MCP . The ACD and CCD amino acid sequences are highly conserved among members of the betaherpesvirus group and appear to have counterparts in the alpha- and gammaherpesvirus pAP homologs . Deleting the ACD from the HCMV pAP, or substituting Ala for a conserved Leu in the ACD, eliminated detectable pAP self-interaction and also substantially reduced MCP binding in the two-hybrid assay . This finding indicates that the pAP self-interaction influences the pAP-MCP interaction . Immunofluorescence studies corroborated the pAP-MCP interaction detected in the GAL4 two-hybrid experiments and showed that nuclear transport of the MCP was mediated by pAP but not AP . We conclude that the pAP interacts with the MCP, that this interaction is mediated by the CCD and is influenced by pAP self-interaction, and that one function of the pAP-MCP interaction may be to provide a controlled mechanism for transporting the MCP into the nucleus.

Blood, 1997 Jan 1, 89(1), 297 - 306
Direct binding of CRKL to BCR-ABL is not required for BCR-ABL transformation; Heaney C et al.; CRKL has previously been shown to be a major tyrosine phosphorylated protein in neutrophils of patients with BCR-ABL+ chronic myelogenous leukemia and in cell lines expressing BCR-ABL CRKL and BCR-ABL form a complex as demonstrated by coimmunoprecipitation and are capable of a direct interaction in a yeast two-hybrid assay . We have mapped the site of interaction of CRKL and BCR-ABL to the amino terminal SH3 domain of CRKL with a proline rich region in the C-terminus of ABL . The proline-rich region was mutated and the effect of this deletion on BCR-ABL transforming function was assayed . Our data show that this deletion does not impair the ability of BCR-ABL to render myeloid cells factor independent for growth . In cells expressing the proline deletion mutation of BCR-ABL, CRKL is still tyrosine phosphorylated and forms a complex with BCR-ABL as demonstrated by coimmunoprecipitation . Our data suggest that the interaction between CRKL and the proline deletion mutant of BCR-ABL is an indirect interaction as CRKL does not interact directly with the proline deletion mutant of BCR-ABL in a gel overlay assay or in a yeast two-hybrid assay . Thus, a direct interaction of CRKL and BCR-ABL is not required for CRKL to become tyrosine phosphorylated by BCR-ABL and suggests that CRKL function may still be required for BCR-ABL function through an indirect interaction.

J Immunol, 1997 Jan 1, 158(1), 376 - 83
Monocyte chemoattractant protein-1 is sufficient for the chemotaxis of monocytes and lymphocytes in transgenic mice but requires an additional stimulus for inflammatory activation; Gunn MD et al.; Monocyte chemoattractant protein-1 (MCP-1), a chemotactic cytokine, acts in vitro as a chemotactic and activating factor for multiple types of leukocytes . To determine the chemotactic and activating effects of MCP-1 in vivo, we constructed transgenic mice that express human MCP-1 in type II alveolar epithelial cells and secrete it into the bronchoalveolar space . We found that MCP-1 overexpression led to a marked increase in the numbers of both monocytes and lymphocytes that could be recovered by bronchoalveolar lavage . This accumulation of mononuclear leukocytes could be reversed by the administration of an MCP-1-blocking Ab . In spite of its chemotactic effect, MCP-1 expression did not cause the inflammatory activation of accumulated leukocytes . Lungs of MCP-1 transgenic mice also showed no morphologic evidence of inflammation . However, MCP-1 mice had an increased sensitivity to other inflammatory stimuli . MCP-1 mice treated with either i.p . LPS or i.v . yeast wall glucan developed consolidated pulmonary infiltrates consisting predominantly of macrophages . Nontransgenic mice developed no such infiltrates . These results demonstrate that MCP-1 is chemotactic for monocytes and lymphocytes in vivo and that MCP-1 expression alone does not cause inflammatory activation of cells, but leads to an enhanced inflammatory response upon treatment with other stimuli.

Infect Immun, 1997 Jan, 65(1), 279 - 84
Development of an in vitro macrophage system to assess Penicillium marneffei growth and susceptibility to nitric oxide; Cogliati M et al.; We investigated the effect of nitric oxide (NO) and reactive nitrogen intermediates on the in vitro growth of Penicillium marneffei both in a cell-free system and in a novel macrophage culture system . In the cell-free system, NO that was chemically generated from NaNO2 in acid media (pH 4 and 5) markedly inhibited the growth of P . marneffei . On the contrary, inhibition of growth did not occur in neutral medium (pH 7.4) in which NO was not produced . P . marneffei conidia were phagocytized by nonstimulated murine J774 macrophages after 2 h of incubation . During the following 24 h, P . marneffei grew as yeast-like cells replicating by fission in the J774 macrophages . The intracellular growth of P . marneffei damaged nonstimulated J774 macrophages, as confirmed by electron microscopy . When J774 cells were stimulated by gamma interferon and lipopolysaccharide, which led to enhanced production of reactive nitrogen intermediates, the percentage of yeast-like cells was significantly reduced and P . marneffei conidia were damaged in the J774 macrophages . The inhibition of NO synthesis by N-monomethyl-L-arginine restored the intracellular growth of P . marneffei . The inverse correlation between intramacrophage growth and the amount of nitrite detected in culture supernatants supports the hypothesis that the L-arginine-dependent NO pathway plays an important role in the murine macrophage immune response against P . marneffei.

Curr Microbiol, 1997 Jan, 34(1), 1 - 5
Induction and Characterization of Laccase in the Ligninolytic Fungus Pleurotus eryngii
Munoz C, Guillen F, Martinez AT, Martinez MJ.
Two protein bands with laccase activity were found after PAGE of culture liquid or mycelium extract of Pleurotus eryngii, grown on glucose-ammonium tartrate-yeast extract medium with and without inducers . A major and a minor laccase band were observed in the basal medium . The intensity of the major band (laccase I) did not change after the addition of inducers . However, the minor band (laccase II), characterized by higher electrophoretic mobility, was strongly induced by wheat-straw alkalilignin and vanillic and veratric acids . Laccase activity in the basal medium had an optimum pH of 4.5 and was stable from pH 3 to 10 during 24 h at room temperature . This enzyme had wide substrate specificity on hydroquinones, methoxy-substituted monophenols, and aromatic amines . In general, laccase activity was found only with compounds having a redox potential lower than 0.5 mV . The highest activity was obtained with methoxy- and methyl-substituted p-hydroquinones and aromatic diamines . Some activity also occurred with the aliphatic compound 3,5-cyclohexadiene-1,2-diol.

DNA Res, 1996 Dec 31, 3(6), 425 - 9
The chromosomal organization of the human endogenous retrovirus-like sequence HERV-H: clustering of the HERV-H sequences in a 300-kb region close to the GRPR locus on the X chromosome; Shiraishi M et al.; Within the haploid genome there are approximately 1,000 copies of the human endogenous retrovirus-like sequence, HERV-H . Although these sequences are scattered throughout the entire genome, in situ hybridization experiments revealed that there are discrete clusters positioned on chromosomes 1 p and 7 q . In this study, we have located three HERV-H sequences which were unexpectedly clustered within a 300-kilobase region close to the GRPR locus on the X chromosome . In previous studies, no clustering of this sequence has been reported at this locus . Our finding demonstrates that, like other repetitive sequences, clustering of HERV-H occurs in the human genome, although these sequences may not always be detected by in situ hybridization methods.

DNA Res, 1996 Dec 31, 3(6), 401 - 6
Assignment of YAC clones spanning rice chromosomes 10 and 12; Shimokawa T et al.; Yeast artificial chromosome (YAC) clones were assigned on rice (Oryza saliva L . cv . Nipponbare) chromosomes 10 and 12 using DNA markers from our high-density linkage map . Out of 1,383 markers localized in this genetic map, 68 and 74 markers were located on chromosomes 10 and 12, respectively . Screening of the YAC genomic library was conducted by colony hybridization and Southern hybridization using restriction fragment length polymorphism (RFLP) markers or by polymerase chain reaction (PCR) using sequence-tagged site (STS) markers . We have completed the screening of 68 markers on chromosomes 10 and 74 markers on chromosome 12 . A total of 134 and 103 YACs were assigned to chromosomes 10 and 12, respectively, with an estimated coverage of more than 60% for chromosome 10 and about 47% for chromosome 12 . As rice is considered a model plant for genome analysis, the ordered YAC clones on chromosomes 10 and 12 as well as other chromosomes will certainly be helpful for isolation of agronomically and biologically important genes and for understanding the genome structure of these chromosomes.

DNA Res, 1996 Dec 31, 3(6), 393 - 400
Physical mapping of rice chromosomes 8 and 9 with YAC clones; Antonio BA et al.; First efforts for physical mapping of rice chromosomes 8 and 9 were carried out by ordering YAC clones of a rice genomic DNA library covering six genome equivalents with mapped DNA markers . A total of 79 and 74 markers from chromosomes 8 and 9, respectively, were analyzed by YAC colony and Southern hybridization using RFLP markers of cDNA and genomic clones, and by polymerase chain reaction (PCR) screening using PCR-derived and sequence-tagged site (STS) markers . As a result, 252 YAC clones were confirmed to contain the mapped DNA fragments on both chromosomes . A contig map was constructed by ordering these YAC clones and about 53% and 43% genome coverage was obtained for chromosomes 8 and 9, respectively, assuming a YAC clone size of 350 kb and overlap between neighboring YACs of 50% . A continuous array of YAC clones with minimum overlap gave a total size of 18.9 Mb for chromosome 8 and 15.6 Mb for chromosome 9, which are close to previous estimates . These contig maps may provide valuable information that can be useful in understanding chromosome structure and isolating specific genes by map-based cloning.

Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15299 - 304
A novel member of the RING finger family, KRIP-1, associates with the KRAB-A transcriptional repressor domain of zinc finger proteins; Kim SS et al.; The Kruppel-associated box A (KRAB-A) domain is an evolutionarily conserved transcriptional repressor domain present in approximately one-third of zinc finger proteins of the Cys2-His2 type . Using the yeast two-hybrid system, we report the isolation of a cDNA encoding a novel murine protein, KRAB-A interacting protein 1 (KRIP-1) that physically interacts with the KRAB-A region . KRIP-1 is a member of the RBCC subfamily of the RING finger, or Cys3HisCys4, family of zinc binding proteins whose other members are known to play important roles in differentiation, oncogenesis, and signal transduction . The KRIP-1 protein has high homology to TIF1, a putative modulator of ligand-dependent activation function of nuclear receptors . A 3.5-kb mRNA for KRIP-1 is ubiquitously expressed among all adult mouse tissues studied . When a GAL4-KRIP-1 fusion protein is expressed in COS cells with a chloramphenicol acetyltransferase reporter construct with five GAL4 binding sites, there is dose-dependent repression of transcription . Thus, KRIP-1 interacts with the KRAB-A region of C2H2 zinc finger proteins and may mediate or modulate KRAB-A transcriptional repressor activity.

J Mol Biol, 1996 Dec 20, 264(5), 1072 - 84
The crystal structure of a hyperthermophilic archaeal TATA-box binding protein; DeDecker BS et al.; This study analyzes the three-dimensional structure of the TATA-box binding protein (TBP) from the hyperthermophilic archaea Pyrococcus woesei . The crystal structure of P . woesei TBP (PwTBP) was solved at 2.2 A by X-ray diffraction and as expected from sequence homology (36% to 41% identical to eukaryotic TBPs) its overall structure is very similar to eukaryotic TBPs . The thermal unfolding transition temperature of this protein was measured by differential scanning calorimetry to be 101 degrees C, which is more than 40 degrees C higher than that of yeast TBP . Preliminary titration calorimetry data show that the affinity of PwTBP for its DNA target, unlike its eukaryotic counterparts, is enhanced by increasing the temperature and salt concentration . The structure reveals possible explanations for this thermostability and these unusual DNA binding properties . The crystal structure of this hyperthermostable protein was compared to its mesophilic homologs and analyzed for differences in the native structure that may contribute to thermostability . Differences found were: (1) a disulfide bond not found in mesophilic counterparts; (2) an increased number of surface electrostatic interactions; (3) more compact protein packing . The presumed DNA binding surface of PwTBP, like its eukaryotic counterparts, is hydrophobic but the electrostatic profile surrounding the protein is relatively neutral compared to the asymmetric positive potential that surrounds eukaryotic TBPs . The total reliance on a hydrophobic interface with DNA may explain the enhanced affinity of PwTBP for its DNA promoter at higher temperatures and increased salt concentration.

J Biol Chem, 1996 Dec 20, 271(51), 33026 - 31
Products of the grg (Groucho-related gene) family can dimerize through the amino-terminal Q domain; Pinto M et al.; The murine grg (Groucho-related gene) products are believed to interact with transcription factors and repress transcription, thereby regulating cell proliferation and differentiation . Most proteins in the grg family contain all of the domains found in the Drosophila Groucho protein, including the S/P (Ser-Pro-rich) domain required for interaction with transcription factors and the WD40 domain, which is thought to interact with other proteins . However, at least two Grg proteins contain only the amino-terminal Q (glutamine-rich) domain . We examined whether the Q domain is used for dimerization between Grg proteins, using the yeast two-hybrid system and binding assays with glutathione S-transferase fusion proteins . We found that Grg proteins are able to dimerize through the Q domain and that dimerization requires a core of 50 amino acids . Surprisingly, the dimerization does not require the leucine zipper located within the Q domain.

J Biol Chem, 1996 Dec 20, 271(51), 32944 - 50
Isolation and reconstitution of the heme-thiolate protein obtusifoliol 14alpha-demethylase from Sorghum bicolor (L.) Moench; Kahn RA et al.; The heme-thiolate (cytochrome P450) enzyme which catalyzes the 14alpha-demethylation of obtusifoliol has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench . The obtusifoliol 14alpha-demethylase is a key enzyme in plant sterol biosynthesis and a target for the design of phyla-specific sterol 14alpha-demethylase inhibitors . Microsomal cytochrome P450s were solubilized by using the detergents Renex 690 and reduced Triton X-100, and the obtusifoliol 14alpha-demethylase was isolated by DEAE ion exchange and dye affinity column chromatography . The isolated enzyme has an absorption spectrum characteristic for low spin cytochrome P450s and produces a Type I binding spectrum with obtusifoliol as substrate . Binding spectra were not obtained with lanosterol, campesterol, sitosterol, or stigmasterol . Obtusifoliol 14alpha-demethylase has an apparent molecular mass of 53 kDa and is estimated to constitute approximately 20% of the total cytochrome P450 content of the microsomal membranes and about 0.2% of the total microsomal protein . Gas chromatography-mass spectrometry analysis of reconstitution experiments with dilauroylphosphatidylcholine micelles containing isolated obtusifoliol 14alpha-demethylase and sorghum NADPHcytochrome P450 oxidoreductase demonstrated the conversion of obtusifoliol (4alpha,14alpha-dimethyl-5alpha-ergosta-8, 24(28)-dien-3beta-ol) to 4alpha-methyl-5alpha-ergosta-8,14, 24(28)-trien3beta-ol, the 14alpha-demethylated product of obtusifoliol with a double bond introduced at the Delta14 position . The N-terminal amino acid sequence of the protein is MDLADIPQ/KQQRLMAGXALVV . Five internal sequences were obtained after endoproteinase Lys-C and Glu-C digestion . The fragment AAGAFSYISFGGGRH aligns with the unique heme binding domain of mammalian and yeast sterol 14alpha-demethylases which belong to the CYP51 family . Therefore it is conceivable that the obtusifoliol 14alpha-demethylase from plants also belongs to the CYP51 family, the only P450 family so far known to be conserved across the phyla.

J Biol Chem, 1996 Dec 20, 271(51), 32937 - 43
Type I phosphatidylinositol-4-phosphate 5-kinases are distinct members of this novel lipid kinase family; Loijens JC et al.; Phosphatidylinositol-4-phosphate 5-kinases (PIP5K) synthesize phosphatidylinositol-4,5-bisphosphate, a key precursor in phosphoinositide signaling that also regulates some proteins and cellular processes directly . Two distinct PIP5Ks have been characterized in erythrocytes, the 68-kDa type I (PIP5KI) and 53-kDa type II (PIP5KII) isoforms . Using peptide sequences from the erythroid 68-kDa PIP5KI, we have isolated cDNAs encoding PIP5KIalpha from human brain . Partial cDNAs obtained for a second isoform, PIP5KIbeta, established that the human STM7 gene encoded a previously unrecognized PIP5KI . However, the peptide sequences demonstrated that erythroid PIP5KI corresponded to PIP5KIalpha . Recombinant, bacterially expressed PIP5KIalpha possessed PIP5K activity and was immunoreactive with erythroid PIP5KI antibodies . By Northern analysis, PIP5KIalpha and PIP5KIbeta had wide tissue distributions, but their expression levels differed greatly . PIP5KIs had homology to the kinase domains of PIP5KIIalpha, yeast Mss4p and Fab1p, and a new Caenorhabditis elegans Fab1-like protein identified in the data base . These new isoforms have refined the sequence requirements for PIP5K activity and, potentially, regulation of these enzymes . Furthermore, the limited homology between PIP5KIs and PIP5KIIalpha, which was almost exclusively within the kinase domain core, provided a molecular basis for distinction between type I and II PIP5Ks.

J Biol Chem, 1996 Dec 20, 271(51), 32923 - 9
FAP48, a new protein that forms specific complexes with both immunophilins FKBP59 and FKBP12 . Prevention by the immunosuppressant drugs FK506 and rapamycin; Chambraud B et al.; We have identified a human gene encoding a 48-kDa protein that specifically interacts with the peptidyl prolyl isomerase FK506-binding protein 59 (FKBP59) and also with the well known FKBP12 . FKBP59 and FKBP12 belong to the large family of immunophilins that bind the macrolide immunosuppressant drugs FK506 and rapamycin . The yeast two-hybrid system was used to isolate target proteins that interact with the immunosuppressant drug binding domain of the rabbit FKBP59 . The cDNA for an as yet unidentified protein was isolated and cloned from a Jurkat cell library . The cDNA sequence of 1804 base pairs reveals an open reading frame of 417 amino acids . In vitro experiments suggest a direct interaction between FKBP59 and this new target protein . This specific association seems to be restricted to the FKBP family, since it also occurs both in vivo and in vitro with FKBP12 but not with cyclophilin 40 . This novel protein was named FKBP-associated protein (FAP48) . The formation of the complexes between FKBP59 or FKBP12 and FAP48 is prevented by FK506 and rapamycin in a dose-dependent manner . These results suggest that FAP48 shares or overlaps the macrolide binding site on FKBP59 as well as on FKBP12 and therefore may represent a natural common ligand of these immunosuppressant drug receptors.

J Biol Chem, 1996 Dec 20, 271(51), 32863 - 8
The nuclear localization sequences of the BRCA1 protein interact with the importin-alpha subunit of the nuclear transport signal receptor; Chen CF et al.; The BRCA1 gene product is a nuclear phosphoprotein that is aberrantly localized in the cytoplasm of most breast cancer cells . In an attempt to elucidate the potential mechanism for the nuclear transport of BRCA1 protein, three regions of highly charged, basic residues, 503KRKRRP508, 606PKKNRLRRKS615, and 651KKKKYN656, were identified as potential nuclear localization signals (NLSs) . These three regions were subsequently mutated to 503KLP508, 607KLS615, and 651KLN656, respectively . Wild-type and mutated proteins were tagged with the flag epitope, expressed in human DU145 cells, and detected with the M2 monoclonal antibody . In DU145 cells, the KLP mutant completely fails to localize in nuclei, whereas the KLS mutant is mostly cytoplasmic with occasional nuclear localization . The KLN protein is always located in nuclei . Consistently, hSRP1alpha (importin-alpha), a component of the NLS receptor complex, was identified in a yeast two-hybrid screen using BRCA1 as the bait . The specificity of the interaction between BRCA1 and importin-alpha was further demonstrated by showing that the 503KRKRRP508 and 606PKKNRLRRKS615 regions, but not 651KKKKYN656, are critical for this interaction . To determine if the cytoplasmic mislocation of endogenous BRCA1 in breast cancer cells is due to a deficiency of the cells, wild-type BRCA1 protein tagged with the flag epitope was ectopically expressed in six breast cancer cell lines . The analysis demonstrated that, in all six, this protein localized in the cytoplasm of these cells . In contrast, expression of the construct in four non-breast cancer cell lines resulted in nuclear localization . These data support the possibility that the mislocation of the BRCA1 protein in breast cancer cells may be due to a defect in the cellular machinery involved in the NLS receptor-mediated pathway of nuclear import.

Oncogene, 1996 Dec 19, 13(12), 2727 - 30
Inverse correlation between RER+ status and p53 mutation in colorectal cancer cell lines; Cottu PH et al.; The high point mutation rate of replication error-prone (RER+) cells could theoretically lead to inactivation of the p53 gene by polyclonal mutations, which might explain the conflicting results that have been published on the p53 status of RER+ colon cancers . To address this issue, we tested the p53 status of 21 human colorectal cancer cell lines, including four showing microsatellite instability (RER+ phenotype) . Denaturing gradient gel electrophoresis (DGGE) followed by sequencing showed that all four RER+ cell lines were wild type for p53 while 15 of the 17 RER- cell lines contained p53 mutations (P=0.001) . Eight cell lines (four RER+ and four RER-) were analysed using three complementary methods to test more rigorously the polyclonal mutation hypothesis . (i) Of 87 single-cell clones (seven to 14 per cell line) examined by DGGE, only those derived from known p53 mutant cell lines showed altered profiles . (ii) Antibody DO-7 stained more than 80% of nuclei from the p53 mutant cell lines, but only 15% of nuclei from the RER+ cell lines . (iii) A yeast functional assay which can simultaneously detect polyclonal mutations at over 500 different sites in the p53 cDNA scored all four RER+ cell lines as containing only transcriptionally active p53 . These data thus do not support the polyclonal mutation hypothesis and instead suggest that mismatch repair deficiency provides a p53-independent pathway for development of colorectal cancers.

EMBO J, 1996 Dec 16, 15(24), 6931 - 42
A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum; Dierks T et al.; Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates . Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins . The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes . Transport was restored by co-reconstitution of the ATP eluate . This eluate showed eight distinct bands in SDS gels . We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5) . In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose . Purified BiP did not stimulate transport activity . Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes . We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.

Nucleic Acids Res, 1996 Dec 15, 24(24), 5054 - 5
A method for high efficiency YAC lipofection into murine embryonic stem cells; Lee JT et al.; We describe a modified protocol for introducing yeast artificial chromosomes (YACs) into murine embryonic stem (ES) cells by lipofection . With a decreased DNA:cell ratio, increased concentration of condensing agents and altered culture conditions, this protocol reduces the requirement for YAC DNA to a few micrograms, improves the recovery of neomycin-resistant ES colonies and increases the yield of clones containing both flanking vector markers and insert . These modifications enable generation of sufficient 'intact' transgenic clones for biological analysis with a single experiment.

Nucleic Acids Res, 1996 Dec 15, 24(24), 4859 - 67
Transcriptional repression by RING finger protein TIF1 beta that interacts with the KRAB repressor domain of KOX1; Moosmann P et al.; Many of the vertebrate zinc finger factors of the Kruppel type (C2H2 zinc fingers) contain in their N-terminus a conserved sequence referred to as the KRAB (Kruppel-associated box) domain that, when tethered to DNA, efficiently represses transcription . Using the yeast two-hybrid system, we have isolated an 835 amino acid RING finger (C3HC4 zinc finger) protein, TIF1 beta (also named KAP-1), that specifically interacts with the KRAB domain of the human zinc finger factor KOX1/ZNF10 . TIF1 beta, TIF1 alpha, PML and efp belong to a characteristic subgroup of RING finger proteins that contain one or two other Cys/His-rich clusters (B boxes) and a putative coiled-coil in addition to the classical C3HC4 RING finger motif (RBCC configuration) . Like TIF1 alpha, TIF1 beta also contains an additional Cys/His cluster (PHD finger) and a bromo-related domain . When tethered to DNA, TIF1 beta can repress transcription in transiently transfected mammalian cells both from promoter-proximal and remote (enhancer) positions, similarly to the KRAB domain itself . We propose that TIF1 beta is a mediator of the transcriptional repression exerted by the KRAB domain.

Exp Cell Res, 1996 Dec 15, 229(2), 272 - 5
Expression of the meiosis-specific synaptonemal complex protein 1 in a heterologous system results in the formation of large protein structures; Yuan L et al.; The synaptonemal complex is a meiosis-specific structure essential for synapsis of homologous chromosomes . The synaptonemal complex protein 1 (SCP1) is a major constituent of the transversal filament, a fibrous structure that connects the central element of the synaptonemal complex with the two lateral elements . The SCP1 protein forms filamentous dimers with the two molecules that have the same polarity, with the C-termini being anchored in the lateral elements and the N-termini reaching into the central element . We investigated whether the SCP1 protein can take part in the formation of higher order protein structures by expressing it in a heterologous system . We find that expression of SCP1 in Swiss-3T3 fibroblast cells results in the formation of large protein structures . These protein structures resemble a higher order protein structure produced by overexpression of a yeast transversal filament protein in meiotic cells . Our results show that SCP1 is a structural protein and that it most likely is directly involved in the assembly of the synaptonemal complex.

Genomics, 1996 Dec 15, 38(3), 442 - 5
Repertoire and organization of human T-cell receptor alpha region variable genes; Wei S et al.; A contig of YAC, PAC, and cosmid clones that spanned the human T-cell receptor alpha variable (AV) gene region on chromosome 14 was assembled . PCR primers corresponding to the members of 32 different subfamilies were used to map AV genes on the genomic clones . Nucleotide sequencing of PCR products derived from different genomic clones was used to discriminate between related AV gene segments that coamplified . The presence of individual AV gene segments on genomic clones was further confirmed by hybridization both to clones and to human genomic DNA from several unrelated individuals . These results suggest that the T-cell receptor alpha (TCRA) region in humans contains at least 46 distinct AV gene segments that can be grouped into 32 subfamilies based on nucleotide homology . Several subfamilies appear to contain additional members detectable by hybridization that do not map to chromosome 14.

Genomics, 1996 Dec 15, 38(3), 435 - 7
Localization of a gene coding for the phospholipase A2-L subtype (PLA2L) to human chromosome 8q24-qter; Meyer AH et al.; Phospholipases A2 (PLA2) form an extended class of enzymes that play an important role in signal transduction . Phospholipase A2-like (PLA2L) belongs to the secreted forms of phospholipases A2, but constitutes a new subgroup . We have assigned the gene for this enzyme to human chromosome 8q24-qter using fluorescence in situ hybridization and radiation hybrid mapping techniques.

Genomics, 1996 Dec 15, 38(3), 432 - 4
A mouse chromosome-specific YAC probe collection for in situ hybridization; Mongelard F et al.; To facilitate the identification of mouse metaphase chromosomes by fluorescence in situ hybridization (FISH), a complete collection of mouse chromosome-specific markers has been established . Yeast artificial chromosome libraries were screened by polymerase chain reaction using primers for known loci . DNAs from positive clones were then tested by FISH . One probe per chromosome was selected on the basis of high specificity (nonchimerism) and strong fluorescence.

Genomics, 1996 Dec 15, 38(3), 331 - 9
Isolation of human and murine homologues of the Drosophila minibrain gene: human homologue maps to 21q22.2 in the Down syndrome "critical region"; Song WJ et al.; The presence of an extra copy of human chromosome 21 (trisomy 21), especially region 21q22.2, causes many phenotypes in Down syndrome, including mental retardation . To study genes potentially responsible for some of these phenotypes, we cloned a human candidate gene (DYRK) from 21q22.2 and its murine counterpart (Dyrk) that are homologous to the Drosophila minibrain (mnb) gene required for neurogenesis and to the rat Dyrk gene (dual specificity tyrosine phosphorylation regulated kinase) . The three mammalian genes are highly conserved, >99% identical at the protein level over their 763-amino-acid (aa) open reading frame; in addition, the mammalian genes are 83% identical over 414 aa to the smaller 542-aa mnb protein . The predicted human DYRK and murine Dyrk proteins both contain a nuclear targeting signal sequence, a protein kinase domain, a putative leucine zipper motif, and a highly conserved 13-consecutive-histidine repeat . Fluorescence in situ hybridization and regional mapping data localize DYRK between markers D21S336 and D21S337 in the 21q22.2 region . Northern blot analysis indicated that both human and murine genes encode approximately 6-kb transcripts . PCR screening of cDNA libraries derived from various human and murine tissues indicated that DYRK and Dyrk are expressed both during development and in the adult . In situ hybridization of Dyrk to mouse embryos (13, 15, and 17 days postcoitus) indicates a differential spatial and temporal pattern of expression, with the most abundant signal localized in brain gray matter, spinal cord, and retina . The observed expression pattern is coincident with many of the clinical findings in trisomy 21 . Its chromosomal locus (21q22 . 2), its homology to the mnb gene, and the in situ hybridization expression patterns of the murine Dyrk combined with the fact that transgenic mice for a YAC to which DYRK maps are mentally deficient suggest that DYRK may be involved in the abnormal neurogenesis found in Down syndrome.

Genomics, 1996 Dec 15, 38(3), 255 - 63
Refined mapping of the Usher syndrome type III locus on chromosome 3, exclusion of candidate genes, and identification of the putative mouse homologous region; Joensuu T et al.; A locus for Usher syndrome type III (USH3; MIM No . 276902) was recently assigned to a 5-cM region on chromosome 3q . We constructed a yeast artificial chromosome contig that allowed us to position novel polymorphisms in the region . These were typed in a total of 32 pedigrees from a geographically isolated Finnish founder population in which a putative single ancestral USH3 mutation segregates . A multipoint linkage analysis assigned USH3 to a 4-cM region between D3S1555 and a novel marker D3S3625 . By analysis of linkage disequilibrium and historical recombinations in 77 USH3 chromosomes, the location of the Finnish USH3 mutation could be narrowed to an approximately 1-cM interval between the markers D3S1299 and D3S3625 . A gene for profilin-2 (PFN2) was mapped in the vicinity and excluded as a candidate for USH3 by sequencing . The putative mouse homolog of PFN2 was mapped to mouse chromosome 3, thus suggesting a localization for the mouse homolog of USH3.

Cancer Res, 1996 Dec 15, 56(24), 5605 - 9
Deletion map of chromosome 16q in ductal carcinoma in situ of the breast: refining a putative tumor suppressor gene region; Chen T et al.; Allelic losses or imbalances affecting chromosome arm 16q appear to be early genomic abnormalities in breast carcinogenesis, because they were observed in a significant number of breast ductal carcinoma in situ lesions in our previous study (Aldaz et al., Cancer Res., 55: 3976-3981, 1995) . To define the minimum region of loss of heterozygosity (LOH), we generated a high-resolution allelotype of 35 ductal carcinoma in situ cases and completed a deletion map of chromosome 16q by means of paraffin-embedded tissue microdissection and PCR microsatellite analysis of 22 markers . We observed a strikingly high frequency of LOH in 16q, with 31 of 35 tumors (89%) affected . We identified three distinctive areas with high LOH . Two areas were described previously and correspond to 16q21 and 16q24.2-qter . The third and most commonly affected area spanned the region from marker D16S515 to marker D16S504 . The most affected locus was at D16S518, in which LOH was observed in 20 of 26 informative cases (77%), and we estimate that it lies in subregion q23.3-q24.1 . The region of highest LOH spanned approximately 2-3 Mb, as determined by a yeast artificial chromosome contig reported to cover this region . Such a high frequency of LOH at a preinvasive stage of breast cancer suggests that a candidate tumor suppressor gene or genes at this location may play an important role in breast carcinogenesis.

Carbohydr Res, 1996 Dec 13, 295, 103 - 16
Purification and characterization of a (1-->3)-beta-D-glucan-binding protein from horseshoe crab (Tachypleus tridentatus) amoebocytes; Tamura H et al.; A novel (1-->3)-beta-D-glucan-binding protein (T-GBP) has been purified from the amoebocyte lysate of the Japanese horseshoe crab, Tachypleus tridentatus . It is a basic protein (pI 9.2) which appears to be a homotetramer composed of subunits with an apparent mol wt of 168000 and with an amino-terminal sequence (20 residues) KSGFILTAPKSLTLGRNNRL . T-GBP exerted an inhibitory effect on the (1-->3)-beta-D-glucan-initiated coagulation cascade reconstituted with purified preparations of factor G and the proclotting enzyme from the lysate . The binding of (1-->3)-beta-D-glucans to T-GBP was evaluated by measuring the residual amidolytic activity of the clotting enzyme, the product of the coagulation cascade, using Boc-Leu-Gly-Arg-4-nitroanilide as the chromogenic substrate . The binding specificity of a wide range of (1-->3)-beta-D-glucans and other polysaccharides towards T-GBP was expressed by the relative inhibition (%) of the activation of factor G, the first protease zymogen in the pathway, which is activated by binding to (1-->3)-beta-D-glucans . T-GBP was found to have a high affinity for linear (1-->3)-beta-D-glucans, e.g . pachyman, curdlan, and paramylon . It was able to bind to (1-->3)-beta-D-glucans with side-chain branches and mixed linkage such as schizophyllan, lentinan, laminarins, yeast beta-D-glucan, and (1-->3),(1-->4)-beta-D-glucans such as lichenin and barley beta-D-glucan . Binding of pachyman to T-GBP was demonstrated by an enzyme-linked immunosorbent assay using a specific antibody (rabbit IgG) raised against T-GBP.

Biochem Pharmacol, 1996 Dec 13, 52(11), 1675 - 85
Amsacrine-promoted DNA cleavage site determinants for the two human DNA topoisomerase II isoforms alpha and beta; Marsh KL et al.; Site-specific DNA cleavage by topoisomerase II (EC 5.99.1.3) is induced by many antitumour drugs . Although human cells express two genetically distinct topoisomerase II isoforms, thus far the role and determinants of drug-induced DNA cleavage have been examined only for alpha . Here we report the first high-resolution study of amsacrine (mAMSA) induced DNA breakage by human topoisomerase II beta (overexpressed and purified from yeast) and a direct comparison with the recombinant alpha isoform . DNA cleavage in plasmid pBR322 and SV40 DNA was induced by alpha or beta in the absence or presence of the antitumour agent mAMSA, and sites were mapped using sequencing gel methodology . Low-resolution studies indicated that recombinant human alpha promoted DNA breakage at sites akin to those of beta, although some sites were only cleaved by one enzyme and different intensities were observed at some sites . However, statistical analysis of 70 drug-induced sites for beta and 70 sites for alpha revealed that both isoforms share the same base preferences at 13 positions relative to the enzyme cleavage site, including a very strong preference for A at +1 . The result for recombinant alpha isoform is in agreement with previous studies using alpha purified from human cell lines . Thus, alpha and beta proteins apparently form similar ternary complexes with mAMSA and DNA . Previous studies have emphasized the importance of DNA topoisomerase II alpha; the results presented here demonstrate that beta is an in vitro target with similar site determinants, strongly suggesting that beta should also be considered a target of mAMSA in vivo.

Cell, 1996 Dec 13, 87(6), 1103 - 14
Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with Topoisomerase II; Bhat MA et al.; We have isolated a Drosophila gene, barren (barr), required for sister-chromatid segregation in mitosis . barr encodes a novel protein that is present in proliferating cells and has homologs in yeast and human . Mitotic defects in barr embryos become apparent during cycle 16, resulting in a loss of PNS and CNS neurons . Centromeres move apart at the metaphase-anaphase transition and Cyclin B is degraded, but sister chromatids remain connected, resulting in chromatin bridging . This phenotype is similar to that described in TOP2 mutants in yeast . Barren protein localizes to chromatin throughout mitosis . Colocalization and biochemical experiments indicate that Barren associates with Topoisomerase II throughout mitosis and alters the activity of Topoisomerase II . We propose that this association is required for proper chromosomal segregation by facilitating the decatenation of chromatids at anaphase.

Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 388 - 95
Interaction of human leukotriene C4 synthase and microsomal glutathione transferase in vivo; Surapureddi S et al.; Microsomal glutathione transferase (mGT) specifically binds leukotriene C4 synthase in the presence of Mg2+ ion (Soderstrom et al., Protein Expression and Purification (1995) 6, 352-356) . To investigate if this interaction occurs in vivo we screened a human lung cDNA library with a bait vector encoding human mGT in the yeast two-hybrid system . One of the five positive clones obtained encoded leukotriene C4 synthase . This clone was expressed in two heterologous systems . The recombinant protein cross-reacted with a guinea pig antibody raised against a Keyhole limpet hemocyanin coupled synthetic peptide corresponding to amino acids 141-150 of human leukotriene C4 synthase.

J Biol Chem, 1996 Dec 13, 271(50), 32411 - 20
Drosophila alpha-catenin and E-cadherin bind to distinct regions of Drosophila Armadillo; Pai LM et al.; Adherens junctions are multiprotein complexes mediating cell-cell adhesion and communication . They are organized around a transmembrane cadherin, which binds a set of cytoplasmic proteins required for adhesion and to link the complex to the actin cytoskeleton . Three components of Drosophila adherens junctions, analogous to those in vertebrates, have been identified: Armadillo (homolog of beta-catenin), Drosophila E-cadherin (DE-cadherin), and alpha-catenin . We carried out the first analysis of the interactions between these proteins using in vitro binding assays, the yeast two-hybrid system, and in vivo assays . We identified a 76-amino acid region of Armadillo that is necessary and sufficient for binding alpha-catenin and found that the N-terminal 258 amino acids of alpha-catenin interact with Armadillo . A large region of Armadillo, spanning six central Armadillo repeats, is required for DE-cadherin binding, whereas only 41 amino acids of the DE-cadherin cytoplasmic tail are sufficient for Armadillo binding . Our data complement and extend results obtained in studies of vertebrate adherens junctions, providing a foundation for understanding how junctional proteins assemble and a basis for interpreting existing mutations and creating new ones.

J Biol Chem, 1996 Dec 13, 271(50), 32008 - 15
Synergy in protein engineering . Mutagenic manipulation of protein structure to simplify semisynthesis; Woods AC et al.; Semisynthesis is a chemical technique of protein engineering that provides a valuable complement to directed mutagenesis . It is the method of choice when the structural modification requires, for example, a noncoded amino acid . The process involves specific and limited protein fragmentation, structural manipulation of the target sequence, and subsequent religation of fragments to give the mutant holoprotein . We suggested and demonstrated that mutagenesis and semisynthesis could be used synergistically to achieve protein engineering goals otherwise unobtainable, if mutagenesis was used to shuffle methionine residues in the yeast cytochrome c sequence (Wallace, C . J . A., Guillemette, J . G., Hibiya, Y., and Smith, M . (1991) J . Biol . Chem . 266, 21355-21357) . These residues can not only be sites of specific cleavage by CNBr but also of spontaneous peptide bond synthesis between fragments in noncovalent complexes, which greatly facilitates the semisynthetic process . We have now used an informed "methionine scan" of the protein sequence to discover other useful sites and to characterize the factors that promote this extraordinary and convenient autocatalytic religation . Of eight sites canvassed, in a wide range of settings, five efficiently provoked peptide bond synthesis . The principal factor determining efficiency seems to be the hydropathy of the religation site . The mutants created have also provided some new insights on structure-function relationships in the cytochrome.

J Biol Chem, 1996 Dec 13, 271(50), 31775 - 8
Physical and functional interaction of rabphilin-3A with alpha-actinin; Kato M et al.; Rabphilin-3A is a downstream target molecule of Rab3A small GTP-binding protein and implicated in Ca2+-dependent neurotransmitter release . Here we have isolated a rabphilin-3A-interacting molecule from a human brain cDNA library by the yeast two-hybrid method and identified it to be alpha-actinin, known to cross-link actin filaments into a bundle . alpha-Actinin interacts with the N-terminal region of rabphilin-3A, with which GTP-Rab3A interacts, and this interaction stimulates the activity of alpha-actinin to cross-link actin filaments into a bundle . The interaction of rabphilin-3A with alpha-actinin is inhibited by guanosine 5'-(3-O-thio)triphosphate-Rab3A . These results suggest that the Rab3A-rabphilin-3A system regulates the alpha-actinin-regulated reorganization of actin filaments . It has been shown that reorganization of actin filaments is also involved in Ca2+-dependent exocytosis . Therefore, rabphilin-3A may serve as a linker for Rab3A and cytoskeleton.

JAMA, 1996 Dec 11, 276(22), 1796 - 8
The heritage of hepatitis B vaccine; Douglas RG Jr; Pioneering development of the plasma-derived and yeast-derived recombinant hepatitis B vaccines represents a major contribution to preventive health care of this century . Current and expanding worldwide application of hepatitis B vaccine promises to eliminate and eventually eradicate hepatitis B infection from the human population and, with it, many cases of cirrhosis and cancer of the liver.

Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14966 - 71
Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants; Flachmann R et al.; A chimeric Lhcb gene encoding light-harvesting chlorophyll a/b-binding protein (LHCII) was expressed in transgenic tobacco plants . To separate native from recombinant LHCII, the protein was extended by six histidines at its C terminus . Recombinant LHCII was isolated by detergent-mediated monomerization of pure trimers followed by affinity-chromatography on Ni(2+)-NTA-agarose (NTA is nitrilotriacetic acid) . Elution with imidazole yielded recombinant monomers that formed trimers readily after dilution of the detergent without further in vitro manipulations . LHCII subunits showed the typical chlorophyll a/b ratio at all steps of purification indicating no significant loss of pigments . Transgenic tobacco overexpressed amounts of recombinant protein that corresponded to about 0.7% of total LHCII . This yield suggested that expression in planta might be an alternative to the expression of eukaryotic membrane proteins in yeast . Recombinant LHCII was able to form two-dimensional crystals after addition of digalactolipids, which diffracted electrons to 3.6-A resolution . LHCII carrying a replacement of Arg-21 with Gln accumulated to only 0.004% of total thylakoid proteins . This mutant was monomeric in the photosynthetic membrane probably due to the deletion of the phosphatidylglycerol binding site and was degraded by the plastidic proteolytic system . Exchange of Asn-183 with Leu impaired LHCII biogenesis in a similar way presumably due to the lack of a chlorophyll a binding site.

Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14884 - 8
Identification of a novel vertebrate circadian clock-regulated gene encoding the protein nocturnin; Green CB et al.; Photoreceptors of the Xenopus laevis retina are the site of a circadian clock . As part of a differential display screen for rhythmic gene products in this system, we have identified a photoreceptor-specific mRNA expressed in peak abundance at night . cDNA cloning revealed an open reading frame encoding a putative 388 amino acid protein that we have named "nocturnin" (for night-factor) . This protein has strong sequence similarity to the C-terminal domain of the yeast transcription factor, CCR4, as well as a leucine zipper-like dimerization motif . Nocturnin mRNA levels exhibit a high amplitude circadian rhythm and nuclear run-on analysis indicates that it is controlled by the retinal circadian clock at the level of transcription . Our observations suggest that nocturnin may function through protein-protein interaction either as a component of the circadian clock or as a downstream effector of clock function.

Biochemistry, 1996 Dec 10, 35(49), 15865 - 9
Conserved residues are functionally distinct within transketolases of different species; Singleton CK et al.; Most of the amino acid residues which interact with thiamine pyrophosphate are highly conserved among enzymes which use this cofactor . The possible roles of several such residues in cofactor binding, catalysis, and/or substrate binding were examined for human transketolase . Mutations in H110 resulted in dramatic reductions to 2% or less of the normal activity . No alterations were found in the K(m)app's for the cofactor or for the donor and acceptor substrates . Alterations in Q428 resulted in a less severe loss of activity and also no changes in the K(m)app's . On the basis of the results, H110, an invariant residue, is proposed to function as a base which abstracts a proton from the protonated 4'-iminopyrimidine ring . The deprotonated 4'-imino moiety is required for generation of the C2-thiazolium carbanion which attacks the donor substrate . Interestingly, the function in the human enzyme of this invariant histidine is distinct from its role in yeast transketolase in which it aids in binding donor substrate and in subsequent catalytic events . Q428 is suggested to play a supportive role by stabilizing and orientating a water molecule which mediates the interaction between the 4'-amino group and H110 . In other TPP-utilizing enzymes, the equivalent residue of Q428 is a histidine and is thought to deprotonate the 4'-amino group.

Biochemistry, 1996 Dec 10, 35(49), 15800 - 6
Control of formation and dissociation of the high-affinity complex between cytochrome c and cytochrome c peroxidase by ionic strength and the low-affinity binding site; Mei H et al.; A new ruthenium photoreduction technique was used to measure the formation and dissociation rate constants kf and kd of the high-affinity complex between yeast iso-1-cytochrome c (yCc) and cytochrome c peroxidase compound I (CMPI) over a wide range of ionic strength . These studies utilized Ru-39-Cc, which contains trisbipyridylruthenium attached to the cysteine residue in the H39C, C102T variant of yCc, and has the same reactivity with CMPI as native yCc . kd and kf were measured by photoreducing a small concentration of Ru-39-Cc in the presence of the oxidized yCcIII: CMPI complex, which must dissociate before Ru-39-CcII can bind to CMPI and reduce the radical action . The value of kd for the 1:1 high-affinity complex is very small at low ionic strength, < 5 s-1 but is increased significantly by binding yCc to a second low-affinity site . However, the low-affinity yCc binding site is not active in direct electron transfer to either the radical cation or the oxyferryl heme in CMPI, and is too weak to play a role in the kinetics at ionic strengths above 70 mM . The value of kd increases to 4000 s-1 at 150 mM ionic strength, while kf decreases from > 3 x 10(9) M-1 s-1 at low ionic strength to 1.3 x 10(9) M-1 s-1 at 150 mM ionic strength . These studies indicate that the rate-limiting step in enzyme turnover is product dissociation below 150 mM ionic strength and intracomplex electron transfer to the oxyferryl heme at higher ionic strength . The interaction between yCc and CcP is optimized at physiological ionic strength to provide the largest possible complex formation rate constant kf without allowing product dissociation to be rate-limiting . The effects of surface mutations on the kinetics provided evidence that the high-affinity binding site used for the reaction in solution is similar to the one identified in the yCc:CcP crystal structure.

FEBS Lett, 1996 Dec 9, 399(1-2), 177 - 82
Kv2.1 and electrically silent Kv6.1 potassium channel subunits combine and express a novel current; Post MA et al.; Heteromultimer formation between Kv potassium channel subfamilies with the production of a novel current is reported for the first time . Protein-protein interactions between Kv2.1 and electrically silent Kv6.1 alpha-subunits were detected using two microelectrode voltage clamp and yeast two-hybrid measurements . Amino terminal portions of Kv6.1 were unable to form homomultimers but interacted specifically with amino termini of Kv2.1 . Xenopus oocytes co-injected with Kv6.1 and Kv2.1 cRNAs exhibited a novel current with decreased rates of deactivation, decreased sensitivity to TEA block, and a hyperpolarizing shift of the half maximal activation potential when compared to Kv2.1 . Our results indicate that Kv channel subfamilies can form heteromultimeric channels and, for the first time, suggest a possible functional role for the Kv6 subfamily.

J Biol Chem, 1996 Dec 6, 271(49), 31379 - 83
Molecular characterization of a novel human hybrid-type receptor that binds the alpha2-macroglobulin receptor-associated protein; Jacobsen L et al.; The 39-40-kDa receptor-associated protein (RAP) binds to the members of the low density lipoprotein receptor gene family and functions as a specialized endoplasmic reticulum/Golgi chaperone . Using RAP affinity chromatography, we have purified a novel approximately 250-kDa brain protein and isolated the corresponding cDNA . The gene, designated SORL1, maps to chromosome 11q 23/24 and encodes a 2214-residue type 1 receptor containing a furin cleavage site immediately preceding the N terminus determined in the purified protein . The receptor, designated sorLA-1, has a short cytoplasmic tail containing a tyrosine-based internalization signal and a large external part containing (from the N-terminal): 1) a segment homologous to domains in the yeast vacuolar protein sorting 10 protein, Vps10p, that binds carboxypeptidase Y, 2) five tandemly arranged YWTD repeats and a cluster of 11 class A repeats characteristic of the low density lipoprotein receptor gene family receptors, and 3) six tandemly arranged fibronectin type III repeats also found in certain neural adhesion proteins . sorLA-1 may therefore be classified as a hybrid receptor . Northern blotting revealed specific mRNA transcripts in brain, spinal cord, and testis but not in several major organs . Both RAP and an antibody against a synthetic peptide derived from a sequence determined in the mature protein detected sorLA-1 in crude human brain extracts . The domain structure suggests that sorLA-1 is an endocytic receptor possibly implicated in the uptake of lipoproteins and of proteases.

J Biol Chem, 1996 Dec 6, 271(49), 31037 - 43
Association of human fas (CD95) with a ubiquitin-conjugating enzyme (UBC-FAP); Wright DA et al.; A novel human ubiquitin conjugating enzyme (UBC) was found to associate with Fas (CD95) . The mRNA for this UBC Fas-associated protein (FAP) was widely expressed in human tissues, and the protein was identified in several mammalian cell lines . UBC-FAP shows strong homology to two recently identified UBCs, Hus5 and Ubc9, which control yeast cell cycle progression . UBC-FAP, but not an active site mutant, complemented ubc9-1(ts) mutants . This suggests that UBC-FAP is a human homologue of Ubc9, possesses ubiquitin conjugating activity, and may play an important role in mammalian cell cycle regulation . A single amino acid substitution in the death domain of Fas that abolishes Fas-mediated apoptosis also abolished Fas association with UBC-FAP, suggesting that UBC-FAP may play a role in Fas signal transduction . The sequence of UBC-FAP is identical to that of HsUbc9, a UBC recently shown to interact with Rad51.

J Biol Chem, 1996 Dec 6, 271(49), 31029 - 32
Protein-protein interaction of zinc finger LIM domains with protein kinase C; Kuroda S et al.; The LIM domain comprising two zinc-finger motifs is found in a variety of proteins and has been proposed to direct protein-protein interactions . During the identification of protein kinase C (PKC)-interacting proteins by a yeast two-hybrid assay, a novel protein containing three LIM domains, designated ENH, was shown to associate with PKC in an isoform-specific manner . Deletion analysis demonstrated that any single LIM domain of ENH associates with the NH2-terminal region of PKC . ENH associated with PKC in COS-7 cells and was phosphorylated by PKC in vitro . Upon treatment of the cells with phorbol ester, ENH in the membrane fraction was translocated to the cytosol fraction in vivo . Other LIM domain-containing proteins, such as Enigma and LIM-kinase 1, also interacted with PKC through their LIM domains . These results suggest that the LIM domain is one of the targets of PKC and that the LIM-PKC interaction may shed light on undefined roles of LIM domain-containing proteins.

Oncogene, 1996 Dec 5, 13(11), 2323 - 30
Inactivation of the cdk inhibitor p27KIP1 by the human papillomavirus type 16 E7 oncoprotein; Zerfass-Thome K et al.; Expression of the E7 oncogene of HPV-16 induces S phase entry of mammalian cells in the presence of antiproliferative signals . In particular, E7 can bypass G0/G1 arrest in response to both serum withdrawal and loss of cell adhesion, two experimental conditions in which cell cycle progression is accompanied by elevated levels of the cdk inhibitor p27KIP1 . We show here that E7 can antagonize the ability of p27KIP1 to block cyclin E-associated kinase in vitro and to inhibit transcription from the cyclin A gene in transfection experiments . E7 associates with p27KIP1 both in a reconstituted in vitro system and in extracts of mammalian cells, and association requires the C-terminal part of E7 . The interaction between p27KIP1 and E7 can also be demonstrated in a yeast two hybrid system . The data suggest that the ability of E7 to override certain forms of G0/G1 arrest is mediated in part by binding to and subsequent inactivation of the cdk inhibitor p27KIP1.

Cancer Lett, 1996 Dec 3, 109(1-2), 155 - 60
A urochordate putative homolog of human EB1, the protein which binds APC1; Pancer Z et al.; The human EBI protein has been cloned by virtue of its interaction with the C-terminus of the APC (adenomatous polyposis coli) protein, whose C-terminal truncated forms have been shown to accompany sporadic and familial forms of colorectal cancer . We have cloned a putative EBI homolog from Botryllus schlosseri (Urochordata . Ascidiacea) . The deduced protein is 287 amino acids long, and is identical with 48% of the residues in human EBI and 24-25% in two yeast hypothetical proteins . We propose that such a high degree of conservation among EBI homologs is indicative of an essential regulatory mechanism in eukaryotic cells.

Mol Biochem Parasitol, 1996 Dec 2, 83(1), 11 - 23
Human and fungal 3' splice sites are used by Trypanosoma brucei for trans splicing; Metzenberg S et al.; In Trypanosoma brucei, pre-mRNAs are joined to a 5' 39 nt spliced leader sequence by trans splicing, a process that has not been well characterized . We have asked whether the 3' splice site regions of human and yeast introns are able to substitute in vivo for the 3' spliced leader acceptor regions of trypanosome pre-mRNA sequences . The ability of heterologous sequences to participate in trans splicing in trypanosomes was assayed by chloramphenicol acetyltransferase (CAT) enzyme activity and/or the detection of spliced CAT mRNA . Four out of the six heterologous 3' splice site regions (human beta-globin intervening sequence (IVS)2, human c-myc IVS2, human factor-VIII IVS1, and yeast actin IVS) functioned as 3' spliced leader acceptor regions in T . brucei, while two did not show significant or detectable levels of CAT activity (human beta-globin IVS1 and human c-myc IVS1) . In the case of the human beta-globin IVS1 however, lengthening of the polypyrimidine tract as a result of single purine to pyrimidine transversions produced an active acceptor in which the spliced leader addition site coincides with the 3' splice site of the beta-globin exon 2 . These studies indicate that some, but not all 3' acceptor regions in humans can function as spliced leader addition sites in trypansomes.

EMBO J, 1996 Dec 2, 15(23), 6662 - 70
Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein; Kubota Y et al.; Repair of a uracil-guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III . The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch . We show that XRCC1 interacts directly with DNA polymerase beta using far Western blotting, affinity precipitation and yeast two-hybrid analyses . In addition, a complex formed between DNA polymerase beta and a double-stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay . The region of interaction with DNA polymerase beta is located within residues 84-183 in the N-terminal half of the XRCC1 protein, whereas the C-terminal region of XRCC1 is involved in binding DNA ligase III . These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision-repair . DNA strand displacement and excessive gap filling during DNA repair were observed in cell-free extracts of an XRCC1-deficient mutant cell line, in agreement with the results from the reconstituted system.

EMBO J, 1996 Dec 2, 15(23), 6629 - 40
Requirement for PP1 phosphatase and 20S cyclosome/APC for the onset of anaphase is lessened by the dosage increase of a novel gene sds23+; Ishii K et al.; Ubiquitin-dependent proteolysis is required for the onset of anaphase . We show that protein dephosphorylation by protein phosphatase 1 (PP1) is also essential for initiating anaphase in fission yeast . PP1 may directly or indirectly regulate the 20S cyclosome/APC (anaphase-promoting complex) required for anaphase-promoting proteolysis . Using anti-phosphopeptide antibodies, PP1 is shown to be dephosphorylated at the C-terminus, upon the onset of anaphase, for reactivation . sds23+, a novel gene, is a multicopy suppressor for mutations in PP1 and the 20S cyclosome/APC, implying that the gene dosage increase can relieve the requirement for PP1 and the cyclosome/APC for the onset of anaphase . The sds23+ gene is not essential for cell viability, but a mutant with the gene deleted cannot form colonies at 22 and 36 degrees C . In the sds23 deletion mutant, the progression of anaphase and cytokinesis is retarded and cell shape is aberrant . These defects are overcome by plasmids carrying the genes encoding subunits of the 20S cyclosome/APC or PP1 . These results demonstrate functions other than promoting anaphase for the components of the 20S cyclosome/APC and also a close functional relationship of Sds23 with PP1 and 20S cyclosome/APC.

EMBO J, 1996 Dec 2, 15(23), 6584 - 94
The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth; Leevers SJ et al.; Phosphoinositide 3-kinases (PI3Ks) have been identified in an evolutionarily diverse range of organisms, including mammals, Drosophila, yeast, plants and Dictyostelium . They are activated by a multitude of extracellular signals and implicated in mitogenesis, differentiation and cell survival, as well as in the control of the cytoskeleton and cell shape . Here we describe the molecular and functional analysis of Drosophila p110 (Dp110) . A full-length Dp110 cDNA was isolated and found to encode a protein homologous throughout its length to the class I mammalian PI3Ks p110alpha and p110beta . Overexpression of Dp110 in wing or eye imaginal discs resulted in flies with enlarged wings or eyes respectively . In contrast, overexpression of Dp110 containing a mutation predicted to result in the loss of catalytic activity resulted in smaller wings and eyes . The alterations in wing size result from changes in both cell size and cell number, whereas in the eye only differences in cell size were detected . These data imply a role for Dp110 in growth control during Drosophila development and have implications for the function of class I PI3Ks in other organisms.

Cell Stress Chaperones, 1996 Dec, 1(4), 215 - 23
The Hsf world: classification and properties of plant heat stress transcription factors; Nover L et al.; Based on the partial or complete sequences of 14 plant heat stress transcription factors (Hsfs) from tomato, soybean, Arabidopsis and maize we propose a general nomenclature with two basic classes, i.e . classes A and B each containing two or more types of Hsfs (HsfA1, HsfA2 etc.) . Despite some plant-specific peculiarities, essential functional domains and modules of these proteins are conserved among plants, yeast, Drosophila and vertebrates . A revised terminology of these parts follows recommendations agreed upon among the authors and representatives from other laboratories working in this field (see legend to Fig . 1) . Similar to the situation with the small heat shock proteins (sHsps), the complexity of the hsf gene family in plants appears to be higher than in other eukaryotic organisms.

Biol Trace Elem Res, 1996 Dec, 55(3), 263 - 77
In vitro and in vivo effects of selenium and selenium with vitamin E on platelet functions in diabetic rats relationship to platelet sorbitol and fatty acid distribution; Douillet C et al.; In vitro 30 min of incubation with selenomethionine (Sm) + vitamin E multiplied by about five platelet selenium (Se) decreased significantly platelet thrombin and ADP-induced aggregation decrease . Four groups of streptozotocin-induced diabetic rats were fed with a supplemented purified diet with an Se-rich yeast (Selenion): DSel, Sm: DSm, Sm alpha-tocopherol: DSmE or unsupplemented diet: D . After 24 wk of supplementation, only a decrease in thrombin-induced aggregation in group DSel compared to DSm and DSmE and D was observed . However, after 24 wk of diet compared to 14 wk, in group D and DSm, a significant increase in thrombin-induced aggregation occurred (p < 0.0001), whereas a significant decrease in groups DSel and DSmE (p < 0.0001, p < 0.03) was noted . After 21 wk of diet, in DSmE, platelet adhesion to fibronectin was significantly decreased compared to group D (p < 0.05) . These changes in DSmE were associated with a significant decrease in platelet sorbitol (p < 0.02) and a very significant increase in platelet Se (p < 0.0005) . Sm associated with vitamin E would appear more efficient to prevent oxidative damage of diabetic platelet membrane and thus to modulate its hyperactivity.

Hautarzt, 1996 Dec, 47(12), 913 - 20
{Fungi of the Trichosporon genus . Identification, epidemiology and significance of dermatologic disease pictures}; Mayser P et al.; Based on molecular data, the genus Trichosporon was recently reclassified . Six human pathogenic species, which are closely related to specific types of infections can be differentiated . By means of commercial test systems and simple key criteria according to Gueho et al . {15}, 44 isolates that had been identified as Tr . cutaneum between 7/92 and 1/96, were reclassified . The evaluation of clinical data also included 66 isolates that had not been preserved, and 27 strains of Tr . inkin . Trichosporon species were found in 4.8% of all yeast isolates (3.8% Tr . cutaneum, 1% Tr . inkin) . Nearly every other isolate of Tr . cutaneum was cultivated from nail material, while Tr . inkin was mainly isolated from skin in the anogenital region . In 38 cases, reclassification revealed Tr . mucoides; Tr . ovoides and Tr . asteroides were identified in 3 cases each, while Tr . asahii, an especially remarkable pathogen in systemic mycoses, was not found . Clinically, isolates of Tr . mucoides were predominant in toenail mycoses which might be considered in the therapy of onychomycosis . Furthermore, isolates from the skin should be evaluated with respect to the increasing incidence of systemic Trichopsoron mycoses.

Development, 1996 Dec, 122(12), 4119 - 29
Xenopus VegT RNA is localized to the vegetal cortex during oogenesis and encodes a novel T-box transcription factor involved in mesodermal patterning; Zhang J et al.; An RNA localized to the vegetal cortex of Xenopus oocytes encodes a novel T-box protein (VegT) capable of inducing either dorsal or posterior ventral mesoderm at different times in development . VegT is a nuclear protein and its C-terminal domain can activate transcription in a yeast reporter assay, observations consistent with VegT functioning as a transcription factor . Zygotic expression is dynamic along the dorsoventral axis, with transcripts first expressed in the dorsal marginal zone . By the end of gastrulation, VegT is expressed exclusively in posterior ventral and lateral mesoderm and is excluded from the notochord . Later expression is confined to a subset of Rohon-Beard cells, a type of primary sensory neuron . In animal cap assays, VegT is capable of converting prospective ectoderm into ventral lateral mesoderm . Such ectopic expression of VegT induces its own expression as well as that of Xwnt-8 in caps, suggesting that a Wnt pathway may be involved . Mis-expression of VegT in dorsal animal blastomeres fated to contribute to brain suppresses head formation . Our results suggest that VegT is a localized transcription factor, which operates sequentially in several developmental pathways during embryogenesis, including dorsoventral and posterior patterning of mesoderm.

Plant J, 1996 Dec, 10(6), 1145 - 8
A transcriptional activation domain of ATMYB2, a drought-inducible Arabidopsis Myb-related protein; Urao T et al.; Trans-activation activity of ATMYB2, a drought-inducible Myb-related protein in Arabidopsis thaliana, was analyzed using a transient assay of Arabidopsis leaf protoplasts . ATMYB2 activated the transcription of a reporter gene from the MYB-binding site in a sequence-specific manner . Deletion of the C-terminal region of ATMYB2 reduced the trans-activation of the reporter gene, indicating that the acidic region at the C-terminus of ATMYB2 is required for transcriptional activation . The domain exchange analysis with the yeast GAL4 revealed that the C-terminal acidic region of ATMYB2 contains a sufficient domain for trans-activation . These results indicate that ATMYB2 acts as a transcriptional activator and that the C-terminal acidic region of ATMYB2 can function as a transcriptional activation domain.

J Biochem (Tokyo), 1996 Dec, 120(6), 1074 - 8
Molecular cloning and characterization of a novel isoform of the human UDP-galactose transporter, and of related complementary DNAs belonging to the nucleotide-sugar transporter gene family; Ishida N et al.; We described recently the molecular cloning of human UDP-galactose transporter 1 (hUGT1) {Miura, N . et al . (1996) J . Biochem . 120, 236-241} . Now we have characterized its isoform, hUGT2, that is most likely generated through the alternative splicing of a transcript derived from the UGT genomic gene, that also codes for hUGT1 . Introduction of the open reading frame sequence of hUGT2 into a mouse cell line, Had-1, that lacks the UDP-galactose transporter, complemented the genetic defect of the mutant, as judged from the lectin-sensitivity spectra of the transformants and the nucleotide-sugar transporting activity of microsomal vesicles isolated from them . UGT-related genes were found through a BLAST search of dbEST based on their significant similarity with hUGT genes . We report here cDNA clones belonging to two subfamilies of the nucleotide-sugar transporter gene family . One is the human CMP-sialic acid transporter gene, and the other is a group of homologous genes with an undefined function that are distributed in man, mouse, and rat, and show significant similarity to the yeast UDP-N-acetylglucosamine transporter.

Int J Gynaecol Obstet, 1996 Dec, 55(3), 231 - 5
Maternal and fetal outcomes in hyperemesis gravidarum; Tsang IS et al.; OBJECTIVE: This study sought to evaluate maternal characteristics and pregnancy outcomes among women with hyperemesis gravidarum . METHODS: We performed a retrospective analysis of pregnancy records of obstetric admissions during a 6-year period . Women treated as out-patients for hyperemesis were also identified . Hyperemesis was defined as excessive nausea and vomiting resulting in dehydration, extensive medical therapy, and/or hospital admission . Statistical analysis was by t-test and chi square . RESULTS: We identified 193 women (1.5%) who developed hyperemesis among 13,053 women . Racial status, marital status, age, and gravidity were similar between the hyperemesis patients and the general population . However, there were less women with hyperemesis who were para 3 or greater . Forty-six women (24%) required hospitalization for hyperemesis, mean hospital stay 1.8 days, range 1-10 days . One patient required parenteral nutrition, two had yeast esophagitis, none had HIV infection, psychiatric pathology or thyroid disease . Pregnancy outcomes between hyperemesis patients and the general population were similar for mean birth weight, mean gestational age, deliveries less than 37 weeks, Apgar scores, perinatal mortality or incidence of fetal anomalies . Our incidence of hyperemesis (1.5%) is similar to that of other published reports . CONCLUSION: Women with hyperemesis have similar demographic characteristics to the general obstetric population, and have similar obstetric outcomes.

Curr Opin Struct Biol, 1996 Dec, 6(6), 736 - 43
Structural and mechanistic studies of enolase; Reed GH et al.; The high-resolution structure of yeast enolase cocrystallized with its equilibrium mixture of substrate and product reveals the stereochemistry of substrate/product binding and therefore the groups responsible for acid/base catalysis and stabilization of the enolate intermediate . Expression and characterization of site-specific mutant forms of the enzyme have confirmed the roles of amino acid side chains in the catalysis of the first and second steps of the reaction . Coordination of both required magnesium ions to the carboxylate of the substrate/product indicates a role for these cations in stabilization of the intermediate.

Curr Opin Genet Dev, 1996 Dec, 6(6), 711 - 4
Cereal genome analysis using rice as a model; Havukkala IJ; Researchers are eagerly waiting for the physical map of rice to become completed and available for use as a model for all cereals . The most significant advances of the past year have been the progress toward positional cloning of genes and the identification of quantitative trait loci (QTL) from detailed restriction fragment length polymorphism maps . Future focus will be: first, the enhanced dissemination and integration of the available data in World Wide Web accessible databases for easy comparison of genetic and physical mapping data across various species; second, the expanded distribution of a wide variety of DNA materials (cDNA clones, yeast artificial chromosomes, bacterial artificial chromosomes and other probes) for use in other cereals on the basis of the rice model map; and third, the applied breeding by locating and isolating sequences corresponding to important agronomic traits, often correlating with QTL.

J Cell Biol, 1996 Dec, 135(6 Pt 2), 1685 - 700
In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition; Robinett CC et al.; We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations . 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor . This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection . Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length . Subsequent experiments demonstrated the potential for more general applications of this labeling technology . Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination . In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA . Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.

Microbiol Rev, 1996 Dec, 60(4), 697 - 721
The secretory pathway of protists: spatial and functional organization and evolution; Becker B et al.; All cells secrete a diversity of macromolecules to modify their environment or to protect themselves . Eukaryotic cells have evolved a complex secretory pathway consisting of several membrane-bound compartments which contain specific sets of proteins . Experimental work on the secretory pathway has focused mainly on mammalian cell lines or on yeasts . Now, some general principles of the secretory pathway have become clear, and most components of the secretory pathway are conserved between yeast cells and mammalian cells . However, the structure and function of the secretory system in protists have been less extensively studied . In this review, we summarize the current knowledge about the secretory pathway of five different groups of protists: Giardia lamblia, one of the earliest lines of eukaryotic evolution, kinetoplastids, the slime mold Dictyostelium discoideum, and two lineages within the "crown" of eukaryotic cell evolution, the alveolates (ciliates and Plasmodium species) and the green algae . Comparison of these systems with the mammalian and yeast system shows that most elements of the secretory pathway were presumably present in the earliest eukaryotic organisms . However, one element of the secretory pathway shows considerable variation: the presence of a Golgi stack and the number of cisternae within a stack . We suggest that the functional separation of the plasma membrane from the nucleus-endoplasmic reticulum system during evolution required a sorting compartment, which became the Golgi apparatus . Once a Golgi apparatus was established, it was adapted to the various needs of the different organisms.

Immunity, 1996 Dec, 5(6), 591 - 604
Functional and physical interactions of Syk family kinases with the Vav proto-oncogene product; Deckert M et al.; Syk family kinases are essential for lymphocyte development and activation . Therefore the identification of their direct effectors is of critical importance . Here, we report that Syk interacts in the yeast two-hybrid system with Vav, a proto-oncogene product exclusively expressed in hematopoietic cells . This interaction was direct, required the catalytic activity of Syk, the SH2 domain of Vav, and tyrosine residues in the linker domain of Syk . Vav also associated with Syk and Zap in antigen receptor-stimulated B or T cells, respectively . Functionally, Vav was phosphorylated by Syk family kinases both in vivo and in vitro . Furthermore, Syk and Vav cooperated to activate NF-AT synergistically . These results indicate that the interaction between Syk family kinases and Vav plays an important role in coupling immune recognition receptors to signaling pathways involved in lymphokine production.

DNA Cell Biol, 1996 Dec, 15(12), 1049 - 56
cDNA cloning of a novel WD repeat protein mapping to the 9q22.3 chromosomal region; Zaphiropoulos PG et al.; To identify expressed sequences from the candidate genomic region of the nevoid basal cell carcinoma syndrome (9q22.3), the CpG island rescue PCR methodology was employed on the yeast artificial chromosome (YAC) ICI-8AD8 that contains microsatellite marker D9S180 . A positive clone (IR10, size 350 bp) was isolated by screening a human epidermal cDNA library with the island rescue PCR products and mapped back to the 9q22.3 region . To obtain additional sequence information from IR10, the rapid amplification of cDNA ends (RACE) methodology was employed . Within the longest IR10 RACE product, two segments having similarities with coronin, a G-like and actin-binding protein of Dictyostelium discoideum, as well as with p57, a recently cloned human actin-binding protein, were revealed . In fact, these two segments have properties of typical exons, suggesting that the RACE products and clone IR10 represent unspliced pre-mRNAs . Using the RACE methodology with primers originating from these two exons, it was shown that they splice together and the sequences corresponding to the processed IR10 mRNA were revealed . This mRNA maps back to 9q22.3 and furthermore was found to code for a novel protein composed of 525 amino acids and containing five WD repeats . The presence of the WD repeat motif, considered to function in protein-protein interactions, implies that the new protein might have a role in intracellular signaling . Moreover, Northern analysis indicated that, in addition to epidermis, the brain is a tissue with relatively high levels of expression of this gene . Finally, Southern analysis showed a functional conservation of the WD repeat protein in various species.

Genome, 1996 Dec, 39(6), 1086 - 92
Genetic and contig map of a 2200-kb region encompassing 5.5 cM on chromosome 1 of Arabidopsis thaliana; Hardtke CS et al.; In the course of the isolation of the MONOPTEROS (MP) gene, required for primary root formation in Arabidopsis thaliana, a yeast artificial chromosome (YAC) contig encompassing approximately 2200 kilobases corresponding to 5.5 cM on the top arm of chromosome 1 was established . Forty-six YAC clones were characterized and 12 new restriction fragment length polymorphism (RFLP) markers are presented . Three new codominant amplified polymorphic sequence (CAPS) markers were generated that enabled high resolution genetic mapping and correlation of physical and genetic distances along the contig . The map contributes to the completion of a physical map of the Arabidopsis genome and should facilitate positional cloning of other genes in the region as well as studies on genome organization . We also present another set of 11 physically linked probes, as well as mapping data for additional RFLP markers within a broader interval of 10.4 cM.

Neuron, 1996 Dec, 17(6), 1209 - 19
The mammalian brain rsec6/8 complex; Hsu SC et al.; rsec6 and rsec8 are two components of a 17S complex in mammalian brain that is homologous to the yeast 834 kDa Sec6/8/15 complex which is essential for exocytosis . Purification and partial amino acid sequencing of the mammalian rsec6/8 complex reveals that it is composed of eight novel proteins with a combined molecular weight of 743 kDa . The complex is broadly expressed in brain and displays a plasma membrane localization in nerve terminals . Membrane associated rsec6/8 complex coimmunoprecipitates with syntaxin, a plasma membrane protein critical for neurotransmission . These data suggest a role for the mammalian rsec6/8 complex in neurotransmitter release via interactions with the core vesicle docking and fusion apparatus.

Plant Mol Biol, 1996 Dec, 32(5), 959 - 68
The heat shock cognate 80 gene of tomato is flanked by matrix attachment regions; Chinn AM et al.; Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains . Although there are several reports on the action of MARs in the context of heterologous genes, information is more limited on the role of MARs associated with plant genes . Transgenic studies suggest that the upstream, intron and downstream regions of the developmentally regulated heat shock cognate 80 gene (HSC80) of tomato participate in chromatin organization . In this study, we tested the in vitro affinity of the HSC80 gene to chromosomal scaffolds prepared from shoot apices of tomato . We found that a 1.5 kb upstream region and a 1.4 kb downstream region, but not the intron region, are MARs . These MARs interact with tomato and pea scaffolds and bind regardless of the expression status of HSC80 in the tissue from which the nuclei were isolated . Comparison to two known yeast MARs, ARS1 and CENIII, showed that the HSC80 5'MAR binds more avidly to tomato scaffolds than ARS1, while no binding of CENIII was observed . Competition binding between the two HSC80 MARs indicated that the 5'MAR can outcompete the 3'MAR and not vice versa . Last, we observed that the interaction of the 3'MAR with the scaffold could result in an electrophoretic mobility shift resistant to SDS, protease, and phenol treatment . In conclusion, MARs whose binding properties can be clearly differentiated are closely flanking the HSC80 gene . The discovery of MARs in regions which have a distinct function in HSC80 transgenes but not in transient expression assays, is consistent with a chromosomal scaffold role in HSC80 gene regulation.

Genetics, 1996 Dec, 144(4), 1757 - 67
Genetic and physical mapping of the mouse Ulnaless locus; Peichel CL et al.; The mouse Ulnaless locus is a semidominant mutation which displays defects in patterning along the proximal-distal and anterior-posterior axes of all four limbs . The first Ulnaless homozygotes have been generated, and they display a similar, though slightly more severe, limb phenotype than the heterozygotes . To create a refined genetic map of the Ulnaless region using molecular markers, four backcrosses segregating Ulnaless were established . A 0.4-cM interval containing the Ulnaless locus has been defined on mouse chromosome 2, which has identified Ulnaless as a possible allele of a Hoxd cluster gene(s) . With this genetic map as a framework, a physical map of the Ulnaless region has been completed . Yeast artificial chromosomes covering this region have been isolated and ordered into a 2 Mb contig . Therefore, the region that must contain the Ulnaless locus has been defined and cloned, which will be invaluable for the identification of the molecular nature of the Ulnaless mutation.

Genetics, 1996 Dec, 144(4), 1639 - 52
Functional and genetic characterization of the oligomerization and DNA binding properties of the Drosophila doublesex proteins; Erdman SE et al.; The doublesex (dsx) gene of Drosophila melanogaster encodes both male-specific (DSXM) and female-specific (DSXF) polypeptides, which are required for normal differentiation of numerous sexually dimorphic somatic traits . The DSX polypeptides are transcription factors and have been shown previously to bind through a zinc finger-like domain to specific sites in an enhancer regulating sex-specific expression of yolk protein genes . We have determined the consensus target sequence for this DNA binding domain to be a palindromic sequence AGNNACTAAATGTNNTC composed of two half-sites around a central (A/T) base pair . As predicted by the symmetric nature of this site, we have found that the DSX proteins exist as dimers in vivo and have mapped two independent dimerization domains by the yeast two-hybrid method; one in the non-sex-specific amino-terminal region of the protein and one that includes the partially sex-specific carboxy-terminal domains of both the male and female polypeptides . We have further identified a missense mutation that eliminates dsx function in female flies, and shown that the same mutation prevents dimerization of DSXF in the yeast two-hybrid system, indicating a critical role for dimerization in dsx function in vivo.

Eur J Immunol, 1996 Dec, 26(12), 3021 - 8
Molecular cloning and chromosomal mapping of a novel human gene, ChemR1, expressed in T lymphocytes and polymorphonuclear cells and encoding a putative chemokine receptor; Samson M et al.; We describe the cloning of a human gene, named ChemR1, encoding a new putative chemokine receptor sharing 48% identity with CC-chemokine receptor (CCR)4 and 44% identity with CCR1 . It displays four extracellular cysteines that are conserved among all other chemokine receptors . ChemR1 transcripts were detected by Northern blotting in the T lymphoblastic cell lines Jurkat and MOLT-4, but not in the pre-B lymphoblastic cell line JM-1 . ChemR1 receptor transcripts were also detected by reverse transcription and polymerase chain reaction analysis in unstimulated CD4+ and CD8+ T cells and polymorphonuclear cells prepared from peripheral blood . The chromosomal localization was performed by radiation hybrid mapping and testing of a panel of yeast artificial chromosome clones . This allowed the assignment of the ChemR1 receptor gene to the p21.3-24 region of human chromosome 3, in close proximity with the functionally characterized CCR . Future work is required to identify the ligand(s) of this new chemokine receptor and to define its role in the recruitment of white blood cell populations.

Genome Res, 1996 Dec, 6(12), 1200 - 6
Structure of the human alpha 2 subunit gene of the glycine receptor--use of vectorette and Alu-exon PCR; Monani U et al.; The alpha subunit of the glycine receptor is encoded by multiple genes that display developmental and tissue-specific expression . The alpha 1 subunit gene is expressed predominantly in the adult brain stem and spinal cord, whereas the alpha 2 subunit gene is expressed in fetal brain and spinal cord . We wished to determine the genomic organization of the human alpha 2 subunit gene as well as to define the 5' ends of the alpha 1 and alpha 2 subunit genes . Gene structure can be defined rapidly from yeast artificial chromosome (YAC) DNA sources by the use of vectorette-exon polymerase chain reaction (PCR) . However, YACs frequently contain small deletions that complicate the determination of the complete exon-intron structure of a gene, and this often necessitates the isolation of additional clones . In this study we have used vectorette-exon PCR from YAC DNA to define exons of the glycine receptor alpha 2 subunit gene . To define those exons that were absent in the isolated YACs, we used Alu-exon PCR on genomic DNA, using nested primers to obtain specificity in the PCR reactions . The alpha 2 subunit gene was found to contain nine exons varying in size from 68 bp (exons 3A and 3B) to 581 bp (exon l) . All of the intron-exon boundary sequences conform to consensus splice donor and acceptor sites . In addition, we have defined the 5' end of this gene as well as that of the alpha 1 subunit gene by RACE-PCR . The structures of the alpha subunit glycine receptor genes in humans are very similar to each other and to the alpha subunit genes in mice.

Genome Res, 1996 Dec, 6(12), 1149 - 59
Long-range mapping and construction of a YAC contig within the cat eye syndrome critical region; McDermid HE et al.; Cat eye syndrome (CES) is typically associated with a supernumerary bisatellited marker chromosome derived from human chromosome 22pter to 22q11.2 . The region of 22q duplicated in the typical CES marker chromosome extends between the centromere and locus D22S36 . We have constructed a long-range restriction map of this region using pulsed-field gel electrophoresis and probes to 10 loci (11 probes) . The map covers -3.6 Mb . We have also used 15 loci to construct a yeast artificial chromosome contig, which encompasses about half of the region critical to the production of the CES phenotype (centromere to D22S57) . Thus, the CES critical region has been mapped and a substantial portion of it cloned in preparation for the isolation of genes in this region.

Biochem J, 1996 Dec 1, 320 ( Pt 2), 643 - 9
Cloning and characterization of a 92 kDa soluble phosphatidylinositol 4-kinase; Nakagawa T et al.; A phosphatidylinositol (PtdIns) 4-kinase cDNA cloned from a rat brain cDNA library encoded a protein of 816 amino acids with a calculated molecular mass of 91654 Da . This molecule contained a lipid-kinase-unique domain and a presumed lipid/ protein kinase homology domain that are found in other PtdIns 4-kinases and PtdIns 3-kinases . Furthermore, this kinase molecule had 43.3% shared identity with the presumed catalytic domain of yeast PtdIns 4-kinase, PtdInsK1, and the two molecules had a region of similarity that is not conserved in other lipid kinases . By examining PtdIns kinase activity in transfected COS-7 cells using epitope tag immunoprecipitation as well as conventional methods, the product PtdIns phosphate was identified as phosphatidylinositol 4-phosphate (PtdIns4P), but not phosphatidylinositol 3-phosphate (PtdIns3P) . The PtdIns 4-kinase activity was recovered predominantly from the soluble fraction and the activity was markedly enhanced in the presence of Triton X-100 and was relatively insensitive to inhibition by adenosine . In addition, the PtdIns 4-kinase activity was completely inhibited in the presence of 10 microM wortmannin . When examined by epitope tag immunocytochemistry, the immunoreactivity for the PtdIns 4-kinase molecule was dominantly aggregated in a cytoplasmic region juxtaposed to the nuclei and was faintly but widely dispersed in the cytoplasm . By in situ hybridization analysis, the mRNA for PtdIns 4-kinase was expressed ubiquitously and was detected in most neurons throughout the grey matter of the brain, with higher expression intensity found in fetal than in adult brain.

Nucleic Acids Res, 1996 Dec 1, 24(23), 4709 - 18
Logitlinear models for the prediction of splice sites in plant pre-mRNA sequences; Kleffe J et al.; Pre-mRNA splicing in plants, while generally similar to the processes in vertebrates and yeast, is thought to involve plant specific cis-acting elements . Both monocot and dicot introns are typically strongly enriched in U nucleotides, and AU- or U-rich segments are thought to be involved in intron recognition, splice site selection, and splicing efficiency . We have applied logitlinear models to find optimal combinations of splice site variables for the purpose of separating true splice sites from a large excess of potential sites . It is shown that plant splice site prediction from sequence inspection is greatly improved when compositional contrast between exons and introns is considered in addition to degree of matching to the splice site consensus (signal quality) . The best model involves subclassification of splice sites according to the identity of the base immediately upstream of the GU and AG signals and gives substantial performance gains compared with conventional profile methods.

Nucleic Acids Res, 1996 Dec 1, 24(23), 4624 - 31
Characterization of a zinc-dependent transcriptional activator from Arabidopsis; de Pater S et al.; The C2-H2 zinc-finger is a widely occurring DNA binding motif, usually present as tandem repeats . The majority of C2-H2 zinc-finger proteins that have been studied are derived from animals . Here, we characterize a member of a distinct class of plant C2-H2 zinc-finger proteins in detail . A cDNA clone encoding a DNA binding protein from Arabidopsis was isolated by SouthWestern screening . The protein, termed ZAP1 (Zinc-dependent Activator Protein-1), is encoded by a single copy gene, which is expressed to similar levels in root and flower, to a somewhat lower level in stem and to low levels in leaf and siliques . The optimal binding site was determined by random binding site selection, and the consensus sequence found is CGTTGACCGAG . The homology between ZAP1 and other DNA binding proteins is restricted to a repeated region of a stretch of 24 highly conserved amino acids followed by a zinc-finger motif (C-X4-C-X22-23-H-X1-H) . The C-terminal zinc-finger region is essential for DNA binding, whereas deletion of the N-terminal one resulted in 2.5-fold reduced binding affinity . Binding of ZAP1 to DNA was abolished by metal-chelating agents . The activation domain as determined in yeast is adjacent to and possibly overlapping with the DNA binding domain . Particle bombardment experiments with plant cells showed that ZAP1 increases expression of a gusA reporter gene that is under control of ZAP1 binding sites . We conclude that ZAP1 is a plant transcriptional activator with a C2-H2 zinc-finger DNA binding domain.

Scand J Immunol, 1996 Dec, 44(6), 643 - 7
Auditory stress induces changes in membrane functions of mouse peritoneal macrophages; Spehner V et al.; Stressful events induce responses in the endocrine and immune systems . The authors analysed the influence of repetitive noise stress on peritoneal macrophage oxidative and phagocytic responses . Plasma corticosterone levels were also measured . Different groups of 6- to 8-week-old C57BL/6 male mice were exposed for 1 night (n = 14) and 3 nights (n = 21) to a sound stress of 110 dB in an audiogenic stress chamber . Control animals were submitted to a sham stress for 1 night (n = 13) and 3 nights (n = 17) . A marked decrease was observed in the phagocytic response to yeast (P = 3 x 10(-4)) while a mild increase in the oxidative response stimulated by opsonized zymosan was noted only after the 3 night stress (P = 0.02) . Corticosterone levels of control and stressed mice did not differ . These results indicate that the stress resulting from repetitive noise causes modifications in peritoneal macrophage activity, and that these changes are dependent on the duration of stress . These functional alterations seem more complex than a simple general suppression or activation.

J Virol, 1996 Dec, 70(12), 8993 - 6
Rapamycin stimulates viral protein synthesis and augments the shutoff of host protein synthesis upon picornavirus infection; Beretta L et al.; The immunosuppressant drug rapamycin blocks progression of the cell cycle at G1 in mammalian cells and yeast . We recently showed that rapamycin inhibits both in vitro and in vivo cap-dependent, but not cap-independent, translation . This inhibition is causally related to reduced phosphorylation and consequent activation of 4E-BP1, a repressor of the function of the cap-binding protein, eIF4E . Two members of the picornavirus family, encephalomyocarditis virus and poliovirus, inhibit phosphorylation of 4E-BP1 . Since translation of picornavirus mRNAs is cap independent, inhibition of phosphorylation of 4E-BP1 could contribute to the shutoff of host protein synthesis . Here, we show that rapamycin augments both the shutoff of host protein synthesis and the initial rate of synthesis of viral proteins in cells infected with encephalomyocarditis virus and poliovirus.

Cutis, 1996 Dec, 58(6), 397 - 401
Chronic paronychia and onycholysis: a thirteen-year experience; Daniel CR 3rd et al.; We report here on two retrospective studies conducted between 1982 and 1995 in 137 patients with clinical evidence of chronic paronychia or onycholysis . The purpose of the studies was to determine what factors played a role in these nail disorders . The culture results indicated that yeast commonly grew from the cultured material . Significant contact irritant exposure was frequently found . These findings and our experience suggest that the resolution of chronic paronychia and onycholysis depends on avoiding exposure to contact irritants and on appropriate treatment of any underlying fungal infection . To accomplish this latter goal, it is important to select a broad-spectrum antifungal agent that is effective against yeasts as well as other fungal pathogens.

Hum Mol Genet, 1996 Dec, 5(12), 1875 - 85
Human huntingtin derived from YAC transgenes compensates for loss of murine huntingtin by rescue of the embryonic lethal phenotype; Hodgson JG et al.; Huntington disease (HD) is caused by expansion of a CAG trinucleotide repeat in exon 1 of a novel gene . The HD protein (huntingtin) plays a critical role in early embryonic development since homozygous targeted disruption of the murine HD gene results in embryonic lethality by day 7.5 . To rescue this phenotype by transgene based huntingtin expression it is therefore essential to express the protein early enough in development in the appropriate cells . Since YAC based transgenes are known to be regulated in an appropriate temporal and tissue-specific manner, we sought to rescue the embryonic lethality by breeding YAC transgenic mice expressing human huntingtin with mice heterozygous for the targeted disruption . We generated viable offspring homozygous for the disrupted murine HD gene but expressing human huntingtin derived from the YAC . This result clearly shows that YAC transgene based expression of huntingtin occurs prior to 7.5 days gestation . Additionally, we show that human huntingtin expression in YAC transgenic mice follows an identical tissue distribution and subcellular localisation pattern as that of the murine endogenous protein and that expression levels of 2-3 times endogenous can be achieved . This shows that human huntingtin under the influence of its native promoter, despite differences to the murine protein, is functional in a murine background and can compensate for loss of the murine protein . These results show that YAC transgenic approaches are a particularly promising route to producing an animal model for disorders associated with CAG expansion.

Mol Pharmacol, 1996 Dec, 50(6), 1463 - 71
Drug sensitivity and sequence specificity of human recombinant DNA topoisomerases IIalpha (p170) and IIbeta (p180); Cornarotti M et al.; Effective anticancer agents, such as epipodophyllotoxins and anthracyclines, exert their antitumor activity through stabilization of cleavable topoisomerase II/DNA complexes, which may result in DNA breakage on detergent addition . Two isozymes (alpha and beta) of DNA topoisomerase II are present in human cells; however, their roles as drug targets have not been completely defined . We determined the in vitro isoenzyme sensitivities to VM-26 (teniposide) and 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin (an anthracycline analog) and established the sequence selectivity of isoenzyme-mediated DNA cleavage . Human topoisomerases IIalpha and IIbeta were purified from yeast cells overexpressing the corresponding plasmid-borne cDNA . Enzyme sensitivities to drugs were measured by a DNA cleavage assay using 32P-labeled simian virus 40 DNA fragments, and cleavage sites were mapped using agarose and sequencing gels . Both isozymes were sensitive to the studied poisons . They stimulated similar cleavage intensity patterns in agarose and sequencing gels; however, minor differences could be detected . The results showed that local base preferences for DNA cleavage without drugs were different at positions -2 and -1 . On the other hand, sequence specificities of VM-26 and 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin were identical for both isozymes and corresponded to those of the native murine enzyme . The identical drug sequence specificities suggested that molecular interactions of the tested drugs in the ternary complex are likely similar between the two isozymes . The current findings indicate that both topoisomerase IIalpha and IIbeta may be in vivo targets of antitumor poisons.

Mol Endocrinol, 1996 Dec, 10(12), 1646 - 55
Two receptor interacting domains in the nuclear hormone receptor corepressor RIP13/N-CoR; Seol W et al.; The thyroid hormone receptor (TR) and the retinoic acid receptor (RAR) act as transcriptional repressors when they are not occupied by their cognate ligands . This repressor function is mediated by proteins called corepressors . One of the nuclear hormone receptor corepressors, N-CoR, was originally isolated as a retinoid X receptor-interacting protein called RIP13 . We have isolated a new potential variant of RIP13/N-CoR that is missing previously described transcriptional repressor domains but is similar in structure to the related corepressor termed SMRT or TRAC-2 . Detailed analysis of the interaction with TR and RAR demonstrates that RIP13/N-CoR contains a new receptor interaction domain, termed ID-II, in addition to the previously described domain, referred to here as ID-I . Both ID-I and ID-II are capable of interacting independently with either TR or RAR, as assessed by the yeast two-hybrid system, by a mammalian two-hybrid system, or by direct in vitro binding . Results with all three approaches confirm that RIP13/N-CoR also interacts with retinoid X receptor, but this interaction is weaker than that with TR or RAR . Together, these results demonstrate that RIP13/N-CoR can interact with several different nuclear hormone receptors via two separate receptor interaction domains . Differences between the interactions observed in the different systems suggest that corepressor function may be modified by additional factors present in various cell types.

Mol Endocrinol, 1996 Dec, 10(12), 1632 - 45
A TATA binding protein-associated factor functions as a coactivator for thyroid hormone receptors; Petty KJ et al.; Transcriptional regulation by thyroid hormone receptors (TRs) requires the TR to interact with various proteins . The TATA binding protein-associated factors (TAFs) are cofactors for several transcription factors and, therefore, are candidate cofactors for the TR . To determine whether one or more of the TAFs are cofactors for TRs, direct protein interactions between human TR beta and several Drosophila TAFs were quantitated in vitro . The human (h) TR beta bound specifically to dTAFII110 and weakly to dTAFII60, but did not bind to dTAFII30 alpha, dTAFII30 beta, dTAFII40, dTAFII80, or dTAFII150 . The dTAFII110:hTR beta interaction required the carboxyl-terminals of both proteins . The dTAFII110 also interacted with the hTR alpha 1 carboxyl-terminus in a yeast two-hybrid system . Thyroid hormone destabilized the dTAFII110:TR interaction in vitro, but had no effect on the interaction in the two-hybrid system . The dTAFII110 did not bind to human retinoid X receptor alpha in vitro, indicating that this TAF interacts differentially with nuclear receptors . The transcriptional function of hTR beta was enhanced by dTAFII110 in transfection assays, indicating that this TAF can function in the thyroid hormone signalling pathway . Thus, TAFII110 functions as a cofactor for TRs, and the interactions between specific TAFs and nuclear receptors may provide another level of selectivity for transcriptional responses to hormones.

Gastroenterol Clin North Am, 1996 Dec, 25(4), 755 - 72
DNA repair and colorectal cancer; Marra G et al.; The mismatch repair system plays a major role in the processing of recombination intermediates and in the repair of errors made during DNA replication or resulting from chemical damage to DNA . Human homologues of the bacterial and yeast mismatch repair genes have been recently identified, and mutations in these genes have been found to show risk for tumor development in hereditary nonpolyposis colorectal cancer syndrome (HNPCC) . Colorectal tumors bearing homozygous mutations in these mismatch repair genes show a hypermutable phenotype, mainly at microsatellite regions of DNA . The temporal relationship between the loss of mismatch repair activity and the cascades of mutations in critical genes involved in the carcinogenesis of HNPCC tumors is unknown.

Genes Dev, 1996 Dec 1, 10(23), 3028 - 40
The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing; Wang ZF et al.; Replication-dependent histone mRNAs are not polyadenylated but end in a conserved 26-nucleotide structure that contains a stem-loop . Much of the cell cycle regulation of histone mRNA is post-transcriptional and is mediated by the 3' end of histone mRNA . The stem-loop binding protein (SLBP) that binds the 3' end of histone mRNA is a candidate for the factor that participates in most, if not all, of the post-transcriptional regulatory events . We have cloned the cDNA for the SLBP from humans, mice, and frogs, using the recently developed yeast three-hybrid system . The human SLBP is a 31-kD protein and contains a novel RNA-binding domain, which has been mapped to a 73-amino-acid region of the protein . The cloned SLBP is the protein bound to the 3' end of histone mRNA as antibodies specific for the SLBP remove all specific binding activity from nuclear and polyribosomal extracts . These depleted extracts do not cleave histone pre-mRNA efficiently, demonstrating that the SLBP is required for efficient histone pre-mRNA processing.

Genomics, 1996 Dec 1, 38(2), 243 - 5
Physical mapping of the human neurotensin gene (NTS) between markers D12S1444 and D12S81 on chromosome 12q21; Marondel I et al.; Neurotensin (NTS) is an endogenous tridecapeptide of the central nervous system and the gastrointestinal tract of different mammalian species including human . The human gene encoding neurotensin has previously been assigned to chromosome 12 but no regional localization was available . We now confirm this assignment and place the NTS gene on the physical and cytogenetic maps . The NTS gene is located on a yeast artificial chromosome contig that contains several polymorphic markers and is close to a polymorphic marker located at 95.8 cM on the Genethon linkage map . NTS is immediately proximal to four polymorphic markers, including D12S81 (AFM102xg9) and D12S88 (AFM158yb4) . Using fluorescence in situ hybridization, we map the gene cytogenetically to band 12q21.

Genomics, 1996 Dec 1, 38(2), 199 - 205
An EST and STS-based YAC contig map of human chromosome 9q22.3; Lench NJ et al.; We have isolated 48 yeast artificial chromosome (YAC) clones from a 4 cM/27 cR region of human chromosome 9q22.3 encompassed by the markers cen-D9S196-D9S173-tel . Within this region, we have assembled a 4.3-Mb YAC contig across the interval cen-FACC-D9S173-tel containing 42 clones . As a first step toward completing the detailed transcription map of the region, we have mapped 9 gene sequences and 10 expressed sequence tags . Fifteen polymorphic microsatellite repeat markers and 17 novel sequence-tagged sites from the region are also described . The mapping of polymorphic simple tandem repeat markers has permitted the integration of existing genetic and physical maps of the region . Together these maps provide a valuable resource for fine structure mapping and DNA sequencing across the region as well as for the identification of disease gene loci and the isolation of novel coding sequences.

Genomics, 1996 Dec 1, 38(2), 166 - 73
A 1.5-megabase physical map encompassing the multiple endocrine neoplasia type-1 (MEN1) locus on chromosome 11q13; Wood TF et al.; Linkage analysis and loss of heterozygosity studies have shown that the gene responsible for the multiple endocrine neoplasia type-1 (MEN1) syndrome localizes to a small interval between D11S427 and D11S460 on chromosome 11q13 . As an initial step to clone this tumor suppressor gene, our group is the first to map the MEN1 region physically using yeast artificial chromosome, bacterial artificial chromosome (BAC), and cosmid contigs . The 1.5-Mb high-resolution, contiguous map extends from PYGM to 300 kb telomeric of D11S460 . Of this, the 1.2-Mb interval between PYGM and D11S460 is isolated in cosmids and BACs and will be useful for the development of genomic sequences and transcription maps of this important region . Nine new sequence-tagged sites (STS) are also characterized from this region . The physical map and the STSs will be valuable tools for the cloning of the MEN1 gene.

Leukemia, 1996 Dec, 10(12), 1891 - 6
Refined chromosomal localization of the human thrombopoietin gene to 3q27-q28 and exclusion as the responsible gene for thrombocytosis in patients with rearrangements of 3q21 and 3q26; Schnittger S et al.; Thrombocytosis is a characteristic clinical feature in patients with myelocytic malignancies and chromosomal rearrangements of 3q21 and 3q26, sometimes called the '3q21q26 syndrome' . The function of thrombopoietin (TPO) in megakaryocytopoiesis and thrombopoiesis as well as its chromosomal location, marked TPO as a candidate gene for malignancies with 3q rearrangements combined with dysmegakaryopoiesis . In this study 12 cases with inv(3)(q21q26) or t(3;3)(q21;q26) were analyzed by means of PFGE, but no rearrangements near the TPO locus were detectable . Six YACs containing the TPO locus were isolated and characterized . By dual color in situ hybridization using a YAC from 3q26 containing the EVI1 gene and a YAC from the TPO locus, the localization of the human TPO gene could be refined to 3q27-q28 about 15-20 Mbp telomeric to the 3q26 breakpoints occurring in myeloid malignancies . TPO levels were analyzed in the serum of three patients and were found to be in the normal range . These results confirm the findings of two previous studies that thrombopoietin expression is not the main cause of thrombocytosis in the 3q21q26 syndrome.

Genes Chromosomes Cancer, 1996 Dec, 17(4), 205 - 14
Molecular cytogenetic characterization and physical mapping of 12q13-15 amplification in human cancers; Elkahloun AG et al.; Amplification of sequences derived from 12q13-15 is frequent in human sarcomas and brain tumors . Detailed mapping studies of the amplified region are necessary for definition of the impact of these amplification events on the tumor cell phenotype . By using the genes in this region and genomic fragments isolated by chromosome microdissection, we have established a series of ordered probes from 12q13-15 for fluorescence in situ hybridization (FISH) and Southern blot analysis . These probes have been used for physical mapping of two portions of the interval from GLI to D12S8 . The centromeric region extends 1.8 Mb from GLI to microclone M79 and contains at least five genes, including the cyclin-dependent kinase gene CDK4 . The more telomeric region includes the p53 regulator MDM2 and covers 1.1 Mb . We used the same group of probes to determine the pattern of amplification in three cell lines and three tumor specimens carrying amplified sequences from 12q13-15 . In addition, we used a yeast artificial chromosome (YAC) contig of several megabases covering the entire region from SAS to D12S8 for FISH to determine the pattern of amplification in the neuroblastoma cell line NGP-127 . The results suggest that the MDM2 and CDK4 regions may be either coamplified or amplified independently, and they illustrate how the map positions of genes and their functions may interact to determine the pattern of DNA amplification in human malignancies.

J Immunol, 1996 Dec 1, 157(11), 5034 - 41
Molecular cloning of a novel protein antigen of Leishmania major that elicits a potent immune response in experimental murine leishmaniasis; Webb JR et al.; BALB/c mice are highly susceptible to infection with the protozoan parasite Leishmania major . This susceptibility has been attributed, in part, to the expansion of parasite-specific CD4+ Th2 cells that antagonize Th1 responses and promote humoral immunity . In the present study, we have utilized sera from L . major-infected BALB/c mice to screen an L . major amastigote cDNA expression library . One of the clones detected encodes a novel Ag designated as L . major stress-inducible 1 (LmSTI1) . LmSTI1 contains six copies of the tetratricopeptide consensus motif and is highly related to a family of stress-inducible proteins that is conserved from yeast to humans . Sera from L . major-infected BALB/c mice have LmSTI1-specific Ab titers in excess of 1:200,000, comprised predominantly of IgG1, IgG2A, and IgG2B isotypes . Recombinant LmSTI1 protein elicited strong proliferative responses from draining lymph node cells of L . major-infected BALB/c mice at both early (10 days) and late (28 days) stages of infection and elicited production of high levels of IFN-gamma and low levels of IL-4 . In contrast, soluble leishmanial lysate elicited high levels of IL-4 and low IFN-gamma production . Thus, we have identified an Ag of Leishmania capable of eliciting a mixed cellular response that is skewed toward a Th1 phenotype in susceptible BALB/c mice with advanced infections . In addition, analyses of sera from human patients with cutaneous, visceral, and post-kala azar visceral leishmaniasis indicated that a majority of individuals from all three clinical groups mounted strong humoral responses against LmSTI1.

Mol Cell Biol, 1996 Dec, 16(12), 7054 - 62
c-ABL tyrosine kinase activity is regulated by association with a novel SH3-domain-binding protein; Zhu J et al.; The c-ABL tyrosine kinase is activated following either the loss or mutation of its Src homology domain 3 (SH3), resulting in both increased autophosphorylation and phosphorylation of cellular substrates and cellular transformation . This suggests that the SH3 domain negatively regulates c-ABL kinase activity . For several reasons this regulation is thought to involve a cellular protein that binds to the SH3 domain . Hyperexpression of c-ABL results in an activation of its kinase, the kinase activity of purified c-ABL protein in the absence of cellular proteins is independent of either the presence or absence of a SH3 domain, and point mutations and deletions within the SH3 domain are sufficient to activate c-ABL transforming ability . To identify proteins that interact with the c-ABL SH3 domain, we screened a cDNA library by the yeast two-hybrid system, using the c-ABL SH3SH2 domains as bait . We identified a novel protein, AAP1 (ABL-associated protein 1), that associates with these c-ABL domains and fails to bind to the SH3 domain in the activated oncoprotein BCRABL . Kinase experiments demonstrated that in the presence of AAP1, the ability of c-ABL to phosphorylate either glutathione S-transferase-CRK or enolase was inhibited . In contrast, AAP1 had little effect on the phosphorylation of glutathione S-transferase-CRK by the activated ABL oncoproteins v-ABL and BCRABL . We conclude that AAP1 inhibits c-ABL tyrosine kinase activity but has little effect on the tyrosine kinase activities of oncogenic BCRABL or v-ABL protein and propose that AAP1 functions as a trans regulator of c-ABL kinase . Our data also indicate that loss of susceptibility to AAP1 regulation correlates with oncogenicity of the activated forms of c-ABL.

Curr Opin Cell Biol, 1996 Dec, 8(6), 773 - 80
The spindle assembly checkpoint; Rudner AD et al.; The spindle assembly checkpoint monitors proper chromosome attachment to spindle microtubules and is conserved from yeast to humans . Checkpoint components reside on kinetochores of chromosomes and show changes in phosphorylation and localization as cells proceed through mitosis . Adaptation to prolonged checkpoint arrest can occur by inhibitory phosphorylation of Cdc2.

Curr Opin Cell Biol, 1996 Dec, 8(6), 788 - 94
Cyclin dependent kinase activating kinases; Sclafani RA; The cyclin dependent kinase activating kinase (CAK) has roles in both cell cycle regulation and transcription . CAK assembly is regulated either by additional protein binding or by phosphorylation . A recent comparison of this kinase from two yeast species shows that different proteins perform distinct roles and that the most studied CAK may function mainly in transcription.

Hum Genet, 1996 Dec, 98(6), 741 - 3
Genetic mapping of RP1 on 8q11-q21 in an Australian family with autosomal dominant retinitis pigmentosa reduces the critical region to 4 cM between D8S601 and D8S285; Xu SY et al.; The locus (RP1) for one form of autosomal dominant retinitis pigmentosa (adRP) was mapped on chromosome 8q11-q22 between D8S589 and D8S285, which are about 8 cM apart, by linkage analysis in an extended family ascertained in the USA . We have studied a multigeneration Australian family with adRP and found close linkage without recombination between the disease locus and D8S591, D8S566, and D8S166 (Zmax = 1.137-4.650 at theta = 0.00), all mapped in the region known to harbor RP1 . Assuming that the mutation of the same gene is responsible for the disease in both families, the analysis of multiply informative meioses in the American and Australian families places the adRP locus between D8S601 and D8S285, which reduces the critical region to about 4 cM, corresponding to approximately 4 Mb, which is completely covered by a yeast artificial chromosome contig assembled recently.

Hum Genet, 1996 Dec, 98(6), 646 - 50
Characterization of a cytogenetic 17q11.2 deletion in an NF1 patient with a contiguous gene syndrome; Riva P et al.; We report on a rare patient screened as a putative carrier of a contiguous gene syndrome on the basis of a complex phenotype characterized by sporadic neurofibromatosis type 1 (NF1), dysmorphism, mental retardation and severe skeletal anomalies . A cytogenetically visible 17q11.2 deletion was detected in the patient's karyotype by high-resolution banding and confirmed by fluorescence in situ hybridization with yeast artificial chromosomes targeting the NF1 region . Analysis of the segregation from parents to proband of 13 polymorphic DNA markers, either contiguous or contained within the NF1 gene, showed that the patient is hemizygous at sites within the NF1 gene-the AAAT-Alu repeat in the 5' region of intron 27b, the CA/GT microsatellite in the 3' region of intron 27b, and the CA/GT microsatellite in intron 38- and at the extragenic D17S798 locus, distal to the 3' end of NF1 . The patient may be an important resource in the identification of genes downstream of NF1 that may contribute to some of his extra-NF1 clinical signs.

J Neurosci, 1996 Dec 1, 16(23), 7407 - 15
Cloning and characterization of postsynaptic density 93, a nitric oxide synthase interacting protein; Brenman JE et al.; Nitric oxide (NO) formation in brain is regulated by the calcium/calmodulin dependence of neuronal NO synthase (nNOS) . Calcium influx through NMDA-type glutamate receptors is efficiently coupled to nNOS activity, whereas many other intracellular calcium pathways are poorly coupled . To elucidate possible mechanisms responsible for this coupling, we performed yeast two-hybrid screening to identify proteins that interact with nNOS . Two nNOS interacting proteins were identified: the postsynaptic density proteins PSD-93 and PSD-95 . Here, we report the cloning and characterization of PSD-93 . PSD-93 is expressed in discrete neuronal populations as well as in specific non-neuronal cells, and it exhibits complex molecular diversity attributable to tissue-specific alternative splicing . PSD-93, like PSD-95, binds to nNOS and to the NMDA receptor 2B . PSD-93, however, is unique among PSD-95/SAP-90 family members in its expression in Purkinje neuron cell bodies and dendrites . We also demonstrate that the PDZ domain at the N terminus of nNOS is required, but it is not sufficient for interaction with PSD-93/95 . Given that PSD-93 and PSD-95 each contain multiple potential binding sites for nNOS and the NMDA receptor, complexes involving oligomers of PSD-93/95 may help account for the functional as well as the physical coupling of nNOS to NMDA receptors.

Nature, 1996 Nov 28, 384(6607), 372 - 5
A death-domain-containing receptor that mediates apoptosis; Kitson J et al.; The cell-killing effects of the cytokines TNF-alpha and FasL are mediated by the distinct cell-surface receptors TNFR1, TNFR2 and Fas (also known as CD95/APO-1), which are all members of a receptor superfamily that is important for regulating cell survival . The cytoplasmic regions of TNFR1 and Fas contain a conserved 'death' domain which is an essential component of the signal pathway that triggers apoptosis and activation of the transcription factor NF-kappaB (refs 5,6) . Here we report the isolation of a 54K receptor that is a new member of the TNFR superfamily, using the death domain of TNFR1 in a yeast two-hybrid system . This protein, WSL-1, is most similar to TNFR1 itself, particularly in the death-domain region . The gene wsl-1 is capable of inducing apoptosis when transfected into 3T3 and 293 cells, and can also activate NF-kappaB in 293 cells . Like TNFR1, WSL-1 will homodimerize in yeast . WSL-1 also interacts specifically with the TNFR1-associated molecule TRADD . The tissue distribution is very restricted and significantly different from that of Fas and TNFR1.

Mol Gen Genet, 1996 Nov 27, 253(1-2), 32 - 41
Chromosome landing at the Arabidopsis TORNADO1 locus using an AFLP-based strategy; Cnops G et al.; The Arabidopsis tornado1 (trn1) mutation causes severe dwarfism combined with twisted growth of all organs . We present a chromosome landing strategy, using amplified restriction fragment length polymorphism (AFLP) marker technology, for the isolation of the TRN1 gene . The recessive trn1 mutation was identified in a C24 transgenic line and is located 5 cM from a T-DNA insertion . We mapped the TRN1 locus to the bottom half of chromosome 5 relative to visible and restriction fragment length polymorphism (RFLP) markers . Recombinant classes within a 3-cM region around TRN1 were used to build a high-resolution map in this region, using the AFLP technique . Approximately 300 primer combinations have been used to test about 26,000 fragments for polymorphisms . Seventeen of these AFLP markers were identified in the 3-cM region around TRN1 . These markers were mapped within this region using individual recombinants . Four of these AFLP markers co-segregate with TRN1 whereas one maps at one recombinant below TRN1 . We isolated and cloned three of these AFLP markers . These markers identified two yeast artificial chromosome (YAC) clones, containing the RFLP marker above and the AFLP marker below TRN1, demonstrating that these YACs span the TRN1 locus and that chromosome landing has been achieved, using an AFLP-based strategy.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 14210 - 3
A conserved repetitive DNA element located in the centromeres of cereal chromosomes; Jiang J et al.; Repetitive DNA sequences have been demonstrated to play an important role for centromere function of eukaryotic chromosomes, including those from fission yeast, Drosophila melanogaster, and humans . Here we report on the isolation of a repetitive DNA element located in the centromeric regions of cereal chromosomes . A 745-bp repetitive DNA clone pSau3A9, was isolated from sorghum (Sorghum bicolor) . This DNA element is located in the centromeric regions of all sorghum chromosomes, as demonstrated by fluorescence in situ hybridization . Repetitive DNA sequences homologous to pSau3A9 also are present in the centromeric regions of chromosomes from other cereal species, including rice, maize, wheat, barley, rye, and oats . Probe pSau3A9 also hybridized to the centromeric region of B chromosomes from rye and maize . The repetitive nature and its conservation in distantly related plant species indicate that the pSau3A9 family may be associated with centromere function of cereal chromosomes . The absence of DNA sequences homologous to pSau3A9 in dicot species suggests a faster divergence of centromererelated sequences compared with the telomere-related sequences in plants.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 14053 - 8
Tumor necrosis factor receptor-associated factor (TRAF)-1, TRAF-2, and TRAF-3 interact in vivo with the CD30 cytoplasmic domain; TRAF-2 mediates CD30-induced nuclear factor kappa B activation; Ansieau S et al.; CD30 is a member of the tumor necrosis factor receptor superfamily, which can transduce signals for proliferation, death, or nuclear factor kappa B (NF-kappa B) activation . Investigation of CD30 signaling pathways using a yeast two-hybrid interaction system trapped a cDNA encoding the tumor necrosis factor receptor-associated factor (TRAF)-2 TRAF homology domain . TRAF-1 and TRAF-3 also interacted with CD30, and > 90% of in vitro-translated TRAF-1 or -2, or 50% of TRAF-3, bound to the CD30 cytoplasmic domain . TRAF-1, -2, and -3 bound mostly, but not exclusively, to the carboxyl-terminal 36 residues of CD30 . The binding was strongly inhibited by a CD30 oligopeptide centered around a PXQXT (where X is any amino acid) motif shared with CD40 and the Epstein-Barr virus transforming protein LMP1, indicating that this motif in CD30 is an important determinant of TRAF-1, -2 or -3 interaction . At least 15% of TRAF-1, -2, or -3 associated with CD30 when coexpressed in 293 cells . The association was not affected by CD30 cross-linking . However, cross-linking of CD30 activated NF-kappa B . NF-kappa B activation was dependent on the carboxyl-terminal 36 amino acids of CD30 that mediate TRAF association . TRAF-2 has been previously shown to have a unique role in TRAF-mediated NF-kappa B activation, and NF-kappa B activation following CD30 cross-linking was blocked by a dominant negative TRAF-2 mutant . These data indicate that CD30 cross-linking-induced NF-kappa B activation is predominantly TRAF-2-mediated.

Biochemistry, 1996 Nov 26, 35(47), 14889 - 98
Cooperative action of Hsp70, Hsp90, and DnaJ proteins in protein renaturation; Schumacher RJ et al.; The proteins required for the repair of damaged proteins in the eukaryotic cytoplasm remain largely uncharacterized . The renaturation of thermally denatured firefly luciferase readily occurs in rabbit reticulocyte lysate by an ATP-dependent process . Earlier studies had shown that this chaperoning activity could be reconstituted, in part, using purified preparations of hsp70 and hsp90 . We have extended the description of this system by clarifying the importance of hsp70 and hsp90 and have tested for additional factors that enhance renaturation . Using mutant hsp70 proteins, we have shown that hsp70 is required for luciferase renaturation . We have also found that hsp70 and hsp90 preparations purified by common procedures were contaminated with low levels of DnaJ proteins that are essential for the renaturing activity . When hsp70 and hsp90 preparations free of DnaJ proteins are used, the system must be supplemented with a DnaJ protein to obtain renaturation activity . The yeast DnaJ protein, YDJ-1, was found to be very effective for this purpose . Although significant renaturation can occur with only hsp70 and DnaJ proteins, hsp90 also contributes to the renaturation process, both in the complex environment of reticulocyte lysate and in a purified system . However, using highly purified hsp90 and geldanamycin, a specific inhibitor of hsp90 function, we have determined that hsp90 is not an essential component of the renaturation system . The contribution of hsp90 to renaturation is only partially blocked by geldanamycin, suggesting that this protein may influence activity in more than one way . This study indicates that hsp70, hsp90, and DnaJ proteins function cooperatively to renature damaged proteins in the eukaryotic cytoplasm and provides a framework by which additional components can be identified and individual chaperone contributions can be investigated.

J Biol Chem, 1996 Nov 22, 271(47), 29903 - 8
Identification of a novel RalGDS-related protein as a candidate effector for Ras and Rap1; Peterson SN et al.; Although Ras and Rap1 share interaction with common candidate effector proteins, Rap1 lacks the transforming activity exhibited by Ras proteins . It has been speculated that Rap antagonizes Ras transformation through the formation of nonproductive complexes with critical Ras effector targets . To understand further the distinct biological functions of these two closely related proteins, we searched for Rap1b-binding proteins by yeast two-hybrid screening . We identified multiple clones that encode the COOH-terminal sequences of a protein that shares sequence identity with RalGDS and RGL, which we have designated RGL2 . A 158-amino acid COOH-terminal fragment of RGL2 (RGL2 C-158) bound to Ras superfamily proteins which shared identical effector domain sequences with Rap1 (Ha-Ras, R-Ras, and TC21) . RGL2 C-158 binding was impaired by effector domain mutations in Rap1b and Ha-Ras . Furthermore, RGL2 C-158 bound exclusively to the GTP-, but not the GDP-bound form of Ha-Ras . Finally, coexpression of RGL2 C-158 impaired oncogenic Ras activation of transcription from a Ras-responsive promoter element and focus-forming activity in NIH 3T3 cells . We conclude that RGL2 may be an effector for Ras and/or Rap proteins.

J Biol Chem, 1996 Nov 22, 271(47), 29876 - 81
Mammalian mitogen-activated protein kinase pathways are regulated through formation of specific kinase-activator complexes; Zanke BW et al.; Mammalian cells contain at least three signaling systems which are structurally related to the mitogen-activated protein kinase (MAPK) pathway . Growth factors acting through Ras primarily stimulate the Raf/MEK/MAPK cascade of protein kinases . In contrast, many stress-related signals such as heat shock, inflammatory cytokines, and hyperosmolarity induce the MEKK/SEK(MKK4)/SAPK(JNK) and/or the MKK3 or MKK6/p38(hog) pathways . Physiological agonists of these pathway types are either qualitatively or quantitatively distinct, suggesting few common proximal signaling elements, although past studies performed in vitro, or in cells using transient over-expression, reveal interaction between the components of all three pathways . These studies suggest a high degree of cross-talk apparently not seen in vivo . We have examined the possible molecular basis of the differing agonist profiles of these three MAPK pathways . We report preferential association between MAP kinases and their activators in eukaryotic cells . Furthermore, using the yeast 2-hybrid system, we show that association between these components can occur independent of additional eukaryotic proteins . We show that SAPK(JNK) or p38(hog) activation is specifically impaired by co-expression of cognate dominant negative MAP kinase kinase mutants, demonstrating functional specificity at this level . Further divergence and insulation of the stress pathways occurs proximal to the MAPK kinases since activation of the MAPK kinase kinase MEKK results in SAPK(JNK) activation but does not cause p38(hog) phosphorylation . Therefore, in intact cells, the three MAPK pathways may be independently regulated and their components show specificity in their interaction with cognate cascade members . The degree of intermolecular specificity suggests that mammalian MAPK signaling pathways may remain distinct without the need for specific scaffolding proteins to sequester components of individual pathways.

Gene, 1996 Nov 21, 180(1-2), 151 - 5
RS cyclophilins: identification of an NK-TR1-related cyclophilin; Nestel FP et al.; We report the isolation of a large cyclophilin protein containing RS (arginine-serine) repeats from a yeast two-hybrid screen using ClK (CDC28/cdc2-like kinase) as a probe . This Clk associating RS-cyclophilin (CARS-Cyp) possesses 39% homology to the NK-TR1 (natural killer tumor recognition protein-1) we have previously characterized (Anderson et al . (1993) Proc . Natl . Acad . Sci . USA 90 (1993) 542-546) . CARS-Cyp is expressed in a variety of tissues and cell types, and codes for a protein with a predicted mass of 89 kDa containing a cyclophilin-related domain, two Nopp140 (nucleolar phosphoprotein of 140 kDa)-related domains, and a large RS domain . The RS-cyclophilins, a novel class of proteins, may play an important role in the regulation of pre-mRNA splicing.

Oncogene, 1996 Nov 21, 13(10), 2255 - 63
Antisense GADD45 expression results in decreased DNA repair and sensitizes cells to u.v.-irradiation or cisplatin; Smith ML et al.; Loss of p53 function in cancer cells commonly results in a condition of genomic instability . This is believed to emanate from a loss of the G1 checkpoint response to DNA damage . While the role of p53 in the induction of a G1 arrest is well-accepted, additional p53 functions are being discovered . Cell cycle checkpoints presumably function to allow additional time for DNA repair after damage is incurred, however, genetic studies in yeast suggest that components of the checkpoint pathway may also be involved in DNA lesion processing (Lydall and Weinert, 1995) . Recent evidence suggests that this may also be the case for p53, as suggested by numerous reports linking p53 function to DNA repair . Thus, loss of p53 function might contribute to genomic instability independent of G1-arrest . In the present study, we explored the effect of p53 disruption and consequences of antisense GADD45 expression on the DNA repair capacity of human colon carcinoma RKO cells . DNA repair was assayed using host-cell reactivation of u.v.-damaged reporter plasmids and unscheduled DNA synthesis experiments in transiently-transfected cells . We show that a number of transfected genes that suppress p53 function reduce the ability of cells to repair u.v.-induced DNA damage . Moreover, cells in which expression of the p53-regulated gene GADD45 was blocked by antisense vectors, also showed altered levels of DNA repair . Blocking Gadd45 expression by constitutive antisense expression sensitized cells to killing by u.v.-radiation or by cis-platinum (II) diamine-dichloride (CDDP, or cisplatin), a cancer chemotherapy drug which produces DNA cross-links . These findings suggest the involvement of downstream effectors of the p53 pathway in the coordination of cell cycle arrest and DNA repair.

Nature, 1996 Nov 21, 384(6606), 276 - 9
20S cyclosome complex formation and proteolytic activity inhibited by the cAMP/PKA pathway; Yamashita YM et al.; The 20S cyclosome complex (also known as the anaphase-promoting complex) has ubiquitin ligase activity and is required for mitotic cyclin destruction and sister chromatid separation . The formation and activation of the 20S cyclosome complex is regulated by an unknown mechanism . Here we show that Cut4 (ref . 6) is an essential component of the cyclosome in fission yeast . Cut4 shares sequence similarity with BimE, a protein that regulates mitosis in Aspergillus nidulans . Mutations in cut4 result in hypersensitivity to cyclic AMP and to stress-inducing heavy metals, inhibition of the onset of anaphase, disruption of the 20S complex, and inhibition of mitotic cyclin ubiquitination . These phenotypes are fully suppressed by cAMP phosphodiesterase and the protein kinase A (PKA) regulatory subunit and weakly suppressed by Sti1 (an activator of the Hsp70 and Hsp90 chaperones) . Suppression correlates with the amount of 20S complex, indicating that cyclosome formation and activation is inhibited by the cAMP/PKA pathway.

Nucleic Acids Res, 1996 Nov 15, 24(22), 4495 - 500
Mapping genes within a YAC by computer-assisted interpretation of partial restriction digestions; Shields DC et al.; Partial restriction digestion is used to map restriction sites and the location of genes within yeast artificial chromosomes (YACs) . Locus-specific probes are hybridised to the partially digested YAC DNA and the fragments to which they hybridise are compared with the pattern of partial digestion products that include each map region . A least squares criterion is presented which allows for error in fragment length determination . This rapidly defines the most likely location of a marker within the restriction map and permits the combination of results from digestions with different restriction enzymes . Approximate confidence intervals may be assigned to gene locations, and tests of goodness-of-fit of the data may be performed . Since the number of erroneously matched fragments increases in proportion to the square of the number of sites, denser maps are not necessarily more informative . Simulations indicate that the optimal number of internal restriction sites given typical experimental error (1% of YAC length) is about five sites; the associated broad support interval (on average one third of YAC length) may be reduced by combining results from different enzyme digestions . Application of a computer implementation of this model to experimental data showed that the model fitted well, and estimates of location were found to be consistent with other evidence.

EMBO J, 1996 Nov 15, 15(22), 6026 - 34
A locus control region at -12 kb of the tyrosinase gene; Montoliu L et al.; We have shown previously that the tyrosinase gene encompassed in a 250 kb yeast artificial chromosome (YAC) is expressed faithfully in transgenic mice . To define the sequences important for this qualitatively and quantitatively correct expression pattern, we have generated transgenic mice with YACs carrying several deletions in the mouse tyrosinase locus . In particular, we wanted to address the in vivo relevance of a regulatory element indicated by a cell-specific DNase I hypersensitive site (HS) located -12 kb upstream of the gene . Wild-type level expression was observed only when the YACs transferred contained this HS . Constructs in which the HS was deleted gave rise to much weaker expression and variable patterns of expression . In conclusion, this HS region appears to harbour the essential regulatory element for the correct expression of the tyrosinase gene . Moreover, it behaves as a locus control region in that it commands the functional status of this expression domain, protecting it from position effects.

Cell, 1996 Nov 15, 87(4), 607 - 17
Absence epilepsy in tottering mutant mice is associated with calcium channel defects; Fletcher CF et al.; Mutations at the mouse tottering (tg) locus cause a delayed-onset, recessive neurological disorder resulting in ataxia, motor seizures, and behavioral absence seizures resembling petit mal epilepsy in humans . A more severe allele, leaner (tg(la)), also shows a slow, selective degeneration of cerebellar neurons . By positional cloning, we have identified an alpha1A voltage-sensitive calcium channel gene that is mutated in tg and tg(la) mice . The alpha1A gene is widely expressed in the central nervous system with prominent, uniform expression in the cerebellum . alpha1A expression does not mirror the localized pattern of cerebellar degeneration observed in tg(la) mice, providing evidence for regional differences in biological function of alpha1A channels . These studies define the first mutations in a mammalian central nervous system-specific voltage-sensitive calcium channel and identify the first gene involved in absence epilepsy.

J Biol Chem, 1996 Nov 15, 271(46), 29436 - 45
Endoplasmic reticulum form of calreticulin modulates glucocorticoid-sensitive gene expression; Michalak M et al.; Calreticulin is a ubiquitously expressed Ca2+-binding protein of the endoplasmic reticulum (ER), which inhibits DNA binding in vitro and transcriptional activation in vivo by steroid hormone receptors . Transient transfection assays were carried out to investigate the effects of different intracellular targeting of calreticulin on transactivation mediated by glucocorticoid receptor . BSC40 cells were transfected with either calreticulin expression vector (ER form of calreticulin) or calreticulin expression vector encoding calreticulin minus leader peptide, resulting in cytoplasmic localization of the recombinant protein . Transfection of BSC40 cells with calreticulin expression vector encoding the ER form of the protein led to 40-50% inhibition of the dexamethasone-sensitive stimulation of luciferase expression . However, in a similar experiment, but using the calreticulin expression vector encoding cytoplasmic calreticulin, dexamethasone-stimulated activation of the luciferase reporter gene was inhibited by only 10% . We conclude that the ER, but not cytosolic, form of calreticulin is responsible for inhibition of glucocorticoid receptor-mediated gene expression . These effects are specific to calreticulin, since overexpression of the ER lumenal proteins (BiP, ERp72, or calsequestrin) has no effect on glucocorticoid-sensitive gene expression . The N domain of calreticulin binds to the DNA binding domain of the glucocorticoid receptor in vitro; however, we show that the N+P domain of calreticulin, when synthesized without the ER signal sequence, does not inhibit glucocorticoid receptor function in vivo . Furthermore, expression of the N domain of calreticulin and the DNA binding domain of glucocorticoid receptor as fusion proteins with GAL4 in the yeast two-hybrid system revealed that calreticulin does not interact with glucocorticoid receptor under these conditions . We conclude that calreticulin and glucocorticoid receptor may not interact in vivo and that the calreticulin-dependent modulation of the glucocorticoid receptor function may therefore be due to a calreticulin-dependent signaling from the ER.

J Biol Chem, 1996 Nov 15, 271(46), 29271 - 8
The inositol 5'-phosphatase SHIP binds to immunoreceptor signaling motifs and responds to high affinity IgE receptor aggregation; Osborne MA et al.; Immunoreceptors such as the high affinity IgE receptor, FcepsilonRI, and T-cell receptor-associated proteins share a common motif, the immunoreceptor tyrosine-based activation motif (ITAM) . We used the yeast tribrid system to identify downstream effectors of the phosphorylated FcepsilonRI ITAM-containing subunits beta and gamma . One novel cDNA was isolated that encodes a protein that is phosphorylated on tyrosine, contains a Src-homology 2 (SH2) domain, inositolpolyphosphate 5-phosphatase activity, three NXXY motifs, several proline-rich regions, and is called SHIP . Mutation of the conserved tyrosine or leucine residues within the FcepsilonRI beta or gamma ITAMs eliminates SHIP binding and indicates that the SHIP-ITAM interaction is specific . SHIP also binds to ITAMs from the CD3 complex and T cell receptor zeta chain in vitro . SHIP protein possesses both phosphatidylinositol-3,4,5-trisphosphate 5'-phosphatase and inositol-1,3,4,5-tetrakisphosphate 5'-phosphatase activity . Phosphorylation of SHIP by a protein-tyrosine kinase, Lck, results in a reduction in enzyme activity . FcepsilonRI activation induces the association of several tyrosine phosphoproteins with SHIP . SHIP is constitutively tyrosine-phosphorylated and associated with Shc and Grb2 . These data suggest that SHIP may serve as a multifunctional linker protein in receptor activation.

J Biol Chem, 1996 Nov 15, 271(46), 28745 - 8
Identification of TRAF6, a novel tumor necrosis factor receptor-associated factor protein that mediates signaling from an amino-terminal domain of the CD40 cytoplasmic region; Ishida T et al.; CD40 signalings play crucial roles in B-cell function . To identify molecules which transduce CD40 signalings, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the cytoplasmic tail of CD40 . A cDNA encoding a putative signal transducer, designated TRAF6, has been molecularly cloned . TRAF6 has a tumor necrosis factor receptor (TNFR)-associated factor (TRAF) domain in its carboxyl terminus and has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other TRAF family proteins . TRAF6 does not associate with the cytoplasmic tails of TNFR2, CD30, lymphotoxin-beta receptor, and LMP1 of Epstein-Barr virus . Deletion analysis showed that residues 246-269 of CD40 which are required for its association with TRAF2, TRAF3, and TRAF5 are dispensable for its interaction with TRAF6, whereas residues 230-245 were required . Overexpression of TRAF6 activates transcription factor NFkappaB, and its TRAF-C domain suppresses NFkappaB activation triggered by CD40 lacking residues 246-277 . These results suggest that TRAF6 could mediate the CD40 signal that is transduced by the amino-terminal domain (230-245) of the CD40 cytoplasmic region and appears to be independent of other known TRAF family proteins.

Biochim Biophys Acta, 1996 Nov 14, 1298(1), 9 - 11
Carp cDNA sequence encoding a putative diazepam-binding inhibitor/endozepine/acyl-CoA-binding protein; Chang JL et al.; A full-length cDNA coding for common carp diazepam-binding inhibitor (DBI)/endozepine (EP)/acyl-CoA-binding protein (ACBP) was isolated and sequenced . The deduced DBI/EP/ACBP is comprised of 87 amino acids (including initiating methionine) without possessing a signal peptide . Common carp DBI/EP/ACBP displays 77%, 78%, 70%, 63%, 61% and 45% identity with human, bovine, rat, frog, duck and yeast DBI/EP/ACBP, respectively.

Mol Biochem Parasitol, 1996 Nov 12, 82(1), 81 - 90
Characterization of ubiquitin genes and -transcripts and demonstration of a ubiquitin-conjugating system in Entamoeba histolytica; Wostmann C et al.; We have investigated the genomic organization of Entamoeba histolytica ubiquitin and looked for the occurrence of a ubiquitin-conjugating system in this organism . Southern blots indicated the presence of > or = 5 ubiquitin-coding regions . One of these, EhUBI1, was cloned and sequenced and found to correspond to a monoubiquitin gene; as shown by a polymerase chain reaction, E . histolytica lacked polyubiquitin genes altogether . Blots of poly(A)+ RNA from exponentially-growing trophozoite cultures exhibited five ubiquitin transcripts, the most prominent and smallest of which corresponded to EhUBI1 mRNA . Expression of the ubiquitin genes was not influenced by heat shock . Although the predicted amino acid sequence of the ubiquitin from E . histolytica differs significantly (in 7-9 amino acid residues) from that of yeast and animals, expression of the coding sequence of EhUBI1 suppressed the heat-sensitive phenotype of a polyubiquitin gene-deficient yeast mutant . In correlation, trophozoite extract catalyzed an ATP-dependent conjugation of radioiodinated bovine ubiquitin to trophozoite proteins . The latter data indicate that E . histolytica contains a functional ubiquitin-conjugating system.

Biochim Biophys Acta, 1996 Nov 11, 1309(1-2), 69 - 72
The cloning and sequencing of a ribosomal L18 protein from an evolutionary divergent eukaryote, Trypanosoma brucei; Coulter LJ et al.; Eukaryotic ribosomal proteins are highly conserved across widely divergent species, suggesting that strong functional constraints prevent divergence of important amino acid motifs . Using this as a basis, an evolutionary approach could be used to identify putative functional motifs . We obtained the DNA sequence of the ribosomal protein L18 from the evolutionary divergent protozoan parasite, Trypanosoma brucei . Analysis of this sequence showed that it had 46% and 43% identity with the human and yeast sequences, respectively, and 30% of amino acid residues were identical across all the species analysed . Using these data, amino acids essential to the structure and function of ribosomal protein L18 can be inferred and could provide valuable information for molecular modelling and mutational studies.

J Biol Chem, 1996 Nov 8, 271(45), 28636 - 40
Novel proteins that interact with the COOH-terminal cytosolic routing determinants of an integral membrane peptide-processing enzyme; Alam MR et al.; The steady state distribution of membrane forms of peptidylglycine alpha-amidating monooxygenase (PAM) in the secretory pathway of neurons and endocrine cells depends on signals in its cytosolic COOH-terminal domain (CD) . Mutagenesis studies yielded catalytically active PAM proteins that are not properly localized or internalized . Employing the yeast two-hybrid system, we isolated two distinct cDNAs whose protein products showed a strong interaction with the CD of PAM . The interaction of these novel PAM COOH-terminal interactor proteins (P-CIPs) did not occur with a misrouted CD mutant as bait in the yeast system . Both proteins, P-CIP2 and P-CIP10, were expressed as fusion proteins that interacted in vitro with solubilized integral membrane PAM . P-CIP2 was homologous to several serine/threonine and dual specificity protein kinases, while P-CIP10 contained spectrin-like repeats . Endogenous P-CIP2 was localized to the Golgi region of AtT-20 corticotrope tumor cells, and expression of integral membrane PAM disrupted the distribution of endogenous P-CIP2 . Both P-CIP2 and P-CIP10 mRNAs were found to be expressed in rat brain neurons also expressing PAM proteins . P-CIP2 and P-CIP10 may be members of a family of cytosolic proteins involved in the routing of membrane proteins that function in the regulated secretory pathway.

J Biol Chem, 1996 Nov 8, 271(45), 28311 - 7
Comparison of binding and block produced by alternatively spliced Kvbeta1 subunits; Wang Z et al.; Voltage-gated K+ (Kv) channels consist of alpha subunits complexed with cytoplasmic Kvbeta subunits . Kvbeta1 subunits enhance the inactivation of currents expressed by the Kv1 alpha subunit subfamily . Binding has been demonstrated between the C terminus of Kvbeta1.1 and a conserved segment of the N terminus of Kv1.4, Kv1.5, and Shaker alpha subunits . Here we have examined the interaction and functional properties of two alternatively spliced human Kvbeta subunits, 1.2 and 1.3, with Kvalpha subunits 1.1, 1.2, 1.4, and 1.5 . In the yeast two-hybrid assay, we found that both Kvbeta subunits interact specifically through their conserved C-terminal domains with the N termini of each Kvalpha subunit . In functional experiments, we found differences in modulation of Kv1alpha subunit currents that we attribute to the unique N-terminal domains of the two Kvbeta subunits . Both Kvbeta subunits act as open channel blockers at physiological membrane potentials, but hKvbeta1.2 is a more potent blocker than hKvbeta1.3 of Kv1.1, Kv1.2, Kv1.4, and Kv1 . 5 . Moreover, hKvbeta1.2 is sensitive to redox conditions, whereas hKvbeta1.3 is not . We suggest that different Kvbeta subunits extend the range over which distinct Kv1alpha subunits are modulated and may provide a variable mechanism for adjusting K+ currents in response to alterations in cellular conditions.

Oncogene, 1996 Nov 7, 13(9), 2027 - 31
ALL-1 interacts with unr, a protein containing multiple cold shock domains; Leshkowitz D et al.; The ALL-1 gene is involved in human acute leukemia through chromosome translocations and fusion to partner genes, or through partial tandem duplications . ALL-1 is the human homologue of Drosophila trithorax which transregulates the homeotic genes of the Antennapedia and bithorax complexes controlling body segment identity . ALL-1 encodes a very large protein of 3968 amino acids which presumably interacts with many proteins . Here we applied yeast two hybrid screening to identify proteins interacting with the N-terminal segment of ALL-1 . One protein obtained in this way was the product of the unr gene . This protein consists of multiple repeats homologous to the cold shock domain (CSD), a motif common to some bacterial and eukaryotic nucleic acids-binding proteins . The minimal region on unr required for the interaction with ALL-1 included two CSD and two intervening polypeptides . The interaction was confirmed by in vitro binding studies, and by coimmunoprecipitation from COS cells overexpressing the relevant segments of the two proteins . These results suggest that unr is involved in an interaction of ALL-1 with DNA or RNA.

Oncogene, 1996 Nov 7, 13(9), 2001 - 8
Detailed deletion mapping with a refined physical map of 7q31 localizes a putative tumor suppressor gene for breast cancer in the region of MET; Lin JC et al.; In breast cancer, loss of heterozygosity (LOH) has been described on the long arm of chromosome 7 at band q31, suggesting the presence of a tumor suppressor gene in this region . To define the deleted region, we analysed 73 cases of breast cancer and matched normal DNAs with 17 polymorphic markers . A minimal area of LOH was identified as the chromosomal interval flanked by markers D7S687 and metH, spanning a segment of 2 Mb on chromosome 7q31 . Of the 73 breast cancer patients studied, all were informative for at least one marker in this region and nine patients showed LOH at one or more loci (12.3%) . To define the physical size of the deletion and to ensure the correct interpretation of the LOH deletion studies, we redefined the physical map of markers within this region of 7q31 . We present a new physical order for markers at 7q31 . More significantly, we have mapped the minimum deletion of 7q31 in the breast cancers studied to date to a physical distance of 1000 kb, contained on a single YAC clone, which includes the MET receptor tyrosine kinase but no other known genes.

Parasite Immunol, 1996 Nov, 18(11), 547 - 58
Influence of adjuvants on murine immune responses against the C-terminal 19 kDa fragment of Plasmodium vivax merozoite surface protein-1 (MSP-1); Yang C et al.; The immunogenicity of a yeast-expressed 19 kDa fragment of P vivax MSP-1 in the presence of different adjuvant formulations was evaluated . ICR mice were immunized with the 19 kDa antigen, using Freund's, alum, and block copolymer P1005 in water-in-oil (W/O) or oil-in-water (O/W) emulsions with or without detoxified lipopolysaccharide (RaLPS) as adjuvants . Five weeks following immunization with the antigen, mice were boosted with asexual blood-stage antigens . Three weeks after the last immunization with the 19 kDa antigen, mice from the Freund's group and most groups that received P1005 as adjuvant had higher total IgG titres than those that received alum as adjuvant or antigen alone . Antibody responses after the antigen immunization were predominantly of the IgG1 isotype, but mice in the Freund's and P1005 (W/O or O/W emulsion with or without RaLPS) groups also had high titres of IgG2a and IgG2b . Antibody titres against merozoites increased in all groups after the parasite antigen boost . IgG2a levels in the group that received antigen in P1005 plus RaLPS in the W/O emulsion were higher than those receiving Freund's, alum or the other copolymer adjuvants . The high IgG2a titres in this group were associated with reduced IL-10 production.

Somat Cell Mol Genet, 1996 Nov, 22(6), 511 - 7
Transcribed sequences encoded in the region involved in contiguous deletion syndrome that comprises X-linked stapes fixation and deafness; Kandpal G et al.; We have used a direct cDNA selection protocol to isolate expressed sequences from yeast artificial chromosome clones that contain approximately 900 Kb of genomic DNA from Xq21 band that is deleted in contiguous gene syndromes comprising of mixed deafness associated with stapes fixation (DFN3) . In addition to identifying Brn4 (POU3f4), a POU domain containing transcription factor that is involved in DFN3 phenotype, we have isolated seven short fragment cDNAs mapping to the deleted region . Some of the selected fragments showed X-chromosome specificity and hybridized to autosomal DNA fragments, indicating the presence of a low abundance interspersed repeat in the cDNAs or their homology to some uncharacterized family of genes . In conformity with the inertness of Xq21 band our results demonstrate that the region encodes far less than the average density of genes in other parts of the genome.

Genes Cells, 1996 Nov, 1(11), 977 - 93
Mouse MCM proteins: complex formation and transportation to the nucleus; Kimura H et al.; BACKGROUND: The members of the MCM protein family, including MCM2, MCM3, Cdc21, CDC46, Mis5 and CDC47, are considered to be involved in the control of a single round of DNA replication during S phase in eukaryotes . They bind to chromatin during G1 and detach from it during S phase as if they license the chromatin to replicate . However, unlike the originally proposed 'licensing factor' and the budding yeast homologues, mammalian MCM2 and P1MCM3 proteins appeared to be localized in the nucleus during the interphase . RESULTS: We purified mCdc21 and its associated proteins from mouse cell extract by anti-mCdc21 immunoaffinity chromatography . Three proteins which co-purified with mCdc21 were identified as mCDC47, mMis5 and mMCM2, all were MCM proteins . Glycerol gradient centrifugation analysis showed that all the mouse MCM proteins were detected at 450-600 kDa, an indication of the sum of their calculated molecular weights from their amino acid sequences . mCdc21 was displaced from replicated chromatin in a similar way to P1MCM3 and MCM2 during S phase . Among the six mouse MCM proteins, only mMCM2 and mP1MCM3 showed nuclear localization when overexpressed in COS cells . CONCLUSIONS: We conclude that in the mouse, six MCM proteins form a single protein complex of molecular weight 450-600 kDa, which may enter the nucleus by nuclear localization signals in the mMCM2 and mP1MCM3 subunits.

Andrologia, 1996 Nov-Dec, 28(6), 327 - 33
Affinity sites for beta-glucuronidase on the surface of human spermatozoa; Barbieri MA et al.; Glycosidases secreted by the epididymis become bound to the surface of spermatozoa during their transit through the epididymal duct . They are believed to play a role in mammalian fertilization . In the present report, we demonstrate that beta-glucuronidase binds to the surface of ejaculated human spermatozoa with high affinity and in a saturable manner . The binding is Ca(2+)-independent, inhibited by either mannose-6-phosphate, phosphomannan fragments from the yeast Hansenula holstii and alpha-mannosidase from the Dictyostelium discoideum, suggesting that phosphomannosyl receptors are involved in the recognition of the enzyme . The catalytic site of the enzyme is not involved in the binding . The localization of the beta-glucuronidase binding-sites is restricted to the surface of the sperm head . These results suggest that the spermatozoa could be the target for glycosidases present in the seminal plasma.

Exp Eye Res, 1996 Nov, 63(5), 493 - 500
Control of Drosophila opsin gene expression by carotenoids and retinoic acid: northern and western analyses; Picking WL et al.; In the fly, thorough retinoid deprivation is possible, to optimize investigation of the effects of vitamin A metabolites and retinoic acid (RA) on visual development . Retinoids had been found to control fly opsin gene transcription, though this finding was contested . Northern blots on Drosophila heads showed that mRNA of Rh1 (the predominant rhodopsin) was high in vitamin A replete controls, very low in deprived flies, and increased upon feeding carrot juice to deprived flies as early as 1 hr . Expression of the ribosomal protein 49 {rp49} gene (the control) was equal both in deprivation and in replacement . Recovery of Rh1 protein upon such carotenoid replacement followed, barely detectable on Western blots at 4 hr but conspicuous by 8 hr . Alternative chromophore deprivation with yeast-glucose food yielded flies with opsin mRNA on Northerns but not rhodopsin, as demonstrated by Western blots, spectrophotometry and the electroretinogram (ERG) . Rh1's mRNA but not Rh1 protein resulted from rearing flies from egg to adult on the otherwise deprivational medium supplemented with RA or beef brain-heart infusion . By comparing results from these different media it was concluded that: {1} deprivation and replacement affect opsin gene transcription; and {2} contradictory conclusions were from chromophore deprivation which does not eliminate all retinoid dependent factors which could affect the opsin promoter . Preliminary evidence shows that carotenoid deprivation decreases two proteins relevant to visual function: {1} phospholipase C (PLC); and {2} Drosophila retinoid binding protein (DRBP).

J Biochem (Tokyo), 1996 Nov, 120(5), 1028 - 33
Protein-RNA and protein-protein interactions of the Drosophila sex-lethal mediated by its RNA-binding domains; Sakashita E et al.; The Drosophila Sex-lethal (Sxl) contains two RNA-binding domains (RBDs) which belong to the RNA recognition motif (RRM) group . Sxl binds to a specific uridine-rich sequence which is believed to be the major cis-acting element for the splicing regulation of the transformer (tra) mRNA precursor . Here we show evidence supporting the previous suggestion that Sxl recognizes the sequence context downstream of the uridine-rich sequence . In addition, by means of UV-crosslinking assays with Sxl deletion constructs, we have demonstrated that Sxl RNA binding requires both of its RBDs for specificity and strength . Moreover, by the yeast two-hybrid analysis, we found that homodimeric interaction occurs between two Sxl molecules . Interestingly, the amino- and carboxy-terminal regions outside of the Sxl RBDs are dispensable for such dimerization, indicating that the protein-protein interaction is also mediated by RBDs . Coprecipitation experiments in vitro showed that the protein-protein interaction seems to be RNA-dependent but greatly enhanced by addition of the specific RNA containing the Sxl binding site, suggesting that the conformational change which is induced on binding to RNA may facilitate the interaction between Sxl molecules.

Plant Mol Biol, 1996 Nov, 32(4), 707 - 16
A family of novel myb-related genes from the resurrection plant Craterostigma plantagineum are specifically expressed in callus and roots in response to ABA or desiccation; Iturriaga G et al.; A cDNA and two genomic clones comprising highly similar genes that encode a protein with a Myb-related DNA-binding domain were isolated from the resurrection plant Craterostigma plantagineum . The structure of cpm5 and cpm10 (Craterostigma plantagineum myb) genes consists of three putative exons encoding a protein of 36.6 kDa . The cDNA of cpm7 encodes a closely related protein of 36.8 kDa . The canonical Myb domain present in transcriptional activators of yeast, animals and plants was localized in the amino terminus of deduced Cpm5, Cpm7 and Cpm10 proteins and corresponds to the two Myb repeats found in plants . The Myb domain of Cpm deduced proteins and a short stretch of amino acids adjacent to this region are closely related to a myb gene from Arabidopsis thaliana which is expressed in response to osmotic stress and ABA . The rest of the deduced protein has no similarity to other reported sequences . The myb-related genes in the Craterostigma genome comprise a small gene family of 6-8 members as estimated by hybridization with a bona fide Myb domain probe . Northern blot experiments showed specific expression of cpm10 in undifferentiated callus tissue up-modulated by ABA and expression of cpm7 mRNA in roots up-regulated by dehydration.

Plant Mol Biol, 1996 Nov, 32(3), 553 - 7
Analysis of the occurrence and nature of repeated DNA in an 850 kb region of Arabidopsis thaliana chromosome 4; Thompson HL et al.; The occurrence and nature of repeated DNA sequences has been analysed within an 850 kb YAC contig on Arabidopsis thaliana chromosome 4 . Hybridization analysis with seven RFLP markers, six cosmid contigs, 29 YAC end probes and eight YAC clones showed that a least 585 kb of the 850 kb contained only low-copy sequences . One YAC end probe, EG15C8LE, hybridized to multiple genomic fragments and contained a sequence with predicted protein homology to cytochrome P450 monooxygenases . Another one, EG11B7RE, was found to be non-contiguous with the other YAC clones and contained a dispersed repetitive sequence associated with centromeric regions.

Zentralbl Veterinarmed B, 1996 Nov, 43(9), 539 - 43
Diagnostic results in animal dermatophytoses; Schmidt A; Superficial mycoses caused by dermatophytes, as well as asymptomatic carriership of dermatophytes, have a high prevalence among domestic animals and pets . We examined 606 clinical specimens from skin lesions of animals with a significant tendency towards superficial mycosis due to their clinical features . Samples were obtained from horses, dogs, cats, small rodents, birds, and rabbits . The specimens were examined by microscopic and cultural techniques . Microscopically, there was no significant difference in the prevalence of structures which may develop fungal elements between the groups culturally positive or negative for dermatophytes and yeasts . Overall, 24.6% of the samples were microscopically positive . In specimens obtained from horses, a high contamination rate of 36%, mostly due to moulds, was found with a cycloheximide supplemented medium, making the examination of these cultures for the growth of dermatophytes impossible . The other animals showed a significantly lower contamination rate, 11% on average . In horses, Trichophyton equinum had the highest prevalence, in small rodents . Trichophyton mentagrophytes, and in cats Microsporum canis . Overall, 10% of the culturally examinable samples were positive for dermatophytal or yeast growth, though yeasts had only a very low isolation frequency.

J Eukaryot Microbiol, 1996 Nov-Dec, 43(6), 468 - 74
Linked genes for calmodulin and E2 ubiquitin-conjugating enzyme in Trichomonas vaginalis; Keeling PJ et al.; In searching the genomes of early-diverging protists to study whether the possession of calmodulin is ancestral to all eukaryotes, the gene for calmodulin was identified in Trichomonas vaginalis . This flagellate is a member of the Parabasalia, one of the earliest lineages of recognized eukaryotes to have diverged . This sequence was used to isolate a homologous 1.250-kb fragment from the T . vaginalis genome by inverse polymerase chain reaction . This fragment was also completely sequenced and shown to contain the 3' end of the single-copy calmodulin gene and the 3' end of a gene encoding a protein with high similarity to E2 ubiquitin-conjugating enzymes, a family which has previously only been identified in animals, plants, and fungi . Phylogenetic analysis of 50 members of the E2 family distinguishes at least nine separate subfamilies one of which includes the T . vaginalis E2-homologue and an uncharacterized gene from yeast chromosome XII.

J Med Vet Mycol, 1996 Nov-Dec, 34(6), 407 - 10
Adhesion of different Candida spp . to plastic: XTT formazan determinations; Hawser S; Adhesion of synchronized yeast-phase Candida cells to tissue culture plastic was investigated using the tetrazolium salt, XTT . The procedure permits the direct enumeration of adherent yeasts following the metabolic conversion of the XTT tetrazolium salt, to its reduced formazan form, by mitochondrial dehydrogenases . Using this procedure, the formation of XTT formazan by Candida cells was typically related to the inoculum size . The adhesion of Candida yeast-phase cells from different Candida spp . to plastic was of the following order: C . krusei (n = 5) > C . albicans (n = 10) > C . glabrata (n = 6) . Furthermore, preliminary experiments with several other species indicated that C . tropicalis (n = 2) may adhere as well as C . albicans and that one strain each of C . guilliermondii and C . parapsilosis appear to adhere to plastic in a similar fashion to C . glabrata . The data indicate the utility of the XTT tetrazolium based assay in enumerating the adhesion of different Candida spp . to plastic.

Hum Hered, 1996 Nov-Dec, 46(6), 329 - 35
X-linked juvenile retinoschisis: localization between (DXS1195, DXS418) and AFM291wf5 on a single YAC; Pawar H et al.; We studied 17 pedigrees with 108 affected males with X-linked juvenile retinoschisis (RS; McKusick No . 31270) and have analyzed all of the known polymorphic markers in the RS region of Xp22.1-p22.2 between DXS987 and DXS41 . By haplotype analyses we found 7 individuals who showed crossovers in this interval surrounding RS . We previously reported AFM291wf5 as the centromeric boundary, and this remains unchanged in the present study . A new recombination was identified on the telomeric side at (DXS1195, DXS418) . Our data support the locus order Xpter--(DXS987, DXS207, DXS1053, DXS43)--(DXS1195, DXS418)--(RS, DXS257, DXS999)--(AFM291wf5, DXS443)--DXS1052--(DXS1226, DXS274, DXS41)--Xcen; loci grouped in parentheses could not be mutually ordered by our genetic data . Physical mapping has indicated a distance of at most 900-1,000 kb between (DXS1195, DXS418) and AFM291wf5 . No recombination was observed between RS and DXS257 which lies in our new interval of interest, but one critical individual was not informative with this marker . Our data now define the smallest RS inclusion interval . This interval is contained on a single YAC from which we have identified expressed sequences as candidate genes for RS.

Clin Exp Allergy, 1996 Nov, 26(11), 1286 - 97
Detection of Pityrosporum orbiculare reactive T cells from skin and blood in atopic dermatitis and characterization of their cytokine profiles; Tengvall Linder M et al.; BACKGROUND: Atopic dermatitis (AD) is associated with increased levels of serum IgE, and T-helper (Th) cells are thought to a play role in the pathogenesis . Individuals with AD often develop IgE antibodies against the yeast Pityrosporum orbiculare, a member of the normal cutaneous flora . OBJECTIVE: The role of P . orbiculare in atopic dermatitis was investigated by examining the T-cell reactivity for P . orbiculare . METHODS: Freshly isolated peripheral blood mononuclear cells (PBMC) were isolated from 10 AD patients with serum IgE antibodies against P . orbiculare, and from six healthy controls . The proliferative response after P . orbiculare stimulation, measured by {3H}thymidine incorporation, was examined in the PBMC and in T-cell clones (TCC) obtained from skin and blood of one patient . The cytokine profile of the TCC was determined by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and reverse transcriptase-polymerase chain reaction (RT-PCR) following challenge with either P . orbiculare extract or anti-CD3 antibodies and phytohaemagglutinin . RESULTS: The PBMC response to P . orbiculare was significantly higher in the AD patients than in the control group (P < 0.05) . Twenty-nine out of 36 tested TCC derived from one responding patient were reactive for P . orbiculare . The clones were CD2+ and CD4+, except for one CD8+ blood clone . A majority of the TCC derived from lesional skin showed a Th2- or Th2/Th0-like cytokine profile . A co-expression of interleukin-5 (IL-5) mRNA and IL-13 mRNA was detected in five out of six P . orbiculare-reactive clones analysed for their cytokine gene expression with RT-PCR . CONCLUSION: Our data suggest that P . orbiculare can induce a T-cell response in AD patients . The Th2-like profile of P . orbiculare-reactive TCC derived from lesional skin indicates that P . orbiculare may play a role in maintaining IgE-mediated skin inflammation in AD.

Plant Cell, 1996 Nov, 8(11), 2105 - 15
MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA; Meier I et al.; The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression . Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato . In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast . DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content . Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein . MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase . Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope.

Plant Cell, 1996 Nov, 8(11), 2057 - 66
Enhancement of somatic intrachromosomal homologous recombination in Arabidopsis by the HO endonuclease; Chiurazzi M et al.; The HO endonuclease promotes gene conversion between mating-type alleles in yeast by a DNA double-strand break at the site of conversion (the MAT-Y/Z site) . As a first step toward understanding the molecular basis of homologous recombination in higher plants, we demonstrate that expression of HO in Arabidopsis enhances intrachromosomal recombination between inverted repeats of two defective beta-glucuronidase (gus) genes (GUS- test construct) . One of these genes has the Y/Z site . The two genes share 2.5 kb of DNA sequence homology around the HO cut site . Somatic recombination between the two repeats was determined by using a histochemical assay of GUS activity . The frequency of Gus+ sectors in leaves of F1 plants from a cross between parents homozygous for the GUS- test construct and HO, respectively, was 10-fold higher than in F1 plants from a cross between the same plant containing the GUS- test construct and a wild-type parent . Polymerase chain reaction analysis showed restoration of the 5' end of the GUS gene in recombinant sectors . The induction of intrachromosomal gene conversion in Arabidopsis by HO reveals the general utility of site-specific DNA endonucleases in producing targeted homologous recombination in plant genomes.

Plant J, 1996 Nov, 10(5), 869 - 52
Alkali cation selectivity of the wheat root high-affinity potassium transporter HKT1; Gassman W et al.; The wheat root high-affinity K+ transporter HKT1 functions as a sodium-coupled potassium co-uptake transporter . At toxic millimolar levels of sodium (Na+), HKT1 mediates low-affinity Na+ uptake while potassium (K+) uptake is blocked . In roots, low-affinity Na+ uptake and inhibition of K+ uptake contribute to Na+ toxicity . In the present study, the selectivity among alkali cations of HKT1 expressed in Xenopus oocytes and yeast was investigated under various ionic conditions at steady state . The data show that HKT1 is highly selective for uptake of the two physiologically significant alkali cations, K+ and Na+ over Rb+, Cs+ and Li+ . In addition, Rb+ and Cs+, and an excess of extracellular K+ over Na+, are shown to partially reduce or block HKT1-mediated K(+)-Na+ uptake . Furthermore, K+, Rb+ and Cs+ also effectively reduce outward currents mediated by HKT1, thereby causing depolarizations . In yeast, HKT1 can produce high-affinity Rb+ uptake at approximately 15-fold lower rates than for K+ . Rb+ influx in yeast can be mediated by the ability of the yeast plasma membrane proton pump to balance the >or= 35-fold lower HKT1 conductance for Rb+ . A model for HKT1 activity is presented involving a high-affinity K+ binding site and a high-affinity Na+ binding site, and competitive interactions of K+, Na+ and other alkali cations for binding to these two sites . Possible implications of the presented results for physiological K+ and Na+ uptake in plants are discussed.

Inflamm Res, 1996 Nov, 45(11), 531 - 40
Predictability of the clinical potency of NSAIDs from the preclinical pharmacodynamics in rats; Mukherjee A et al.; OBJECTIVE AND DESIGN: Relevance of the preclinical pharmacodynamic, toxicity and pharmacokinetic parameters predicting the clinical potency of nonsteroidal antiinflammatory drugs (NSAIDs) was evaluated . MATERIAL: Data for oral potencies of 24 NSAIDs in rats were collected from the literature and from New Drug Applications with respect to the following parameters: antiinflammatory, analgesic, antipyretic, acute ulcerogenic activities, acute toxicity, in vitro inhibition of prostaglandin synthesis, acid dissociation constant (pKa), octanol-water partition coefficient and elimination half-life . TREATMENT: Data for most of the in vivo parameters in rats were collected following single dose administration with the exception of adjuvant arthritis . Single and daily clinical doses were considered . All of these NSAIDs have been approved for marketing although not all have been sold in the USA . METHODS: The preclinical data were compared to human dose (unit or daily doses) using single and multiple stepwise regression analyses . RESULTS: Analyses suggest that NSAIDs are effective in all models of preclinical tests for fever, pain and inflammation, however, carrageenin-induced rat paw edema model is clearly the best predictor of human dose . Rank order of preclinical models for predicting human dose is carrageenin > yeast induced fever > pressure induced pain = adjuvant arthritis in rats . The analysis suggested that the pain and adjuvant arthritis models in rats may also involve a prostaglandin independent mechanism . Of the two physicochemical factors tested, pKa contributed best to the carrageenin model towards predicting the clinical potency of NSAIDs . Mathematical relationships between human dose, carrageenin ED50 and pKa were established that may assist in the future clinical development of NSAIDs . CONCLUSIONS: Carrageenin-induced paw edema model in rats is the most robust predictor of the clinical potency of NSAIDs . Acid dissociation constant (pKa) appears to be a secondary contributor to the potency of NSAIDs . The relevance of the data analyses for developing cyclooxygenase-2 (COX-2) selective NSAIDs is discussed.

Genes Chromosomes Cancer, 1996 Nov, 17(3), 156 - 65
Additional copies of a 25 Mb chromosomal region originating from 17q23.1-17qter are present in 90% of high-grade neuroblastomas; Meddeb M et al.; Neuroblastoma shows remarkable heterogeneity, ranging from spontaneous regression to progression toward highly malignant tumors . In search of genetic abnormalities that could explain this variability, we have characterized neuroblastoma tumors by using multiple fluorescent hybridizations . Our results indicate that chromosome 17 is rearranged very frequently in the form of unbalanced translocations with numerous chromosomal partners, all leading to the presence of supernumerary copies of a 25 Mb chromosomal region originating from 17q23.1-qter . Additional 17q material was detected in more than 90% of untreated high-grade neuroblastomas and, along with 1p36 deletion, should represent the most frequent genetic abnormality of neuroblastoma observed until now.

Genes Chromosomes Cancer, 1996 Nov, 17(3), 151 - 5
Localization of a novel t(1;7) translocation associated with Wilms' tumor predisposition and skeletal abnormalities; Reynolds PA et al.; Cytogenetic analysis of predisposition syndromes has played a critical role in the elucidation of the genetics of Wilms' tumor (WT) . Therefore, we became interested in a patient who presented with a WT and a nephrogenic rest in the contralateral kidney (suggestive of a predisposition) and a de novo t(1;7)(q42;p15) constitutional translocation as the only visible cytogenetic abnormality . He also had bilateral radial aplasia and other skeletal abnormalities, but there was no manifestation of any syndrome previously associated with WT . In the tumor, the translocation was retained, and the other 7p region was lost by the formation of an isochromosome i(7q) . Here, we report the localization of the chromosome 7 breakpoint within a yeast artificial chromosome (YAC) contig by using fluorescence in situ hybridization (FISH), localizing the breakpoint between markers sWSS355 and sWSS1449 . A number of YACs span the breakpoint and, thus, contain the region that is disrupted by the translocation . This may represent the site of a novel tumor suppressor gene that is involved in WT and also in normal renal development.

Eur J Biochem, 1996 Nov 1, 241(3), 779 - 86
Computational method to predict mitochondrially imported proteins and their targeting sequences; Claros MG et al.; Most of the proteins that are used in mitochondria are imported through the double membrane of the organelle . The information that guides the protein to mitochondria is contained in its sequence and structure, although no direct evidence can be obtained . In this article, discriminant analysis has been performed with 47 parameters and a large set of mitochondrial proteins extracted from the SwissProt database . A computational method that facilitates the analysis and objective prediction of mitochondrially imported proteins has been developed . If only the amino acid sequence is considered, 75-97% of the mitochondrial proteins studied have been predicted to be imported into mitochondria . Moreover, the existence of mitochondrial-targeting sequences is predicted in 76-94% of the analyzed mitochondrial precursor proteins . As a practical application, the number of unknown yeast open reading frames that might be mitochondrial proteins has been predicted, which revealed that many of them are clustered.

Genomics, 1996 Nov 1, 37(3), 381 - 5
Chromosomal localisation of the human envoplakin gene (EVPL) to the region of the tylosis oesophageal cancer gene (TOCG) on 17q25; Ruhrberg C et al.; Envoplakin is a membrane-associated precursor of the epidermal cornified envelope . Envoplakin is homologous to desmoplakin I and desmoplakin II (DPI/II), bullous pemphigoid antigen 1 (BPAG1), and plectin and is proposed to link desmosomes and keratin filaments to the cornified envelope . We describe the isolation of cosmids and yeast artificial chromosomes containing the complete human envoplakin gene (EVPL) and show, by analysis of somatic cell hybrids and chromosomal in situ hybridisation, that the envoplakin gene, unlike the genes encoding BPAG1 and DPI/II, maps to 17q25 and is physically linked to D17S1603 . This sequence-tagged site segregates with the autosomal dominant human disease focal nonepidermolytic palmoplantar keratosis (NEPKK; "tylosis"), which is associated with an increased risk of oesophageal cancer . The chromosomal localisation of the envoplakin gene, the homology of the encoded protein to keratin-binding proteins, and its expression in epidermal and oesophageal keratinocytes all raise the possibility that loss of envoplakin function could be responsible for this form of palmoplantar keratoderma.

Genomics, 1996 Nov 1, 37(3), 354 - 65
Definition of the minimal MEN1 candidate area based on a 5-Mb integrated map of proximal 11q13 . The European Consortium on Men1, (GENEM 1; Groupe d'Etude des Néoplasies Endocriniennes Multiples de type 1); Courseaux A et al.; Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder with a high penetrance characterized by tumors of the parathyroid glands, the endocrine pancreas, and the anterior pituitary . The MEN1 gene, a putative tumor suppressor gene, has been mapped to a 3- to 8-cM region in chromosome 11q13 but it remains elusive as yet . We have combined the efforts and resources from four laboratories to form the European Consortium on MEN1 with the aims of establishing the genetic and the physical maps of 11q13 and of further narrowing the MEN1 region . A 5-Mb integrated map of the region was established by fluorescence in situ hybridization on both metaphase chromosomes and DNA fibers, by hybridization to DNA from somatic cell hybrids containing various parts of human chromosome 11, by long-range restriction mapping, and by characterization of YACs and cosmids . Polymorphic markers were positioned and ordered by physical mapping and genetic linkage in 86 MEN1 families with 452 affected individuals . Two critical recombinants identified in two affected cases placed the MEN1 gene in an approximately 2-Mb region around PYGM, flanked by D11S1883 and D11S449.

Genomics, 1996 Nov 1, 37(3), 345 - 53
Chromosomal localization in mouse and human of the vasoactive intestinal peptide receptor type 2 gene: a possible contributor to the holoprosencephaly 3 phenotype; Mackay M et al.; The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) have been shown to act on a wide range of tissue and cell types, both in the central nervous system and in the periphery . Two distinct receptors for VIP, the VIP receptor type 1 (VIPR1) and the VIP receptor type 2 (VIPR2), have recently been cloned, each of which binds PACAP and VIP with equal affinity . We report here the chromosomal mapping of the human and mouse VIPR2 genes by fluorescence in situ hybridization . The VIPR2 gene maps to the human chromosomal region 7q36.3 and to the F2 region of mouse chromosome 12 . Our localization of the human gene places it in the region where the locus for the craniofacial defect holoprosencephaly type 3 (HPE3) maps . Further mapping experiments, carried out on cell lines derived from patients with HPE or HPE microforms and associated 7q deletions, have led us to redefine the distal extent of the HPE3 minimal critical region, originally characterized by Gurrieri et al . (1993, Nature Genet . 3: 247-251.) The VIPR2 gene lies within this new HPE3 minimal critical region . Our results suggest that deletion of the VIPR2 gene is not the sole factor responsible for the HPE3 phenotype . However, it is possible that monosomy at the VIPR2 locus may contribute to the phenotype observed in many cases of HPE3.

Genomics, 1996 Nov 1, 37(3), 316 - 26
Systematic sequencing of the human HLA-A/HLA-F region: establishment of a cosmid contig and identification of a new gene cluster within 37 kb of sequence; Lepourcelet M et al.; The class I region of the human histocompatibility complex is characterized by a high density of genes and pseudogenes and a complex structural organization . To elucidate the complete structure of the HLA-A/HLA-F region with a view to defining its contents in genes and pseudogenes, we developed a strategy of systematic sequencing . This report describes the establishment of a cosmid contig spanning most of the region and the analysis of a 37-kb sequence from one of the cosmids . Four new genes, organized with the HCG-V gene in a clustered structure, have been identified . Two of these contain a zinc finger motif characteristic of DNA-binding proteins . The former, a member of the C3HC4 protein family, is highly expressed in prostate and contains a B30-2-like sequence identified in several genes mapped within the class I region . The latter, which is ubiquitously expressed, is the human equivalent of the yeast polymerase IA12.2 subunit and of the murine tctex6 gene . Of the two other genes, one remains an anonymous gene with no particular feature, while the fourth, specifically expressed in testis, is the human equivalent of the murine tctex4 gene . This cluster, located in a region corresponding to a syntenic unit between mouse and human, appears to be highly conserved.

Genomics, 1996 Nov 1, 37(3), 295 - 302
Genetic analysis of the epidermal differentiation complex (EDC) on human chromosome 1q21: chromosomal orientation, new markers, and a 6-Mb YAC contig; Marenholz I et al.; The epidermal differentiation complex (EDC) unites a remarkable number of structurally, functionally, and evolutionarily related genes that play an important role in terminal differentiation of the human epidermis . It is localized within 2.05 Mb of region q21 on human chromosome 1 . We have identified and characterized 24 yeast artificial chromosome (YAC) clones by mapping individual EDC genes, sequence-tagged site (STS) markers (D1S305, D1S442, D1S498, D1S1664), and 10 new region-specific probes (D1S3619-D1S3628) . Here we present a contig that covers about 6 Mb of 1q21 including the entire EDC . Fluorescence in situ hybridization on metaphase chromosomes with two YACs flanking the EDC determined its chromosomal orientation and established, in conjunction with physical mapping results, the following order of genes and STSs: 1cen-D1S442-D1S498-S100A10-THH-FLG- D1S1664-IVL-SPRR3-SPRR1-SPRR2-LOR- S100A9-S100A8-S100A7-S100A6-S100A5-S100 A4- S100A3-S100A2-S100A1-D1S305-1qtel . These integrated physical, cytogenetic, and genetic mapping data will be useful for linkage analyses of diseases associated with region 1q21 and for the identification of novel genes and regulatory elements in the EDC.

Genome Res, 1996 Nov, 6(11), 1093 - 102
Mapping the RP2 locus for X-linked retinitis pigmentosa on proximal Xp: a genetically defined 5-cM critical region and exclusion of candidate genes by physical mapping; Thiselton DL et al.; Genetic linkage studies have implicated at least two loci for X-linked retinitis pigmentosa (XLRP) on proximal Xp . We now report a defined genetic localization for the RP2 locus to a 5-cM interval in Xp11.3-11.23 . Haplotype analysis of polymorphic markers in recombinant individuals from two XLRP families has enabled us to identify DXS8083 and DXS6616 as the new distal and proximal flanking markers for RP2 . Using STS-content and YAC end-clone mapping, an approximately 1.2 Mb YAC contig has been established encompassing the proximal RP2 boundary and extending from T1MP1 to DXS1240 in Xp11.23 . Several ESTs have been positioned and ordered on this contig, one of which is novel to the region, identified by sequence data-base match to a physically mapped YAC insert terminal STS . Integration of the genetic and physical data has placed four retinally expressed genes proximal to DXS6616, and thereby excluded them from a causitive role in RP2 . This work now provides a much needed focus for positional cloning approaches to isolation of the defective gene.

Genome Res, 1996 Nov, 6(11), 1056 - 69
Long-range map of a 3.5-Mb region in Xp11.23-22 with a sequence-ready map from a 1.1-Mb gene-rich interval; Schindelhauer D et al.; Most of the yeast artificial chromosomes (YACs) isolated from the Xp11.23-22 region have shown instability and chimerism and are not a reliable resource for determining physical distances . We therefore constructed a long-range pulsed-field gel electrophoresis map that encompasses approximately 3.5 Mb of genomic DNA between the loci TIMP and DXS146 including a CpG-rich region around the WASP and TFE-3 gene loci . A combined YAC-cosmid contig was constructed along the genomic map and was used for fine-mapping of 15 polymorphic microsatellites and 30 expressed sequence tags (ESTs) or sequence transcribed sites (STSs), revealing the following order: tel-(SYN-TIMP)-(DXS426-ELK1)-ZNF(CA) n-L1-DXS1367-ZNF81-ZNF21-DXS6616- (HB3-OATL1pseudogenes-DXS6950)-DXS6949-DXS694 1-DXS7464E(MG61)-GW1E(EBP)- DXS7927E(MG81)-RBM- DXS722-DXS7467E(MG21)-DXS1011E-WASP-DXS6940++ +-DXS7466E(MG44)-GF1- DXS226-DXS1126-DXS1240-HB1- DXS7469E-(DXS6665-DXS1470)-TFE3-DXS7468E-+ ++SYP-DXS1208-HB2E-DXS573-DXS1331- DXS6666-DXS1039-DXS 1426-DXS1416-DXS7647-DXS8222-DXS6850-DXS255++ +-CIC-5-DXS146-cen . A sequence-ready map was constructed for an 1100-kb gene-rich interval flanked by the markers HB3 and DXS1039, from which six novel ESTs/STSs were isolated, thus increasing the number of markers used in this interval to thirty . This precise ordering is a prerequisite for the construction of a transcription map of this region that contains numerous disease loci, including those for several forms of retinal degeneration and mental retardation . In addition, the map provides the base to delineate the corresponding syntenic region in the mouse, where the mutants scurfy and tattered are localized.

Plant Physiol, 1996 Nov, 112(3), 1229 - 36
Sugar regulates mRNA abundance of H(+)-ATPase gene family members in tomato; Mito N et al.; The plant plasma membrane H(+)-ATPase energizes the secondary uptake of nutrients and may facilitate cell expansion by acidifying the cell wall . In yeast, Glc stimulates the accumulation of H(+)-ATPase mRNA, and the growth rate supported by various sugars is correlated with H(+)-ATPase protein abundance . Expression of three H(+)-ATPase genes, LHA1, LHA2, and LHA4, was previously detected in tomato (Lycopersicon esculentum) . We have characterized the sequence of the LHA4 gene and examined the expression of these three tomato H(+)-ATPase genes in growing tissues and in response to exogenous sugars . LHA4 is a member of the H(+)-ATPase subfamily, including the Arabidopsis thaliana genes AHA1, AHA2, and AHA3 . The 5' untranslated region of the deduced LHA4 cDNA contains a short, open reading frame very similar to that in the Nicotiana plumbaginifolia gene PMA1 . LHA4 transcript abundance in seedlings is correlated with cell growth, being 2.5 times greater in hypocotyls of dark- versus light-grown plants . The accumulation of both LHA4 and LHA2 mRNAs is induced by the addition of exogenous sugars and this induction appears to be dependent on sugar uptake and metabolism, because mannitol and 3-O-methylglucose do not stimulate mRNA accumulation . These results suggest that the induction of expression of H(+)-ATPase genes by metabolizable sugars may be part of a generalized cellular response to increased cell growth and metabolism promoted by the availability of an abundant carbon source.

Neuron, 1996 Nov, 17(5), 921 - 9
The timSL mutant of the Drosophila rhythm gene timeless manifests allele-specific interactions with period gene mutants; Rutila JE et al.; To identify new components of the Drosophila circadian clock, we screened chemically mutagenized flies for suppressors or enhancers of the long periods characteristic of the period (per) mutant allele perL . We isolated a novel mutant that maps to the rhythm gene timeless (tim) . This novel allele, timSL, alters the temporal pattern of perL protein nuclear localization and restores temperature compensation to perL flies . timSL more generally manifests specific interactions with different per alleles . The identification of this first period-altering tim allele provides further evidence that TIM is a major component of the clock, and the allele-specific interactions with PER provide evidence that the PER/TIM heterodimer is a unit of circadian function . Although timSL fails to restore PER-L/TIM temperature insensitivity in yeast, it alters the TIM phosphorylation pattern during the late night . The effects on phosphorylation suggest that timSL functions as a partial bypass suppressor of perL and provide evidence that the TIM phosphorylation program contributes to the circadian timekeeping mechanism.

J Cell Sci, 1996 Nov, 109 ( Pt 11), 2715 - 26
Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site; Hemmings L et al.; We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein . Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent . By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541 . The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p . Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin . The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin . Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions . The former was readily solubilised, and the latter was resistant to Triton extraction . The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect . However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions . The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.

Mol Biol Cell, 1996 Nov, 7(11), 1759 - 69
Novel members of the cdc2-related kinase family in Drosophila: cdk4/6, cdk5, PFTAIRE, and PITSLRE kinase; Sauer K et al.; In addition to the previously identified Drosophila cdc2 and cdc2c genes, we have identified four additional cdc2-related genes with low stringency and polymerase chain reaction approaches . Sequence comparisons suggest that the four putative kinases represent the Drosophila homologues of vertebrate cdk4/6, cdk5, PCTAIRE, and PITSLRE kinases . Although the similarity between human and Drosophila homologues is extensive in the case of cdk5, PCTAIRE, and PITSLRE kinases (78%, 58%, and 65% identity in the kinase domain), only limited conservation is observed for Drosophila cdk4/6 (47% identity) . However, like vertebrate cdk4 and cdk6, Drosophila cdk4/6 binds also to a D-type cyclin according to the results of two-hybrid experiments in yeast . Northern blot analysis indicated that the four Drosophila kinases are expressed throughout embryogenesis . Expression in early embryogenesis appeared to be ubiquitous according to in situ hybridization . Abundant expression already at the start of embryogenesis and long before neuron differentiation was also observed in the case of cdk5 protein, which has been described as predominantly neuron specific in mice . Sequence conservation and expression pattern, therefore, suggest that all of these kinases perform important cellular functions.

J Anim Sci, 1996 Nov, 74(11), 2711 - 8
Effect of inorganic or organic selenium at two dietary levels on reproductive performance and tissue selenium concentrations in first-parity gilts and their progeny; Mahan DC et al.; A 2 x 2 factorial arrangement of treatments in a randomized complete block design was conducted at two time periods using a total of 43 first-parity gilts . Two sources of Se (selenite or Se-enriched yeast) were added at .1 or .3 ppm to corn-soybean meal diets to evaluate reproductive performance and gilt and progeny tissue Se contents . Treatment diets were initially provided approximately 60 d before breeding . Gilts were bled at periodic intervals and serum glutathione peroxidase (GSH-Px) activity and Se concentrations were determined . Milk was collected at parturition and at weekly intervals to weaning (21 d) for Se analysis . Liver and loin tissues were collected from stillborn (n = 17) and neonatal pigs (n = 19) before colostrum consumption . Three pigs from each litter were bled at weaning, and six pigs per treatment group were killed (two/litter) and tissue (liver, loin, kidney) collected . Three to four sows per treatment were killed at weaning and tissue (loin, liver, pancreas, kidney) collected . Tissues collected from each were analyzed for Se . Dietary Se level or Se source had no effect (P > .15) on gilt reproductive performance . Gilt serum GSH-Px activity was generally similar at the .1 and .3 ppm Se level for either Se source, whereas serum Se was consistently higher when the dietary Se level was .3 ppm . Colostrum Se content was unaffected by Se source and Se level, but milk Se increased as the dietary Se level increased and when the Se-enriched yeast source was fed, resulting in an interaction response (P < .01) . Loin tissue had similar Se contents between stillborn and neonatal pigs . Loin Se content was higher when dietary Se level increased (P < .05) and when the Se-enriched yeast source (P < .01) was fed to gestating gilts . Weanling pig lion Se content increased as dietary Se level increased (P < .01) and when the Se-enriched yeast source was fed (P < .01) . A higher liver Se content in weaned pigs also resulted when the dietary Se level was .3 ppm (P < .08) and when the Se-yeast (P < .01) was provided . Weanling pig serum GSH-Px activity was similar regardless of the Se level or Se source fed to the dam, but serum Se increased when the .3 ppm Se level and the Se-yeast was fed to the gilt . If GSH-Px activity is used as the criterion to evaluate Se adequacy, then .1 ppm Se from either Se source was adequate, but if higher milk Se or pig tissue content is desired, then a .3 ppm Se level from the Se-enriched yeast source was superior to inorganic Se.

Eur J Immunol, 1996 Nov, 26(11), 2595 - 600
Dendritic cells process exogenous viral proteins and virus-like particles for class I presentation to CD8+ cytotoxic T lymphocytes; Bachmann MF et al.; Previous reports have indicated that both dendritic cells and macrophages have the ability to induce cytotoxic T lymphocyte (CTL) and T helper (Th) cell responses in vivo . Dendritic cells process exogenous antigens conventionally for presentation on major histocompatibility complex (MHC) class II molecules . However, unconventional processing of exogenous antigens in vitro for presentation on MHC class I molecules is still an open question . In this study, we report that a cloned dendritic cell line (D2SC/1) is able to present cell debris-associated exogenous viral proteins to MHC class I-restricted CTL in vitro . The dendritic cell line was very efficient in processing recombinant lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) and presenting the class I-restricted epitope to CTL primed in vivo . Peritoneal macrophages could also process the recombinant LCMV NP for subsequent MHC class I presentation, but were less efficient compared to the dendritic cells . Furthermore, recombinant yeast-derived virus-like particles carrying the HIV-1 V3 loop (V3-VLP), which are protenaceous and do not contain any lipid, were also found to be efficiently processed by the dendritic cell line for presentation of the class I-restricted epitope . These results clearly indicate that viral proteins, in particulate form or associated with cell debris, are processed by dendritic cells for CTL induction.

Am J Vet Res, 1996 Nov, 57(11), 1556 - 8
Evaluation of recombinant bluetongue virus antigens, using dot immunobinding assay with serum from sheep and cattle; Naresh A et al.; OBJECTIVE: To evaluate 2 genetically engineered group-specific antigens: baculovirus- and yeast-expressed VP7 and the conventionally produced group-specific antigen of bluetongue virus (BTV), using dot immunobinding assay (DIA) . SAMPLE POPULATION AND PROCEDURE: A total of 260 serum samples of sheep and cattle from various livestock farms in different Indian states (eg, Haryana, Himachal Pradesh, Jammu and Kashmir, Punjab, and Rajasthan) were tested for the presence of BTV antibodies by DIA . RESULTS: Of 260 sera tested, 92 (35%) were positive for BTV antibodies using the baculovirus-expressed antigen, 96 (37%) were positive using the yeast-expressed antigen, and 103 (40%) were positive by use of conventionally produced antigen . CONCLUSION AND CLINICAL RELEVANCE: The overall agreement for all the 3 antigens was 92%, indicating that the recombinant group-specific proteins can be used for serodiagnosis of BTV, using DIA.

Genetics, 1996 Nov, 144(3), 1087 - 95
Subunit interactions in the mariner transposase; Lohe AR et al.; We have studied the Mos1 transposase encoded by the transposable element mariner . This-transposase is a member of the "D,D(35)E" superfamily of proteins exhibiting the motif D,D(34)D . It is not known whether this transposase, or other eukaryote transposases manifesting the D,D(35)E domain, functions in a multimeric form . Evidence for oligomerization was found in the negative complementation of Mos1 by an EMS-induced transposase mutation in the catalytic domain . The transposase produced by this mutation has a glycine-to-arginine replacement at position 292 . The G292R mutation strongly interferes with the ability of wild-type transposase to catalyze excision of a target element . Negative complementation was also observed for two other EMS mutations, although the effect was weaker than observed with G292R . Results from the yeast two-hybrid system also imply that Mos1 subunits interact, suggesting the possibility of subunit oligomerization in the transposition reaction . Overproduction of Mos1 subunits through an hsp70 promoter also inhibits excision of the target element, possibly through autoregulatory feedback on transcription or through formation of inactive or less active oligomers . The effects of both negative complementation and overproduction may contribute to the regulation of mariner transposition.

Mol Pharmacol, 1996 Nov, 50(5), 1157 - 66
Rat 17 beta-hydroxysteroid dehydrogenase type IV is a novel peroxisome proliferator-inducible gene; Corton JC et al.; To better understand the molecular mechanisms of the pleiotropic responses induced by exposure to peroxisome proliferator chemicals (PPCs), we conducted a systematic search for genes whose mRNA levels are modulated by the PPC WY-14,643 (WY) in rat liver . The sequence of one up-regulated cDNA (2480 bp) was predicted to encode a protein of 735 aa with 82% identity to the porcine 17 beta-hydroxysteroid dehydrogenase type IV (HSD IV) . Like the porcine enzyme, the rat HSD IV contains' a region homologous to yeast hydratase-dehydrogenase-epimerases and to sterol carrier proteins, indicating that the rat HSD IV has broad substrate specificity and contributes to cholesterol metabolism . The rat HSD IV was regulated by diverse PPCs via two distinct mechanisms . Induction of HSD IV and acyl-CoA oxidase (ACO) proteins in rat liver at different treatment times and concentrations of gemfibrozil and di-n-butyl phthalate were almost identical, indicating that HSD IV mRNA induction involves the peroxisome proliferator-activated receptor alpha, a regulator of ACO . In contrast, HSD IV protein levels were only weakly induced by WY, a strong inducer of ACO protein, even though the levels of HSD IV and ACO mRNA were strongly stimulated by WY and gemfibrozil . Thus, HSD IV protein levels were uniquely regulated pretranslationally by WY via a novel mechanism . Increased conversion of estradiol to the less-active estrone by HSD IV induction may explain how phthalate exposure leads to decreases in serum estradiol levels and suppression of ovulation.

J Biol Chem, 1996 Nov 1, 271(44), 27802 - 9
A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes; Sun J et al.; Using a polymerase chain reaction strategy we identified a serine proteinase inhibitor (serpin) in human bone marrow that is related to the cellular serpin proteinase inhibitor 6 (PI-6) and the viral serpin cytokine response modifier A (CrmA) . This serpin, proteinase inhibitor 9 (PI-9), has an unusual reactive center P1(Glu)-P1'(Cys), which suggests that it inhibits serine proteinases that cleave after acidic residues . The only known serine proteinase with this specificity is granzyme B, a granule cytotoxin produced by cytotoxic lymphocytes . To test the interaction of PI-9 with granzyme B we prepared recombinant hexa-histidine tagged PI-9 in a yeast expression system . Addition of the recombinant protein to native granzyme B resulted in an SDS-resistant complex typical of serpin-serine proteinase interactions . Further analysis showed that complex formation followed bimolecular kinetics with a second order rate constant of 1.7 +/- 0.3 x 10(6) M-1 s-1, which is in the range for a physiologically significant serpin-proteinase interaction . Recombinant PI-9 also completely abrogated granzyme B and perforin-mediated cytotoxicity in vitro . Examination of PI-9 mRNA distribution demonstrated that it is expressed in immune tissue, primarily in lymphocytes . The highest levels of PI-9 mRNA and protein were observed in natural killer cell leukemia cell lines and in interleukin-2 stimulated peripheral blood mononuclear cells, which also produce granzyme B . Like PI-6, PI-9 was shown to be a cytosolic protein that is not secreted . Fractionation of natural killer cells and stimulated peripheral blood mononuclear cells demonstrated that PI-9 is in a separate subcellular compartment to granzyme B . These results suggest that PI-9 serves to inactivate misdirected granzyme B following cytotoxic cell degranulation . This may explain why cytotoxic cells are not damaged by their own granzyme B during destruction of abnormal cells.

J Biol Chem, 1996 Nov 1, 271(44), 27209 - 12
The GTPase-activating protein RGS4 stabilizes the transition state for nucleotide hydrolysis; Berman DM et al.; RGS proteins constitute a newly appreciated group of negative regulators of G protein signaling . Discovered by genetic screens in yeast, worms, and other organisms, two mammalian RGS proteins, RGS4 and GAIP, act as GTPase-activating proteins for members of the Gi family of G protein alpha subunits . We have purified recombinant RGS4 to homogeneity and demonstrate that it acts catalytically to stimulate GTP hydrolysis by Gi proteins . Furthermore, RGS4 stabilizes the transition state for GTP hydrolysis, as evidenced by its high affinity for the GDP-AlF4--bound forms of Goalpha and Gialpha and its relatively low affinity for the GTPgammaS- and GDP-bound forms of these proteins . Consequently, RGS4 is most likely not a downstream effector for activated Galpha subunits . All members of the Gi subfamily of proteins tested are substrates for RGS4 (including Gtalpha and Gzalpha); the protein has lower affinity for Gqalpha, and it does not stimulate the GTPase activity of Gsalpha or G12alpha.

Am J Pathol, 1996 Nov, 149(5), 1565 - 73
Fluorescence in situ hybridization evaluation of chromosome deletion patterns in prostate cancer; Huang SF et al.; Various nonrandom chromosomal aberrations have been identified in prostate carcinoma . These aberrations include deletions of several chromosome regions, particularly the chromosome 8 short arm . Large-scale numerical aberrations, reflected in aberrant DNA ploidy, are also found in a minority of cases . However, it is unclear whether prostate carcinomas contain aberrations of certain chromosome regions that are deleted frequently in other common types of cancer . In this study, we performed dual-color fluorescence in situ hybridization on intact nuclei from touch preparations of 16 prostate cancers . Chromosome copy number was determined using pericentromeric probes, whereas potential chromosome arm deletions were evaluated using yeast artificial chromosome (YAC) and P1 probes . Two YAC probes targeted chromosome 8 short arm regions known to be deleted frequently in prostate cancer . Other YACs and P1s were for chromosome regions, including 1p22, 3p14, 6q21, 9p21, and 22q12, that are deletion targets in a variety of cancers although not extensively studied in prostate cancer . Hybridization efficiencies and signal intensities were excellent for both repeat sequence (alpha-satellite) and single, copy (YAC and P1) fluorescence in situ hybridization probes . Of 16 prostate cancers, 11 had clonal aberrations of 1 or more of the 13 chromosome regions evaluated, and 10 cases (62.5%) had 8p deletions, including 4 cases with 8p deletion in virtually all cells and aneuploidy in only a subset of those deleted cells . Deletions at 3p14, 6q21, and 22q12 were identified in 2, 1, and 1 case, respectively, and each of those cases had a similarly sized cell population with 8p deletion . These studies confirm 8p deletion in the majority of prostate carcinomas . 8p deletions appear to be early events in prostate tumorigenesis, often antedating aneuploidy . Fluorescence in situ hybridization strategies incorporating pericentromeric and single-copy regional chromosome probes offer a powerful and efficient means for determining frequency and progression of oncogenetic events in prostate cancer.

Am J Hum Genet, 1996 Nov, 59(5), 999 - 1005
Tourette syndrome in a pedigree with a 7;18 translocation: identification of a YAC spanning the translocation breakpoint at 18q22.3; Boghosian-Sell L et al.; Tourette syndrome is a neuropsychiatric disorder characterized by the presence of multiple, involuntary motor and vocal tics . Associated pathologies include attention deficit disorder and obsessive-compulsive disorder (OCD) . Extensive linkage analysis based on an autosomal dominant mode of transmission with reduced penetrance has failed to show linkage with polymorphic markers, suggesting either locus heterogeneity or a polygenic origin for Tourette syndrome . An individual diagnosed with Tourette syndrome has been described carrying a constitutional (7;18) chromosome translocation (Comings et al . 1986) . Other family members carrying the translocation exhibit features seen in Tourette syndrome including motor tics, vocal tics, and OCD . Since the disruption of specific genes by a chromosomal rearrangement can elicit a particular phenotype, we have undertaken the physical mapping of the 7;18 translocation such that genes mapping at the site of the breakpoint can be identified and evaluated for a possible involvement in Tourette syndrome . Using somatic cell hybrids retaining either the der(7) or the der(18), a more precise localization of the breakpoints on chromosomes 7 and 18 have been determined . Furthermore, physical mapping has identified two YAC clones that span the translocation breakpoint on chromosome 18 as determined by FISH . These YAC clones will be useful for the eventual identification of genes that map to chromosomes 7 and 18 at the site of the translocation.

Cell, 1996 Nov 1, 87(3), 459 - 70
A specialized nucleosome modulates transcription factor access to a C . glabrata metal responsive promoter; Zhu Z et al.; The ability of DNA binding transcription factors to access cis-acting promoter elements is critical for transcriptional responses . We demonstrate that rapid transcriptional autoactivation by the Amt1 Cu metalloregulatory transcription factor from the opportunistic pathogenic yeast Candida glabrata is dependent on rapid metal-induced DNA binding to a single metal response element (MRE) . In vivo footprinting and chromatin-mapping experiments demonstrate that the MRE and a homopolymeric (dA x dT) element adjacent to the MRE are packaged into a positioned nucleosome that exhibits homopolymeric (dA x dT)-dependent localized distortion . This distortion is critical for rapid Amt1 binding to the MRE, for Cu-dependent AMT1 gene transcription, and for C . glabrata cells to mount a rapid transcriptional response to Cu for normal metal detoxification . The AMT1 promoter represents a novel class of specialized nucleosomal structures that links rapid transcriptional responses to the biology of metal homeostasis.

Nat Genet, 1996 Nov, 14(3), 307 - 11
Identification and mutation analysis of the complete gene for Chediak-Higashi syndrome; Nagle DL et al.; Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder characterized by hypopigmentation, severe immunologic deficiency with neutropenia and lack of natural killer (NK) cells, a bleeding tendency and neurologic abnormalities . Most patients die in childhood . The CHS hallmark is the occurrence of giant inclusion bodies and organelles in a variety of cell types, and protein sorting defects into these organelles . Similar abnormalities occur in the beige mouse, the proposed model for human CHS . Two groups have recently reported the identification of the beige gene, however the two cDNAs were not at all similar . Here we describe the sequence of a human cDNA homologous to mouse beige, identify pathologic mutations and clarify the discrepancies of the previous reports . Analysis of the CHS polypeptide demonstrates that its modular architecture is similar to the yeast vacuolar sorting protein, VPS15.

Leukemia, 1996 Nov, 10(11), 1844 - 6
An interstitial 11q23 deletion proven to be a rearrangement interrupting the MLL gene in an infant with acute myeloblastic leukemia; Leblanc T et al.; Chromosome studies of an infant with acute myeloblastic leukemia (AML), classified as M2 in the FAB nomenclature revealed an unusual karyotype with del(11)(q23) and a marker chromosome resembling a small chromosomal fragment present in all metaphase cells examined . Fluorescence in situ hybridization (FISH) showed the splitting of a YAC probe containing a part of MLL between the del(11) and mar chromosomes . Painting showed that the mar chromosome contained DNA sequences from chromosome 11, but that the centromeric region was not marked by a chromosome 11-specific alphoid probe . The chromosomal breakpoint was located within the MLL gene by Southern blot experiments . The deletion of 11q was thus interstitial . This case illustrates the importance of associating cytogenetics, several FISH techniques, and molecular studies to analyze unusual karyotypes in leukemia.

Mol Cell Biol, 1996 Nov, 16(11), 6132 - 40
TC21 causes transformation by Raf-independent signaling pathways; Graham SM et al.; Although the Ras-related protein TC21/R-Ras2 has only 55% amino acid identity with Ras proteins, mutated forms of TC21 exhibit the same potent transforming activity as constitutively activated forms of Ras . Therefore, like Ras, TC21 may activate signaling pathways that control normal cell growth and differentiation . To address this possibility, we determined if regulators and effectors of Ras are also important for controlling TC21 activity . First, we determined that Ras guanine nucleotide exchange factors (SOS1 and RasGRF/CDC25) synergistically enhanced wild-type TC21 activity in vivo and that Ras GTPase-activating proteins (GAPs; p120-GAP and NF1-GAP) stimulated wild-type TC21 GTP hydrolysis in vitro . Thus, extracellular signals that activate Ras via SOS1 activation may cause coordinate activation of Ras and TC21 . Second, we determined if Raf kinases were effectors for TC21 transformation . Unexpectedly, yeast two-hybrid binding analyses showed that although both Ras and TC21 could interact with the isolated Ras-binding domain of Raf-1, only Ras interacted with full-length Raf-1, A-Raf, or B-Raf . Consistent with this observation, we found that Ras- but not TC21-transformed NIH 3T3 cells possessed constitutively elevated Raf-1 and B-Raf kinase activity . Thus, Raf kinases are effectors for Ras, but not TC21, signaling and transformation . We conclude that common upstream signals cause activation of Ras and TC21, but activated TC21 controls cell growth via distinct Raf-independent downstream signaling pathways.

Mamm Genome, 1996 Nov, 7(11), 835 - 42
Degeneracy in human multicopy RBM (YRRM), a candidate spermatogenesis gene; Prosser J et al.; In order to search for mutations in the multicopy RBM genes that might be associated with male infertility, we have used sequence data from the reported cDNA clone to determine the intron exon boundaries of the YRRM 1 gene . This gene has 12 exons, three of which encode the putative RNA binding domain of the protein . Different copies of the gene contain sequence variations and, additionally, give rise to transcripts with different numbers of copies of the repeated SRGY motif . Since mutations in the RNA binding domain would seem likely to have an effect on the activity of the protein, we have scanned these exons for mutations by SSCP on DNA from normal and infertile men . Sequence differences in the exon encoding the N-terminal part of the RNA binding domain account for at least four different classes of the gene and give rise to different SSCP conformers . Sequence analysis shows that one of these classes is a pseudogene and that the members of another class are nonfunctional . RT-PCR shows that all classes are transcribed and that the A class is most abundant . We have found a point mutation that alters the highly conserved RNP2 motif in one infertile patient . This mutation is also found in his father . We have used PCR followed by SSCP analysis to map RBM on a Y Chromosome (Chr) YAC contig and have demonstrated a distribution that spans a major part of this chromosome's euchromatin.

J Mol Evol, 1996 Nov, 43(5), 484 - 516
Classification and phylogeny of the MADS-box multigene family suggest defined roles of MADS-box gene subfamilies in the morphological evolution of eukaryotes; Theissen G et al.; The MADS-box encodes a novel type of DNA-binding domain found so far in a diverse group of transcription factors from yeast, animals, and seed plants . Here, our first aim was to evaluate the primary structure of the MADS-box . Compilation of the 107 currently available MADS-domain sequences resulted in a signature which can strictly discriminate between genes possessing or lacking a MADS-domain and allowed a classification of MADS-domain proteins into several distinct subfamilies . A comprehensive phylogenetic analysis of known eukaryotic MADS-box genes, which is the first comprising animal as well as fungal and plant homologs, showed that the vast majority of subfamily members appear on distinct subtrees of phylogenetic trees, suggesting that subfamilies represent monophyletic gene clades and providing the proposed classification scheme with a sound evolutionary basis . A reconstruction of the history of the MADS-box gene subfamilies based on the taxonomic distribution of contemporary subfamily members revealed that each subfamily comprises highly conserved putative orthologs and recent paralogs . Some subfamilies must be very old (1,000 MY or more), while others are more recent . In general, subfamily members tend to share highly similar sequences, expression patterns, and related functions . The defined species distribution, specific function, and strong evolutionary conservation of the members of most subfamilies suggest that the establishment of different subfamilies was followed by rapid fixation and was thus highly advantageous during eukaryotic evolution . These gene subfamilies may have been essential prerequisites for the establishment of several complex eukaryotic body structures, such as muscles in animals and certain reproductive structures in higher plants, and of some signal transduction pathways . Phylogenetic trees indicate that after establishment of different subfamilies, additional gene duplications led to a further increase in the number of MADS-box genes . However, several molecular mechanisms of MADS-box gene diversification were used to a quite different extent during animal and plant evolution . Known plant MADS-domain sequences diverged much faster than those of animals, and gene duplication and sequence diversification were extensively used for the creation of new genes during plant evolution, resulting in a relatively large number of interacting genes . In contrast, the available data on animal genes suggest that increase in gene number was only moderate in the lineage leading to mammals, but in the case of MEF2-like gene products, heterodimerization between different splice variants may have increased the combinatorial possibilities of interactions considerably . These observations demonstrate that in metazoan and plant evolution, increased combinatorial possibilities of MADS-box gene product interactions correlated with the evolution of increasingly complex body plans.

Science, 1996 Nov 1, 274(5288), 765 - 8
Nested retrotransposons in the intergenic regions of the maize genome; SanMiguel P et al.; The relative organization of genes and repetitive DNAs in complex eukaryotic genomes is not well understood . Diagnostic sequencing indicated that a 280-kilobase region containing the maize Adh1-F and u22 genes is composed primarily of retrotransposons inserted within each other . Ten retroelement families were discovered, with reiteration frequencies ranging from 10 to 30,000 copies per haploid genome . These retrotransposons accounted for more than 60 percent of the Adh1-F region and at least 50 percent of the nuclear DNA of maize . These elements were largely intact and are dispersed throughout the gene-containing regions of the maize genome.

J Neurosci, 1996 Nov 1, 16(21), 6695 - 702
A murine neural-specific homolog corrects cholinergic defects in Caenorhabditis elegans unc-18 mutants; Gengyo-Ando K et al.; Caenorhabditis elegans UNC-18 protein, homologous to yeast Sec1p, is important in neurotransmitter release, because the unc-18 mutation leads to severe paralysis and presynaptic acetylcholine (ACh) accumulation . To examine the functional conservation in mammals, we tried to isolate unc-18 isoforms from mouse and human brain cDNA libraries and obtained two classes of isoforms-neural genes and ubiquitous genes . Neural genes were identical to Munc-18 (also known as n-Sec1 or rbSec1), identified in rat and bovine brains as a syntaxin-binding protein . According to "Munc-18" terminology, we call the neural genes Munc-18-1 and the ubiquitous genes Munc-18-3 . These mammalian isoforms exhibit 58% (Munc-18-1) and 42-43% (Munc-18-3) amino acid sequence identity with UNC-18 . Next, we constructed transgenic unc-18 mutants to test biological activity of mouse Munc-18-1 and Munc-18-3 under the control of C . elegans unc-18 promoter . Munc-18-1 compensates for severe locomotion disability and cholinergic defects, e.g., abnormal sensitivities to cholinesterase inhibitors and cholinergic receptor agonists in unc-18 mutants, but Munc-18-3 fails . These data suggest that Munc-18-1 and C . elegans unc-18 may play positive roles in ACh release and that the molecular mechanism of neuronal regulated secretion has been partially conserved from nematodes to mammals.

DNA Res, 1996 Oct 31, 3(5), 297 - 302
Construction of YAC contigs on rice chromosome 5; Saji S et al.; A physical map of rice chromosome 5 was constructed with yeast artificial chromosome (YAC) clones along a high-resolution molecular linkage map carrying 118 DNA markers distributed over 123.7 cM of genomic DNA . YAC clones have been identified by colony and Southern hybridization for 105 restriction fragment length polymorphism (RFLP) markers and by polymerase chain reaction (PCR) screening for 8 sequence-tagged site (STS) markers and 5 randomly amplified polymorphic DNA (RAPD) markers . Of 458 YACs, 235 individual YACs with an average insert length of 350 kb were selected and ordered on chromosome 5 from the YAC library . Forty-eight contigs covering nearly 21 Mb were formed on the chromosome 5; the longest one was 6 cM and covered 1.5 Mb . The length covered with YAC clones corresponded to 62% of the total length, of chromosome 5 . There were many multicopy sequences of expressed genes on chromosome 5 . The distribution of many copies of these expressed gene sequences was determined by YAC Southern hybridization and is discussed . A physical map with these characteristics provides a powerful tool for elucidation of genome structure and extraction of useful genetic information in rice.

Carbohydr Res, 1996 Oct 31, 293(2), 195 - 204
Calystegine B4, a novel trehalase inhibitor from Scopolia japonica; Asano N et al.; GLC-MS analysis has been developed for screening plants of the family Solanaceae for new calystegines . GLC-MS analyses of the extract of Scopolia japonica showed the presence of a new tetrahydroxy-nor-tropane alkaloid in addition to the known calystegines A3, A5, B1, B2, B3, and C1 . We gave this new alkaloid the trivial name calystegine B4 . The structure of calystegine B4 was determined as 1 alpha, 2 beta, 3 alpha, 4 alpha-tetrahydroxy-nor-tropane from a variety of NMR spectral data . Calystegines B1, B2, and C1 are potent competitive inhibitors with Ki values ranging from 10(-6) to 10(-7) M for almond beta-glucosidase, while calystegine B4 inhibited this enzyme in a competitive manner, with a Ki value of 7.3 microM . Calystegine B2 is also a potent inhibitor of green coffee bean alpha-galactosidase, whereas calystegine B4 exhibited no significant activity for this enzyme . Among rat intestinal glycosidases, only trehalase was potently inhibited by calystegine B4, with an IC50 value of 9.8 microM . Furthermore, calystegine B4 potently inhibited pig kidney trehalase in a competitive manner, with a Ki value of 1.2 microM, but it was almost inactive against yeast and fungal trehalases.

Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12267 - 71
Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF; Perkins AC et al.; Globin genes are subject to tissue-specific and developmental stage-specific regulation . A switch from human fetal (gamma)-to adult (beta)-globin expression occurs within erythroid precursor cells of the adult lineage . Previously we and others showed by targeted gene disruption that the zinc finger gene, erythroid Kruppel-like factor (EKLF), is required for expression of the beta-globin gene in mice, presumably through interaction with a high-affinity binding site in the proximal promoter . To examine the role of EKLF in the developmental regulation of the human gamma-globin gene we interbred EKLF heterozygotes (+/-) with mice harboring a human beta-globin yeast artificial chromosome transgene . We find that in the absence of EKLF, while human beta-globin expression is dramatically reduced, gamma-globin transcripts are elevated approximately 5-fold . Impaired silencing of gamma-globin expression identifies EKLF as the first transcription factor participating quantitatively in the gamma-globin to beta-globin switch . Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the beta-globin gene promoter and the locus control region that excludes the gamma-globin gene.

Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12240 - 4
Mutations in copper-zinc superoxide dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein; Lyons TJ et al.; A series of mutant human and yeast copper-zinc superoxide dismutases has been prepared, with mutations corresponding to those found in familial amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease) . These proteins have been characterized with respect to their metal-binding characteristics and their redox reactivities . Replacement of Zn2+ ion in the zinc sites of several of these proteins with either Cu2+ or Co2+ gave metal-substituted derivatives with spectroscopic properties different from those of the analogous derivative of the wild-type proteins, indicating that the geometries of binding of these metal ions to the zinc site were affected by the mutations . Several of the ALS-associated mutant copper-zinc superoxide dismutases were also found to be reduced by ascorbate at significantly greater rate than the wild-type proteins . We conclude that similar alterations in the properties of the zinc binding site can be caused by mutations scattered throughout the protein structure . This finding may help to explain what is perhaps the most perplexing question in copper-zinc superoxide dismutase-associated familial ALS-i.e., how such a diverse set of mutations can result in the same gain of function that causes the disease.

J Biol Chem, 1996 Oct 25, 271(43), 27013 - 7
SMAP, an Smg GDS-associating protein having arm repeats and phosphorylated by Src tyrosine kinase; Shimizu K et al.; Smg GDS is a regulator having two activities on a group of small G proteins including the Rho and Rap1 family members and Ki-Ras; one is to stimulate their GDP/GTP exchange reactions, and the other is to inhibit their interactions with membranes . Structurally, it has 11 Arm repeats, a protein interaction motif, found in the Drosophila Armadillo protein, a homolog of mammalian beta-catenin . We have isolated here an Smg GDS-interacting protein from a human brain cDNA library by use of the yeast two-hybrid method and named it SMAP (Smg GDS-associated protein) . SMAP was a protein with a Mr of 91,189 and 792 amino acids . SMAP had 9 Arm repeats . Recombinant SMAP interacted with recombinant Smg GDS but did not affect the two activities of Smg GDS on RhoA . SMAP was tyrosine phosphorylated by v-Src, and this phosphorylation reduced the affinity of SMAP for Smg GDS . Tissue and subcellular distribution analyses indicated that SMAP was ubiquitously expressed and highly concentrated at the endoplasmic reticulum area . Searches for sequence homology to SMAP revealed that SMAP was significantly homologous to sea urchin SpKAP115, suggesting that SMAP is a mammalian counterpart of SpKAP115 or its related protein . SpKAP115 is an accessory subunit of sea urchin kinesin II, an ATPase motor that transports vesicles along microtubules . These results suggest that SMAP serves as an adaptor for both Smg GDS and kinesin II or its related protein and links them with both the Smg GDS-regulated small G protein and Src tyrosine kinase signalings.

J Biol Chem, 1996 Oct 25, 271(43), 26981 - 8
Purification and identification of a major activator for p38 from osmotically shocked cells . Activation of mitogen-activated protein kinase kinase 6 by osmotic shock, tumor necrosis factor-alpha, and H2O2; Moriguchi T et al.; A stress-activated, serine/threonine kinase, p38 (also known as HOG1 or MPK2) belongs to a subgroup of mitogen-activated protein kinase (MAPK) superfamily molecules . An activity to activate p38 (p38 activator activity) as well as p38 activity itself were greatly stimulated by hyperosmolar media in mouse lymphoma L5178Y cells . The activator activity has been purified by sequential chromatography . A 36-kDa polypeptide that was coeluted with the activity in the final chromatography step was identified as MAPK kinase 6 (MAPKK6) by protein microsequencing analysis . Monoclonal and polyclonal antibodies raised against recombinant MAPKK6 recognized specifically the 36-kDa MAPKK6 protein but did not cross-react with MKK3 proteins . The use of these anti-MAPKK6 antibodies revealed that two major peaks of the p38 activator activity in the first chromatography step reside in the activated MAPKK6 . Using a genetic screen in yeast, we isolated MKK3b, an alternatively spliced form of MKK3 . Like MKK3 and MAPKK6, MKK3b was shown to be a specific activator for p38 and was activated by osmotic shock when expressed in COS7 cells . Immunoblotting analysis revealed that MAPKK6 is expressed highly in HeLa and KB cells and scarcely in PC12 cells, whereas MKK3 and MKK3b are expressed in all cells examined . Immunodepletion of MAPKK6 from the extracts obtained from L5178Y cells and KB cells exposed to hyperosmolar media depleted them of almost all of the p38 activator activity, indicating that MAPKK6 is a major activator for p38 in an osmosensing pathway in these cells . In addition, MAPKK6 was activated strongly by tumor necrosis factor-alpha, H2O2, and okadaic acid and moderately by cycloheximide in KB cells . Thus, there are at least three members of p38 activator, MKK3, MKK3b, and MAPKK6, and MAPKK6 may function as a major activator for p38 when expressed.

J Biol Chem, 1996 Oct 25, 271(43), 26971 - 80
Sodium salicylate decreases intracellular ATP, induces both heat shock factor binding and chromosomal puffing, but does not induce hsp 70 gene transcription in Drosophila; Winegarden NA et al.; Sodium salicylate has long been known to be an inducer of the heat shock puffs and presumably heat shock gene transcription in the polytene chromosomes of Drosophila salivary gland cells . Stress-induced transcription of the heat shock genes is mediated by the transcription factor known as Heat Shock Factor (HSF) . In yeast, sodium salicylate has been reported to induce the DNA binding of HSF but not heat shock gene transcription itself, and similar findings have been reported in human cells . This apparent discrepancy in the induction of certain aspects of the heat shock response between these organisms prompted us to carefully reexamine the induction of the heat shock response in Drosophila salivary gland cells of third instar larvae and Drosophila tissue culture (SL2) cells . Sodium salicylate (3-30 mM) decreases intracellular ATP levels in SL2 cells and induces HSF binding activity in SL2 and salivary gland cells in a dose-dependent manner . Despite the induction of HSF binding and heat shock puffs in polytene chromosomes, we found no evidence for increased hsp 70 gene transcription suggesting that chromosomal puffing and gene transcription may be separable events . Salicylate did not induce the HSF hyperphosphorylation that is normally associated with HSF activation . Furthermore, salicylate (30 mM) prevented heat-induced hyperphosphorylation of HSF and hsp 70 gene transcription indicating that salicylate's inhibitory effect on hsp 70 transcription may be independent of its effect on HSF binding activity . We propose that the reduction in intracellular ATP caused by the addition of salicylate likely plays a role in the activation of HSF binding and the inhibition of both HSF hyperphosphorylation and hsp 70 gene transcription.

J Biol Chem, 1996 Oct 25, 271(43), 26931 - 8
Expression studies of delta-globin gene alleles associated with reduced hemoglobin A2 levels in Greek Cypriots; Trifillis P et al.; We previously identified five delta-globin gene alleles associated with reduced hemoglobin (Hb) A2 (Trifillis, P., Ioannou, P., Schwartz, E., and Surrey, S . (1991) Blood 78, 3298-3305) . We have now evaluated functional consequences of the changes after expression in COS-1 cells to monitor effects on RNA splicing . In addition, variant Hb A2 tetramers were expressed in yeast to assess effects of amino acid changes on oxygen binding and stability to heat and mechanical agitation . The G --> T change at codon 27 and the A --> G change in IVS-2 both affect RNA splicing, whereas the C --> T change at codon 97 and the AT deletion in IVS-2 have no effect . Oxygen equilibrium curves of the Hb A2 variants expressed in yeast were similar to that of wild type Hb A2 . None of the three variant Hb A2 tetramers (Thr --> Ile at codon 4 (Hb deltaT4I), Ala --> Ser at codon 27 (Hb deltaA27S), and Arg --> Cys at codon 116 (Hb deltaR116C)) showed decreased heat stability compared with Hb A2, whereas the Hb deltaT4I variant showed highest instability to mechanical agitation . Co-expression in yeast of alpha-globin chain and the delta-chain variant containing a Leu --> Pro change at codon 141 yielded no identifiable tetramers, suggesting lack of assembly or severe tetramer instability . These studies show the probable cause for decreased Hb A2 for two alleles is due to defective splicing, whereas decreased protein stability, increased tetramer association with red cell membranes, increased interdisulfide bond formation of delta-chains, which inhibits assembly with alpha-chains, and/or reduced assembly is suggested for the other three alleles.

Science, 1996 Oct 25, 274(5287), 540 - 6
A gene map of the human genome; Schuler GD et al.; The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags . An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library . More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers . The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease . The integrated resource is available through a site on the World Wide Web at http://www.ncbi.nlm.nih.gov/SCIENCE96/.

Nature, 1996 Oct 24, 383(6602), 707 - 10
Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia; Joutel A et al.; Stroke is the third leading cause of death, and vascular dementia the second cause of dementia after Alzheimer's disease . CADASIL (for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) causes a type of stroke and dementia whose key features include recurrent subcortical ischaemic events and vascular dementia and which is associated with diffuse white-matter abnormalities on neuroimaging . Pathological examination reveals multiple small, deep cerebral infarcts, a leukoencephalopathy, and a non-atherosclerotic, non-amyloid angiopathy involving mainly the small cerebral arteries . Severe alterations of vascular smooth-muscle cells are evident on ultrastructural analysis . We have previously mapped the mutant gene to chromosome 19 . Here we report the characterization of the human Notch3 gene which we mapped to the CADASIL critical region . We have identified mutations in CADASIL patients that cause serious disruption of this gene, indicating that Notch3 could be the defective protein in CADASIL patients.

Mutat Res, 1996 Oct 18, 364(2), 81 - 9
Self-association of human RAD52 protein; Shen Z et al.; The yeast RAD52 protein is required for both homologous DNA recombination and repair of DNA double-strand breaks . RAD52 can bind to the yeast RAD51 protein, which shares a functional similarity with the bacterial RecA protein . The gene encoding the human homolog of the yeast RAD52 protein shares significant N-terminus amino acid homology with the yeast RAD52 protein . Using a yeast two hybrid system and purified GST-RAD52 fusion protein, we demonstrate that the human RAD52 protein self-associates both in vivo and in vitro . The region of RAD52 required for its self-interaction, mapped here as amino acid residues 65-165, has significant homology with the yeast RAD52 (52% identity, and 89% similarity), suggesting the importance of self-association for RAD52's function.

Cell, 1996 Oct 18, 87(2), 287 - 96
Interaction between the origin recognition complex and the replication licensing system in Xenopus; Rowles A et al.; The origin recognition complex (ORC) binds to origins of replication in budding yeast . We have cloned a Xenopus homolog of the largest ORC polypeptide (XORC1) . Immunodepletion of XOrc1 from Xenopus egg extracts blocks the initiation of DNA replication . We have purified Xenopus ORC, consisting of a protein complex similar to yeast ORC . In Xenopus egg extracts, ORC associates with chromatin throughout G1 and S phases . RLF-M, a component of the replication licensing system, also associates with chromatin early in the cell cycle but dissociates during S phase . We show that the assembly of RLF-M onto chromatin is dependent on the presence of chromatin-bound ORC, leading to sequential assembly of initiation proteins onto replication origins during the cell cycle.

J Biol Chem, 1996 Oct 18, 271(42), 25823 - 9
Signal anchor sequence insertion into the outer mitochondrial membrane . Comparison with porin and the matrix protein targeting pathway; Millar DG et al.; We have addressed the question of overlap between the pathways for protein insertion into the outer mitochondrial membrane and import to the matrix compartment, using competition studies in vitro . A synthetic peptide corresponding to the matrix-targeting signal of pre-ornithine carbamyl transferase competed for outer membrane insertion of porin but did not compete for membrane insertion of outer membrane signal anchor-containing proteins . Conversely, however, a synthetic peptide corresponding to the signal anchor sequence of Tom70 competed for import of all proteins examined . Both peptides competed for a step beyond receptor binding . Import of all precursors examined was inhibited by antibodies raised against the import receptor Tom20 . Following binding to the surface of the organelle, outer membrane integration of porin was sensitive to depletion of nucleoside triphosphates by apyrase, whereas signal anchor protein insertion was not . The results demonstrate that outer membrane signal anchor insertion overlaps with a general insertion pathway . However, it exhibits both properties and steps that differ from the pathway followed by porin and matrix-targeted protein.

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