Yeasts

Science, 1997 Nov 7, 278(5340), 1064 - 8
Integrating genetic approaches into the discovery of anticancer drugs; Hartwell LH et al.; The discovery of anticancer drugs is now driven by the numerous molecular alterations identified in tumor cells over the past decade . To exploit these alterations, it is necessary to understand how they define a molecular context that allows increased sensitivity to particular compounds . Traditional genetic approaches together with the new wealth of genomic information for both human and model organisms open up strategies by which drugs can be profiled for their ability to selectively kill cells in a molecular context that matches those found in tumors . Similarly, it may be possible to identify and validate new targets for drugs that would selectively kill tumor cells with a particular molecular context . This article outlines some of the ways that yeast genetics can be used to streamline anticancer drug discovery.

Nature, 1997 Oct 30, 389(6654), 963 - 6
Mutation of an axonemal dynein affects left-right asymmetry in inversus viscerum mice; Supp DM et al.; The development of characteristic visceral asymmetries along the left-right (LR) axis in an initially bilaterally symmetrical embryo is an essential feature of vertebrate patterning . The allelic mouse mutations inversus viscerum (iv) and legless (lgl) produce LR inversion, or situs inversus, in half of live-born homozygotes . This suggests that the iv gene product drives correct LR determination, and in its absence this process is randomized . These mutations provide tools for studying the development of LR-handed asymmetry and provide mouse models of human lateralization defects . At the molecular level, the normally LR asymmetric expression patterns of nodal and lefty are randomized in iv/iv embryos, suggesting that iv functions early in the genetic hierarchy of LR specification . Here we report the positional cloning of an axonemal dynein heavy-chain gene, left/right-dynein (lrd), that is mutated in both lgl and iv . lrd is expressed in the node of the embryo at embryonic day 7.5, consistent with its having a role in LR development . Our findings indicate that dynein, a microtubule-based motor, is involved in the determination of LR-handed asymmetry and provide insight into the early molecular mechanisms of this process.

Infect Immun, 1997 Nov, 65(11), 4790 - 4
The major surface glycoprotein of Pneumocystis carinii induces release and gene expression of interleukin-8 and tumor necrosis factor alpha in monocytes; Benfield TL et al.; Recent studies suggest that interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) may play a central role in host defense and pathogenesis during Pneumocystis carinii pneumonia . In order to investigate whether the major surface antigen (MSG) of human P . carinii is capable of eliciting the release of IL-8 and TNF-alpha, human monocytes were cultured in the presence of purified MSG . MSG-stimulated cells released significant amounts of IL-8 within 4 h, and at 20 h, cells stimulated with MSG released 45.5 +/- 9.3 ng of IL-8/ml versus 3.7 +/- 1.1 ng/ml for control cultures (P = 0.01) . In a similar fashion, MSG elicited release of TNF-alpha . Initial increases were also seen at 4 h, and at 20 h, TNF-alpha levels reached 6.4 +/- 1.1 ng/ml, compared to 0.08 +/- 0.01 ng/ml for control cultures (P < 0.01) . A concentration-dependent increase in IL-8 and TNF-alpha secretion was observed at 20 h with 0.2 to 5 microg of MSG/ml (P < 0.01) . Secretion of IL-8 and TNF-alpha from MSG-stimulated monocytes at 20 h was inhibited by 60 and 86%, respectively, after coincubation with soluble yeast mannan (P = 0.01) . With an RNase protection assay, increases in steady-state mRNA levels for IL-8 and TNF-alpha were detectable at 4 h . These data show that recognition of MSG by monocytes involves a mannose-mediated mechanism and results in the release of the proinflammatory cytokines IL-8 and TNF-alpha.

J Bacteriol, 1997 Nov, 179(21), 6680 - 7
Purification and properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel flavin adenine dinucleotide-dependent monooxygenase from Candida parapsilosis CBS604; Eppink MH et al.; A novel flavoprotein monooxygenase, 4-hydroxybenzoate 1-hydroxylase (decarboxylating), from Candida parapsilosis CBS604 was purified to apparent homogeneity . The enzyme is induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, or 3,4-dihydroxybenzoate as the sole carbon source . The purified monooxygenase is a monomer of about 50 kDa containing flavin adenine dinucleotide as weakly bound cofactor . 4-Hydroxybenzoate 1-hydroxylase from C . parapsilosis catalyzes the oxidative decarboxylation of a wide range of 4-hydroxybenzoate derivatives with the stoichiometric consumption of NAD(P)H and oxygen . Optimal catalysis is reached at pH 8, with NADH being the preferred electron donor . By using (18)O2, it was confirmed that the oxygen atom inserted into the product 1,4-dihydroxybenzene is derived from molecular oxygen . 19F nuclear magnetic resonance spectroscopy revealed that the enzyme catalyzes the conversion of fluorinated 4-hydroxybenzoates to the corresponding hydroquinones . The activity of the enzyme is strongly inhibited by 3,5-dichloro-4-hydroxybenzoate, 4-hydroxy-3,5-dinitrobenzoate, and 4-hydroxyisophthalate, which are competitors with the aromatic substrate . The same type of inhibition is exhibited by chloride ions . Molecular orbital calculations show that upon deprotonation of the 4-hydroxy group, nucleophilic reactivity is located in all substrates at the C-1 position . This, and the fact that the enzyme is highly active with tetrafluoro-4-hydroxybenzoate and 4-hydroxy-3-nitrobenzoate, suggests that the phenolate forms of the substrates play an important role in catalysis . Based on the substrate specificity, a mechanism is proposed for the flavin-mediated oxidative decarboxylation of 4-hydroxybenzoate.

Eur J Clin Microbiol Infect Dis, 1997 Sep, 16(9), 637 - 43
Diagnostic laparoscopy in patients with acute leukemia and suspected hepatic candidiasis; Anttila VJ et al.; To assess the value of laparoscopy in the diagnosis of suspected hepatosplenic candidiasis in patients with acute leukemia, a retrospective analysis of 28 laparoscopies was conducted . In all but two cases, imaging of the liver showed focal lesions before laparoscopy . Diagnosis of hepatic candidiasis was established significantly more often when the biopsy was targeted at white nodules (in 12 of 22 laparoscopies) than when targeted randomly or at scars (0 of 6 laparoscopies) (p = 0.017, chi-square test) . Yeast was detected more often if the laparoscopy was performed during the three-week period after recovery from neutropenia (in 8 of 12 laparoscopies) than when performed later (in 4 of 16 laparoscopies) (p = 0.028, chi-square test) . In addition to the 12 laparoscopically diagnosed patients, eight (29%) patients were diagnosed with disseminated Candida infection by other methods . In another eight (29%) patients the causative agent was not identified . No bleeding or other problems occurred after the laparoscopy . Laparoscopy-guided liver biopsy is most useful if biopsies are targeted to macroscopic lesions and if laparoscopy is performed soon after recovery from neutropenia.

Curr Top Dev Biol, 1998, 37, 1 - 35
Recombination in the mammalian germ line; Pittman DL et al.; Elucidation of meiotic recombination mechanisms in mammals faces many obstacles . Much of our understanding has been built upon studies in the fungi, which have served to guide experimental design in mammalian cells and mice . A clearer picture is now emerging which reveals that many of the general principles of recombination are conserved across this evolutionary divide . A number of genes critical to meiotic recombination in yeast also exist in mammals . Transgenic technologies, in addition to advances in molecular biology, now provide several strategies to investigate the properties and regulation of mammalian recombination . This chapter reviews the current state of knowledge regarding recombination in the mammalian germ line, covering topics such as gene conversion, recombination mechanics, recombination-based genetic mutation, crossing over, and genes involved in meiotic recombination.

Biochimie, 1997 Jul, 79(7), 387 - 95
Chlamydomonas U2, U4 and U6 snRNAs . An evolutionary conserved putative third interaction between U4 and U6 snRNAs which has a counterpart in the U4atac-U6atac snRNA duplex; Jakab G et al.; The spliceosomal UsnRNAs U2, U4 and U6 from the green alga Chlamydomonas reinhardtii (Cre) were sequenced using a combination of RNA and cDNA sequencing methods and were compared to other sequenced UsnRNAs . The lengths of Cre U6 and Cre U2 RNAs are similar to those of their higher plant equivalents . Cre U4 RNA is shorter (139 nt) than its counterpart from higher plants (150-154 nt), and contains stem IV and loop D which are absent, with the exception of the Tetrahymena U4 RNA, from the U4 RNAs of other unicellular organisms studied to date . Base-pairing interactions between U6 and U4 RNAs and between U6 and U2 RNAs, identical to those described for mammalian and yeast systems, are structurally feasible in the Cre system . In addition, based on comparative analyses of the predicted U4/U6 RNA duplex from various species, an evolutionary conserved third putative U6-U4 interaction was found . Interestingly, it can also be formed with the recently discovered U6atac and U4atac RNAs . This is a strong support in favor of the possible biological significance of this third putative interaction . Based on comparative analysis, an extension of the earlier described U6-U2 interaction patterns is also proposed.

Biochem Mol Biol Int, 1997 Oct, 43(3), 489 - 98
Design of novel analogue peptides with potent fungicidal but low hemolytic activity based on the cecropin A-melittin hybrid structure; Lee DG et al.; In order to design synthetic peptides with potent antifungal activity but low cytotoxic activity under physiological conditions, several analogues of the previously reported cecropin A (CA)-melittin (ME) hybrid peptide, CA(1-8)-ME(1-12), were synthesized . These analogues were designed by analysis of the alpha-helical wheel diagram of CA(1-8)-ME(1-12) . Antifungal activities were measured by growth inhibition of the yeast Trichosporon beigelii and by hemolytic assay with human red blood cells, respectively . Substitution of Thr for Lys at position 18 and 19 of CA(1-8)-ME(1-12) caused a dramatic reduction in hemolytic activity . Two analogue peptides (analogue I and III) showed more potent antifungal and lower hemolytic activity than the original peptide . To study the antifungal mechanism of these peptides, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed with the most powerful antifungal analogue I peptide designed in the present study . As determined by propidium iodide staining, fungal cells treated with analogue I or melittin showed higher fluorescence intensity than those treated with the weak antifungal peptide, cecropin A . By confocal microscopy the analogue I was detected in the intracellular region as well as the in cell membrane . These facts suggested that the antifungal function of this novel peptide analogue acts by pore formation in the cell membrane.

Plant J, 1997 Sep, 12(3), 711 - 30
Construction of an approximately 2 Mb contig in the region around 80 cM of Arabidopsis thaliana chromosome 2; Wang ML et al.; A method for construction of bacterial artificial chromosome (BAC) contigs from a yeast artificial chromosome (YAC) physical map is described . An approximately 2 Mb contig, consisting of two large BAC contigs linked by a small YAC, has been assembled in the region around 80 cM of Arabidopsis thaliana chromosome 2 . Clones from this contig will facilitate gene isolation in the region and can be used directly as substrates for DNA sequencing.

J Clin Microbiol, 1997 Nov, 35(11), 3004 - 6
Comparison of charcoal- and starch-based media for testing susceptibilities of Legionella species to macrolides, azalides, and fluoroquinolones; Pendland SL et al.; We compared growth characteristics of 46 Legionella strains grown on buffered charcoal yeast extract alpha (BCYE alpha) agar and buffered starch yeast extract (BSYE) agar and MICs of macrolides, azalides, and fluoroquinolones for these organisms . Growth was poor and not reproducible on BSYE agar . Growth was excellent on BCYE alpha, and MICs were easy to interpret . BCYE alpha is superior to BSYE for testing susceptibilities of Legionella species by agar dilution.

Mol Cell Biochem, 1997 Oct, 175(1-2), 21 - 7
Ribonuclease activity dependent cytotoxicity of Asp fl, a major allergen of A . fumigatus; Madan T et al.; A major allergen/antigen, Asp fl, secreted by Aspergillus fumigatus exhibits cytotoxicity towards eukaryotic cell lines . Asp fl inhibited protein synthesis in RAW cells with an IC50 of 4.5 nM and also degraded ribosomal RNA of RAW cells at a similar concentration . Ribosomal inactivation by Asp fl may be the probable mechanism for protein synthesis inhibition . Specific ribonuclease activity of Asp fl was observed to be 100,000 U/mg . Presence of strong RNase activity in Asp fl was further confirmed by agar gels containing yeast RNA . Electrophoretic run on agarose gels showed that Asp fl degrades all species of naked RNA . Modification of histidine residues of Asp fl with diethyl pyrocarbonate and alkylation of cysteines with iodoacetamide resulted in loss of ribonuclease activity and cytotoxicity of Asp fl . The current study establishes the ribonuclease activity of a purified major allergen of A . fumigatus that inhibits protein synthesis and kills the eukaryotic cells.

J Cell Biol, 1997 Nov 3, 139(3), 773 - 84
The amino-terminal domain of desmoplakin binds to plakoglobin and clusters desmosomal cadherin-plakoglobin complexes; Kowalczyk AP et al.; The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell-cell adhesion . Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin . Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome . However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified . To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components . When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution . However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP . In addition, plakoglobin and DP-NTP were recruited to cell-cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome . In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin . Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1 . These results identify a role for desmoplakin in organizing the desmosomal cadherin-plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.

Blood, 1997 Oct 15, 90(8), 2916 - 20
Amino acids responsible for decreased 2,3-biphosphoglycerate binding to fetal hemoglobin; Adachi K et al.; To clarify the role of gammaN-terminal Gly, gamma5 Glu, and gamma143 Ser in 2,3-biphosphosphoglycerate (BPG) binding to fetal hemoglobin (Hb F), we engineered and produced normal human Hb F and two Hb F variants (Hb F gammaG1V, gammaS143H, and Hb F gammaG1V, gammaE5P, gammaS143H) using a yeast expression system and then compared their oxygen-binding properties with those of native human Hb F and adult Hb (Hb A) . Oxygen affinity of Hb F gammaG1V, gammaS143H in the absence of 2,3-BPG was slightly higher than that of normal Hb F . The decrease in oxygen affinities for Hb F gammaG1V, gammaS143H with increasing 2,3-BPG concentrations was larger than that of normal Hb F, but significantly less than that of Hb A . In contrast, oxygen affinities of Hb F gammaG1V, gammaE5P, gammaS143H in the absence and presence of 2,3-BPG were much lower than those of Hb F gammaG1V, gammaS143H and were similar to those of Hb A . These results indicate that differences between Pro and Glu at the A2 position in the A helix in Hb A and Hb F, respectively, are critical for reduced binding of 2,3-BPG to Hb F, even though beta5 Pro does not interact directly with 2,3-BPG in Hb A . Hb F variants such as Hb F gammaG1V, gammaE5P, gammaS143H, which exhibit reduced oxygen affinity, should facilitate design of efficient antisickling fetal Hb variants for potential use in gene therapy for sickle cell disease.

Biochemistry, 1997 Oct 14, 36(41), 12535 - 41
Identification of the predominant non-native histidine ligand in unfolded cytochrome c; Colon W et al.; The heme and its two axial ligands, His18 and Met80, play a central role in the folding/unfolding mechanism of cytochrome c . Because of the covalent heme attachment, His18 remains bound under typical denaturing conditions, while the more labile Met80 ligand is replaced by an alternate histidine ligand . To distinguish between the two possible non-native histidine ligands in horse cytochrome c, variants with a His26 to Gln or His33 to Asn substitution were prepared using a yeast expression system . Protonation of the non-native histidine ligand in the GuHCl-denatured state results in a pronounced blue shift of the Soret heme absorbance band (low-spin to high-spin transition) . While substitution of His26 has no effect on the apparent pKa of this transition (5.7 +/- 0.05), the H33N variant exhibits a substantially higher pKa (6.1 +/- 0.05), indicating that His33 is the dominant sixth heme ligand in denatured cytochrome c and that His26 (or another nitrogenous group) acts as a ligand in the absence of a histidine at position 33 . The kinetics of the pH-induced ligand dissociation shows two phases which were assigned to each of the two histidine ligands on the basis of their distinct temperature dependence . Despite their nearly identical equilibrium unfolding transitions, the two histidine mutants show differences in their folding kinetics . While the kinetic behavior of H26Q cyt c is very similar to that of the wild-type, the H33N mutation leads to loss of a kinetic phase with a rate in the 2-10 s-1 range that has previously been attributed to the rate-limiting dissociation of a trapped non-native histidine, which is thus identified as His33.

Environ Health Perspect, 1997 Aug, 105(8), 802 - 11
The estrogenic activity of phthalate esters in vitro; Harris CA et al.; A large number of phthalate esters were screened for estrogenic activity using a recombinant yeast screen . a selection of these was also tested for mitogenic effect on estrogen-responsive human breast cancer cells . A small number of the commercially available phthalates tested showed extremely weak estrogenic activity . The relative potencies of these descended in the order butyl benzyl phthalate (BBP) > dibutyl phthalate (DBP) > diisobutyl phthalate (DIBP) > diethyl phthalate (DEP) > diisiononyl phthalate (DINP) . Potencies ranged from approximately 1 x 10(6) to 5 x 10(7) times less than 17beta-estradiol . The phthalates that were estrogenic in the yeast screen were also mitogenic on the human breast cancer cells . Di(2-ethylhexyl) phthalate (DEHP) showed no estrogenic activity in these in vitro assays . A number of metabolites were tested, including mono-butyl phthalate, mono-benzyl phthalate, mono-ethylhexyl phthalate, mon-n-octyl phthalate; all were wound to be inactive . One of the phthalates, ditridecyl phthalate (DTDP), produced inconsistent results; one sample was weakly estrogenic, whereas another, obtained from a different source, was inactive . analysis by gel chromatography-mass spectometry showed that the preparation exhibiting estrogenic activity contained 0.5% of the ortho-isomer of bisphenol A . It is likely that the presence of this antioxidant in the phthalate standard was responsible for the generation of a dose-response curve--which was not observed with an alternative sample that had not been supplemented with o,p'-bisphenol A--in the yeast screen; hence, DTDP is probably not weakly estrogenic . The activities of simple mixtures of BBP, DBP, and 17beta-estradiol were assessed in the yeast screen . No synergism was observed, although the activities of the mixtures were approximately additive . In summary, a small number of phthalates are weakly estrogenic in vitro . No data has yet been published on whether these are also estrogenic in vitro . No data has yet been published on whether these are also estrogenic in vivo; this will require tests using different classes of vertebrates and different routes of exposure.

Mikrobiol Z, 1997 May-Jun, 59(3), 41 - 6
Certain peculiarities of biological action of the stachybotryotoxin preparations; Andriienko OV et al.; Some biological and toxigenic properties of 47 Stachybotrys alternans strains, isolated from different ecological niches have been studied . On the basis of antifungal, phytotoxic and dermacidic properties the strains studied can be divided into several characteristic groups: strains with clearly expressed dermacidic reaction and without other properties studied (19.1%); strains displayed only antifungal activity to a number of yeast test cultures (4.3%); strains with complex action (antifungal, phytotoxic and dermacidic activity) (46.8%) . Considerable portion of strains (25.5%) did not show any biocidal activity tested . No clear dependence of strain activity on the term of storage under laboratory conditions (from 5 to 40 years) has been revealed.

J Biol Chem, 1997 Oct 31, 272(44), 27678 - 85
TbetaRI phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling; Abdollah S et al.; Mothers against Dpp-related or Smad proteins are essential components of serine/threonine kinase receptor signaling pathways that are regulated by phosphorylation . Recently, it was demonstrated that Smad2 interacts transiently with and is a direct substrate of the transforming growth factor-beta (TGF-beta) type I receptor, TbetaRI . Phosphorylation sites on Smad2 were localized to a carboxyl-terminal fragment containing three serine residues at positions 464, 465, and 467 . In this report, we show that TbetaRI specifically phosphorylates Smad2 on serines 465 and 467 . Serine 464 is not a site of phosphorylation, but is important for efficient phosphorylation of Smad2 . Phosphorylation at both sites is required to mediate association of Smad2 with Smad4 in mammalian cells, while in yeast, Smad2 interacts directly with Smad4 and does not require phosphorylation . Mutation of either serine residue 465 or 467 prevents dissociation of Smad2 from activated TbetaRI and blocks TGF-beta-dependent signaling and Smad2 transcriptional activity . These results indicate that receptor-dependent phosphorylation of Smad2 on serines 465 and 467 is required in mammalian cells to permit association with Smad4 and to propagate TGF-beta signals.

Asian Pac J Allergy Immunol, 1997 Jun, 15(2), 115 - 22
Parasites elicited cross-reacting antibodies to Opisthorchis viverrini; Sakolvaree Y et al.; Two batches of crude antigens extracted from adult Opisthorchis viverrini worms were compared . One was derived from adult worms harvested from the livers of laboratory infected hamsters and another was obtained from worms sedimented from the faeces of opisthorchiasis patients following treatment with Praziquantel . SDS-PAGE and Coomassie brilliant blue staining revealed that the two preparations had similar protein components of which the predominant ones were the 17-18 kDa doublet . The antigens were used in an indirect ELISA for the detection of antibodies against O . viverrini in the sera of four groups of patients, ie . patients with opisthorchiasis (group 1), patients with mixed infections of O . viverrini and other parasites (group 2), patients with other parasitic infections (group 3), and normal-heathy, parasite-free individuals (group 4) . The sensitivity of the test was high (91-92%), regardless of the batch of the antigen used . However, its specificity was relatively low (70-80%) . Cross-reaction was observed with patients infected with Paragonimus heterotremus, Schistosoma spp.; Taenia spp.; Trichinella spiralis; Strongyloides stercoralis; hookworms; Plasmodium spp.; hookworms and Plasmodium spp.; S . stercoralis, Blastocystis hominis and yeast; and hookworms, Ascaris lumbricoides, Trichuris trichiura and P . falciparum . Western blot analysis revealed that sera of patients infected with these heterologous organisms contained antibodies reactive to O . viverrini antigenic components ranging from Mr 15.5 to 144.

Cell, 1997 Oct 17, 91(2), 253 - 62
GRASP65, a protein involved in the stacking of Golgi cisternae; Barr FA et al.; NEM prevents mitotic reassembly of Golgi cisternae into stacked structures . The major target of NEM is a 65 kDa protein conserved from yeast to mammals . Antibodies to this protein and a recombinant form of it block cisternal stacking in a cell-free system, justifying its designation as a Golgi ReAssembly Stacking Protein (GRASP65) . One of the two minor targets of NEM is GM130, previously implicated in the docking of transport vesicles and mitotic fragmentation of the Golgi stack . GRASP65 is complexed with GM130 and is tightly bound to Golgi membranes, even under mitotic conditions when both are heavily phosphorylated . These results link vesicle docking, stacking of Golgi cisternae, and the disruption of both of these interactions during mitosis.

Biochem Biophys Res Commun, 1997 Oct 9, 239(1), 217 - 22
Functional elements in molecular chaperone alpha-crystallin: identification of binding sites in alpha B-crystallin; Sharma KK et al.; alpha-Crystallin, the predominant eye lens protein with sequence homology to small heat shock proteins, acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins . To gain an insight into the amino acid sequences in alpha-crystallin involved in chaperone-like function, we used a cleavable, fluorescent, photoactive, crosslinking agent, sulfosuccinimidyl-2 (7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3' dithiopropionate (SAED), to derivatize yeast alcohol dehydrogenase (ADH) and allowed it to complex with bovine alpha-crystallin at 48 degrees C . The complex was photolyzed and reduced with DTT and the subunits of alpha-crystallin, alpha A- and alpha B-, were separated . Fluorescence analysis showed that both alpha A- and alpha B-crystallins interacted with ADH during chaperone-like function . Tryptic digestion, amino acid sequencing, and mass spectral analysis of alpha B-crystallin revealed that APSWIDTGLSEMR (57-69) and VLGDVIEVHGKHEER (93-107) sequences were involved in binding with ADH.

Biochem Biophys Res Commun, 1997 Oct 20, 239(2), 480 - 2
Presenilin 1 binds to amyloid precursor protein directly; Waragai M et al.; Mutations in the presenilin genes are associated with early onset familial Alzheimer's disease and lead to accumulation of beta-amyloid peptide in the brain of patients, suggesting that presenilin abnormalities induce pathological processing of amyloid precursor protein (APP) in Alzheimer's disease . For the understanding of pathogenesis in this type of familal Alzheimer disease, it is important to know whether presenilins are directly involved in the metabolism of beta-amyloid or not . To test whether presenilin 1 (PS1) directly binds to APP, we performed two-hybrid interaction assays between these proteins in yeast cells by using bait plasmids for normal and mutant PS1 and prey plasmids for APP fragments corresponding to the different molecular portions . Positive interaction was observed in any combination between PS1 bait plasmids and APP prey plasmids . Therefore, our results show that PS1 binds to APP directly and suggest that the PS1 protein itself is involved in the metabolism of beta-amyloid peptide .

Genomics, 1997 Oct 15, 45(2), 421 - 4
Exon-intron organization of the human dystrophin gene; Nobile C et al.; Analysis of the exon-intron organization of the human dystrophin gene has been hampered by its enormous size . By using a YAC-based exon mapping approach and long PCR, we have succeeded in defining the size of the gene and its organization . Our results, compared with data on the distribution of deletion breakpoints by intron, elucidate the topography of the intragenic deletion-prone regions . Within the central high-frequency deletion region, the small, 6.6-kb, intron 49 shows a much higher density of deletion breakpoints than intron 44, which was previously believed to coincide with the most mutable zone of the gene . On the other hand, in the proximal part of the gene, deletion breakpoints do not preferentially occur in a few introns, but are spread over a large DNA segment containing introns 2 to 42 .

Genomics, 1997 Oct 15, 45(2), 407 - 11
Detailed physical analysis of a 1.5-megabase YAC contig containing the MXI1 and ADRA2A genes; Manca A et al.; The distal long arm of chromosome 10 harbors genes of biomedical interest such as MXI1, a putative tumor suppressor gene, and those encoding the adrenergic receptors alpha2A (ADRA2A) and beta1 (ADRB1) . As part of a physical and genetic study of this genomic region, we constructed a 1.5-Mb YAC contig mapping to 10q25 that contains MXI1 and ADRA2A as well as a number of STSs . Rare cutting restriction site analysis of overlapping YACs allowed fine mapping of these genes and markers along the contig and revealed the presence of four CpG islands . MXI1 and ADRA2A appear to be about 600 kb apart, whereas ADRB1 is separated from ADRA2A by a distance larger than previously reported .

Genomics, 1997 Oct 15, 45(2), 402 - 6
PMS2-related genes flank the rearrangement breakpoints associated with Williams syndrome and other diseases on human chromosome 7; Osborne LR et al.; The human PMS2 mismatch repair gene and a family of at least 17 other related genes (named human PMSR or PMS2L genes) have been localized to human chromosome 7 . Human PMS2 has been mapped previously to 7p22 and shown to be causative in hereditary nonpolyposis colon cancer (HNPCC), but the human PMS2L genes have not been positioned in the context of the physical or genetic map of chromosome 7 . In this study we have used various mapping methodologies to determine the precise location of the human PMS2L genes at 7q11.22, 7q11.23, and 7q22 . Within 7q11.23, human PMS2L genes were found to be present at at least three sites as part of duplicated genomic segments that flank the most common rearrangement breakpoints in Williams syndrome .

Genomics, 1997 Oct 15, 45(2), 297 - 303
Chromosome localization and structure of the murine cyclin G1 gene promoter sequence; Jensen MR et al.; Cyclins play an essential role in the control of the cell cycle . In this study the murine cyclin G1 gene expression, structure, and chromosomal localization were examined . Genes with high homology to murine cyclin G1 were detected in various mammals, including human, monkey, rat, dog, cow, and rabbit, but not in yeast or chicken . Cyclin G1 gene was expressed in all murine tissues examined, with the highest levels in cardiac and skeletal muscle . A 10,366-bp genomic DNA fragment encompassing the promoter region and the 5'-flanking region of the gene was cloned and sequenced . Three putative binding sites for the myocyte enhancer factor-2 family of transcription factors were revealed . Furthermore, an upstream p53-binding site was localized to nucleotides -252 to -233 and a new putative p53-binding site was identified in the first intronic region at nucleotides 275 to 294 . By fluorescence in situ hybridization, the cyclin G1 gene was mapped to mouse chromosome 11B1.1 . This region is homologous with human chromosome 5q31-q32, consistent with the recent mapping of the human cyclin G1 gene to chromosome 5q32-q34 . Localization of murine cyclin G1 will facilitate determination of gene linkage and the identification of synteny groups in mammals and of DNA elements in or near this gene that mediate its tissue expression or development-specific pattern of expression .

Genomics, 1997 Oct 15, 45(2), 250 - 8
Positional cloning of novel skin-specific genes from the human epidermal differentiation complex; Zhao XP et al.; The epidermal differentiation complex, located on human chromosomal band 1q21, contains at least 20 genes expressed during epidermal differentiation . We constructed a 1.2-Mb YAC contig spanning the SPRR and S100 gene clusters . Restriction mapping and FISH confirmed the colinearity of the contig with the genomic restriction map (A . Volz et al., 1993, Genomics 18:92-99) . However, the YAC clones revealed several additional restriction sites not previously detected in genomic DNA, presumably due to CpG methylation . Making use of cDNA selection, we have identified three novel cDNAs, all of which map to the SPRR/IVL region . All three transcripts are expressed at high levels in normal and psoriatic skin, but not in cultured keratinocytes or in a variety of cell lines and human tissues . The molecular cloning of this region provides a valuable tool for identifying additional epidermal differentiation genes and for elucidating the relationship between chromatin structure and gene expression during terminal differentiation .

Fungal Genet Biol, 1997 Aug, 22(1), 39 - 53
Cerato-ulmin, a hydrophobin secreted by the causal agents of Dutch elm disease, is a parasitic fitness factor; Temple B et al.; Dutch elm disease is caused by the aggressive Ophiostoma novo-ulmi and the nonaggressive O . ulmi . Both secrete the protein cerato-ulmin (CU) . To determine what role CU plays in the pathology of Dutch elm disease, we constructed a CU overexpression mutant of the nonaggressive O . ulmi H5 . Stable integration of a single copy of the cu gene from the aggressive O . novo-ulmi into the genome of the nonaggressive isolate resulted in increased secretion of CU protein . Trials with American elm, Ulmus americana, suggested no alteration of virulence of this overexpressing transformant . Using aggressive and nonaggressive wild types, the cu overexpressing mutant, and our cu- mutant (Bowden et al., 1996), we have demonstrated that CU production is correlated with an altered phenotype and more hydrophobic and adherent yeast-like cells . Our results also demonstrate that CU has a role in protecting infectious propagules from desiccation . These biological roles for CU would affect transmission of Dutch elm disease, and we therefore propose that this hydrophobin acts as a parasitic fitness factor .

Fungal Genet Biol, 1997 Aug, 22(1), 1 - 12
The duplication cycle in Aspergillus nidulans; Harris SD; The duplication cycle encompasses the spectrum of events required for the growth and division of individual cells within a fungal hyphae . Recent advances in understanding the mechanisms which underlie nuclear division and cellular morphogenesis in the filamentous fungus Aspergillus nidulans have shown that in many respects, the duplication cycle differs significantly from the cell cycles of both budding and fission yeast . The purpose of this review is to summarize these advances and to highlight the fundamental differences between the duplication cycle and the yeast cell cycles . In addition, it is argued that the duplication cycle is controlled by cellular regulatory networks which integrate the processes of nuclear division, cellular morphogenesis, and cell growth with each other . Functional dissection of these networks should help to reveal features that are unique to the hyphal mode of growth .

Arch Biochem Biophys, 1997 Oct 1, 346(1), 28 - 36
Wheat germ initiation factor 2 (WG x eIF2) decreases the inhibition in protein synthesis and eIF2B activity of reticulocyte lysates mediated by eIF2alpha phosphorylation; Krishna VM et al.; Phosphorylation of serine 51 residue in the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) impairs the guanine nucleotide exchange (GNE) activity of eIF2B protein and thereby inhibits protein synthesis in mammalian systems, insects and yeast . It is not known if phosphorylation of plant eIF2 can inhibit an eIF2B-like activity . Interestingly purified wheat germ eIF2 (WG x eIF2) can exchange guanine nucleotides in vitro without the addition of any protein factor like eIF2B . It is not clear if this is due to a contaminant eIF2B-like activity associated with WG x eIF2 or because the affinity of WG x eIF2 for GDP and GTP is not markedly different . Our observations here indicate that the GNE activity of WG x eIF2 is not inhibited upon phosphorylation of the p41-42 doublet subunit in WG x eIF2 by reticulocyte eIF2alpha kinases, or in the presence of reticulocyte eIF2(alphaP) in which serine 51 residue is phosphorylated . Further, addition of WG x eIF2 reduces the inhibition in eIF2B activity, protein synthesis, and also the formation of 15S complex that occurs between reticulocyte eIF2(alphaP) and eIF2B protein in heme-deficient or poly(IC)-treated reticulocyte lysates, presumably by a mechanism of competition between wheat germ and reticulocyte eIF2 for phosphorylation . Unlike reticulocyte eIF2(alphaP), phosphorylated WG x eIF2 is unable to interact with reticulocyte eIF2B to form a 15S complex . The ability of WG x eIF2 to exchange guanine nucleotides independent of an eIF2B like protein and the inability of phosphorylated WG x eIF2 to interact with reticulocyte eIF2B suggests that WG x eIF2 is different from mammalian eIF2 and these differences may have occurred in evolution probably due to some changes in the amino acid sequences around the phosphorylation site in eIF2alpha.

Arterioscler Thromb Vasc Biol, 1997 Sep, 17(9), 1741 - 5
Effects of estrus cycle, ovariectomy, and treatment with estrogen, tamoxifen, and progesterone on apolipoprotein(a) gene expression in transgenic mice; Zysow BR et al.; Plasma levels of lipoprotein(a) (Lp(a)), are regulated by the synthetic rate of apolipoprotein(a) (apo(a)), a major protein component of this atherogenic lipoprotein . Exogenously administered sex steroid hormones are potent regulators of plasma Lp(a) concentrations . We utilized a recently developed apo(a) yeast artificial chromosome (YAC) transgenic mouse model to study the effects of ovariectomy, estrus cycle, and exogenous administration of ethinyl-estradiol, the partial estrogen receptor agonist, tamoxifen, and progesterone on circulating apo(a) plasma levels . Analysis of liver RNA revealed that estrogen and tamoxifen exerts their plasma apo(a) lowering effect at the level of apo(a) mRNA . This action of estrogen and tamoxifen may contribute to their antiatherosclerotic and cardiovascular protective effect.

Bioconjug Chem, 1997 Sep-Oct, 8(5), 714 - 23
Synthesis and biological properties of mannosylated starburst poly(amidoamine) dendrimers; Page D et al.; Starburst PAMAM dendrimers ending with mannopyranoside residues were readily synthesized in large scale and good yields from commercially available dendrimers bearing high-density amine functionality on their surface and p-isothiocyanatophenyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranoside . The first four generations of this novel class of monodispersed neoglycoconjugates having up to 32 mannoside units were evaluated as ligands for the phytohemagglutinins from concanavalin A (Con A) and Pisum sativum (pea lectin) using enzyme-linked lectin assay (ELLA) and turbidimetric analyses . The binding properties of these glycodendrimers, together with reference monosaccharides, were determined using yeast mannan as a coating antigen and peroxidase-labeled lectins . These mannosylated dendrimers were demonstrated to be potent inhibitors with IC50 values 400 times better than those of monomeric methyl alpha-D-mannopyranoside taken as a standard . Their lipophilic character was shown to be sufficient for their direct use as coating antigens in microtiter plate assays . Moreover, their ability to bind and form insoluble carbohydrate-lectin complexes was also demonstrated by radial double immunodiffusion and turbidimetric analyses . Furthermore, the ability of these ligands to selectively precipitate a mannose-binding protein (Con A) from a crude lectin mixture was also demonstrated using polyacrylamide gel electrophoresis (SDS-PAGE) . These multivalent neoglycoconjugates were shown to constitute novel biochromatography materials of high affinity for the easy isolation of carbohydrate-binding proteins.

Nat Genet, 1997 Oct, 17(2), 226 - 30
Deregulation of MUM1/IRF4 by chromosomal translocation in multiple myeloma; Iida S et al.; The pathogenesis of multiple myeloma (MM), an incurable tumour causing the deregulated proliferation of terminally differentiated B cells, is unknown . Chromosomal translocations (14q1) affecting band 14q32 and unidentified partner chromosomes are common in this tumour, suggesting that they may cause the activation of novel oncogenes . By cloning the chromosomal breakpoints in an MM cell line, we show that the 14q+ translocation represents a t(6;14)x(p25;q32) and that this aberration is recurrent in MM, as it was found in two of eleven MM cell lines . The translocation juxtaposes the immunoglobulin heavy-chain (IgH) locus to MUM1 (multiple myeloma oncogene 1)/IRF4 gene, a member of the interferon regulatory factor (IRF) family known to be active in the control of B-cell proliferation and differentiation . As a result, the MUM1/IRF4 gene is overexpressed--an event that may contribute to tumorigenesis, a MUM1/IRF4 has oncogenic activity in vitro . These findings identify a novel genetic alteration associated with MM, with implications for the pathogenesis and diagnostics of this tumour.

Nat Genet, 1997 Oct, 17(2), 185 - 9
Identification of PAHX, a Refsum disease gene; Mihalik SJ et al.; Refsum disease is an autosomal recessive disorder characterized by retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia and increased cerebrospinal fluid protein . Biochemically, the disorder is defined by two related properties: pronounced accumulation of phytanic acid and selective loss of the peroxisomal dioxygenase required for alpha-hydroxylation of phytanoyl-CoA2 . Decreased phytanic-acid oxidation is also observed in human cells lacking PEX7, the receptor for the type-2 peroxisomal targetting signal (PTS2; refs 3,4), suggesting that the enzyme defective in Refsum disease is targetted to peroxisomes by a PTS2 . We initially identified the human PAHX and mouse Pahx genes as expressed sequence tags (ESTs) capable of encoding PTS2 proteins . Human PAHX is targetted to peroxisomes, requires the PTS2 receptor for peroxisomal localization, interacts with the PTS2 receptor in the yeast two-hybrid assay and has intrinsic phytanoyl-CoA alpha-hydroxylase activity that requires the dioxygenase cofactor iron and cosubstrate 2-oxoglutarate . Radiation hybrid data place PAHX on chromosome 10 between the markers D10S249 and D10S466, a region previously implicated in Refsum disease by homozygosity mapping . We find that both Refsum disease patients examined are homozygous for inactivating mutations in PAHX, demonstrating that mutations in PAHX can cause Refsum disease.

Nat Genet, 1997 Oct, 17(2), 154 - 63
Homologous recombination of a flanking repeat gene cluster is a mechanism for a common contiguous gene deletion syndrome; Chen KS et al.; Smith-Magenis syndrome (SMS), caused by del(17)p11.2, represents one of the most frequently observed human microdeletion syndromes . We have identified three copies of a low-copy-number repeat (SMS-REPs) located within and flanking the SMS common deletion region and show that SMS-REP represents a repeated gene cluster . We have isolated a corresponding cDNA clone that identifies a novel junction fragment from 29 unrelated SMS patients and a different-sized junction fragment from a patient with dup(17)p11.2 . Our results suggest that homologous recombination of a flanking repeat gene cluster is a mechanism for this common microdeletion syndrome.

Genomics, 1997 Sep 15, 44(3), 350 - 4
Human XPMC2H: cDNA cloning, mapping to 9q34, genomic structure, and evaluation as TSC1; Kwiatkowska J et al.; XPMC2 is a Xenopus gene identified on the basis of its ability to correct a mitotic defect in fission yeast . Here we report the identification of cDNA clones for human XPMC2H, its mapping to the tuberous sclerosis gene TSC1 region on 9q34, determination of genomic structure, and identification of several coding region polymorphisms . The predicted protein has strong sequence similarity to the Xenopus gene . Through SSCP and heteroduplex analysis of genomic DNA, we found two intragenic polymorphisms but no evidence for significant mutations in patients with tuberous sclerosis in this gene.

Genomics, 1997 Sep 15, 44(3), 321 - 9
A high-resolution PAC and BAC map of the SCA2 region; Nechiporuk T et al.; The spinocerebellar ataxia type 2 (SCA2) gene has been localized to chromosome 12q24.1 . To characterize this region and to aid in the identification of the SCA2 gene, we have constructed a 3.9-Mb physical map, which covers markers D12S1328 and D12S1329 known to flank the gene . The map comprises a contig of 84 overlapping yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) onto which we placed 82 PCR markers . We localized eight genes and expressed sequence tags on this map, many of which had not been precisely mapped before . In contrast to YACs, which showed a high degree of chimerism and deletions in this region, PACs and BACs were stable . Only 1 in 65 PACs contained a small deletion, and 2 in 18 BACs were chimeric . The high-resolution physical map, which was used in the identification of the SCA2 gene, will be useful for the positional cloning of other disease genes mapped to this region.

Genomics, 1997 Sep 15, 44(3), 300 - 8
High-resolution physical map of the X-linked retinoschisis interval in Xp22; Walpole SM et al.; X-linked retinoschisis (RS) is the leading cause of macular degeneration in young males and has been mapped to Xp22 between DXS418 and DXS999 . To facilitate identification of the RS gene, we have constructed a yeast artificial chromosome (YAC) contig across this region comprising 28 YACs and 32 sequence-tagged sites including seven novel end clone markers . To establish the definitive marker order, a PAC contig containing 50 clones was also constructed, and all clones were fingerprinted . The marker order is: Xpter-DXS1317-(AFM205yd12-DXS7175-DXS7992) -60N8-T7-DXS1195-DXS7993-DXS7174 -60N8-SP6-DXS418-DXS7994-DXS7995-DXS7996-+ ++HYAT2-25HA10R-HYAT1-DXS7997-DXS7998- DXS257-434E8R-3542R-DXS6762-DXS7999-DXS 6763-434E8L-DXS8000-DXS6760-DXS7176- DXS8001-DXS999-3176R-PHKA2-Xcen . A long-range restriction map was constructed, and the RS region is estimated to be 1300 kb, containing three putative CpG islands . An unstable region was identified between DXS6763 and 434E8L . These data will facilitate positional cloning of RS and other disease genes in Xp22.

J Inherit Metab Dis, 1997 Sep, 20(5), 633 - 42
Biochemical characterization of the S135L allele of galactose-1-phosphate uridylyltransferase associated with galactosaemia; Wells L et al.; Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosaemia . The S135L mutation, which accounts for almost 50% of the GALT alleles in galactosaemia patients of African-American descent, has been associated with activities ranging from null to wild-type by different investigators examining cell lysates representing different tissues or model systems . Because of the crude nature of the lysates examined, however, and the absence of quantitative measures concerning GALT abundance in most of those lysates, the available data do not distinguish between differences in GALT enzyme expression/abundance, specific activity, or kinetic constants in these different tissues or systems . In an effort to overcome this uncertainty and investigate the biochemical impact of the S135L substitution on human GALT function under defined conditions, we have overexpressed both wild-type and S135L-mutant GALT sequences in a null-background yeast expression system, and purified both proteins to near homogeneity . Abundance of the wild-type and mutant proteins in crude yeast lysates differed by approximately 2-fold . Kinetic studies of the purified proteins, however, demonstrated that although K(m) values differed by < 2-fold, specific activities differed by 10-fold . Temperature-activity profiles revealed no significant differences, and coprecipitation studies demonstrated that S135L-hGALT subunits remained competent to self-associate in vivo . We conclude that the S135L substitution causes either steric or electrochemical changes sufficiently close to the active site in human GALT to result in partial impairment of the transferase reaction.

Plant Physiol, 1997 Sep, 115(1), 93 - 100
Phosphorylation by a cyclin-dependent kinase modulates DNA binding of the Arabidopsis heat-shock transcription factor HSF1 in vitro; Reindl A et al.; Phosphorylation is one of the mechanisms controlling the activity of heat-shock transcription factors in yeast and mammalian cells . Here we describe partial purification, identification, and characterization of a protein kinase that phosphorylates the Arabidopsis heat-shock factor AtHSF1 at multiple serine residues . The HSF1 kinase forms a stable complex with AtHSF1, which can be detected by kinase pull-down assays using a histidine-tagged AtHSF1 substrate . The HSF1 kinase interacts with the cell-cycle control protein Suc1p and is immunoprecipitated by an antibody specific for the Arabidopsis cyclin-dependent CDC2a kinase . Phosphorylation by CDC2a in vitro inhibits DNA binding of AtHSF1 to the cognate heat-shock elements, suggesting a possible regulatory interaction between heat-shock response and cell-cycle control in plants.

Cytogenet Cell Genet, 1997, 78(1), 65 - 8
Experimental assessment of the detection limit of genomic amplification by comparative genomic hybridization CGH; Parente F et al.; Artificial amplicons of known size, constructed by use of YACs featuring human 8p12 and 12q13, were analyzed by comparative genomic hybridization (CGH) . A minimum of 15 Mb of overrepresented DNA sequences could be detected . The sensitivity is (1) not dependent on the chromosome site and (2) related to the size of the amplicon, decreasing with decreasing size.

Cytogenet Cell Genet, 1997, 78(1), 12 - 9
Complex FISH probes for the subtelomeric regions of all human chromosomes: comparative hybridization of CEPH YACs to chromosomes of the Old World monkey Presbytis cristata and great apes; Kingsley K et al.; We have generated a human subtelomere probe panel, utilizing well characterized CEPH YACs, for the investigation of human chromosome pathology and evolution through fluorescent in situ hybridization (FISH) . Region-specific FISH probes will be extremely valuable for detecting cytogenetically cryptic telomere abnormalities . Here, we present the first comparative mapping study (with 29 subtelomere probes and 6 chromosome paints) to the Old World monkey Presbytis cristata, followed by hybridizations to the great apes, gorilla and orangutan, when rearrangements were detected . We observed that the position of telomere-associated genomic sequences has been only moderately conserved during primate evolution . YAC 364f9, specific for the subtelomeric long arm of human chromosome 3, contains an evolutionary inversion breakpoint that was involved in independent chromosome rearrangements in P . cristata and gorilla.

Oncogene, 1997 Sep 25, 15(13), 1587 - 97
The E1B 19K protein associates with lamins in vivo and its proper localization is required for inhibition of apoptosis; Rao L et al.; Expression of the E1B 19K protein is required to inhibit apoptosis induced by E1A during adenovirus infection and transformation . E1B 19K is homologous to Bcl-2 in function and the two proteins also share limited amino acid sequence homology . Consequently, the E1B 19K and Bcl-2 proteins bind to and inhibit the cellular death-inducing proteins Bax, Bak and Nbk/Bik . Both E1B 19K and Bcl-2 localize to membranes of the nucleus and the endoplasmic reticulum . In addition to membrane association, and unlike Bcl-2, the E1B 19K protein is found associated with intermediate filament proteins in the cytoplasm and the nuclear lamina and copurifies with the lamins both during infection and transformation . While a membrane targeting domain at the C-terminus of Bcl-2 ensures its proper localization, the mechanism by which the E1B 19K protein localizes is unknown . Not surprisingly, lamin A fragments were cloned from a yeast two-hybrid screen for E1B 19K-interacting proteins . The interaction was demonstrated in yeast and mammalian cells in vivo and in vitro and was unique and specific to E1B 19K, with no interaction evident between Bcl-2 and lamin A . Mutants of lamin A/C which localized inappropriately in the cytoplasm or nucleus but retained E1B 19K binding, interfered with the nuclear envelope and cytoplasmic membrane targeting of the E1B 19K protein . Improper localization impaired the ability of the E1B 19K protein to inhibit apoptosis . Thus, proper localization of the E1B 19K protein is required for its function and the interaction of the E1B 19K protein with lamin A/C may represent a means for nuclear envelope localization.

Oncogene, 1997 Sep 25, 15(13), 1565 - 72
Identification of a second Grb2 binding site in the v-Fms tyrosine kinase; Mancini A et al.; Tyrosine autophosphorylation of the v-Fms oncogene product results in the formation of high-affinity binding sites for cellular proteins containing Src homology 2 (SH2) domains . These proteins transduce various mitogenic and morphogenic signals . As reported previously, Y696KNI in the kinase insert domain of v-Fms binds to the growth factor receptor bound protein 2 (Grb2), a stimulator of the Ras/Raf1 pathway . Here, we mapped Y921TNL within the C-terminal domain of Fms as a novel autophosphorylation site . We demonstrate that this site constitutes a second Grb2 binding site: a recombinant fusion protein (residues 904-944) containing phosphorylated Y921 bound Grb2 from FDCP-1Mac11 cell extracts significantly more efficiently than a corresponding protein (residues 617-759) containing Y696 . A yeast two-hybrid system which allowed the formation of a functional Fms tyrosine kinase was employed to quantify binding of Grb2 . Fms-protein containing either one of the two phosphorylation sites bound Grb2 equally well, binding was increased for proteins carrying both sites . In contrast, the simultaneous substitution of Y696 and Y921 by phenylalanines abolished Grb2 binding . Mouse NIH3T3 cells expressing the Y921F mutant Fms-protein showed a substantially higher content of fibronectin network than wild-type transformed cells and had largely lost their serum independent growth phenotype.

Med Klin (Munich), 1997 Sep 15, 92 Suppl 3, 42 - 5
Reduction of cancer mortality and incidence by selenium supplementation; Combs GF Jr et al.; PATIENTS AND METHOD: In order to test the hypothesis that a dietary supplement of selenium (Se) may reduce cancer risk, 1312 patients with histories of basa/squamous cell carcinomas of the skin were assigned in random, double-blind fashion to daily oral supplements of either Se-enriched yeast (200 micrograms Se/day), or a low-Se yeast placebo . Patients were recruited in 1983 to 1990 and were followed with regular dermatologic examinations through, 1993 for a total of 8269 person-years of observation . Skin cancer diagnoses were confirmed histologically and plasma Se concentration was determined at 6 to 12 months intervals . All deaths and patient-reported illnesses were confirmed and documented by consultation with the patient medical care providers . RESULTS: Results showed that Se-supplementation did not significantly affect the incidences of recurrent basal/squamous cell carcinomas of the skin . However, Se-treatment was associated with reductions in total cancer mortality and in the incidences of lung, colorectal, prostate and total cancers . These effects were consistent over time and between study clinics . CONCLUSION: The results strongly suggest benefits of Se-supplementation for this cohort of patients and support the hypothesis that supplemental Se can reduce risks to at least some types of cancer.

Mol Cell Biol, 1997 Nov, 17(11), 6645 - 52
Processing of targeted psoralen cross-links in Xenopus oocytes; Segal DJ et al.; Psoralen cross-links have been shown to be both mutagenic and recombinagenic in bacterial, yeast, and mammalian cells . Double-strand breaks (DSBs) have been implicated as intermediates in the removal of psoralen cross-links . Recent work has suggested that site-specific mutagenesis and recombination might be achieved through the use of targeted psoralen adducts . The fate of plasmids containing psoralen adducts was evaluated in Xenopus oocytes, an experimental system that has well-characterized recombination capabilities and advantages in the analysis of intermediates in DNA metabolism . Psoralen adducts were delivered to a specific site by a triplex-forming oligonucleotide . These lesions are clearly recognized and processed in oocytes, since mutagenesis was observed at the target site . The spectrum of induced mutations was compared with that found in similar studies in mammalian cells . Plasmids carrying multiple random adducts were preferentially degraded, perhaps due to the introduction of DSBs . However, when DNAs carrying site-specific adducts were examined, no plasmid loss was observed and removal of cross-links was found to be very slow . Sensitive assays for DSB-dependent homologous recombination were performed with substrates with one or two cross-link sites . No adduct-stimulated recombination was observed with a single lesion, and only very low levels were observed with paired lesions, even when a large proportion of the cross-links was removed by the oocytes . We conclude that DSBs or other recombinagenic structures are not efficiently formed at psoralen adducts in Xenopus oocytes . While psoralen is not a promising reagent for stimulating site-specific recombination, it is effective in inducing targeted mutations.

Mol Cell Biol, 1997 Nov, 17(11), 6633 - 44
Identification of SH2-Bbeta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling; Rui L et al.; Activation of the tyrosine kinase JAK2 is an essential step in cellular signaling by growth hormone (GH) and multiple other hormones and cytokines . Murine JAK2 has a total of 49 tyrosines which, if phosphorylated, could serve as docking sites for Src homology 2 (SH2) or phosphotyrosine binding domain-containing signaling molecules . Using a yeast two-hybrid screen of a rat adipocyte cDNA library, we identified a splicing variant of the SH2 domain-containing protein SH2-B, designated SH2-Bbeta, as a JAK2-interacting protein . The carboxyl terminus of SH2-Bbeta (SH2-Bbetac), which contains the SH2 domain, specifically interacts with kinase-active, tyrosyl-phosphorylated JAK2 but not kinase-inactive, unphosphorylated JAK2 in the yeast two-hybrid system . In COS cells coexpressing SH2-Bbeta or SH2-Bbetac and murine JAK2, both SH2-Bbetac and SH2-Bbeta coimmunoprecipitate to a significantly greater extent with wild-type, tyrosyl-phosphorylated JAK2 than with kinase-inactive, unphosphorylated JAK2 . SH2-Bbetac also binds to immunoprecipitated wild-type but not kinase-inactive JAK2 in a far Western blot . In 3T3-F442A cells, GH stimulates the interaction of SH2-Bbeta with tyrosyl-phosphorylated JAK2 both in vitro, as assessed by binding of JAK2 in cell lysates to glutathione S-transferase (GST)-SH2-Bbetac or GST-SH2-Bbeta fusion proteins, and in vivo, as assessed by coimmunoprecipitation of JAK2 with SH2-Bbeta . GH promoted a transient and dose-dependent tyrosyl phosphorylation of SH2-Bbeta in 3T3-F442A cells, further suggesting the involvement of SH2-Bbeta in GH signaling . Consistent with SH2-Bbeta being a substrate of JAK2, SH2-Bbetac is tyrosyl phosphorylated when coexpressed with wild-type but not kinase-inactive JAK2 in both yeast and COS cells . SH2-Bbeta was also tyrosyl phosphorylated in response to gamma interferon, a cytokine that activates JAK2 and JAK1 . These data suggest that GH-induced activation and phosphorylation of JAK2 recruits SH2-Bbeta and its associated signaling molecules into a GHR-JAK2 complex, thereby initiating some as yet unidentified signal transduction pathways . These pathways are likely to be shared by other cytokines that activate JAK2.

J Virol, 1997 Nov, 71(11), 8928 - 32
Bovine herpesvirus 4 BORFE2 protein inhibits Fas- and tumor necrosis factor receptor 1-induced apoptosis and contains death effector domains shared with other gamma-2 herpesviruses; Wang GH et al.; Fas- and tumor necrosis factor receptor 1 (TNFR1)-induced apoptosis is mediated by the interaction of FADD with caspase-8 . Here, we report that the bovine herpesvirus 4 (BHV4) BORFE2 gene encodes a protein that inhibits Fas- and TNFR1-induced apoptosis and contains death effector domains (DEDs) . Using the yeast two-hybrid system, we found that the BORFE2 protein interacts with the prodomain of caspase-8 . Furthermore, we show that BHV4 BORFE2 is a member of a family of DED-containing proteins that includes other gamma-2 herpesviruses, such as Kaposi's sarcoma-associated herpesvirus and herpesvirus saimiri.

J Virol, 1997 Nov, 71(11), 8856 - 9
Hepatitis C virus nonstructural region 5A protein is a potent transcriptional activator; Kato N et al.; The hepatitis C virus (HCV) nonstructural region 5A (NS5A) protein, without its 146 amino-terminal amino acids and fused to the DNA-binding domain of GAL4, strongly activates transcription in yeast and human hepatoma cells . Transcriptional activation by the HCV NS5A protein may play a role in viral replication and hepatocarcinogenesis.

J Chromatogr B Biomed Sci Appl, 1997 Sep 12, 697(1-2), 111 - 21
Capillary electrophoresis of recombinant proteins; Denton KA et al.; Many naturally occurring proteins which are used therapeutically have been cloned and expressed in large quantities in bacterial, yeast or mammalian systems . Purification of these proteins by column chromatography generates high purity products with low levels of host protein contaminants . However, isoforms of the desired protein may be present at variable concentrations . Analysis of these variant forms has been enhanced by the utilisation of capillary electrophoresis (CE), a highly efficient, widely applicable technique which is increasingly used in the field of biotechnology . The role of CE in the analysis of recombinant proteins is reviewed with respect to microcharacterisation, comparison of natural and recombinant proteins, separation of mutant or variant forms and analysis of glycoforms . Examples of these applications are described and illustrated with analysis of recombinant human albumin . The rapid development of CE, further enhancing its versatility, and its use with complementary analytical techniques is also discussed.

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 12053 - 8
Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2; Kuziel WA et al.; CC chemokine receptor 2 (CCR2) is a prominent receptor for the monocyte chemoattractant protein (MCP) group of CC chemokines . Mice generated by gene targeting to lack CCR2 exhibit normal leukocyte rolling but have a pronounced defect in MCP-1-induced leukocyte firm adhesion to microvascular endothelium and reduced leukocyte extravasation . Constitutive macrophage trafficking into the peritoneal cavity was not significantly different between CCR2-deficient and wild-type mice . However, after intraperitoneal thioglycollate injection, the number of peritoneal macrophages in CCR2-deficient mice did not rise above basal levels, whereas in wild-type mice the number of macrophages at 36 h was approximately 3.5 times the basal level . The CCR2-deficient mice showed enhanced early accumulation and delayed clearance of neutrophils and eosinophils . However, by 5 days neutrophils and eosinophils in both CCR2-deficient and wild-type mice had returned to near basal levels, indicating that resolution of this inflammatory response can occur in the absence of macrophage influx and CCR2-mediated activation of the resident peritoneal macrophages . After intravenous injection with yeast beta-glucan, wild-type mice formed numerous large, well-defined granulomas throughout the liver parenchyma, whereas CCR2-deficient mice had much fewer and smaller granulomas . These results demonstrate that CCR2 is a major regulator of induced macrophage trafficking in vivo.

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 12036 - 40
Incomplete penetrance of familial retinoblastoma linked to germ-line mutations that result in partial loss of RB function; Otterson GA et al.; To study the molecular basis for the clinical phenotype of incomplete penetrance of familial retinoblastoma, we have examined the functional properties of three RB mutations identified in the germ line of five different families with low penetrance . RB mutants isolated from common adult cancers and from classic familial retinoblastoma (designated as classic RB mutations) are unstable and generally do not localize to the nucleus, do not undergo cyclin-dependent kinase (cdk)-mediated hyperphosphorylation, show absent protein "pocket" binding activity, and do not suppress colony growth of RB(-) cells . In contrast, two low-penetrant alleles (661W and "deletion of codon 480") retained the ability to localize to the nucleus, showed normal cdk-mediated hyperphosphorylation in vivo, exhibited a binding pattern to simian virus 40 large T antigen using a quantitative yeast two-hybrid assay that was intermediate between classic mutants (null) and wild-type RB, and had absent E2F1 binding in vitro . A third, low-penetrant allele, "deletion of RB exon 4," showed minimal hyperphosphorylation in vivo but demonstrated detectable E2F1 binding in vitro . In addition, each low-penetrant RB mutant retained the ability to suppress colony growth of RB(-) tumor cells . These findings suggest two categories of mutant, low-penetrant RB alleles . Class 1 alleles correspond to promoter mutations, which are believed to result in reduced or deregulated levels of wild-type RB protein, whereas class 2 alleles result in mutant proteins that retain partial activity . Characterization of the different subtypes of class 2 low-penetrant genes may help to define more precisely functional domains within the RB product required for tumor suppression.

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 12030 - 5
A gene spans the pseudoautosomal boundary in mice; Palmer S et al.; The X and Y chromosomes of the mouse, like those of other mammals, are heteromorphic over most of their length, but at the distal ends of the chromosomes is a region of sequence identity, the pseudoautosomal region (PAR), where the chromosomes pair and recombine during male meiosis . The point at which the PAR diverges into X- and Y-specific sequences is called the pseudoautosomal boundary . We have completed a genomic walk from the X-specific Amelogenin gene to the PAR . Analysis of this region revealed that the pseudoautosomal boundary of mice is located within an intron of a transcribed gene that encodes a novel RING finger protein . The first three of the exons of the gene are located on the X chromosome whereas the 3' exons of the gene are located on both X and Y chromosomes . This unusual arrangement may indicate that the gene is in a state of transition from pseudoautosomal to X-unique and provides evidence for a process of attrition of the pseudoautosomal region on the Y chromosome.

Development, 1997 Sep, 124(18), 3621 - 32
Imprinting of Igf2 and H19 from a 130 kb YAC transgene; Ainscough JF et al.; A stringent test for imprint control elements is to examine their function at ectopic loci in transgenic experiments . Igf2 and H19 are part of a larger imprinting region and as a first step, we examined these reciprocally imprinted genes in transgenic experiments using a 130 kb YAC clone . After paternal inheritance, H19 was appropriately repressed and Igf2 was expressed, irrespective of copy number or genetic background . After maternal inheritance H19 was consistently expressed, albeit with some variability . The levels of H19 expression per copy of the transgene inversely correlated with Igf2 (-lacZ) expression in cis . The consistent imprinting of H19 from this YAC contrasts with the previously described imprinting of mini-H19 transgenes, which only occurs at multi-copy loci, is inconsistent, and is prone to genetic background effects . We propose a novel model in which silencing of the H19 gene is the default state and its activation after maternal inheritance is the key mechanistic event for imprinting in this region . In addition, in situ analysis of the Igf2-lacZ reporter indicates that additional mesoderm-specific enhancers are present within the YAC clone . No obvious phenotype was detected from the excess gene dosage of H19.

Hum Genet, 1997 Oct, 100(5-6), 595 - 600
A third neurofibromatosis type 1 (NF1) pseudogene at chromosome 15q11.2; Kehrer-Sawatzki H et al.; Sequences related to the neurofibromatosis type 1 (NF1) gene have been identified on several human chromosomes . In the centromeric region of chromosomes 14 and 15, two NF1 pseudogenes have been described . Sequence comparison between NF1-related exons amplified from two yeast artificial chromosome clones hybridizing to chromosomal region 15q11.2 and published NF1-related sequences localized at 15q11.2 suggested that a third NF1 pseudogene resides in this chromosomal region . The previous localization of an NF1-related locus to the telomeric part of chromosome 15 could not be confirmed by us . Our findings further support pericentromeric spreading of partial NF1 gene copies at chromosome 15q11.2 during evolution.

Hum Genet . 1997 Oct;100(5-6):569-72 ;|
AFX1 and p54nrb: fine mapping, genomic structure, and exclusion as candidate genes of X-linked dystonia parkinsonism; Peters U et al.; We have mapped AFX1 and p54nrb to a yeast artificial chromosome (YAC) contig of Xq13.1 that harbors the X-linked dystonia parkinsonism (XDP) locus DYT3 . AFX1 is flanked by loci DXS7116 and Il2R gamma, and p54nrb by loci DXS6673E and DXS7120 . The exon-intron structure of both genes was analyzed . AFX1 is composed of three exons with most of exon 3 being untraslated . p54nrb is made up of 12 exons ranging in size from 40 bp to 1227 bp . The start codon is in exon 3 and the stop codon in exon 12 . Both genes are expressed in the brain, among other tissues . AFX1 and p54nrb were excluded as candidates of DYT3 by sequencing of the exons and the flanking intronic sequences in an XDP patient and a control, and by Northern blot analysis.

J Biol Chem, 1997 Oct 24, 272(43), 26893 - 8
Expression and secondary structure determination by NMR methods of the major house dust mite allergen Der p 2; Mueller GA et al.; There exists a strong correlation between asthma and sensitization to indoor allergens . This study reports on the secondary structure of the major house dust mite allergen Der p 2, determined using heteronuclear NMR methods . The DNA was subcloned from the yeast expression vector pSAY1 into the high yield bacterial expression vector pET21a, resulting in yields of 50 mg/liter . The recombinant protein was shown to have immunoreactivity comparable with that of the natural mite protein using competitive inhibition enzyme-linked immunosorbent assay (ELISA) and a modified monoclonal radioallergosorbent test (RAST) . The secondary structure was determined by examining chemical shifts, short and long range NOESYs, JHN-HA coupling constants, and amide exchange rates . From these data, it is clear that Der p 2 is composed of beta-sheets and random coil . Based on long range distance constraints, a number of beta-strands were aligned into two three-stranded, anti-parallel beta-sheets.

J Biol Chem, 1997 Oct 24, 272(43), 27178 - 82
SOCS-1/JAB/SSI-1 can bind to and suppress Tec protein-tyrosine kinase; Ohya K et al.; Tec is the prototype of a recently emerging subfamily among nonreceptor type protein-tyrosine kinases and is known to become tyrosine-phosphorylated and activated by a wide range of cytokine stimulations in hematopoietic cells . Although Tec was recently shown to be involved in the cytokine-driven activation mechanism of c-fos transcription, it is yet obscure how Tec relays the signals from cell surface receptors to the nucleus . To identify signaling molecules acting downstream of Tec, we have looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid system . Here we report the identification and characterization of a novel protein, TIP3, which has been simultaneously identified by other groups as SOCS-1, JAB, or SSI-1 . TIP3 carries one Src homology 2 domain with a sequence similarity to that of CIS . In 293 cells, TIP3 associates with Tec and suppresses its kinase activity . Interestingly, TIP3 can also down-regulate the activity of Jak2 but not that of Lyn . We propose that SOCS-1/JAB/SSI-1/TIP3 is a novel type of negative regulator to a subset of protein-tyrosine kinases.

Mol Cells, 1997 Aug 31, 7(4), 567 - 71
Isolation of molecular markers for salt stress responses in Arabidopsis thaliana; Pih KT et al.; Characterization of many osmotic stress-induced genes has greatly contributed to the understanding of the physiological responses of plant cells to osmotic stress at the molecular level . In this study we constructed a subtraction library and generated 15 salt stress-inducible ESTs from this library to use as molecular markers that reflect the cellular responses to salt stress responses in Arabidopsis . The sequence analysis showed that 5 salt stress-inducible ESTs were identical to previously identified genes in Arabidopsis, 6 cDNAs were homologous to known genes found in plants as well as yeast, and 4 cDNAs were new genes . To confirm that expression of these clones are induced by salt stress, we carried out Northern blot analysis . When we examined for 15 cDNA clones, they were indeed induced by NaCl treatment . The induction level was variable among these genes ranging from approximately 2-fold to more than 50-fold . Also, Northern blot analysis revealed that these genes can be divided into three different induction patterns: early induction, late induction, and continuous induction.

Mol Cells, 1997 Aug 31, 7(4), 482 - 8
The effect of dietary threonine on Adh expression during the development of Drosophila melanogaster; Kang SJ et al.; The alcohol dehydrogenase (ADH) activity and ADH cross reacting material (ADH CRM) were measured and Northern blot analysis was carried to define the function and the regulation mechanism of the Adh gene . This study examined how dietary threonine affects the expression of the Adh gene during development of Drosophila melanogaster . Two wild type strains, one homozygous for Adh(F) and one for Adh(S) from Chunan, Korea were used . The ADH activity and CRMs of the Adh(F) strain were 2.1 times higher than those of Adhs strain, and ADH activity was higher with isopropanol (secondary alcohol) than with ethanol (primary alcohol) as a substrate in both Adh(F) and Adh(S) strains . When the larvae, pupae, newly emerged adults (0-1 day), and adults (5-7 days) of Drosophila melanogaster Adh(F) and Adh(S) strains were fed on a defined low yeast and threonine medium, ADH activity and ADH CRM levels were increased . Northern blot analyses indicated that the production of mRNA of the larvae, young adults (0-1 day), and adults (5-7 days) was increased by dietary threonine . ADH activity and ADH CRM increases in Drosophila melanogaster fed on threonine were as a result of threonine-stimulated alteration in the amount of ADH mRNA . The elevated level of the ADH mRNA transcribed from the proximal and distal promoters of threonine-fed larvae and adults showed that there was an induction.

Regul Toxicol Pharmacol, 1997 Aug, 26(1 Pt 1), 96 - 101
Failure to confirm estrogenic activity for benzoic acid and clofibrate: implications for lists of endocrine-disrupting agents; Ashby J et al.; Earlier reports that benzoic acid is uterotrophic to the rat and mouse and that clofibrate is uterotrophic to the rat have not been confirmed . The studies reported here involved the use of a range of test protocols and dose levels, including the protocols/dose levels used by the original investigators . In addition, both chemicals were inactive in a human estrogen receptor (hER alpha) yeast estrogenicity assay . It is concluded that benzoic acid and clofibrate are not estrogenic in the assays used here . This conclusion has implications for the compilation of lists of endocrine-disrupting chemicals.

Genomics, 1997 Oct 1, 45(1), 140 - 6
Mapping of 228 ESTs and 26 genes into an integrated physical and genetic map of human chromosome 17; Plummer SJ et al.; We have integrated genetic and physical mapping data for chromosome 17 subdivided into 26 bins, by using a panel of chromosome 17 deletion somatic cell hybrids . One hundred four short tandem repeat and STS markers have been localized into these bins and have enabled the ordering of 288 ESTs and 26 genes, including 142 ESTs that had not been previously sublocalized on chromosome 17 . The mapping information of several genetic maps, as well as information obtained by radiation hybrid and STS content mapping of YACs, has been integrated using this hybrid panel . Although existing mapping information for chromosome 17 was generally consistent for many ESTs previously mapped, the map presented here further refines the location of ESTs, as well as demonstrating a number of discrepancies found in the 17q24-q25 region . We attribute these discrepancies to the fact that the current radiation hybrid panels were selected for retention of the thymidine kinase gene at 17q25, as well as to a low concentration of YAC contigs in this region . These data illustrate the benefit of combining multiple mapping techniques to obtain the greatest accuracy . The integration of maps developed by different methods will generate the most accurate genome maps, which may then be used for the generation of large insert clone contigs for chromosome sequencing . Additionally, accurate transcript maps generated by ESTs will greatly speed the isolation of genes linked to disease loci.

Genomics, 1997 Oct 1, 45(1), 88 - 96
Novel genes mapping to the critical region of the 5q- syndrome; Boultwood J et al.; The 5q- syndrome is a myelodysplastic syndrome with specific hematological features and a good prognosis . Using molecular mapping techniques, we have previously defined the critical region of gene loss of the 5q- chromosome in the 5q- syndrome as the approximately 5-Mb region at 5q31-q33 flanked by the genes for FGF1 and IL12B . This region is completely represented by a series of overlapping YACs, and we are currently generating a transcription map with the aim of identifying the tumor-suppressor gene associated with the development of the 5q- syndrome . In this study two techniques have been used: first, the screening of full-length cDNA libraries with radiolabeled YACs and second, the mapping of chromosome 5-specific expressed sequence tags (ESTs) to a YAC contig . A 1-Mb YAC contig encompassing the CSF1R gene has been used to screen a fetal brain cDNA library, and this has resulted in the identification of two genes comprising one known gene previously localized to the region (ADRB2) and one known gene previously unlocalized . Six of 135 chromosome 5-specific ESTs were localized by PCR screening to the YAC contig mapping to the critical region of the 5q- syndrome . IMAGE cDNA clones for each of the six ESTs have been obtained . These seven (excluding ADRB2) newly assigned cDNA clones were subjected to further analysis . The expression patterns of each of the cDNA clones have been established in a range of human tissues, including bone marrow . Six of seven cDNAs are expressed in human bone marrow . Six of seven cDNAs have no known homology to any deposited human sequences, and one (C29) is dihydropyrimidinase-related protein-3, a member of a novel gene family . Genomic localization and expression patterns would suggest that these newly assigned cDNAs represent potential candidate genes for the 5q- syndrome.

Genomics, 1997 Oct 1, 45(1), 78 - 87
Estimation of the age of the ancestral arginine3500-->glutamine mutation in human apoB-100; Myant NB et al.; Familial defective apoB-100 (R3500Q) {FDB (R3500Q)} is caused by a mutation in the apoB gene (2p23.24) . Almost all individuals with this disorder are of European descent, and in almost all cases the mutation is on a chromosome with a rare haplotype (194) at the apoB locus, suggesting that all FDB (R3500Q) probands are descended from a common ancestor in whom the original mutation occurred . The distribution of the mutation is consistent with an origin in Europe 6000-7000 years ago . We have estimated the amount of recombination between the apoB gene and markers on chromosome 2 in 34 FDB (R3500Q) probands in whom the mutation is on a 194 haplotype . Significant linkage disequilibrium was found between the apoB gene and marker D2S220 . We have identified three YACs that contain the apoB gene and D2S220 . The shortest restriction fragment common to the three YACs that contained both loci was 240 kb long . No shorter fragments with both loci were identified . On the assumption that 1000 kb corresponds to 1 cM, we deduce that the recombination distance between D2S220 and the apoB gene is about 0.24 cM . Combining this value with the linkage disequilibrium observed between the two loci in the probands, we estimate that the ancestral mutation occurred about 270 generations ago . We postulate that the original mutation occurred in the common ancestor of living FDB (R3500Q) probands, who lived in Europe about 6750 years ago . The errors in this estimate are discussed.

Bull Cancer, 1997 Jul, 84(7), 763 - 6
{Ataxia-telangiectasia and cancer: an open question}; Rodriguez C et al.; Ataxia-telangiectasia is a rare recessive disorder which, among other clinical signs, is characterized by an extreme sensitivity to ionising radiation . Cells isolated from AT patients show radioresistant DNA synthesis and this has lead to the hypothesis that the product of the genetic determinant of AT may play a role in the detection, signalling or repair of double stranded DNA breaks . The gene to AT, called ATM has been recently cloned and characterized . It codes for a large RNA transcript of 13,000 bp of which a 3,500 aa protein is translated . The gene itself covers 150 kb, spread over 64 exons . The amino acid sequence has revealed the existence, at the carboxyterminal end of the protein, of a domain presenting homology to PI-3 kinase . This characteristic has allowed the description of a new family of nuclear protein, in yeast, drosophila an human, functionally involved in DNA damage signalling . It is interesting to note that a vast majority of mutations described in AT patients lead to the truncation of the protein and consequently to the elimination of the PI-3K domain, thus suggesting an important role in the normal function of the protein . An important question linked to AT mutation concerns the cancer risk associated to heterozygous mutations . It is well established that AT patients, homozygous for the mutation, present a 100-200 fold increased risk of cancer . Epidemiological studies have described a 3-5 fold increase risk of cancer (particular breast cancer in women) associated to the heterozygous mutation . Knowing that the incidence of the heterozygotes can be estimated to range 0.5 to 1% in the general population this question is of great importance in terms of public health.

Bull Cancer, 1997 Jul, 84(7), 735 - 40
{Li-Fraumeni syndrome}; Frebourg T; The Li-Fraumeni syndrome is an autosomal dominant syndrome representing a genetic predisposition to a wide spectrum of tumours including sarcomas, breast carcinomas, brain tumors and adrenocortical carcinomas . In most of the cases, tumours will develop in children and young adults . Germline mutations of the tumor suppressor gene p53 have been identified in approximately 50% of the families . In most of the cases, germline p53 mutations are missense mutations, located between exon 5 and exon 8, within the DNA-binding domain of p53 . Since these mutations inactivate the transcriptional activity of the protein, they can easily be detected by analyzing in yeast the transcriptional competence of p53 cDNA derived from lymphocytes . The presence of a germline p53 mutations must be considered in: (1) families including two first degree relatives with cancers belonging to the Li-Fraumeni spectrum, one relative being affected before age 45; (2) children or young adults with a rare tumour of in the general population, belonging to the Li-Fraumeni spectrum, such as adrenocortical carcinoma; and (3) children or young adults under age 45 with multiple primary tumours of the Li-Fraumeni spectrum . Identification of a germline p53 mutation in an affected subject allows to establish the diagnosis of the Li-Fraumeni syndrome on a molecular basis.

Plant Cell, 1997 Sep, 9(9), 1633 - 46
Recombination occurs uniformly within the bronze gene, a meiotic recombination hotspot in the maize genome; Dooner HK et al.; The bronze (bz) gene is a recombinational hotspot in the maize genome: its level of meiotic recombination per unit of physical length is > 100-fold higher than the genome's average and is the highest of any plant gene analyzed to date . Here, we examine whether recombination is also unevenly distributed within the bz gene . In yeast genes, recombination (conversion) is polarized, being higher at the end of the gene where recombination is presumably initiated . We have analyzed products of meiotic recombination between heteroallelic pairs of bz mutations in both the presence and absence of heterologies and have sequenced the recombination junction in 130 such Bz intragenic recombinants . We have found that in the absence of heterologies, recombination is proportional to physical distance across the bz gene . The simplest interpretation for this lack of polarity is that recombination is initiated randomly within the gene . Insertion mutations affect the frequency and distribution of intragenic recombination events at bz, creating hotspots and coldspots . Single base pair heterologies also affect recombination, with fewer recombination events than expected by chance occurring in regions of the bz gene with a high density of heterologies . We also provide evidence that meiotic recombination in maize is conservative, that is, it does not introduce changes, and that meiotic conversion tracts are continuous and similar in size to those in yeast.

Plant Cell, 1997 Sep, 9(9), 1595 - 606
A conserved family of WD-40 proteins binds to the retinoblastoma protein in both plants and animals; Ach RA et al.; In mammalian cells, the retinoblastoma (RB) protein regulates G1 progression and functions through its association with various cellular proteins . Two closely related mammalian RB binding proteins, RbAp48 and RbAp46, share sequence homology with the Msi1 protein of yeast . MSI1 is a multicopy suppressor of a mutation in the IRA1 gene involved in the Ras-cAMP pathway that regulates cellular growth . Human RbAp48 is present in protein complexes involved in histone acetylation and chromatin assembly . We report the cloning of cDNAs encoding four plant RbAp48- and Msi1-like proteins: one from tomato, LeMSI1, and three from Arabidopsis . Complementation studies confirm that LeMSI1 can function as a multicopy suppressor of the yeast ira1 mutant phenotype . The LeMSI1 protein localizes to the nucleus and binds to a 65-kD protein in wild-type as well as ripening inhibitor (rin) and Neverripe (Nr) tomato fruit . LeMSI1 also binds to the human RB protein and the RB-like RRB1 protein from maize, indicating that this interaction is conserved between plants and animals.

Rev Argent Microbiol, 1997 Jul-Sep, 29(3), 131 - 6
Nutritional requirements for growth of an endophyte: Ceratopycnidium baccharidicola; Bertoni MD et al.; The carbon sources, nitrogen sources and vitamin requirements for the growth of Ceratopycnidium baccharidicola (an endophyte of Baccharis coridifolia) were studied . The fungus utilized several carbon sources: pectin, sucrose, fructose, glucose, maltose, cellobiose, xylose, arabinose, mannitol, mannose and sorbitol . Sucrose and fructose were found to be the best carbon sources . Nitrogen sources utilized by the endophyte included: nitrates, ammonium and amino acids such as proline, asparagine and glycine . Undefined complex nitrogen sources such as soytone, tryptone, yeast extract and casamino acids supported excellent growth . In a defined medium, thiamine was the only vitamin required for growth . Under optimum conditions the vegetative growth of C . baccharidicola was enhanced six fold over its growth in glucose-asparagine medium.

Nature, 1997 Oct 16, 389(6652), 745 - 9
Imprinted expression of the Igf2r gene depends on an intronic CpG island; Wutz A et al.; Gametic imprinting is a developmental process that induces parental-specific expression or repression of autosomal and X-chromosome-linked genes . The mouse Igf2r gene (encoding the receptor for insulin- like growth factor type-2) is imprinted and is expressed from the maternal allele after embryonic implantation . We previously proposed that methylation of region 2, a region rich in cytosine-guanine doublets (a 'CpG island') in the second intron of Igf2r, is the imprinting signal that maintains expression of the maternal allele . Here we use mouse transgenes to test the role of region 2 and the influence of chromosome location on Igf2r imprinting . Yeast artificial chromosome transgenes successfully reproduced the imprinted methylation and expression pattern of the endogenous Igf2r gene; deletion of region 2 from these transgenes caused a loss of imprinting and restored biallelic Igf2r expression . These results define a primary role for region 2 and a negligible role for chromosomal location in Igf2r imprinting; they also show that methylation imprints can maintain allelic expression . Short transgenes containing only region 2 and yeast artificial chromosome transgenes with an inactive Igf2r promoter do not attract parental-specific methylation . All transgenes showing paternal-specific repression of Igf2r produced an antisense RNA whose transcription was dependent on region 2 . The production of an antisense RNA by the repressed parental allele is reminiscent of the imprinting of the Igf2/H19 gene pair and may indicate that expression competition could play a general role in imprinting.

Blood, 1997 Oct 15, 90(8), 3130 - 5
Abnormalities of chromosome band 8p11 in leukemia: two clinical syndromes can be distinguished on the basis of MOZ involvement; Aguiar RC et al.; Two distinct leukemia syndromes are associated with abnormalities of chromosome band 8p11 . First, a myeloproliferative disorder with features characteristic of both chronic myeloid leukemia and non-Hodgkin's lymphoma and second, an acute myeloid leukemia (AML) with French-American-British (FAB) M4/5 morphology and prominent erythrophagocytosis . The two syndromes are exemplified by a t(8;13)(p11;q12) and a t(8;16)(p11;p13), respectively, but cytogenetic variants of both have been described . Recently, the t(8;16) has been cloned and shown to fuse the MOZ gene at 8p11 to the CBP gene at 16p13 . We have used fluorescence in situ hybridization (FISH), Southern blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) to refine the 8p11 breakpoint in three cases with t(8;13)(p11;q12) and in a single case of AML-M5 with a clinical picture apparently identical to that found in patients with a t(8;16), but characterized by an inv(8)(p11q13) . FISH analysis was performed with several 8p11 CEPH yeast artificial chromosome (YAC) clones . YAC 782H11 was centromeric to the one case with t(8;13) tested, but was telomeric to the inv(8) . YAC 847B12 was telomeric to both the t(8;13) and the inv(8), whereas YAC 829D12 was centromeric to the t(8;13), but split by the inv(8) . Southern blotting and PCR of YAC 829D12 showed that it contained the MOZ gene . A 900-bp MOZ fragment encompassing the published t(8;16) breakpoint was amplified by PCR from normal peripheral blood leukocyte cDNA and used to probe Southern blots of patient DNA . A rearrangement was detected in the case with inv(8), but not in any of the three cases with t(8;13) . Southern blotting with a CBP probe and RT-PCR with MOZ and CBP primers suggested that the inv(8) does not result in a cryptic MOZ-CBP fusion . It is likely, therefore, that MOZ is fused to a novel gene at 8q13 in this case . We conclude that the t(8;13) breakpoint is flanked by YACs 782H11 and 847B12 and is at least 1 Mb telomeric to MOZ . MOZ is involved, however, in a new variant of the t(8;16).

Cancer Surv, 1997, 29, 263 - 84
Telomerase, checkpoints and cancer; Harley CB et al.; Telomere dynamics and changes in telomerase activity are consistent elements of cellular alterations associated with changes in proliferative state . In particular, the highly specific correlations and early causal relationships between telomere loss in the absence of telomerase activity and replicative senescence or crisis, on the one hand, and telomerase reactivation and cell immortality, on the other, point to a new and important paradigm in the complementary fields of ageing and cancer . Although the signalling pathways between telomeres and transcriptional and cell cycle machinery remain undefined, recently described homologies between telomeric proteins and lipid/protein kinase activities important in chromosome stability provide evidence for the existence of pathways transducing signals originating in chromosome structure to cell cycle regulatory processes . Similarities between cell cycle arrest at senescence and the response of mortal cells to DNA/oxidative damage suggest overlap in the signal transduction mechanisms culminating in irreversible and stable cell cycle arrest . The feasibility of targeting telomeres/telomerase as a strategy for antiproliferative therapeutics has been shown in studies in yeast, in which mutations in specific telomere associated genes result in delayed cell death . Similarly, antisense oligonucleotide inhibition of telomerase activity in human tumour cells (HeLa) results in delayed cell death . The mechanism of cell death and possible escape from this fate require further study . In human cells, however, it would seem reasonable to predict that in these circumstances, apoptosis is induced in the vast majority of cells either directly in response to a DNA damage signal arising from critically shortened telomeres or as a secondary consequence of genetic instability.

Cancer Surv, 1997, 29, 151 - 82
Mammalian G1 and G2 phase checkpoints; O'Connor PM; This present review explores the mechanisms for DNA damage induced G1 and G2 arrest in mammalian cells . The complexity of the TP53 pathway is attested to by the variety of genes regulated by TP53, many of which require further investigation to bring their importance into focus . One gene intensely studied, p21, has been linked to the G1 arrest mechanism and may, like TP53, be involved in some aspect of DNA repair . The outcome of TP53 activation for cell survival is equally complex and relies much upon cellular context and the type of DNA damaging agent employed . Although TP53 may participate in sensing DNA damage, additional components are likely to be required . Much of the focus on defining the mechanism of G2 arrest in mammalian cells has concentrated on the cyclin B1/CDC2 kinase . Activation of this kinase is suppressed by DNA damage, and this may result from the imposition of inhibitory phosphorylations on the CDC2 kinase as well as downregulation of cyclin B1 levels . The logical point where the G2 checkpoint interacts with the CDC2-CDC25C autocatalytic loop to prevent CDC2 activation remains to be defined and could involve inhibition of CDC25C-CDC2 interaction . It is hoped that moving upstream of CDC2 towards the point where DNA damage is sensed by the cell will uncover homologues of yeast components implicated in G2 checkpoint control . The finding that certain G2 checkpoint abrogators preferentially synergize with DNA damaging agents in cells with defective TP53 provides a potential pharmacological route through which TP53 defective cells might be targeted for destruction . Further exploration of this vulnerability might prove useful for future anti-cancer drug discovery efforts.

Cancer Surv, 1997, 29, 75 - 90
The DNA replication licensing system; Thommes P et al.; The Xenopus cell free system has proved a good model system to study in vitro DNA replication and the mechanism preventing rereplication in a single cell cycle . Studies using this system resulted in the development of a model postulating the existence of a replication licensing factor (RLF), which binds to the chromatin before the G1-S transition of the cell cycle and is displaced during replication . The nuclear envelope prevents rebinding of RLF and hence relicensing . Nuclear envelope breakdown at mitosis is required to allow another round of replication . Protein kinase inhibitors block licensing factor activity and arrest Xenopus extracts in a G2 like state . These kinase inhibitors have allowed the development of an in vitro assay leading to the biochemical purification of RLF components . RLF can be separated into RLF-B and RLF-M, the latter consisting of several members of the MCM/P1 class of replication proteins . In Xenopus as well as in many other eukaryotes, the binding of MCM/P1 proteins to chromatin before S phase is essential for replication to occur . The proteins are then displaced as replication proceeds . These changes in subnuclear distribution are reflected by changes in the phosphorylation status . MCM/P1 proteins do not bind to the DNA on their own but need RLF-B to be loaded onto the chromatin . Their cycling behaviour is reminiscent of the existence of a prereplicative complex at the origins of replication in yeast, suggesting that the licensing mechanism is ubiquitous in eukaryotes.

Biochem J, 1997 Aug 15, 326 ( Pt 1), 21 - 9
The third member of the Tetrahymena CCT subunit gene family, TpCCT alpha, encodes a component of the hetero-oligomeric chaperonin complex; Soares H et al.; The sequence of a third member of the Tetrahymena pyriformis chaperonin CCT ('chaperonin containing TCP1') subunit gene family is presented . This gene, designated TpCCT alpha, is the orthologue of the mouse chaperonin gene TCP1/CCT alpha . To characterize the CCT complex in this ciliate, we have produced polyclonal antibodies against synthetic peptides based on C-terminal sequences deduced from the primary sequences of the TpCCT alpha, TpCCT gamma and TpCCT eta subunits . We have also used polyclonal antibodies produced against recombinant yeast CCT alpha and CCT beta subunits . Using these antibodies, we show that Tetrahymena cells contain a hetero-oligomeric CCT chaperonin comprising at least seven distinct subunits . Three of these were assigned to specific TpCCT genes, whereas a fourth was recognized by the polyclonal antibody against yeast CCT beta, suggesting that this gene is also present in the ciliate . The CCT complex also contains other unidentified proteins that were recognized by the polyclonal antibody UM-1, raised against the putative ATP binding domain of the chaperonin proteins . TpCCT alpha gene expression was shown in exponentially growing cells and cells regenerating their cilia for different periods to have a similar pattern to the previously identified genes TpCCT gamma and TpCCT eta, and also to tubulin genes.

Genes Chromosomes Cancer, 1997 Oct, 20(2), 196 - 200
Isolation of osteosarcoma-associated amplified DNA sequences using representational difference analysis; Simons A et al.; Comparative genomic hybridization analysis of a primary osteosarcoma and its metastasis revealed two regions of DNA amplification, one at 17p11.2-12 and one at 19q12-13 . Subsequent representational difference analysis of the primary tumor resulted in the isolation of two distinct tumor-amplified DNA fragments originating from chromosome 19 . A YAC clone corresponding to one of the two isolated DNA fragments was used for fluorescence in situ hybridization on normal human lymphocyte metaphases and tumor-derived nuclei . This resulted in the localization of this YAC to 19q12-13.1 and confirmed the amplification status of the isolated fragment in the tumors . The availability of such RDA-isolated sequences may be instrumental in the search for genes relevant for tumor development.

Nucleic Acids Res, 1997 Nov 1, 25(21), 4278 - 86
Parameters controlling the rate of gene targeting frequency in the protozoan parasite Leishmania; Papadopoulou B et al.; In this study we investigated the role of several parameters governing the efficiency of gene targeting mediated by homologous recombination in the protozoan parasite Leishmania . We evaluated the relative targeting frequencies of different replacement vectors designed to target several sequences within the parasite genome . We found that a decrease in the length of homologous sequences <1 kb on one arm of the vector linearly influences the targeting frequency . No homologous recombination was detected, however, when the flanking homologous regions were <180 bp . A requirement for a very high degree of homology between donor and target sequences was found necessary for efficient gene targeting in Leishmania , as targeted recombination was strongly affected by base pair mismatches . Targeting frequency increased proportionally with copy number of the target only when the target was part of a linear amplicon, but remained unchanged when it was present on circles . Different chromosomal locations were found to be targeted with significantly variable levels of efficiency . Finally, different strains of the same species showed differences in gene targeting frequency . Overall, gene targeting mediated by homologous recombination in Leishmania shares similarities to both the yeast and the mammalian recombination systems.

Genetics, 1997 Oct, 147(2), 815 - 21
Analysis of recombination sites within the maize waxy locus; Okagaki RJ et al.; Genetic fine structure analysis of the maize wx locus has determined that the ratio of genetic to physical distance within wx was one to two orders of magnitude higher than the average for the maize genome . Similar results have been found at other maize loci . In this study, we examined several mechanisms that could account for this pattern . First, crossovers in two other maize genes resolve preferentially at specific sites . By mapping exchanges between wx-B1 and wx-I relative to a polymorphic SstI site, we found no evidence for such a hotspot at wx . Second, deletion of promoter sequences from wx alleles had little effect on recombination frequencies, in contrast to results in yeast where promoter sequences are important for initiating recombination in some genes . Third, high levels of insertion polymorphism may suppress intergenic recombination . However, the presence of a 2-kb Ds element 470 bp upstream of the wx transcription start site did not further suppress recombination between Ds insertions in nearby wx sequences . Thus, none of these mechanisms is sufficient to explain the difference between intergenic and intragenic recombination rates at wx.

Genetics, 1997 Oct, 147(2), 801 - 7
Detailed comparative mapping of cereal chromosome regions corresponding to the Ph1 locus in wheat; Foote T et al.; Detailed physical mapping of markers from rice chromosome 9, and from syntenous (at the genetic level) regions of other cereal genomes, has resulted in rice yeast artificial chromosome (YAC) contigs spanning parts of rice 9 . This physical mapping, together with comparative genetic mapping, has demonstrated that synteny has been largely maintained between the genomes of several cereals at the level of contiged YACs . Markers located in one region of rice chromosome 9 encompassed by the YAC contigs have exhibited restriction fragment length polymorphism (RFLP) using deletion lines for the Ph1 locus . This has allowed demarcation of the region of rice chromosome 9 syntenous with the ph1b and ph1c deletions in wheat chromosome 5B . A group of probes located in wheat homoeologous group 5 and barley chromosome 5H, however, have synteny with rice chromosomes other than 9 . This suggests that the usefulness of comparative trait analysis and of the rice genome as a tool to facilitate gene isolation will differ from one region to the next, and implies that the rice genome is more ancestral in structure than those of the Triticeae.

Genetics, 1997 Oct, 147(2), 787 - 99
Murine albino-deletion complex: high-resolution microsatellite map and genetically anchored YAC framework map; Rikke BA et al.; The murine albino-deletion complex developed as part of the Oak Ridge specific-locus test covers 6-11 cM of chromosome 7 . This complex has proven to be a valuable resource for localizing traits to a small target region for positional cloning . In this study, we mapped the endpoints of deletions in this complex using all of the available Mit simple-sequence length polymorphism (SSLP) markers . Concurrently, this mapping has determined the map order of nearly all of the SSLP markers, most of which were previously unresolved . The SSLP-based deletion map was confirmed and genetic distances were determined using the European Collaborative Interspecific Backcross panel of nearly a thousand mice . The average SSLP marker resolution is 0.3-0.4 cM, comparable to the cloning capacity of yeast artificial chromosomes (YACs) . The SSLP markers were then used to construct a genetically anchored YAC framework map that further confirms the deletion map . We find that the largest deleted region distal to Tyr is about two to three times larger than the largest proximal deletion region, and the original C3H/101 regions flanking the deletions (moved to an St2A cch/cch background) are smaller than anticipated, which we suggest may result from increased recombination rates immediately flanking the deleted regions.

Biochemistry, 1997 Oct 21, 36(42), 12672 - 82
The substrate binding site of human liver cytochrome P450 2C9: an NMR study; Poli-Scaife S et al.; Purified recombinant human liver cytochrome P450 2C9 was produced, from expression of the corresponding cDNA in yeast, in quantities large enough for UV-visible and 1H NMR experiments . Its interaction with several substrates (tienilic acid and two derivatives, lauric acid and diclofenac) and with a specific inhibitor, sulfaphenazole, was studied by UV-visible and 1H NMR spectroscopy . At 27 degrees C, all those substrates led to an almost complete conversion of CYP 2C9 to high-spin (S = 5/2) CYP 2C9-substrate complexes characterized by a Soret peak at 390 nm; their KD values varied between 1 and 42 microM . On the contrary, sulfaphenazole led to a low-spin (S = 1/2) CYP 2C9 complex upon binding of its NH2 group to CYP 2C9 iron . Interactions of the five substrates with the enzyme were studied by paramagnetic relaxation effects of CYP 2C9-iron(III) on the 1H NMR spectrum of each substrate . Distances between the heme iron atom and substrate protons were calculated from the NMR data, and the orientation of the substrate relative to iron was determined from those distances . Finally, a model for substrate positioning in the CYP 2C9 active site was constructed by molecular modeling studies under the constraint of the iron-proton distances . It points out two structural characteristics for a compound to be selectively recognized by CYP 2C9: (i) the presence of an anionic site able to establish an ionic bond with a putative cationic residue of the protein and (ii) the presence of an hydrophobic zone between the substrate hydroxylation site and the anionic site . Sulfaphenazole was easily included in that model; its very high affinity for CYP 2C9 is due to a third structural feature, the presence of its NH2 function which binds to CYP 2C9 iron.

J Bacteriol, 1997 Oct, 179(20), 6318 - 24
Characterization of two heat shock genes from Haloferax volcanii: a model system for transcription regulation in the Archaea; Kuo YP et al.; The expression of two heat-responsive cct (chaperonin-containing Tcp-1) genes from the archaeon Haloferax volcanii was investigated at the transcription level . The cct1 and cct2 genes, which encode proteins of 560 and 557 amino acids, respectively, were identified on cosmid clones of an H . volcanii genomic library and subsequently sequenced . The deduced amino acid sequences of these genes exhibited a high degree of similarity to other archaeal and eucaryal cct family members . Expression of the cct genes was characterized in detail for the purpose of developing a model for studying transcription regulation in the domain Archaea . Northern (RNA) analysis demonstrated that the cct mRNAs were maximally induced after heat shock from 37 to 55 degrees C and showed significant heat inducibility after 30 min at 60 degrees C . Transcription of cct mRNAs was also stimulated in response to dilute salt concentrations . Transcriptional analysis of cct promoter regions coupled to a yeast tRNA reporter gene demonstrated that 5' flanking sequences up to position -233 (cct1) and position -170 (cct2) were sufficient for promoting heat-induced transcription . Transcript analysis indicated that both basal transcription and stress-induced transcription of the H . volcanii cct genes were directed by a conserved archaeal consensus TATA motif (5'-TTTATA-3') centered at -25 relative to the mapped initiation site . Comparison of the cct promoter regions also revealed a striking degree of sequence conservation immediately 5' and 3' of the TATA element.

Nat Biotechnol, 1997 Oct, 15(10), 992 - 6
Plant resistance to fungal infection induced by nontoxic pokeweed antiviral protein mutants; Zoubenko O et al.; Pokeweed antiviral protein (PAP), a 29-kD protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes . Transgenic plants expressing PAP are resistant to a broad spectrum of plant viruses . Nontoxic PAP mutants have been isolated by random mutagenesis and selection in yeast . One of these mutants, PAP-X, had a point mutation at the active-site (E176V) that abolished enzymatic activity, and another mutant, delta C25PAP, had a nonsense mutation near the C-terminus (W237stop) that deleted 25 C-terminal amino acids . Unlike the wild-type PAP, expression of neither mutant was toxic to transgenic plants . We show that both class I (basic) and class II (acidic) isoforms of pathogenesis-related (PR) proteins are overexpressed in transgenic plants expressing PAP and the nontoxic PAP mutants . Although PR-proteins are constitutively expressed, no increase in salicylic acid levels was detected . Homozygous progeny of transgenic plants expressing either PAP or the nontoxic PAP mutants displayed resistance to the fungal pathogen Rhizoctonia solani . These results show that expression of PAP or the nontoxic PAP mutants activates multiple plant defense pathways independently of salicylic acid and confers resistance to fungal infection . The C-terminal 25 amino acids of PAP, which are required for toxicity in vivo, are not critical for resistance to viral or fungal infection, indicating that toxicity of PAP can be separated from pathogen resistance.

J Cell Biol, 1997 Oct 20, 139(2), 507 - 15
Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat motifs; Xia H et al.; PDZ motifs are protein-protein interaction domains that often bind to COOH-terminal peptide sequences . The two PDZ proteins characterized in skeletal muscle, syntrophin and neuronal nitric oxide synthase, occur in the dystrophin complex, suggesting a role for PDZ proteins in muscular dystrophy . Here, we identify actinin-associated LIM protein (ALP), a novel protein in skeletal muscle that contains an NH2-terminal PDZ domain and a COOH-terminal LIM motif . ALP is expressed at high levels only in differentiated skeletal muscle, while an alternatively spliced form occurs at low levels in the heart . ALP is not a component of the dystrophin complex, but occurs in association with alpha-actinin-2 at the Z lines of myofibers . Biochemical and yeast two-hybrid analyses demonstrate that the PDZ domain of ALP binds to the spectrin-like motifs of alpha-actinin-2, defining a new mode for PDZ domain interactions . Fine genetic mapping studies demonstrate that ALP occurs on chromosome 4q35, near the heterochromatic locus that is mutated in fascioscapulohumeral muscular dystrophy.

Genes Dev, 1997 Oct 1, 11(19), 2456 - 67
The PAG gene product, a stress-induced protein with antioxidant properties, is an Abl SH3-binding protein and a physiological inhibitor of c-Abl tyrosine kinase activity; Wen ST et al.; Biochemical and genetic evidence suggests that the tyrosine kinase activity of c-Abl is tightly regulated in vivo by a cellular factor binding to the Src homology 3 (SH3) domain of Abl . We used the yeast two-hybrid system to identify a gene, PAG, whose protein product (Pag) interacts specifically with the Abl SH3 domain . Pag, also known as macrophage 23-kD stress protein (MSP23), is a member of a novel family of proteins with antioxidant activity implicated in the cellular response to oxidative stress and in control of cell proliferation and differentiation . In a co-expression assay, Pag associates with c-Abl in vivo and inhibits tyrosine phosphorylation induced by overexpression of c-Abl . Inhibition requires the Abl SH3 and kinase domains and is not observed with other Abl SH3-binding proteins . Expression of Pag also inhibits the in vitro kinase activity of c-Abl, but not SH3-mutated Abl or v-Abl . When transfected in NIH-3T3 cells, Pag is localized to nucleus and cytoplasm and rescues the cytostatic effect induced by c-Abl . These observations suggest Pag is a physiological inhibitor of c-Abl in vivo.

J Biol Chem, 1997 Oct 17, 272(42), 26669 - 77
Structure of the 32-kDa galectin gene of the nematode Caenorhabditis elegans; Arata Y et al.; Galectins are a family of soluble beta-galactoside-binding lectins distributed in both vertebrates and invertebrates and, more recently, found also in fungus . The 32-kDa galectin isolated from the nematode Caenorhabditis elegans (Hirabayashi, J., Satoh, M., and Kasai, K . (1992) J . Biol . Chem . 267, 15485-15490) was the first "tandem repeat-type" galectin, containing two homologous carbohydrate-binding sites . Here, we report the structure of the nematode 32-kDa galectin gene . Physical mapping by yeast artificial chromosome polytene filter hybridization revealed that the 32-kDa galectin gene is located on chromosome II . Analysis of the transcript (1.4 kilobases) showed the presence at its 5'-end of a 22-nucleotide trans-spliced leader sequence (SL1) . The entire genomic structure spanning >5 kilobase pairs (kbp), including the 5'-noncoding region, two intervening sequences (introns 1 and 2), and the 3'-noncoding region, was completely determined by the combination of genomic polymerase chain reaction and conventional colony hybridization . Intron 1 was relatively long (2.4 kbp) and was found to be inserted after the ninth codon (TAC) from the initiation codon . This position proved to be almost homologous to the conserved first intron insertion position in the vertebrate galectin genes (i . e . genes of mammalian galectin-1, -2, and -3 and chick 14-kDa galectin) . On the other hand, intron 2 was much shorter (0.6 kbp), and it was inserted into the central region of the second carbohydrate-binding site . Although such an insertion pattern has never been observed in the vertebrate galectin genes, it seems to be common in C . elegans tandem repeat-type galectin genes, as predicted by the C . elegans genome project (Coulson, A., and the C . elegans Genome Consortium (1996) Biochem . Soc . Trans . 24, 289-291) . Based on extensive sequence comparison, the origin and molecular evolution of the tandem repeat-type galectins are discussed.

J Biol Chem, 1997 Oct 17, 272(42), 26604 - 10
The Groucho/transducin-like enhancer of split transcriptional repressors interact with the genetically defined amino-terminal silencing domain of histone H3; Palaparti A et al.; Groucho is a transcriptional repressor implicated in Notch signaling and involved in neural development and segmentation in Drosophila . We are investigating the molecular mechanisms underlying the functions of Groucho and its mammalian homologs, the transducin-like Enhancer of split (TLE) proteins . We report that Groucho/TLEs are associated with chromatin in live cells and that they co-purify with isolated histones . Affinity chromatography and far Western blotting studies show further that native Groucho/TLE proteins interact specifically with histone H3 and not with other core histones . This interaction is mediated by the H3 amino-terminal domain previously shown by genetic analysis in yeast to be essential for the role of H3 in transcriptional silencing . We also demonstrate that Groucho/TLEs form oligomeric structures in vivo . These combined findings suggest that transcription complexes containing Groucho/TLEs may associate with chromatin through interactions with the amino terminus of histone H3 and that these interactions may be propagated along the chromosome due to the ability of Groucho/TLEs to participate in higher order structures.

J Biol Chem, 1997 Oct 17, 272(42), 26522 - 9
Type VI collagen anchors endothelial basement membranes by interacting with type IV collagen; Kuo HJ et al.; Type VI collagen filaments are found associated with interstitial collagen fibers, around cells, and in contact with endothelial basement membranes . To identify type VI collagen binding proteins, the amino-terminal domains of the alpha1(VI) and alpha2(VI) chains and a part of the carboxyl-terminal domain of the alpha3(VI) chain were used as bait in a yeast two-hybrid system to screen a human placenta library . Eight persistently positive clones were identified, two coding the known matrix proteins fibronectin and basement membrane type IV collagen and the rest coding new proteins . The amino-terminal domain of alpha1(VI) was shown to interact with the carboxyl-terminal globular domain of type IV collagen . The specificity of this interaction was further studied using the yeast two-hybrid system in a one-on-one format and confirmed by using isolated protein domains in immunoprecipitation, affinity blots, and enzyme-linked immunosorbent assay-based binding studies . Co-distribution of type VI and type IV collagens in human muscle was demonstrated using double labeling immunofluorescent microscopy and immunoelectron microscopy . The strong interaction of type VI collagen filaments with basement membrane collagen provided a possible molecular pathogenesis for the heritable disorder Bethlem myopathy.

J Biol Chem, 1997 Oct 17, 272(42), 26375 - 81
Differential modes of nuclear localization signal (NLS) recognition by three distinct classes of NLS receptors; Miyamoto Y et al.; The targeting of karyophilic proteins to nuclear pores is mediated via the formation of a nuclear pore-targeting complex, through the interaction of nuclear localization signal (NLS) with its NLS receptor . Recently, a novel human protein, Qip1, was identified from a yeast two-hybrid system with DNA helicase Q1 . This study demonstrates that Qip1 is a novel third class of NLS receptor that efficiently recognizes the NLS of the helicase Q1 . Moreover, the data obtained in this study show that the specific interaction between Qip1 and the NLS of the helicase Q1 requires its upstream sequence of the minimal essential NLS . By using purified recombinant proteins alone in the digitonin-permeabilized cell-free transport system, it was demonstrated that the two known human NLS receptors, Rch1 and NPI-1, are able to transport all the tested NLS substrates into the nucleus, while Qip1 most efficiently transports the helicase Q1-NLS substrates, which contain its upstream sequence in so far as we have examined the system . Furthermore, in HeLa cell crude cytosol, it was found that endogenous Rch1 binds to all the tested NLS substrates, while the binding of endogenous NPI-1 is restricted to only some NLSs, despite the fact that NPI-1 itself shows binding activity to a variety of NLSs . These results indicate that at least three structurally and functionally distinct NLS receptors exist in the human single cell population, and suggest that the nuclear import of karyophilic proteins may be controlled in a complex manner at the NLS recognition step by the existence of a variety of NLS receptors with various specificities to each NLS.

Langenbecks Arch Chir, 1997, 382(4 Suppl 1), S5 - 8
{Importance of mycoses in intra-abdominal infections}; Blinzler L et al.; Although there is a 20% yeast colonization in the gastrointestinal tract of the population, fungal infections appear only rarely in secondary peritonitis . The risk of severe mycosis increases after a major operation and when a patient is taking broad-spectrum antibiotics, is on total parenteral nutrition, is catheterized, and/or is immune-suppressed . In the past years the incidence of nosocomial fungal infections (usually Candida spp.) has risen significantly . Five percent of CAPD-related peritonitis is caused by fungi . In enteral anastomosis breakdown, invasive mycosis occurs more often, with an accompanying lethality of up to 80% . In severe pancreatitis, up to 5% of peripancreatic necrosis is infected with fungi . The clinical course of severe mycosis, like the septic syndrome, is associated with fungemia in up to 50% of cases . As most of the facultative pathogenic fungi are part of the physiological flora, it is difficult to interpret mycological cultures . In order to diagnose invasive fungal infections, histopathological techniques and serologic tests for antigens and antibodies are available . Three antifungal agents (amphotericin B, flucytosine, fluconazole) are available for intravenous administration . Amphotericin B is given at doses of up to 1 mg/kg per day, in liposomal galenism up to 3 mg/kg per day . Combining amphotericin B with flucytosine (150-200 mg/kg per day) a synergistic effect is reached . Fluconazole at a dosage of 200-800 mg per day represents an alternative with similar antifungal activity and lower side effects.

Oncogene, 1997 Sep 18, 15(12), 1489 - 95
Modulation of ETS-1 transcriptional activity by huUBC9, a ubiquitin-conjugating enzyme; Hahn SL et al.; Ets transcription factors have Ets DNA binding domains, that have a winged helix-turn-helix structure . ETS-1, the founding member of the family, is regulated by the Ras and Ca2+ signaling pathways and is implicated in various physiological processes leading to cell growth, differentiation and apoptosis . We have identified ETS-1 interacting factors with a yeast two-hybrid screen . The majority of the positive clones turned out to encode the human homologue of the yeast ubiquitin-conjugating enzymes UBC9 and Hus5 . In two different yeast assays, ETS-1 interacted with huUBC9 . In an in vitro GST 'pull-down' assay, ETS-1 and several other Ets family members complexed with huUBC9 . Interestingly, in mammalian cells, coexpression of huUBC9 resulted in a substantial increase in the transcriptional activity of ETS-1 . Coexpressed huUBC9 did not affect the ETS-1 protein level, and moreover, a point mutation at Cys93, an amino acid known to be essential for ubiquitination, did not abolish the stimulation of the ETS-1 transcriptional activity . Our results indicate that the modulation of ETS-1 activity by huUBC9 results from processes other than ubiquitination and ETS-1 stabilization.

Clin Infect Dis, 1997 Aug, 25(2), 281 - 4
Histoplasmosis and kidney disease in patients with AIDS; Burke DG et al.; Renal disease in patients infected with human immunodeficiency virus (HIV) often presents with significant proteinuria and progressive renal failure; focal glomerulosclerosis is the most common renal pathology identified . To our knowledge, we report the first case of nephrotic-range proteinuria and preserved renal function in an HIV-infected patient in association with disseminated histoplasmosis . The initial level of proteinuria was 12.5 g/24 h . The patient developed a concomitant lesion on his neck, which was biopsied and identified as Histoplasma capsulatum by fungal stains and culture . The serum CF titer of antibody against yeast antigens of H . capsulatum was 1:8 . The level of serum albumin decreased to 2.0 g/dL, and the level of serum cholesterol increased to 284 mg/dL . Immunohistochemical staining of renal biopsy tissue demonstrated immune complexes within the mesangium; H . capsulatum antigen was also demonstrated in the mesangium . Therapy with oral itraconazole resulted in marked clinical improvement . The findings in this case emphasize the need to rule out treatable causes of the nephrotic syndrome in AIDS, especially in cases of immune-complex glomerulonephritis.

Gene, 1997 Sep 15, 197(1-2), 225 - 9
A CHD1 gene is Z chromosome linked in the chicken Gallus domesticus; Griffiths R et al.; Chromo-helicase-DNA binding 1 (CHD1) is a conserved protein with a putative role in chromatin architecture . Single homologues have been found in mouse, Drosophila and yeast . In birds the situation is different as they possess two homologues . One is known to be W-linked, we show the second, closely related gene is linked to the Z sex chromosome . The basic structure of the Z-linked gene is similar to the homologous genes, however, it does possess an additional, internal 88 amino acid hydrophilic domain, rich in glutamic acid and lysine . Studies on pairs of genes sex-linked in mammals suggests rapid divergence of DNA sequence and function . We suggest the DNA sequences of CHD-W and CHD-Z do not follow this pattern.

Gene, 1997 Sep 15, 197(1-2), 195 - 204
Cloning of a human multispanning membrane protein cDNA: evidence for a new protein family; Chluba-de Tapia J et al.; We report the cloning of a human cDNA encoding a protein of calculated 68.8 kDa molecular mass, named hMP70 . The deduced protein sequence shows a large N-terminal hydrophilic part and a C-terminal part with nine putative hydrophobic regions characteristic of integral transmembrane domains . Computer searches with sequence databases revealed homologies with three complete yeast proteins and with at least 19 human, 10 plant and one nematode short unidentified protein sequences translated from Expressed Sequence Tags (ESTs) . Remarkably, this hMP70 protein retains between 27 and 31% overall sequence identity with the yeast proteins . We propose that hMP70 and related genes have evolved from a common ancestral gene and form a new multispanning membrane protein family which we call the MP70 protein family . Gene expression of hMP70 appears to be ubiquitous, as the mRNA is detectable in all human tissues analysed so far, as shown by Northern blot analysis . Furthermore, a protein of about 70 kDa is detectable in different mammalian cell lines, as shown by immunoblot analysis . From its widespread expression and conservation from yeast, plants to mammals, it is likely that hMP70 has a fundamental biological function in the cell.

Gene, 1997 Sep 15, 197(1-2), 73 - 81
CASP, a novel, highly conserved alternative-splicing product of the CDP/cut/cux gene, lacks cut-repeat and homeo DNA-binding domains, and interacts with full-length CDP in vitro; Lievens PM et al.; Human CDP/cut and its murine counterpart, cux1/CDP are homeodomain repressor proteins in the family of Drosophila Cut . Northern blot analysis reveals complex alternative splicing, including forms too small to encode the full 1505 amino acid protein . We have characterized a CDP/cut alternatively spliced cDNA (CASP) of 3.4 kb . Human CASP, a predicted 678 amino acid polypeptide, shares 400 amino acids with CDP, but has an alternate N terminal exon of 20 aa, and the C-terminal 258 amino acids diverge from CDP/cut entirely . As the unique C-terminus of CASP lacks the three 'cut-repeats' and homeodomain of CDP/cut, we predict it does not bind DNA . Murine CASP, 96% similar to human, shares these features . Database searches identify homologs in chicken (86% identical to human CASP) and yeast (29% identical to human) . Murine CASP mRNA is ubiquitous in mouse tissues and in tissue-culture cell lines . We generated a specific antiserum against the unique C-terminus of CASP, and used this reagent to demonstrate that CASP protein is expressed as an approx . 80 kDa protein in human and murine cells . Co-translation of in vitro-translated CDP and CASP mRNA, followed by immunoprecipitation with specific anti-CASP IgG, shows that CASP polypeptide can from a complex with CDP . Studies of the intron/exon structure of the murine cux/CDP/mCASP locus (>> 100 kb) reveal that the unique 3' exons of CASP are interposed between cut-repeats 2 and 3 of the cux gene . We speculate that a primordial CASP-like gene captured a cut-repeat-homeobox gene to give rise to the eukaryotic Cut/CDP family of proteins.

Cancer Res, 1997 Oct 1, 57(19), 4153 - 7
Characterization of a 4-Mb region at chromosome 6q21 harboring a replicative senescence gene; Morelli C et al.; A 4-Mb region containing a senescence gene was defined at 6q21 by fluorescence in situ hybridization and deletion mapping after transfer of a normal human chromosome 6 to a BK virus-transformed mouse cell line . By screening three different yeast artificial chromosome (YAC) libraries, a YAC contig was constructed that covers the deleted region at 6q21 . The contig is composed of 18 overlapping YACs with a size of 250-1800 kb and contains 3 CpG islands and 10 expressed sequence tags . By sequencing YACs and P1 artificial chromosomes, nine new sequence tagged sites and three new expressed sequence tags were detected that enrich the genetic resources of the region . The contig may also contain a fragile site, FRA6F, located close to a CpG island, which could be a landmark to localize the senescence gene . This YAC contig will be used to detect expressed sequences to clone and characterize the senescence gene at 6q21.

Somat Cell Mol Genet, 1997 Mar, 23(2), 153 - 7
Generation of a contig comprising YACs and BACs within chromosome region 1p13.1; Brintnell B et al.; Chromosome region 1p13 is known to show loss of heterozygosity (LOH) in a number of human tumor types, including breast . We have generated a contig comprising YACs and BACs spanning part of 1p13.1 which includes the smallest region of overlapping loss identified in our earlier studies . The contig is anchored to the genetic map by a number of microsatellite markers, and by the use of CEPH YACs . We have excluded a number of candidate genes from this region, and we have oriented the contig with respect to the centromere and a number of other genes and markers on 1p13 . This resource will be valuable in mapping the target for LOH in breast and other tumors, and may also be useful for the genetic analysis of other genes or diseases known to map to this region.

Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11731 - 5
Arabidopsis thaliana mutants altered in homologous recombination; Masson JE et al.; Homologous recombination contributes both to the generation of allelic diversity and to the preservation of genetic information . In plants, a lack of suitable experimental material has prevented studies of the regulatory and enzymatic aspects of recombination in somatic and meiotic cells . We have isolated nine Arabidopsis thaliana mutants hypersensitive to x-ray irradiation (xrs) and examined their recombination properties . For the three xrs loci described here, single recessive mutations were found to confer simultaneous hypersensitivities to the DNA-damaging chemicals mitomycin C (MMCs) and/or methyl methanesulfonate (MMSs) and alterations in homologous recombination . Mutant xrs9 (Xrays, MMSs) is reduced in both somatic and meiotic recombination and resembles yeast mutants of the rad52 epistatic group . xrs11 (Xrays, MMCs) is deficient in the x-ray-mediated stimulation of homologous recombination in somatic cells in a manner suggesting a specific signaling defect . xrs4 (Xrays, MMSs, MMCs) has a significant deficiency in somatic recombination, but this is accompanied by meiotic hyper-recombination . A corresponding phenotype has not been reported in other systems and thus this indicates a novel, plant-specific regulatory circuit linking mitotic and meiotic recombination.

Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11534 - 9
Functional cleavage of the common cytokine receptor gamma chain (gammac) by calpain; Noguchi M et al.; The small subunit of calpain, a calcium-dependent cysteine protease, was found to interact with the cytoplasmic domain of the common cytokine receptor gamma chain (gammac) in a yeast two-hybrid interaction trap assay . This interaction was functional as demonstrated by the ability of calpain to cleave in vitro-translated wild-type gammac, but not gammac containing a mutation in the PEST (proline, glutamate, serine, and threonine) sequence in its cytoplasmic domain, as well as by the ability of endogenous calpain to mediate cleavage of gammac in a calcium-dependent fashion . In T cell receptor-stimulated murine thymocytes, calpain inhibitors decreased cleavage of gammac . Moreover, in single positive CD4(+) thymocytes, not only did a calpain inhibitor augment CD3-induced proliferation, but antibodies to gammac blocked this effect . Finally, treatment of cells with ionomycin could inhibit interleukin 2-induced STAT protein activation, but this inhibition could be reversed by calpain inhibitors . Together, these data suggest that calpain-mediated cleavage of gammac represents a mechanism by which gammac-dependent signaling can be controlled.

Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11333 - 8
MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclXL and initiates cell death; Han DK et al.; Activation of the cascade of proteolytic caspases has been identified as the final common pathway of apoptosis in diverse biological systems . We have isolated a gene, termed MRIT, that possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases . However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC(R/Q)G . Nonetheless MRIT activates caspase-dependent death . Using yeast two-hybrid assays, we demonstrate that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD . In addition, MRIT simultaneously and independently interacts with BclXL and FLICE in mammalian cells . Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member.

J Clin Pathol, 1997 Jul, 50(7), 563 - 5
Use of CHROMagar Candida for genital specimens in the diagnostic laboratory; Houang ET et al.; OBJECTIVE: To evaluate CHROMagar Candida (CA), a new yeast differential medium, for yeast isolation in a clinical laboratory for the routine examination of high vaginal swabs . METHODS: Results of high vaginal swab cultures processed in a standard manner on plates containing equal halves of Sabouraud dextrose agar (SDA) and CA were compared . Non-Candida albicans yeast isolates were further speciated with API 20C AUX or API 32C . To assess the ease of use of CA, laboratory staff lacking in experience of the medium were asked to identify 23 unlabelled yeast cultures on CA by referring to six labelled reference plates . RESULTS: Of the 1784 swab cultures processed, yeasts were isolated from 373 SDA and 368 CA . Of the 78 non-albicans isolates further speciated, CA identified correctly all cultures of C krusei and C tropicalis, and 82% of C glabrata . All the 38 inexperienced laboratory staff achieved 100% accuracy for C albicans and over 90% for C krusei and C tropicalis . CONCLUSIONS: CA is a satisfactory isolation medium for genital specimens, allowing immediate and correct identification of the commonly encountered yeasts and easy recognition of mixed cultures.

Nucleic Acids Res, 1997 Oct 1, 25(19), 3959 - 61
Marked improvement of PAC and BAC cloning is achieved using electroelution of pulsed-field gel-separated partial digests of genomic DNA; Strong SJ et al.; We describe a simple electroelution method for purifying large, gel-fractionated DNA molecules that alleviates the need for melting of the agarose and subsequent enzymatic agarose digestion . The method yields DNA that is visibly more intact than that purified from a standard agarose-digestion protocol and is more amenable to large-fragment cloning with PAC and BAC vectors . These findings are notable in that PAC and BAC library construction is a very labor-intensive and costly procedure, such that any net improvement in cloning efficiency is highly advantageous . This method also should prove useful towards other applications which require purification of very large DNA molecules, such as YAC cloning.

Cell, 1997 Sep 19, 90(6), 1013 - 21
The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins; Liu Q et al.; Spinal muscular atrophy (SMA), one of the most common fatal autosomal recessive diseases, is characterized by degeneration of motor neurons and muscular atrophy . The SMA disease gene, termed Survival of Motor Neurons (SMN), is deleted or mutated in over 98% of SMA patients . The function of the SMN protein is unknown . We found that SMN is tightly associated with a novel protein, SIP1, and together they form a specific complex with several spliceosomal snRNP proteins . SMN interacts directly with several of the snRNP Sm core proteins, including B, D1-3, and E . Interestingly, SIP1 has significant sequence similarity with Brr1, a yeast protein critical for snRNP biogenesis . These findings suggest a role for SMN and SIP1 in spliceosomal snRNP biogenesis and function and provide a likely molecular mechanism for the cause of SMA.

Gene, 1997 Sep 1, 196(1-2), 1 - 8
Genomic structure and organization of kringles type 3 to 10 of the apolipoprotein(a) gene in 6q26-27; Mihalich A et al.; Apolipoprotein(a) {apo(a)} is a highly polymorphic glycoprotein covalently linked to the apolipoprotein B-100 of LDL in a particle called lipoprotein(a) {Lp(a)} . High plasma levels of Lp(a) are associated with coronary as well as peripheral atherosclerosis . Plasma levels of Lp(a) show a remarkable variation ranging from 0.1 mg/dl to over 100 mg/dl . The apo(a) gene shows a size polymorphism which resides in the variable number of kringle domains which resemble plasminogen kringle IV . Ten different types of kringle IV repeats have been described, nine of which (kringle IV type 1 and type 3-10) are each supposed to be present in a single copy . The other kringles, namely kringle IV type 2 repeats, vary in number from 3 to 42 between apo(a) alleles and form the basis for the apo(a) size polymorphism . Although an inverse relationship has been observed between the number of kringle type 2 repeats and plasma levels of Lp(a), there are exceptions to this general finding . Indeed, several individuals have been described with similar apo(a) size alleles but very different plasma levels of Lp(a) . Genetic studies have linked these differences to the apo(a) locus on 6q26-27, outlining the importance, besides the kringle type 2 repeats, of other regions of the apo(a) gene in contributing to the interindividual differences in the plasma concentration of Lp(a) . One of the candidate regions is represented by the non-repeated type-3 to type-10 kringles which are invariably present in each apo(a) allele and whose structural integrity is playing a critical role in the correct assembly of the Lp(a) particle . Biochemical studies with recombinant wild type and mutagenized apo(a) cDNAs with several alterations of the non-repeated kringles have well documented this latter point . As a starting point to search for genetic variations in these kringles associated with different levels of Lp(a), we are presenting the genome organization of type-3 to 10 kringle along with specific PCR primers for easy analysis from genomic DNA . Restriction as well as partial sequencing analyses of the type-3 to 10 kringles region has also provided interesting clues as to the different evolutionary origin of these types of kringle with respect to the polymorphic type-2 kringles.

Anal Chem, 1997 Oct 1, 69(19), 3995 - 4001
A strategy for rapid, high-confidence protein identification; Qin J et al.; A procedure is described for rapid, high-confidence identification of proteins using matrix-assisted laser desorption/ionization tandem ion trap mass spectrometry in conjunction with a genome database searching strategy . The procedure involves excision of copper-stained bands or spots from electrophoretic gels, in-gel trypsin digestion of the proteins, single-stage mass spectrometric analysis of the resultant mixture of tryptic peptides, followed by tandem ion trap mass spectrometric analysis of selected individual peptides, and database searching of the relevant genomic database using the program PepFrag . The scheme provides sensitive, real-time protein identification as well as facile identification of modifications . A single operator can unambiguously identify 5-10 proteins/day from an organism whose genome is known at a level of > 0.5 pmol of protein loaded on a gel . The utility of the technique was demonstrated by the identification and characterization of a band from a human HTLV-I preparation and 11 different proteins from a yeast RNA polymerase II C-terminal repeat domain-affinity preparation . The technology has great potential for postgenome biological science, where it promises to facilitate the dissection and anatomy of macromolecular assemblages, the definition of disease state markers, and the investigation of protein targets in biological processes such as the cell cycle and signal transduction.

Proc Int Conf Intell Syst Mol Biol, 1997, 5, 294 - 302
The Gene-Finder computer tools for analysis of human and model organisms genome sequences; Solovyev V et al.; We present a complex of new programs for promoter, 3'-processing, splice sites, coding exons and gene structure identification in genomic DNA of several model species . The human gene structure prediction program FGENEH, exon prediction-FEXH and splice site prediction-HSPL have been modified for sequence analysis of Drosophila (FGENED, FEXD and DSPL), C.elegance (FGENEN, FEXN and NSPL), Yeast (FEXY and YSPL) and Plant (FGENEA, FEXA and ASPL) genomic sequences . We recomputed all frequency and discriminant function parameters for these organisms and adjusted organism specific minimal intron lengths . An accuracy of coding region prediction for these programs is similar with the observed accuracy of FEXH and FGENEH . We have developed FEXHB and FGENEHB programs combining pattern recognition features and information about similarity of predicted exons with known sequences in protein databases . These programs have approximately 10% higher average accuracy of coding region recognition . Two new programs for human promoter site prediction (TSSG and TSSW) have been developed which use Gosh (1993) and Wingender (1994) data bases of functional motifs, respectively . POLYAH program was designed for prediction of 3'-processing regions in human genes and CDSB program was developed for bacterial gene prediction . We have developed a new approach to predict multiple genes based on double dynamic programming, that is very important for analysis of long genomic DNA fragments generated by genome sequencing projects . Analysis of uncharacterized sequences based on our methods is available through the University of Houston, Weizmann Institute of Science email servers and several Web pages at Baylor College of Medicine.

Nucleic Acids Res, 1997 Oct 15, 25(20), 4167 - 8
A dicistronic construct allows easy detection of human CFTR expression from YAC DNA in human cells; Vassaux G et al.; We have made a dicistronic construct where the picornaviral internal ribosome-entry site (IRES) driving the expression of the beta-geo gene has been inserted into the 3'untranslated region of the human CFTR gene present in a YAC . When introduced into the human cell line Caco-2 expressing the CFTR gene, the expression of the dicistronic gene can be detected by lacZ staining and follows the accumulation of the endogenous CFTR mRNA upon differentiation of the cells . These data demonstrate that this IRES-based approach presents an alternative to mRNA in situ hybridisation and allows detection of expression in an autologous system.

Mamm Genome, 1997 Oct, 8(10), 760 - 6
Localization and expression analysis of a novel conserved brain expressed transcript, Brx/BRX, lying within the Xic/XIC candidate region; Simmler MC et al.; The X inactivation center candidate region (Xic/XIC in mouse and human) is poorly characterized for the presence of transcription units . Only two conserved genes have been isolated to date, Xist/XIST and Cdx4/CDX4 . The other known gene lying within this region, Tsx, has been identified so far only in rodents by analyzing the complete genomic sequence of a 94-kb region distal to Xist . Here, we report the characterization of an additional gene lying within this 94-kb sequenced region . Brx, for Brain X-linked gene, is a rare transcript preferentially expressed in the brain . It is normally X-inactivated in the mouse . Localisation of BRX, its human homolog has shown the gene to be located within the orthologous but inverted human CDX4-XIST segment . These results suggest that the gene order of the region encompassing the Cdx4-Xist interval in the mouse is similar in human . Comparison of the Xist-Brx and Brx-Cdx4 regions in mouse and human indicates that these intervals are three times longer in human than in mouse . BRX is a new potential candidate for one of the X-linked mental retardation syndromes mapped within the pericentromeric region of the human X Chromosome (Chr).

Mamm Genome, 1997 Oct, 8(10), 742 - 4
Myostatin maps to the interval containing the bovine mh locus; Smith TP et al.; Myostatin (GDF-8) is a member of the transforming growth factor-beta superfamily and plays a role in muscle growth and development . Mice having targeted disruption of this gene display marked increases in muscle mass, a phenotype similar to the muscular hypertrophy (mh) in several cattle breeds . Physical mapping data developed from YAC clones indicate the bovine myostatin gene lies close to the centromere of bovine Chromosome (Chr) 2 (BTA2) at 2q11, indistinguishable from the cytogenetic location of the mh locus . In addition, a polymorphism in the second intron of the gene was used to show that myostatin maps within the interval previously shown to contain mh . These data suggest myostatin may be the gene causing muscular hypertrophy in cattle.

J Clin Microbiol, 1997 Oct, 35(10), 2660 - 2
A new medium for the presumptive identification of dermatophytes; Salkin IF et al.; A new medium, Dermatophyte Identification Medium (DIM) (trade mark pending), was specifically developed to eliminate problems of false-positive results associated with commercially marketed media, such as dermatophyte test medium (DTM) . Previous investigations had demonstrated that DTM only partially suppressed growth of nondermatophytes and that several of these nondermatophytic fungi that were morphologically similar to dermatophytes caused false-positive results . Presumptive identification of an unknown isolate as a dermatophyte required only the transfer of a portion of the suspected colony recovered from the specimen to DIM . Positive results, evidenced by a change in the color of the medium, were observed within 24 to 48 h . In studies of over 500 isolates of dermatophytes and common nondermatophyte molds, as well as close to 600 yeast isolates, false-positive results were always associated with bacterial contamination of the mold isolates while false negatives were only observed with occasional isolates of Trichophyton verrucosum . DIM culture was an inexpensive, rapid, and accurate method for the presumptive identification of dermatophytes in the clinical mycology laboratory.

J Pharmacol Exp Ther, 1997 Sep, 282(3), 1465 - 72
Involvement of human CYP1A isoenzymes in the metabolism and drug interactions of riluzole in vitro; Sanderink GJ et al.; Cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) isoenzymes involved in riluzole oxidation and glucuronidation were characterized in (1) kinetic studies with human hepatic microsomes and isoenzyme-selective probes and (2) metabolic studies with genetically expressed human CYP isoenzymes from transfected B-lymphoblastoid and yeast cells . In vitro incubation of {14C}riluzole (15 microM) with human hepatic microsomes and NADPH or UDPGA cofactors resulted in formation of N-hydroxyriluzole (K(m) = 30 microM) or an unidentified glucuroconjugate (K(m) = 118 microM) . Human microsomal riluzole N-hydroxylation was most strongly inhibited by the CYP1A2 inhibitor alpha-naphthoflavone (IC50 = 0.42 microM) . Human CYP1A2-expressing yeast microsomes generated N-hydroxyriluzole, whereas human CYP1A1-expressing yeast microsomes generated N-hydroxyriluzole, two additional hydroxylated derivatives and an O-dealkylated derivative . CYP1A2 was the only genetically expressed human P450 isoenzyme in B-lymphoblastoid microsomes to metabolize riluzole . Riluzole glucuronidation was inhibited most potently by propofol, a substrate for the human hepatic UGT HP4 (UGT1.8/9) isoenzyme . In vitro, human hepatic microsomal hydroxylation of riluzole (15 microM) was weakly inhibited by amitriptyline, diclofenac, diazepam, nicergoline, clomipramine, imipramine, quinine and enoxacin (IC50 approximately 200-500 microM) and cimetidine (IC50 = 940 microM) . Riluzole (1 and 10 microM) produced a weak, concentration-dependent inhibition of CYP1A2 activity and showed competitive inhibition of methoxyresorufin O-demethylase . Thus, riluzole is predominantly metabolized by CYP1A2 in human hepatic microsomes to N-hydroxyriluzole; extrahepatic CYP1A1 can also be responsible for the formation of several other metabolites . Direct glucuronidation is a relatively minor metabolic route . In vivo, riluzole is unlikely to exhibit significant pharmacokinetic drug interaction with coadministered drugs that undergo phase I metabolism.

Biomed Environ Sci, 1997 Sep, 10(2-3), 227 - 34
Reduction of cancer risk with an oral supplement of selenium; Combs GF Jr et al.; The hypothesis that a dietary supplement of selenium (Se) may reduce cancer risk was tested experimentally in humans . Patients with histories of basal/squamous cell carcinomas of the skin were assigned randomly in double-blind fashion to daily oral supplements of either Se-enriched yeast (200 micrograms Se/day), or a low-Se yeast placebo . A total of 1312 patients recruited in 1983-1990 were followed with regular dermatologic examinations through 1993 for a total of 8269 person-years of observation . Skin cancer diagnoses were confirmed histologically . Plasma Se concentration was determined at 6-12 months intervals . All deaths and patient-reported illnesses were recorded; reported cancers were confirmed and documented by consultation with the patient medical care providers . The results indicate that Se did not significantly affect the primary endpoints: incidences of recurrent basal/squamous cell carcinomas of the skin . However, Se-treatment was associated with reductions in several secondary endpoints: total mortality, mortality from all cancers combined, as well as the incidence of all cancers combined, lung cancer, colorectal cancer and prostate cancer . The consistencies of these associations over time, between study clinics and for the leading cancer sites strongly suggests benefits of Se-supplementation for this cohort of patients, supporting the hypothesis that supplemental Se can reduce cancer risk . Although Se did not shown protective effects against non-melanoma skin cancers, the suggested reductions in risks to other frequent cancers demand further evaluation in well controlled clinical intervention trials.

Biochemistry, 1997 Oct 7, 36(40), 12346 - 54
Restructuring the active site of fumarase for the fumarate to malate reaction; Rose IA; Changes in the active site of fumarase (yeast fumarase II) that occur when fumarate is converted to malate (E.F --> E.M) must be reversed for another cycle of reaction to take place . As shown here, recycling of the enzyme includes two proton transfers and one conformational change . These events, together with the M-off step, are variously rate-determining depending on the medium . In very low salt the release of M is limited by the conformational change . Thus, (V/Km)F decreases with increased viscosity, shown with glycerol . A variety of simple anions, such as Cl- at approximately 50 mM and F itself at low concentration, activate the dissociation of M . This nonspecific anion effect is the basis for the >4-fold apparent cooperative activation by substrate . The M-off step and the conformational change are independent and random-order events . Thus, even when M-off is made rapid the rate of recycling is inhibited by glycerol, which in 100 mM NaCl inhibits Vmax but not V/Km . The enzyme form that results when M is released is M-specific, Em . Thus mesotartarate, competitive toward M, is noncompetitive toward F . The slow conformational change required for recycling of Em is activated by Pi and chaotropic anions such as azide and thiocyanate, giving rise to a nonspecific intermediate, Emf (mesotartarate becomes competitive toward F and Britton's countertransport property disappears with these activators) . Evidence is presented for the locations and rates of the two proton transfer steps required to complete the cycle.

Mol Cell Biol, 1997 Oct, 17(10), 6184 - 90
A new member of the I kappaB protein family, I kappaB epsilon, inhibits RelA (p65)-mediated NF-kappaB transcription; Li Z et al.; A novel member of the I kappaB family has been identified as a protein that associated with the p50 subunit of NF-kappaB in a yeast two-hybrid screen . Similar to previously known I kappaB proteins, this member, I kappaB epsilon, has six consecutive ankyrin repeats . I kappaB epsilon mRNA is widely expressed in different human tissues, with highest levels in spleen, testis, and lung . I kappaB epsilon interacts with different NF-kappaB proteins, including p65 (RelA), c-Rel, p50, and p52, in vitro and in vivo and inhibits the DNA-binding activity of both p50-p65 and p50-c-Rel complexes effectively . Endogenous and transfected NF-kappaB (RelA-dependent) transcriptional activation is inhibited by I kappaB epsilon . I kappaB epsilon mRNA is expressed at different levels in specific cell types and is synthesized constitutively in transformed B-cell lines . It also displays differential induction in response to tumor necrosis factor alpha, interleukin-1, or phorbol ester stimulation compared to I kappaB alpha in non-B-cell lines . Therefore, I kappaB epsilon represents a novel I kappaB family member which provides an alternative mechanism for regulation of NF-kappaB-dependent transcription.

Mol Cell Biol, 1997 Oct, 17(10), 5976 - 86
SNF2beta-BRG1 is essential for the viability of F9 murine embryonal carcinoma cells; Sumi-Ichinose C et al.; The yeast and animal SNF-SWI and related multiprotein complexes are thought to play an important role in processes, such as transcription factor binding to regulatory elements, which require nucleosome remodeling in order to relieve the repressing effect of packaging DNA in chromatin . There are two mammalian homologs of the yeast SNF2-SWI2 subunit protein, SNF2alpha-brm and SNF2beta-BRG1, and overexpression of either one of them has been shown to enhance transcriptional activation by glucocorticoid, estrogen, and retinoic acid (RA) receptors in transiently transfected cells . We have investigated here the function of SNF2beta-BRG1 in the RA receptor-retinoid X receptor-mediated transduction of the retinoid signal in F9 embryonal carcinoma (EC) cells which differentiate into endodermal-like cells upon RA treatment . The two SNF2beta-BRG1 alleles have been targeted by homologous recombination and subsequently disrupted by using a conditional Cre recombinase . We show that F9 EC cells inactivated on both SNF2beta alleles are not viable and that heterozygous mutant cells are affected in proliferation but not in RA-induced differentiation . Thus, in F9 EC cells, SNF2beta-BRG1 appears to play an essential role in basal processes involved in cell proliferation, in addition to its putative role in the activation of transcription mediated by nuclear receptors.

Mol Cell Biol, 1997 Oct, 17(10), 5748 - 57
IRF-7, a new interferon regulatory factor associated with Epstein-Barr virus latency; Zhang L et al.; The Epstein-Barr virus (EBV) BamHI Q promoter (Qp) is the only promoter used for the transcription of Epstein-Barr virus nuclear antigen 1 (EBNA-1) mRNA in cells in the most restricted (type I) latent infection state . However, Qp is inactive in type III latency . With the use of the yeast one-hybrid system, a new cellular gene has been identified that encodes proteins which bind to sequence in Qp . The deduced amino acid sequence of the gene has significant homology to the interferon regulatory factors (IRFs) . This new gene and products including two splicing variants are designated IRF-7A, IRF-7B, and IRF-7C . The expression of IRF-7 is predominantly in spleen, thymus, and peripheral blood leukocytes (PBL) . IRF-7 proteins were identified in primary PBL with specific antiserum against IRF-7B protein . IRF-7s can bind to interferon-stimulated response element (ISRE) sequence and repress transcriptional activation by both interferon and IRF-1 . Additionally, a functional viral ISRE sequence, 5'-GCGAAAACGAAAGT-3', has been identified in Qp . Finally, the expression of IRF-7 is consistently high in type III latency cells and almost undetectable in type I latency, corresponding to the activity of endogenous Qp in these latency states and the ability of the IRF-7 proteins to repress Qp-reporter constructs . The identification of a functional viral ISRE and association of IRF-7 with type III latency may be relevant to the mechanism of regulation of Qp.

Exp Gerontol, 1997 Jul-Oct, 32(4-5), 383 - 94
The heterochromatin loss model of aging; Villeponteau B; There are significant changes in gene expression that occur with cellular senescence and organismic aging . Genes residing in compacted heterochromatin domains are typically silenced due to an altered accessibility to transcription factors . Heterochromatin domains and gene silencing are set up in early development and were initially believed to be maintained for the remainder of the lifespan . Recent data suggest that there may be a net loss of heterochromatin with advancing age in both yeast and mice . The gradual loss of heterochromatin-induced gene silencing could explain the changes in gene expression that are closely linked with aging . A general model is proposed for heterochromatin loss as a major factor in generating alterations in gene expression with age . The heterochromatin loss model is supported by several lines of evidence and suggests that a fundamental genetic mechanism underlies most of the changes in gene expression observed with senescence.

Crit Rev Food Sci Nutr, 1997 Aug, 37(5), 443 - 8
The effect of selenium on antioxidant system in erythrocytes and liver of the carp (Cyprinus carpio L.)
Jovanovic A, Grubor-Lajsic G, Djukic N, Gardinovacki G, Matic A, Spasic M.
The effect of selenium-supplemented diet (sodium selenate and selenium yeast) on antioxidant in erythrocytes and liver of the carp (Cyprinus carpio L.) fingerlings was studied . With this goal, the activities of glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione-S-transferase (GST), as well as glutathione (GSH + GSSG) level, were determined . In the group supplemented with sodium selenate, no significant changes in the activity of the above enzymes were recorded in both the erythrocytes and in the liver, with the exception of GST activity that was significantly reduced in the plasma compared with the controls . Glutathione content was at the control level . In the group supplemented with selenium-yeast, the activities of GSH-Px, CAT, and SOD were significantly increased in erythrocytes, whereas GST activity and plasma content of GSH + GSSG were reduced compared with the controls . At the same time, the activities of hepatic SOD and GST were increased compared with the controls . These results demonstrate that organically bound selenium (selenium-yeast) acts more efficiently on antioxidant system of the carp fingerlings than inorganic selenium salts.

Oncogene, 1997 Sep, 15(11), 1303 - 7
Is Cdk7/cyclin H/MAT1 the genuine cdk activating kinase in cycling Xenopus egg extracts?
Fesquet D, Morin N, Doree M, Devault A.
Formation of active cdk (cyclin dependent kinase)/ cyclin kinases involves phosphorylation of a conserved threonine residue in the T loop of the cdk catalytic-subunit by CAK (Cdk Activating Kinase) . CAK was first purified biochemically from higher eukaryotes and identified as a trimeric complex containing a cdk7 catalytic subunit, cyclin H and MAT1 (Menage a trois), a member of the RING finger family . The same trimeric complex is also part of basal transcription factor TFIIH . In budding yeast, the closest homologs of cdk7 and cyclin H, KIN28 and CCL1, respectively, also associate with TFIIH . However, the KIN28/CCL1 complex does not display CAK activity and a distinct protein kinase able to phosphorylate monomeric CDC28 and GST-cdk2 was recently identified, challenging the identification of cdk7 as the physiological CAK in higher eukaryotes . Here we demonstrate that immunodepletion of cdk7 suppresses CAK activity from cycling Xenopus egg extracts, and arrest them before M-phase . We also show that specific translation of mRNAs encoding Xenopus cdk7 and its associated subunits restores CAK activity in cdk7-immunodepleted Xenopus egg extracts . Hence, the cdk7 complex is necessary and sufficient for activation of cdk-cyclin complexes in cycling Xenopus egg extracts.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10762 - 7
Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein; Dhordain P et al.; The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas . It encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal domain, termed BTB/POZ (bric-a-brac tramtrack broad complex/pox viruses and zinc fingers) . Using a yeast two-hybrid screen, we show here that the LAZ3/BCL6 BTB/POZ domain interacts with the SMRT (silencing mediator of retinoid and thyroid receptor) protein . SMRT originally was identified as a corepressor of unliganded retinoic acid and thyroid receptors and forms a repressive complex with a mammalian homolog of the yeast transcriptional repressor SIN3 and the HDAC-1 histone deacetylase . Protein binding assays demonstrate that the LAZ3/BCL6 BTB/POZ domain directly interacts with SMRT in vitro . Furthermore, DNA-bound LAZ3/BCL6 recruits SMRT in vivo, and both overexpressed proteins completely colocalize in nuclear dots . Finally, overexpression of SMRT enhances the LAZ3/BCL6-mediated repression . These results define SMRT as a corepressor of LAZ3/BCL6 and suggest that LAZ3/BCL6 and nuclear hormone receptors repress transcription through shared mechanisms involving SMRT recruitment and histone deacetylation.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10624 - 9
B cell receptor-associated protein alpha4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A; Murata K et al.; Recently, TAP42 was isolated as a high copy suppressor of sit4-, a yeast phosphatase related to protein phosphatase 2A (PP2A) . TAP42 is related to the murine alpha4 protein, which was discovered independently by its association with Ig-alpha in the B cell receptor complex . Herein we show that a glutathione S-transferase (GST)-alpha4 fusion protein bound the catalytic subunit (C) of human PP2A from monomeric or multimeric preparations of PP2A in a "pull-down" assay . In an overlay assay, the GST-alpha4 protein bound to the phosphorylated and unphosphorylated forms of C that were separated in two-dimensional gels and immobilized on filters . The results show direct and exclusive binding of alpha4 to C . This is unusual because all known regulatory B subunits, or tumor virus antigens, bind stably only to the AC dimer of PP2A . The alpha4-C form of PP2A had an increased activity ratio compared with the AC form of PP2A when myelin basic protein phosphorylated by mitogen-activated protein kinase and phosphorylase a were used as substrates . Recombinant alpha4 cleaved from GST was phosphorylated by p56(lck) tyrosine kinase and protein kinase C . A FLAG-tagged alpha4 expressed in COS7 cells was recovered as a protein containing phosphoserine and coimmunoprecipitated with the C but not the A subunit of PP2A . Treatment of cells with rapamycin prevented the association of PP2A with FLAG-alpha4 . The results reveal a novel heterodimer alpha4-C form of PP2A that may be involved in rapamycin-sensitive signaling pathways in mammalian cells.

J Biochem (Tokyo), 1997 Aug, 122(2), 251 - 7
GPI-anchor synthesis in mammalian cells: genes, their products, and a deficiency; Kinoshita T et al.; Protein GPI anchors are ubiquitous in eukaryotic cells . More than 50 mammalian proteins are anchored to the membrane via GPI . GPI anchoring is a posttranslational modification occurring in the endoplasmic reticulum where preassembled GPI anchor precursors are transferred to proteins bearing a C-terminal GPI signal sequence . The GPI anchor precursors are synthesized in the endoplasmic reticulum by sequential addition of sugar and other components to phosphatidylinositol . More than ten genes participate in this biosynthetic pathway, eleven of the mammalian genes having been cloned by means of complementation of mutant cells that are defective in this pathway or based on sequence homology to previously cloned yeast counterparts . A somatic mutation in one of those genes, PIG-A, involved in the first reaction step, is responsible for the hemolytic disease, paroxysmal nocturnal hemoglobinuria.

Cell Signal, 1997 Sep, 9(6), 403 - 10
Stress-activated protein kinases: activation, regulation and function; Paul A et al.; The response of cells to extracellular stimuli is mediated in part by a number of intracellular kinase and phosphatase enzymes . Within this area of research the activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinases have been extensively described and characterised as central components of the signal transduction pathways stimulated by both growth factors and G-protein-coupled receptor agonists . Signaling events mediated by these kinases are fundamental to cellular functions such as proliferation and differentiation . More recently, homologues of the p42 and p44 isoforms of MAP kinase have been described, namely the stress-activated protein kinases (SAPKs) or alternatively the c-jun N-terminal kinases (JNKs) and p38 MAP kinase (the mammalian homologue of yeast HOG1) . These MAP kinase homologues are integral components of parallel MAP kinase cascades activated in response to a number of cellular stresses including inflammatory cytokines (e.g., Interleukin-1 (Il-1) and tumour necrosis factor-alpha (TNF-alpha), heat and chemical shock, bacterial endotoxin and ischaemia/cellular ATP depletion . Activation of these MAP kinase homologues mediates the transduction of extracellular signals to the nucleus and are pivotal events in the regulation of the transcription events that determine functional outcome in response to such stresses . In this review we highlight the identification and characterisation of the stress-activated MAP kinase homologues, their role as components of parallel MAP kinase pathways and the regulation of cellular responses following exposure to cellular stress.

Genome Res, 1997 Sep, 7(9), 887 - 96
High-resolution mapping and transcript identification at the progressive epilepsy with mental retardation locus on chromosome 8p; Ranta S et al.; Progressive epilepsy with mental retardation (EPMR) is an autosomal recessive central nervous system disorder characterized by childhood onset epilepsy and subsequent mental retardation . The locus for EPMR has been mapped to human chromosome 8p23 . We recently reported the construction of a YAC contig across the 4 centimorgan minimum genetic region that harbors the disease locus . We now report further delineation of the critical region to <700 kb . Our mapping strategy relied on the identification of nine novel microsatellite markers and the construction of a complete BAC contig across the critical region . Several partial gene sequences have been identified from the region and are being analyzed as candidate genes for EPMR.

Semin Respir Infect, 1997 Sep, 12(3), 189 - 95
Virulent isolates and mutants of Blastomyces in mice: a legacy for studies of pathogenesis; Stevens DA et al.; This article provides information on the history of Blastomyces dermatitidis isolates and spontaneous mutants, the relationship with maintenance of cultures, and their virulence as quantified in murine models . Virulent isolates have been obtained from soil or from patients . Regardless of origin, mutants attenuated in virulence have sometimes arisen from storage as frozen yeast, frozen mycelia, or refrigerated yeast or serial passage in vitro at 35 degrees to 37 degrees C . Once virulence is lost, reversion to the virulent phenotype (even after serial passage in animals) has not been seen . These data, methods, and isolates provide a basis for future studies of virulence factors.

EMBO J, 1997 Sep 15, 16(18), 5742 - 51
Clusters of multiple different small nucleolar RNA genes in plants are expressed as and processed from polycistronic pre-snoRNAs; Leader DJ et al.; Small nucleolar RNAs (snoRNAs) are involved in many aspects of rRNA processing and maturation . In animals and yeast, a large number of snoRNAs are encoded within introns of protein-coding genes . These introns contain only single snoRNA genes and their processing involves exonucleolytic release of the snoRNA from debranched intron lariats . In contrast, some U14 genes in plants are found in small clusters and are expressed polycistronically . An examination of U14 flanking sequences in maize has identified four additional snoRNA genes which are closely linked to the U14 genes . The presence of seven and five snoRNA genes respectively on 2.05 and 0.97 kb maize genomic fragments further emphasizes the novel organization of plant snoRNA genes as clusters of multiple different genes encoding both box C/D and box H/ACA snoRNAs . The plant snoRNA gene clusters are transcribed as a polycistronic pre-snoRNA transcript from an upstream promoter . The lack of exon sequences between the genes suggests that processing of polycistronic pre-snoRNAs involves endonucleolytic activity . Consistent with this, U14 snoRNAs can be processed from both non-intronic and intronic transcripts in tobacco protoplasts such that processing is splicing independent.

EMBO J, 1997 Sep 15, 16(18), 5710 - 21
StuAp is a sequence-specific transcription factor that regulates developmental complexity in Aspergillus nidulans; Dutton JR et al.; The Aspergillus nidulans Stunted protein (StuAp) regulates multicellular complexity during asexual reproduction by moderating the core developmental program that directs differentiation of uninucleate, terminally differentiated spores from multinucleate, vegetative hyphae . StuAp is also required for ascosporogenesis and multicellular development during sexual reproduction . StuAp is a member of a family of fungal transcription factors that regulate development or cell cycle progression . Further, StuAp characterizes a sub-family possessing the conserved APSES domain . We demonstrate for the first time that the APSES domain is a sequence-specific DNA-binding domain that can be modeled as a basic helix-loop-helix (bHLH)-like structure . We have found that StuAp response elements (A/TCGCGT/ANA/C) are located upstream of both critical developmental regulatory genes and cell cycle genes in A.nidulans . StuAp is shown to act as a transcriptional repressor in A.nidulans, but as a weak activator in budding yeast . Our data suggest that the differentiation of pseudohyphal-like sterigmatal cells during multicellular conidiophore development requires correct StuAp-regulated expression of both developmental and cell cycle genes in A.nidulans . The budding pattern of sterigmata may involve processes related to those controlling pseudohyphal growth in budding yeast.

Science, 1997 Oct 3, 278(5335), 141 - 4
Evidence for a role of CRM1 in signal-mediated nuclear protein export; Ossareh-Nazari B et al.; Chromosome maintenance region 1 (CRM1), a protein that shares sequence similarities with the karyopherin beta family of proteins involved in nuclear import pathway, was shown to form a complex with the leucine-rich nuclear export signal (NES) . This interaction was inhibited by leptomycin B, a drug that prevents the function of the CRM1 protein in yeast . To analyze the role of the CRM1-NES interaction in nuclear export, a transport assay based on semipermeabilized cells was developed . In this system, which reconstituted NES-, cytosol-, and energy-dependent nuclear export, leptomycin B specifically blocked export of NES-containing proteins . Thus, the CRM1 protein could act as a NES receptor involved in nuclear protein export.

Int J Food Microbiol, 1997 Jul 22, 37(2-3), 231 - 5
Mycoflora of paddy and milled rice produced in the region of northeastern Argentina and southern Paraguay; Tonon SA et al.; Thirty samples of paddy rice and twenty-five of milled rice were obtained from processing centers located in two northern Provinces of Argentina and one southern Province of Paraguay . Contaminating fungi were enumerated by direct plating on dichloran rose bengal chloramphenicol agar and oxytetracycline glucose yeast extract agar before and after surface disinfection . All fungi were isolated and identified to the genus level and percentage infection of samples calculated . Those belonging to the genera Penicillium, Aspergillus and Fusarium were identified to species level . The surface mycoflora was dominated by storage fungi, notably Penicillium citrinum (73% of samples), P . islandicum (60% of samples), Aspergillus niger, A . flavus and Fusarium semitectum . The major fungi found as internal contaminants of paddy rice were, again, Penicillium citrinum (66% of samples) and P . islandicum (50% of samples) . Milled rice showed a lower level of contamination, but with a similar species distribution, Penicillium citrinum and P . islandicum again being the main contaminants . The presence of these species suggests a potential for mycotoxin production . Further studies are needed to establish the mycotoxin quality of rice from this region.

Eur J Biochem, 1997 Aug 15, 248(1), 187 - 92
Molecular cloning of the hamster CMP-sialic acid transporter; Eckhardt M et al.; Chinese hamster ovary (CHO) glycosylation mutants of the Lec2 complementation group are unable to express sialylated glycoproteins and glycolipids due to a defect in the Golgi CMP-sialic acid transporter (CMP-Sia-Tr) . Using an expression cloning strategy, we isolated a cDNA encoding the hamster CMP-Sia-Tr which complements the Lec2 phenotype . The deduced amino acid sequence of the cloned cDNA shows 95% identity to the recently cloned murine CMP-Sia-Tr . The expression of a hamster CMP-Sia-Tr fusion protein with an N-terminal MDYKDDDDK (FLAG) sequence revealed Golgi localisation of the transporter . Amino acid sequence comparison revealed strong similarity (44.6% identity and 19.3% similarity) of CMP-Sia-Tr to the recently cloned human UDP-galactose transporter (UDP-Gal-Tr) . In contrast, sequence similarities to the yeast UDP-N-acetylglucosamine transporter (UDP-GlcNAc-Tr) and the GDP-mannose transporter (GDP-Man-Tr) of Leishmania donovani are restricted to a region encoding the two most C-terminally located transmembrane helices . A computer-based structural analysis of CMP-Sia-Tr proposes an eight transmembrane helix model with the N- and C-termini located on the cytosolic side of the Golgi membrane.

Eur J Biochem, 1997 Aug 15, 248(1), 15 - 23
The structural organisation of LAMA4, the gene encoding laminin alpha4; Richards A et al.; We have determined the complete structural arrangement of LAMA4, the gene encoding the laminin alpha4 chain . Using both yeast artificial chromosome clones and total human genomic DNA and primers derived from the cDNA sequence, regions of the gene were amplified and sequenced to determine the splice donor and acceptor sites . The introns were sized by agarose gel electrophoresis of the PCR products . The gene consisted of 39 exons spanning 122 kb . All of the splice sites conformed to the GT/AG rule, except intron 7 which possessed a GC dinucleotide at the donor splice site . The intron/exon ratio was large at 17.8:1, mainly due to large introns at the 5' end of the gene . Regions at both the 5' and 3' end of the gene were subcloned from the yeast artificial chromosomes to enable untranscribed DNA to be sequenced . The gene represents the second of the laminin A gene family to be characterised and its structural organisation is similar to the equivalent regions of the LAMA2 gene.

Eur J Biochem, 1997 Aug 15, 248(1), 1 - 9
Linking protein kinase C to cell-cycle control; Livneh E et al.; Protein kinase C (PKC) isoenzymes are involved in diverse cellular functions, including differentiation, growth control, tumor promotion, and cell death . In recent years, evidence has began to emerge suggesting a role for PKC in cell cycle control . A paper published recently, demonstrating a functional link between PKC and cell cycle control in yeast (Marini, N . J., Meldrum, E., Buehrer, B., Hubberstey, A . V., Stone, D . E., Traynor-Kaplan, A . & Reed, S . I . (1996) EMBO J . 15, 3040-3052), strengthens this data . Thus, the existence of cell-cycle-regulated pathways involving PKC in both yeast and mammals indicate that PKC may be a conserved regulator of cell cycle events that links signal transduction pathways and the cell-cycle machinery . In this paper, we will review current data on the cell cycle components that are targets for PKC regulation . PKC enzymes appear to operate as regulators of the cell cycle at two sites, during G1 progression and G2/M transition . In G1, the overall effect of PKC activation is inhibition of the cell cycle at mid to late G1 . This cell cycle inhibition correlates with a blockage in the normal phosphorylation of the tumor suppressor retinoblastoma Rb protein, presumably through an indirect mechanism . The reduced activity of the cyclin-dependent kinase, Cdk2, appears to be the major effect of PKC activation in various cell systems . This may also underlie the inhibition of Rb phosphorylation exhibited by PKC activation . Several mechanisms were described in different studies on the regulation of Cdk2 activity by PKC; reduced Cdk-activating kinase activity, diminished expression of the Cdk2 partners cyclins E or A, and the increased expression of the cyclin-dependent inhibitors, p21WAF1 and p27KIP1, which are capable of binding to cyclin/Cdk2 complexes . PKC enzymes were also shown to play a role in G2/M transition . Among the suggested mechanisms is suppression of Cdc2 activity . However, most of the published data strongly implicate PKC in lamin B phosphorylation and nuclear envelope disassembly.

Gan To Kagaku Ryoho, 1997 Sep, 24(11), 1442 - 7
{F-actin cappers}; Maruta H; Members of a large protein family that cap the barbed (fast-growing) end of actin filament (F-actin) are called F-actin "Cappers" . The first F-actin capper called Cap 28/31 is a heterodimer of 28 kDa and 31 kDa proteins, and was isolated from a soil amoeba called Acanthamoeba (Isenberg et al., 1980) . F-actin cappers are present in any eucaryotes from yeast to human, and block actin polymerization by capping the fast-growing end of F-actin . In non-stimulated cells, most of the fast-growing ends of actin filaments are capped by an 1:1 complex of actin monomer (G-actin) and profilin, a PIP2-binding protein . When cells are stimulated by one of the mitogenic cytokines such as EGF and PDGF, Ras is activated, and consequently Rac is activated . Rac in turn activates PI-4 kinase which produces PIP2 . PIP2 then binds profilin, and dissociates the profilin/G-actin complex, leading to uncapping of the fast-growing end of actin filament, and induces a rapid actin polymerization . Eventually, this results in the induction of membrane ruffling . We found that (1) the Ras/Rac-induced uncapping is required for oncogenicity of Ras, and (2) either capping at the fast-growing end by F-actin cappers such as tensin and cytochalasins, or sequestering PIP2 by PIP2-binders such as cofilin mutants (blocking the uncapping) is sufficient to suppress the malignant transformation caused by oncogenic Ras mutants such as v-Ha-Ras.

Arch Biochem Biophys, 1997 Sep 15, 345(2), 175 - 84
Homologous and heterologous protein-protein interactions of human DNA topoisomerase IIalpha; Kroll DJ; DNA topoisomerase II (topo II; EC 5.99.1.3) is a nuclear enzyme whose DNA decatenating activity on newly replicated DNA is essential to successful cell division . Topo II catalytic activity proceeds by a concerted DNA breakage-reunion reaction coordinated between two interacting, homologous subunits . Human and yeast topo II have recently been shown to enter into heterologous protein-protein interactions and some of these interactions appear necessary for successful chromosomal segregation . In the present study, the sequences mediating homologous and heterologous protein-protein interactions have been investigated biochemically using various truncated peptides from the major alpha form of human topo II . From nonreducing gel electrophoresis and solid-phase protein-protein binding (Far Western) assays, topo II homodimerization appeared to be minimally governed by the region between amino acids 951 and 1042 . However, maximal homodimerization and multimerization required sequences C-terminal to position 1042 . Topo II peptides were also able to interact with 10-12 nuclear proteins from HeLa cells, termed topo II-interactive proteins or TIPs . Interestingly, small topo II peptides between residues 808 and 951 that did not homodimerize with topo II (857-1447) were nonetheless capable of binding to HeLa TIPs . These interactions were confirmed by use of topo II affinity chromatography for isolation of specific TIPs from HeLa nuclear extracts . Taken together, these data confirm that human topo II is also capable of heterologous interactions with nuclear proteins and that the region governing these interactions is distinct from, but has some overlap with, sequences directing topo II homodimerization.

Mol Biol Cell, 1997 Sep, 8(9), 1777 - 87
An isoform of the Golgi t-SNARE, syntaxin 5, with an endoplasmic reticulum retrieval signal; Hui N et al.; The early Golgi t-SNARE (target-membrane-associated soluble-N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 5 is thought to specify the docking site for both COPI and COPII coated vesicles originating from the endoplasmic reticulum (ER) and COPI vesicles on the retrograde pathway . We now show that there are two forms of syntaxin 5 that appear to be generated from the same mRNA by alternative initiation of translation . The short form (35 kDa) corresponds to the published sequence . The long form (42 kDa) has an N-terminal cytoplasmic extension containing a predicted type II ER retrieval signal . When grafted onto a reporter molecule, this signal localized the construct to the ER . Biochemical fractionation and immunofluorescence microscopy showed that there was less of the long form in the Golgi apparatus and more in peripheral punctate structures, some of which colocalized with markers of the intermediate compartment . The predicted absence of the long form in budding yeast points to a function unique to higher organisms.

J Biol Chem, 1997 Sep 26, 272(39), 24508 - 13
A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex; Ishimi Y; All six minichromosome maintenance (MCM) proteins have DNA-dependent ATPase motifs in the central domain which is conserved from yeast to mammals . Our group purified MCM protein complexes consisting of MCM2, -4 (Cdc21), -6 (Mis5), and -7 (CDC47) proteins from HeLa cells by using histone-Sepharose column chromatography (Ishimi, Y., Ichinose, S., Omori, A., Sato K., and Kimura, H . (1996) J . Biol . Chem . 271, 24115-24122) . The present study revealed that both ATPase activity and DNA helicase activity that displaces oligonucleotides annealed to single-stranded circular DNA are associated with an MCM protein complex . Both ATPase and DNA helicase activities were co-purified with a 600-kDa protein complex that is consisted of equal amounts of MCM4, -6, and -7 proteins . An immunodepletion of the MCM protein complex from the purified fraction using anti-MCM4 antibody resulted in the severe reduction of the DNA helicase activity . Displacement of DNA fragments by the DNA helicase suggested that it migrated along single-stranded DNA in the 3' to 5' direction, and the DNA helicase activity was detected only in the presence of hydrolyzable ATP or dATP . These results suggest that this helicase may be involved in the initiation of DNA replication as a DNA unwinding enzyme.

J Biol Chem, 1997 Sep 26, 272(39), 24494 - 8
Functional dissection of the pro-apoptotic protein Bik . Heterodimerization with anti-apoptosis proteins is insufficient for induction of cell death; Elangovan B et al.; Bik is a potent pro-apoptotic protein, which complexes with various anti-apoptotic proteins such as Bcl-2, Bcl-xL, 19-kDa adenovirus E1B, and EBV-BHRF1 . The mechanism by which Bik promotes cell death is not known . It shares a conserved domain, BH3, with other pro-apoptotic proteins, Bax, Bak, Bid, and Hrk, and certain anti-apoptosis proteins such as Bcl-2 and Bcl-xL . Mutations within the BH3 domain of Bik abrogate its ability to induce cell death and to complex with anti-apoptosis proteins . This result is consistent with the hypothesis that Bik may promote cell death by complexing with and antagonizing the activity of endogenous cellular anti-apoptosis proteins such as Bcl-2 and Bcl-xL . To elucidate the relationship between protein complex formation and induction of cell death, we have identified the minimal sequences of Bik, from a library of N-terminal and C-terminal deletion mutants, required for interaction with Bcl-2 and Bcl-xL and for inducing efficient cell death . Two-hybrid analysis in yeast and immunoprecipitation analysis of proteins expressed in mammalian cells indicate that a 52-amino acid region (amino acids 43-94) of Bik, encompassing the BH3 domain, is sufficient for efficient heterodimerization with Bcl-2 and Bcl-xL . Protein interaction studies further reveal that an 18-amino acid region, encompassing the BH3 domain (residues 57-74), constitutes the core heterodimerization domain . Functional analysis indicates that a Bik deletion mutant expressing residues 43-120, which efficiently heterodimerizes with the anti-apoptosis proteins Bcl-2 and Bcl-xL, is defective in eliciting cell death . In contrast, a mutant expressing additional C-terminal sequences (amino acids 43-134) interacts with the survival proteins and elicits efficient cell death . Our results suggest that for Bik-mediated cell death, the heterodimerization activity encoded by the BH3 domain alone is insufficient and raise the possibility that Bik may induce cell death autonomous of heterodimerization with survival proteins such as Bcl-2 and Bcl-xL.

J Biol Chem, 1997 Sep 26, 272(39), 24387 - 92
Three vha genes encode proteolipids of Caenorhabditis elegans vacuolar-type ATPase . Gene structures and preferential expression in an H-shaped excretory cell and rectal cells; Oka T et al.; The proteolipids of the vacuolar-type H+-ATPase (V-ATPase) are major components of the integral membrane sector . The vha-1 and vha-2 (vacuolar-type H+-ATPase) genes in Caenorhabditis elegans encode putative 16-kDa proteolipids and are tandemly localized on chromosome III . The vha-2 gene has three exons, whereas vha-1 has no introns . The deduced amino acid sequences of the two genes exhibit about 60% identity with the homologues from yeast, mouse, and cow . The mRNAs of both vha genes are trans-spliced to spliced leaders, suggesting that these genes constitute a polycistronic transcriptional unit . The vha-4 gene consists of four exons and is very similar to the yeast VMA16 gene that codes for the 23-kDa proteolipid . This is the first example of three distinct V-ATPase proteolipids being identified in higher eukaryotes . Northern blot and transgenic analyses show that the three vha genes may be highly expressed in the H-shaped excretory cell, rectum, and a pair of cells posterior to the anus . These results suggest that the V-ATPase activity may be important for exporting toxic compounds or metabolic wastes in this organism.

J Biol Chem, 1997 Sep 26, 272(39), 24129 - 32
Uncoupling protein-3 is a mediator of thermogenesis regulated by thyroid hormone, beta3-adrenergic agonists, and leptin; Gong DW et al.; Mitochondrial uncoupling proteins (UCPs) are transporters that are important for thermogenesis . The net result of their activity is the exothermic movement of protons through the inner mitochondrial membrane, uncoupled from ATP synthesis . We have cloned a third member of the UCP family, UCP3 . UCP3 is expressed at high levels in muscle and rodent brown adipose tissue . Overexpression in yeast reduced the mitochondrial membrane potential, showing that UCP3 is a functional uncoupling protein . UCP3 RNA levels are regulated by hormonal and dietary manipulations . In contrast, levels of UCP2, a widely expressed UCP family member, showed little hormonal regulation . In particular, muscle UCP3 levels were decreased 3-fold in hypothyroid rats and increased 6-fold in hyperthyroid rats . Thus UCP3 is a strong candidate to explain the effects of thyroid hormone on thermogenesis . White adipose UCP3 levels were greatly increased by treatment with the beta3-adrenergic agonist, CL214613, suggesting another pathway for increasing thermogenesis . UCP3 mRNA levels were also regulated by dexamethasone, leptin, and starvation, albeit differently in muscle and brown adipose tissue . Starvation caused increased muscle and decreased BAT UCP3, suggesting that muscle assumes a larger role in thermoregulation during starvation . The UCP3 gene is located close to that encoding UCP2, in a chromosomal region implicated in previous linkage studies as contributing to obesity.

Hum Mol Genet, 1997 Sep, 6(9), 1505 - 11
Mutations in the MTM1 gene implicated in X-linked myotubular myopathy . ENMC International Consortium on Myotubular Myopathy . European Neuro-Muscular Center; Laporte J et al.; X-linked recessive myotubular myopathy (XLMTM) is characterized by severe hypotonia and generalized muscle weakness, with impaired maturation of muscle fibres . The gene responsible, MTM1, was identified recently by positional cloning, and encodes a protein (myotubularin) with a tyrosine phosphatase domain (PTP) . Myotubularin is highly conserved through evolution and defines a new family of putative tyrosine phosphatases in man . We report the identification of MTM1 mutations in 55 of 85 independent patients screened by single-strand conformation polymorphism for all the coding sequence . Large deletions were observed in only three patients . Five point mutations were found in multiple unrelated patients, accounting for 27% of the observed mutations . The possibility of detecting mutations and determining carrier status in a disease with a high proportion of sporadic cases is of importance for genetic counselling . More than half of XLMTM mutations are expected to inactivate the putative enzymatic activity of myotubularin, either by truncation or by missense mutations affecting the predicted PTP domain . Additional mutations are missenses clustered in two regions of the protein . Most of these affect amino acids conserved in the homologous yeast and Caenorhabditis elegans proteins, thus indicating the presence of other functional domains.

EMBO J, 1997 Aug 15, 16(16), 4851 - 8
Linkage of the ubiquitin-conjugating system and the endocytic pathway in ligand-induced internalization of the growth hormone receptor; Govers R et al.; The major function of the ubiquitin-conjugating system is the targeting of cytosolic and nuclear proteins for degradation by the proteasome . Recently, ubiquitin conjugation has been implicated in the downregulation of signalling receptors such as the mammalian growth hormone receptor (GHR) and the alpha-factor receptor in yeast . By examining truncated receptors, the internalization-deficient receptor mutant F327A and conditions under which clathrin-mediated GHR endocytosis is inhibited, we show here that GHR ubiquitination and ligand-induced GHR internalization are coupled events . Previously, we had shown that GHR endocytosis is dependent on an intact ubiquitination system . Here we present evidence that GHR ubiquitination depends on an intact endocytic pathway . Our data indicate that the ubiquitin-conjugating system and the endocytic pathway interact at the cytoplasmic tail of the GHR at the plasma membrane, where they cooperate to regulate internalization of the GHR.

Gen Pharmacol, 1996 Dec, 27(8), 1395 - 400
Evaluation of the antiinflammatory activity of sodium valproate in rats and mice; Raza M et al.; 1 . Treatment of rats with sodium valproate (100, 200 and 400 mg/kg i.p.) reduced the paw oedema induced by carrageenan by 36, 15 and 48%, respectively, within 3 h . 2 . The effect produced by the higher dose (400 mg/kg) was equivalent to that produced by indomethacin (100 mg/kg) . At 100 and 400 mg/kg, sodium valproate decreased the granuloma formation by a significant level . Similar doses of sodium valproate did not affect the rectal temperature in yeast-fevered mice, except with a dose of 200 mg/kg, which showed a significant decrease at 180 and 240 min posttreatment . 3 . In comparison, sodium salicylate reduced the hyperthermia very significantly throughout the study period . 4 . In normothermic mice, the rectal temperature changed only with a 400 mg/kg dose, but did not respond to lower SV doses . 5 . The results indicate that sodium valproate, useful clinically as an epileptic drug, may have a potential therapeutic use as a mild antiinflammatory agent.

Genes Dev, 1997 Sep 1, 11(17), 2239 - 49
Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module; Salcini AE et al.; EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode . We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif . Direct screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction . Among these interactors were the human homolog of NUMB, a developmentally reguated gene of Drosophila, and RAB, the cellular cofactor of the HIV REV protein . We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB . Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a family of EH-containing proteins in mammals . Based on the characteristics of EH-containing and EH-binding proteins, we propose that EH domains are involved in processes connected with the transport and sorting of molecules within the cell.

Int Arch Allergy Immunol, 1997 Sep, 114(1), 15 - 22
Epitope analysis of human T-cell response to MSP-1 of Plasmodium falciparum in malaria-nonexposed individuals; Ohta N et al.; BACKGROUND: MSP-1 of Plasmodium falciparum induces strong proliferative T cell responses even in malaria-nonexposed individuals . Epitopes recognized by malaria-nonimmune T cells have not been identified, and immunological mechanisms inducing such T cell responses remain to be uncovered . MSP-1 is a vaccine candidate, and it should be understood whether those epitopes have any roles in MSP-1-mediated protective immunity . The T epitopes-inducing malaria-naive T cell response was analyzed in the hope of understanding the underlying mechanisms . METHODS: Human T cell lines and clones reactive to MSP-1 of P . falciparum were established from malaria-nonexposed Japanese donors in vitro, and epitope peptides were identified . Sequences of those epitope peptides were compared to unrelated peptides in the data base . One of those peptides was tested for both binding to HLA-DR molecules and inducing proliferative responses of MSP-1-reactive T cells . RESULTS: There are at least 6 epitopes recognized by malaria-naive T cells under the restriction by HLA-DRB1*1502 or 0802 . Important amino acids for the T cell recognition were identified for an MSP-1 peptide . A yeast peptide which shared those residues induced proliferative responses of MSP-1-reactive T cells . CONCLUSION: We identified T epitopes in the N-terminal region of MSP-1, some of which showed molecular similarities with unrelated environmental antigens, suggesting the presence of cross-reactive T epitopes in MSP-1 . Cytokine production in response to those epitopes suggests regulatory functions of those T cells during primary infection with P . falciparum.

Dev Comp Immunol, 1997 Jul-Aug, 21(4), 349 - 62
LPS-induced stimulation of phagocytosis in the sipunculan worm Themiste petricola: possible involvement of human CD14, CD11B and CD11C cross-reactive molecules; Blanco GA et al.; Coelomocytes of Themiste petricola, a marine invertebrate of the phylum Sipuncula, were exposed in vitro to bacterial lipopolysaccharides (LPS), and the phagocytic activity against heat-killed yeast (Saccharomices cerevisiae) was evaluated using a flow cytometric assay . An increase of phagocytic activity was observed following pre-incubation of coelomocytes over 20 h with either 5 micrograms/mL LPS or 1.5 micrograms/mL phorbol 12-myristate 13-acetate (PMA) . The phagocytic enhancement induced by LPS was blocked by co-incubation with polymixin B, a ligand for the lipid A region of LPS . In a 72 h stimulation assay, LPS was also found to enhance phagocytosis . The enhancement was significantly higher when coelomocytes were incubated with LPS plus coelomic plasma . Using mAbs directed against human CD14 and components of the human LFA-1 complex, we identified coelomocyte surface antigens cross-reactive with CD14, CD11b and CD11c . The expression of CD11b and CD11c antigens was augmented by LPS treatment of coelomocytes . By double fluorescence assays, using mAb Leu-M3 and fluorescein labeled yeast, phagocytic coelomocytes were found to be mainly anti-CD14 positive . No cross-reactions were detected with mAbs against CD11a and CD18 . Enzymatic treatment of coelomocytes with phosphatidyl inositol phospholipase C (PI-PLC) reduced the expression of the CD14-like antigen . The presence, in sipunculan coelomocytes, of antigens cross-reactive with CD14, the alpha chain of CR3 and of p150,95 raises the possibility that molecules related, although not necessary homologous, to the mammalian counterparts may have a role in the defense systems of these animals.

Nat Struct Biol, 1997 Sep, 4(9), 717 - 24
Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein; Matsuo H et al.; eIF4E, the mRNA cap binding protein, is a master switch that controls eukaryotic translation . To be active, it must bind eIF4G and form the eIF4F complex, which also contains eIF4A . Translation is downregulated by association of eIF4E with 4E-BP, which occupies the eIF4G binding site . Signalling events acting on 4E-BP cause it to dissociate from eIF4E, and eIF4E is then free to bind eIF4G to form the active eIF4F complex . We have solved the structure of the yeast eIF4E/m7Gpp complex in a CHAPS micelle . We determined the position of the second nucleotide in a complex with m7GpppA, and identified the 4E-BP binding site . eIF4E has a curved eight-stranded antiparallel beta-sheet, decorated with three helices on the convex face and three smaller helices inserted in connecting loops . The m7G of the cap is intercalated into a stack of tryptophans in the concave face . The 4E-BP binding site is located in a region encompassing one edge of the beta-sheet, the adjacent helix a2 and several regions of non-regular secondary structure . It is adjacent to, but does not overlap the cap-binding site.

Hum Mol Genet, 1997 Oct, 6(11), 1817 - 22
Cloning of a novel transcription factor-like gene amplified in human glioma including astrocytoma grade I; Fischer U et al.; Gene amplification, which is generally considered to occur late in tumor development, is a common feature of high grade glioma . Up until now, there have been no reports on amplification in astrocytoma grade I . In this study, we report cloning and sequencing of a cDNA termed glioma-amplified sequence (GAS41) which was identified recently in a glioblastoma cell line by microdissection-mediated cDNA capture . This technique is tailored to isolate amplified genes from human tumors . An increased copy number of GAS41 was found in glioblastoma multiforme and astrocytoma III, and at a high frequency in astrocytoma grades I and II . Sequence comparison indicates a high homology between the GAS41 protein, the yeast and human AF-9 and the human ENL proteins . Both AF-9 and ENL belong to a new class of transcription factors, indicating that GAS41 might also represent a transcription factor . With GAS41 being the first gene found with increased copy number in low grade glioma, this study provides the first evidence that gene amplification can occur in early tumor development.

J Pak Med Assoc, 1997 Jul, 47(7), 191 - 2
Candidiasis in cancer patients; Zai S et al.; One hundred clinical specimens from hospitalized cancer patients were examined microscopically for evidence of yeast cells and cultured for Candida colonization . Candida cells were observed microscopically in both unstained and Gram-stained preparations and culture in 60% of specimens.

Biosci Biotechnol Biochem, 1997 Aug, 61(8), 1370 - 2
NADPH-dependent reduction of ethyl acetoacetate coupled with ethanol oxidation in Kloeckera magna; Kometani T et al.; We evaluated the catalytic ability of 29 yeast strains to reduce ethyl acetoacetate (EA) in the presence of ethanol or glucose . In 18 yeast strains, the reduction in the presence of ethanol proceeded as well as in the presence of glucose . Among them, Kloeckera magna (AKU 4704) effectively catalyzed the NADPH-dependent reduction of EA in the presence of ethanol . In this reduction, 1 mol of EA was reduced by consuming 1 mol of ethanol . We found that the NADPH regeneration system responsible for EA reduction in K . magna was coupled with oxidation of acetaldehyde to acetic acid catalyzed by an NADP(+)-dependent aldehyde dehydrogenase.

Plant J, 1997 Aug, 12(2), 471 - 9
Fine-scale molecular genetic (RFLP) and physical mapping of a 8.9 cM region on the top arm of Arabidopsis chromosome 5 encompassing the male sterility gene, ms1; Thorlby GJ et al.; Fine-scale molecular mapping has been conducted using 183 recombinants between the markers lutescens (lu; 17.6 cM) and transparent testa glabra (ttg; 35.5 cM) on the top arm of Arabidopsis thaliana chromosome 5 . This region contains a number of genes involved in floral development including Ms1, a gene required for the post-meiotic development of pollen . In homozygous ms1 mutant plants, pollen development is aborted soon after microspore release, regardless of environmental conditions . The ms1 mutation is located at 29.8 +/- 0.8 cM on chromosome 5 . Markers have been identified which co-segregate with ms1 and should lie within 39 kb of the gene . The fine-scale map of the lu-ms1-ttg region that has been generated is significantly different from the published integrated map and provides substantially more accurate and higher marker density than the current recombinant inbred map for this region . Using clones derived from four yeast artificial chromosome libraries, a contig has been established between the RFLP markers 4111 and 4556, which encompasses the ms1 gene . This covers a genetic distance of 8.9 cM which corresponds to a physical distance of approximately 1.44 Mb, representing about 1.5-2.0% of the Arabidopsis genome . In this region, 1 cM represents a physical distance of approximately 160 kb.

Biol Chem, 1997 Aug, 378(8), 803 - 13
Lipid mobilization and gluconeogenesis in plants: do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis; Escher CL et al.; Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves . They are the site of beta-oxidation and of the glyoxylate cycle . These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids . In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors . The necessity of NAD+ regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles . Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system . Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD+ regeneration) . Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.

J Immunol, 1997 Sep 15, 159(6), 2789 - 94
Activation and transdominant suppression of MHC class II and HLA-DMB promoters by a series of C-terminal class II transactivator deletion mutants; Chin KC et al.; The class II transactivator (CIITA) is a highly specific transcription factor that activates only genes known to be involved in the class II MHC processing pathway, including class II MHC, invariant chain, and HLA-DMA/B genes . In this work, we show the requirement of a new region in CIITA that is critical for its function . Deletion mutants lacking varying length of the C terminus show that the C-terminal 41 amino acids of CIITA are indispensable for the activation of both the conventional DRA and nonconventional DMB promoters . This region contains a highly charged stretch of amino acids that is homologous to a yeast transcription factor, repression activator protein-1 (RAP1)-interacting factor 1 . Mutants lacking the C terminus were tested in a transdominant-negative assay to examine their capacity to block the wild-type CIITA function . Two of these deletion mutants suppressed the activity of endogenously expressed wild-type CIITA to activate both DRA and DMB promoters . It may be possible to utilize these mutants to modulate MHC class II and DM gene expression.

Protein Sci, 1997 Sep, 6(9), 1825 - 34
Identification of electrostatic interaction sites between the regulatory and catalytic subunits of cyclic AMP-dependent protein kinase; Gibson RM et al.; Two classes of molecules inhibit the catalytic subunit (C) of the cyclic AMP-dependent protein kinase (cAPK), the heat-stable protein kinase inhibitors (PKIs) and the regulatory (R) subunits . Basic sites on C, previously identified as important for R/C interaction in yeast TPK1 and corresponding to Lys213, Lys217, and Lys189 in murine C alpha, were replaced with either Ala or Thr and characterized for their kinetic properties and ability to interact with RI and PKI . rC(K213A) and rC(K217A) were both defective in forming holoenzyme with RI but were inhibited readily with PKI . This contrasts with rC(R133A), which is defective in binding PKI but not RI (Wen & Taylor, 1994) . Thus, the C-subunit employs two distinct electrostatic surfaces to achieve high-affinity binding with these two types of inhibitory molecules even though all inhibitors share a common consensus site that occupies the active site cleft . Unlike TPK1, mutation of Lys189 had no effect . The mutant C subunits that were defective in binding RI, rC(K213A) and rC(K217A), were then paired with three RI mutants, rRI(D140A), rRI(E143A), and rRI(D258A), shown previously to be defective in recognition of C . Although the mutations at Asp140 and Asp258 in RI were additive with respect to the C mutations . rC(K213A) and rRI(E143A) were compensatory, thus identifying a specific electrostatic interaction site between RI and C . The results are discussed in terms of the RI and C crystal structures and the sequence homology between the yeast and mammalian enzymes.

J Mol Biol, 1997 Aug 8, 271(1), 124 - 38
The mouse histone H1 genes: gene organization and differential regulation; Wang ZF et al.; There are six mouse histone H1 genes present in the histone gene cluster on mouse chromosome 13 . These genes encode five histone H1 variants expressed in somatic cells, H1a to H1e, and the testis-specific H1t histone . Two of the genes that have not been assigned previously to the five somatic H1 subtypes have been identified as encoding the H1b and H1d subtypes . Three of the H1 genes, H1a, H1c and H1t, are present on an 80 kb segment of DNA that contains nine core histone genes . Two others, H1d and H1e, are present in a second patch, while the H1b gene is at least 500 kb away in a patch containing 14 core histone genes . The histone H1 genes are differentially expressed . All five genes for the somatic histone H1 proteins are expressed in exponentially growing cells . However, the levels of H1a, H1b and H1d mRNAs are greatly reduced in cells that are terminally differentiated or arrested in G0, while the H1c and H1e mRNAs continue to be expressed . In addition to the major RNA that ends at the stem-loop, the H1c gene expresses a longer, polyadenylated mRNA in differentiated cells, although in varying amounts . None of the other histone H1 genes encodes detectable amounts of polyadenylated mRNAs.

Biochem Biophys Res Commun, 1997 Sep 18, 238(2), 590 - 4
A20 inhibits NF-kappaB activation independently of binding to 14-3-3 proteins; De Valck D et al.; The A20 protein, which belongs to a class of Cys2/Cys2 zinc finger proteins, has been characterized as an inhibitor of NF-kappaB activation . In order to clarify its molecular mechanism of action, the yeast two-hybrid system was used to screen for interacting proteins . We report that different isoforms of 14-3-3 proteins, viz . eta and zeta, are able to bind A20, involving the 14-3-3-binding motif RSKSDP located between zinc fingers 3 and 4 . However, A20 mutants that no longer associated with 14-3-3 proteins could still fully inhibit NF-kappaB activation induced by tumor necrosis factor, interleukin-1beta or phorbol 12-myristate 13-acetate, thus excluding a crucial role for 14-3-3 interaction in this A20 function .

Genomics, 1997 Sep 1, 44(2), 214 - 21
A high-resolution STS, EST, and gene-based physical map of the hereditary paraganglioma region on chromosome 11q23; Baysal BE et al.; The genes responsible for hereditary paragangliomas (glomus tumors, MIM No . 168000) have been mapped to two distinct loci on the long arm of chromosome 11 . Most of the informative families appear to be linked to the distal locus on chromosome 11q23 (PGL1), which has been previously confined to a 2-cM interval by haplotype analysis in an extended Dutch pedigree . To facilitate the identification of the PGL1 disease gene, we constructed an approximately 4-Mb ordered clone contig map of Sequence tagged sites, expressed sequence tags (ESTs), and known genes that spans the PGL1 critical region on chromosome 11q23 . Among 29 new positional candidate ESTs, only two (EST100999 and EST241777) mapped within the PGL1 critical region . We further characterized the genomic organization of the promyelocytic leukemia zinc finger (PLZF) gene that maps within the PGL1 critical region and physically excluded the serotonin receptor type 3 (5HT3R) gene . Finally, we identified a common, silent, single-base substitution polymorphism in the 5HT3R gene and characterized the allele sets of two new highly polymorphic microsatellite repeats within the PGL1 critical region .

Chronobiol Int, 1997 Sep, 14(5), 455 - 68
Insights into the molecular mechanisms of temperature compensation from the Drosophila period and timeless mutants; Price JL; The relative constancy of the circadian period over a wide range of temperatures is a general property of circadian rhythms . Insights into the molecular mechanisms of temperature compensation are emerging from genetic and molecular genetic studies of the period (per) and timeless (tim) genes in Drosophila . These genes encode proteins that are thought to be part of a negative feedback cycle, which results in circadian oscillations of both per and tim mRNA, as well as a complex of the two proteins . Complex formation is temporally regulated and apparently necessary for nuclear localization of both per and tim proteins . While insights into the roles of per and tim in temperature compensation have been intriguing, they have also been somewhat perplexing . For instance, the interaction of wild-type per peptides is relatively insensitive to temperature in the yeast two-hybrid assay or in assays employing in-vitro-translated peptides, while the interaction of perL mutant peptides is reduced at a high temperature . Apparently, the perL mutation increases an intramolecular interaction between different parts of the per peptide in these assays, and this interaction reduces the amount of per homodimer . On the other hand, the same assays show that the intermolecular interaction between the per and tim peptides is reduced at a high temperature by the perL mutation; this reduction does not require the competing intramolecular interaction . Despite this difference, in all of the experiments employing these assays the perL mutation has rendered per-per and per-tim peptide interactions sensitive to high temperature, so it is likely that one or both of these reduced interactions contribute to the longer circadian periods at high temperature in perL mutant flies . However, the timSL and perS mutations, as well as deletion of the Thr-Gly repeats from per, affect temperature compensation but have not been shown to affect these molecular interactions of per and tim . Finally, a recent report of oscillating per and tim proteins in the cytoplasm (rather than the nuclei) of silk moth neurons may suggest an alternative mechanism for per and tim function in these cells.

Antonie Van Leeuwenhoek, 1997 Aug, 72(2), 141 - 7
A new variety of Aureobasidium pullulans characterized by exopolysaccharide structure, nutritional physiology and molecular features; Yurlova NA et al.; The black yeast Aureobasidium pullulans (de Bary) Arnaud is known to synthesize the exopolysaccharide pullulan, a poly-alpha-1,6-maltotriose . Nine strains were found to produce additional aubasidan-like EPS, i.e . glucans with alpha-1,4-D-, beta-1,6-D- and beta-1,3-D-glycosidic bonds . These strains had previously been found to deviate in genotypic characters . Additional physiological differences were found: the optimal nitrogen source for exopolysaccharide production in liquid medium was NaNO3 for aubasidan-producing strains, and (NH4)2SO4 for the remaining strains . A new variety, A . pullulans var . aubasidani Yurlova, is described for the strains producing aubasidan-like components . The new variety can be distinguished from A . pullulans var . pullulans by the absence of assimilation of methyl-alpha-D-glucoside and lactose.

Antonie Van Leeuwenhoek, 1997 Aug, 72(2), 119 - 26
Penicillium discolor, a new species from cheese, nuts and vegetables; Frisvad JC et al.; The new species Penicillium discolor, frequently isolated from nuts, vegetables and cheese is described . It is characterised by rough, dark green conidia, synnemateous growth on malt agar and the production of the secondary metabolites chaetoglobosins A, B and C, palitantin, cyclopenin, cyclopenol, cyclopeptin, dehydrocyclopeptin, viridicatin and viridicatol . It also produces the mouldy smelling compounds geosmin and 2-methyl-isoborneol, and a series of specific orange to red pigments on yeast extract sucrose agar, hence the epithet discolor . P . discolor resembles P . echinulatum morphologically but on basis of the secondary metabolites is also related to P . expansum, P . solitum and P . crustosum.

Antonie Van Leeuwenhoek, 1997 Aug, 72(2), 111 - 7
Genetics and electrophoretic karyotyping of wild-type and astaxanthin mutant strains of Phaffia rhodozyma; Cifuentes V et al.; In this work we establish the chromosomal composition of a wild-type, one astaxanthin and two beta-carotene overproducer strains of the red yeast Phaffia rhodozyma . The method used has been pulsed field gel electrophoresis, which has determined 9 DNA chromosomal bands in the yeast genome . The two largest bands are triplets and two other bands, VI and VIII, seem to be doublets . The size of the chromosomal bands varies between 0.35 and 2.5 Mb, suggesting a genome size of 25 Mb . The technique used, complemented with hybridization assays using specific DNA probes, provides direct information about the genomic organization of P . rhodozyma . We have also cloned and located in chromosomal bands different DNA sequences that code for the translation elongation factor 1 alpha (ef-1 alpha), a 7.6 kb BamHI fragment of repetitive DNA (possibly rDNA) and a randomly chosen fragment (named locus R2) . Additionally, we have detected a chromosomal length polymorphism between wild-type strains and mutant strains affecting carotenogenesis obtained in our laboratory.

Lett Appl Microbiol, 1997 Jun, 24(6), 463 - 6
Identification of cheese-associated fungi using selected ion monitoring of volatile terpenes; Larsen TO; The cheese-associated fungi Penicillium commune, P . roqueforti, P.solitum, P . discolor and Aspergillus versicolor have been investigated for production of volatile terpenes for chemical identification, when grown on yeast extract agar . Volatiles were collected by headspace solid-phase microextraction . Selected ion monitoring of four to seven of the most characteristic ions of mainly sesquiterpenes made it possible to identify the fungi to species level within 2 d . In a mixed culture of P . roqueforti and P . commune, inoculated in a ratio of 1000:1, volatiles from both fungi could be detected within 3 d, making identification possible.

Nature, 1997 Sep 11, 389(6647), 149 - 52
Regulation of transcription by proteins that control the cell cycle; Dynlacht BD; In eukaryotes, progression of a cell through the cell cycle is partly controlled at the level of transcriptional regulation . Yeast and mammalian cells use similar mechanisms to achieve this regulation . Although gaps still remain, progress has been made recently in connecting the links between the cell's cycle and its transcriptional machinery.

Arzneimittelforschung, 1997 Aug, 47(8), 917 - 27
Role of caffeine in combined analgesic drugs from the point of view of experimental pharmacology; Engelhardt G et al.; The interactions of caffeine (CAS 58-08-2) with acetylsalicylic acid (CAS 50-78-2, ASA) and paracetamol (CAS 103-90-2) were investigated with regard to the analgesic, antiphlogistic, antipyretic and other properties . The inhibitory effect of paracetamol and ASA on the prostaglandin biosynthesis in a cyclooxygenase preparation from bovine brain in vitro was not affected by the addition of caffeine . Caffeine additively increases the antinociceptive effect of paracetamol with regard to the heat-induced pain in the mouse, as does aminophenazone . The antinociceptive effect of aminophenazone on the mechanically induced pain in the mouse is also additively increased by caffeine . In contrast to the effect of aminophenazone on the inflammatory pain in the rat, the effect of ASA is not increased by caffeine and that of paracetamol only negligibly . The antipyretic effect of paracetamol is additively increased by caffeine in the normothermic rat . The antipyretic effect of ASA and paracetamol on the yeast-induced pyrexia of the rat is not affected by caffeine . Caffeine additively increases the acute antiexudative effect of ASA and aminophenazone on the carageenin-induced oedema of the hind paw of the rat . The increase in locomotor activity caused by caffeine in mouse and rat is neutralised or diminished when the caffeine is given in combination with paracetamol . This effect is maintained even if the rats are pretreated with the combination of active ingredients for 3 weeks . The ulcerogenic effect of ASA in the stomach of the rat is not increased by caffeine . The protective effect of ASA against the hepatotoxic effect of paracetamol in the mouse is not influenced by the addition of caffeine . The plasma levels after the oral administration of 20 mg/caffeine/ kg and 80 mg paracetamol/kg in the rat are not significantly changed when the substances are given in combination . The toxicological advantages resulting from combining ASA and paracetamol with caffeine are discussed.

Pathol Biol (Paris), 1997 Mar, 45(3), 240 - 4
Colon cancer . Molecular biology of the APC protein; White RL; The discovery of a tumor suppressor gene opens a new pathway to discovery of the fundamental mechanisms that underlie tumor initiation and progression . An inherited tumor suppressor gene is of special interest in that it defines a step in the tumorigenesis pathway that can be rate limiting in development of that tumor type . In the case of colon cancer, we were fortunate in identifying an inherited tumor suppressor gene, the APC gene, that plays a major etiologic role in both the inherited disease, familial adenomatous polyposis (FAP), and in sporadic colon polyps . Characterization of the molecular biology of that gene, and the underlying mechanisms that result in the development of colon tumors, could provide new approaches to both colon cancer diagnostics, therapeutics and chemopreventives . We have embarked, therefore, on a series of exploratory studies designed to provides clues to possible functional roles for the APC protein . We have found through immunocytochemistry that APC protein is distributed throughout the cell, in both the cytoplasm and nucleus . Furthermore, within the nucleus much of the APC protein seems associated with the nucleoli . The cytoplasmic label is distributed in a punctate pattern, with concentrations at the leading edge of migrating cells at the ends of microtubules . Furthermore, following an extraction of the cells that leaves behind primarily cytoskeletal and nuclear scaffold structures, we see strong APC staining of these structures . The yeast two-hybrid system has offered a number of potentially interacting partners for APC, including a new binding site for alpha-tubulin . These results, and others recent discoveries concerning APC, suggest a rather global role for APC protein, modulating cellular activity and signal transduction pathway from the cell periphery to the nucleus.

Pathol Biol (Paris), 1997 Mar, 45(3), 205 - 8
{Mapping and human genome sequence program}; Weissenbach J; Until recently, human genome programs focused primarily on establishing maps that would provide signposts to researchers seeking to identify genes responsible for inherited diseases, as well as a basis for genome sequencing studies . Preestablished gene mapping goals have been reached . The over 7,000 microsatellite markers identified to date provide a map of sufficient density to allow localization of the gene of a monogenic disease with a precision of 1 to 2 million base pairs . The physical map, based on systematically arranged overlapping sets of artificial yeast chromosomes (YACs), has also made considerable headway during the last few years . The most recently published map covers more than 90% of the genome . However, currently available physical maps cannot be used for sequencing studies because multiple rearrangements occur in YACs . The recently developed sets of radioinduced hybrids are extremely useful for incorporating genes into existing maps . A network of American and European laboratories has successfully used these radioinduced hybrids to map 15,000 gene tags from large-scale cDNA library sequencing programs . There are increasingly pressing reasons for initiating large scale human genome sequencing studies.

J Neurosci, 1997 Oct 1, 17(19), 7425 - 32
The swiss cheese mutant causes glial hyperwrapping and brain degeneration in Drosophila; Kretzschmar D et al.; Swiss cheese (sws) mutant flies develop normally during larval life but show age-dependent neurodegeneration in the pupa and adult and have reduced life span . In late pupae, glial processes form abnormal, multilayered wrappings around neurons and axons . Degeneration first becomes evident in young flies as apoptosis in single scattered cells in the CNS, but later it becomes severe and widespread . In the adult, the number of glial wrappings increases with age . The sws gene is expressed in neurons in the brain cortex . The conceptual 1425 amino acid protein shows two domains with homology to the regulatory subunits of protein kinase A and to conceptual proteins of yet unknown function in yeast, worm, and human . Sequencing of two sws alleles shows amino acid substitutions in these two conserved domains . It is suggested that the novel SWS protein plays a role in a signaling mechanism between neurons and glia that regulates glial wrapping during development of the adult brain.

J Biol Chem, 1997 Sep 19, 272(38), 24088 - 95
Characterization of human activating transcription factor 4, a transcriptional activator that interacts with multiple domains of cAMP-responsive element-binding protein (CREB)-binding protein; Liang G et al.; We demonstrate that human activating transcription factor 4 (hATF4), a member of the activating transcription factor/cAMP-responsive element-binding protein (ATF/CREB) family of transcription factors, is a potent transcriptional activator in both mammalian cells and yeast . The N-terminal 113 amino acids of hATF4 activate transcription efficiently, and unexpectedly, the C-terminal bZip DNA binding domain of hATF4 also activates transcription, albeit weakly . Our results indicate that hATF4 interacts with several general transcription factors: TATA-binding protein, TFIIB, and the RAP30 subunit of TFIIF . In addition, hATF4 interacts with the coactivator CREB-binding protein (CBP) at four regions: 1) the KIX domain, 2) a region that contains the third zinc finger and the E1A-interacting domain, 3) a C-terminal region that contains the p160/SRC-1-interacting domain, and 4) the recently identified histone acetyltransferase domain . Interestingly, both the N-terminal and C-terminal regions of hATF4 interact with the above general transcription factors and CBP, providing a mechanistic explanation for their ability to activate transcription . Consistent with its role as a coactivator, CBP potentiates the ability of hATF4 to activate transcription . The potential significance of the interaction between hATF4 and multiple factors is discussed.

J Biol Chem, 1997 Sep 19, 272(38), 23799 - 804
Ligand-dependent heterodimerization of thyroid hormone receptor and retinoid X receptor; Kakizawa T et al.; The thyroid hormone receptors (TR) bind to cis-acting DNA elements as heterodimers with the retinoid X receptors (RXR) . These heterodimers display distinct specificities in mediating the hormonal response to target gene transcription . We characterized the interaction between TRalpha1 and RXRalpha via their ligand binding domains (LBDs) and the effect of ligands on the interaction using a yeast two-hybrid system . The DNA binding domain (BD) of yeast Gal4 fusion to the LBD of TRalpha1 had no transcriptional activity on its own, but when it was coexpressed with the activation domain (AD) of yeast Gal4 fusion to LBD of RXRalpha conferred activation to a reporter gene harboring a Gal4 binding site, indicating that LBDs of TRalpha1 and RXRalpha interact with each other in solution . Furthermore, T3 and 9-cis-RA increased the reporter activity, and an additive effect was observed when both ligands were added, indicating that the TRalpha1.RXRalpha heterodimerization is augmented by their respective ligands in vivo . Using an in vitro pull-down experiment, we confirmed the ligand-dependent interaction observed in the yeast system . Matrix-bound glutathione S-transferase-RXRalpha specifically coprecipitated the 35S-labeled TRalpha1 above the control, and associated 35S-labeled TRalpha1 was increased by the addition of T3 and 9-cis-RA . These results imply a complex, sensitive cross-talk in vivo among nuclear receptors and their respective ligands through distinct hormonal signaling pathways.

J Biol Chem, 1997 Sep 19, 272(38), 23616 - 22
Transgenic mice expressing human ApoB95 and ApoB97 . Evidence that sequences within the carboxyl-terminal portion of human apoB100 are important for the assembly of lipoprotein; McCormick SP et al.; The structural features of apolipoprotein (apo) B that are important for its covalent linkage to apo(a) to form lipoprotein(a) (Lp(a)) are incompletely understood . Although apoB100 cysteine 4326 is required for the disulfide linkage with apo(a), other structural features, aside from a single free cysteine residue, must be important for apoB's initial interaction with apo(a) and for facilitating the formation of the disulfide bond . To determine if sequences carboxyl-terminal to cysteine 4326 affect the efficiency of Lp(a) formation, we used "pop-in, pop-out" gene targeting in a human apoB yeast artificial chromosome to introduce nonsense mutations into exon 29 of the apoB gene . The mutant yeast artificial chromosomes, which coded for the truncated versions of human apoB, apoB95, and apoB97, were then used to express these mutant forms of apoB in transgenic mice . As judged by in vitro assays of Lp(a) formation, apoB95 (4330 amino acids) formed a small amount of Lp(a) but did so slowly . In contrast, apoB97 (4397 amino acids) formed Lp(a) rapidly, although not quite as rapidly as the full-length apoB100 (4536 amino acids) . These results were supported by an analysis of double-transgenic mice expressing both human apo(a) and either apoB95 or apoB97 . In mice expressing both apoB95 and apo(a), there was only a trace amount of Lp(a) in the plasma, and most of the apo(a) was free, whereas in mice expressing both apoB97 and apo(a), virtually all of the apo(a) was bound to apoB97 in the form of Lp(a) . These results show that sequences carboxyl-terminal to apoB95 (amino acids 4331-4536) are not absolutely required for Lp(a) formation, but this segment of the apoB molecule, particularly residues 4331-4397, is necessary for the efficient assembly of Lp(a).

Proteins, 1997 Sep, 29(1), 68 - 76
Pseudocontact shifts as constraints for energy minimization and molecular dynamics calculations on solution structures of paramagnetic metalloproteins; Banci L et al.; The pseudocontact shifts of NMR signals, which arise from the magnetic susceptibility anisotropy of paramagnetic molecules, have been used as structural constraints under the form of a pseudopotential in the SANDER module of the AMBER 4.1 molecular dynamics software package . With this procedure, restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations can be performed on structural models by using pseudocontact shifts . The structure of the cyanide adduct of the Met80Ala mutant of the yeast iso-1-cytochrome c has been used for successfully testing the calculations . For this protein, a family of structures is available, which was obtained by using NOE and pseudocontact shifts as constraints in a distance geometry program . The structures obtained by REM and RMD calculations with the inclusion of pseudocontact shifts are analyzed.

Oncogene, 1997 Sep 4, 15(10), 1151 - 9
K-ras is essential for the development of the mouse embryo; Koera K et al.; ras genes encode members of the small GTP-binding proteins . Ras protein in highly conserved in various species from yeast to humans and plays a key role in signal transduction . Ras is related to cell proliferation and differentiation . While, in addition, mutations in the ras genes are implicated in a variety of tumors . However, the physiological functions and specific roles of each ras gene, H-ras, K-ras and N-ras, are still not fully understood . To clarify the role of the K-Ras in vivo, we generated K-ras mutant mice by gene targeting . In contrast to the findings that H-Ras-deficient mice and N-Ras-deficient mice are born and grow normally, the K-Ras-deficient embryos die progressively between embryonic day 12.5 and term . At embryonic day 15.5, their ventricular walls are extremely thin . Besides, at embryonic day 11.5, they demonstrate increased cell death of motoneurons in the medulla and the cervical spinal cord . Our results thus indicate K-Ras to be essential for normal development in mice and residual Ras composed of H-Ras and N-Ras cannot compensate for the loss of K-Ras function in the mutant mice.

Proc Natl Acad Sci U S A, 1997 Sep 16, 94(19), 10479 - 84
CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants; Wedel N et al.; CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1 . 13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle) . Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12 . The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12 . Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts . Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop . Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation . Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH . From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).

J Clin Invest, 1997 Sep 15, 100(6), 1507 - 12
Tissue- and development-specific expression of multiple alternatively spliced transcripts of rat neuronal nitric oxide synthase; Lee MA et al.; Nitric oxide (NO) functions as an intercellular messenger and mediates numerous biological functions . Among the three isoforms of NO synthase that produce NO, the ubiquitously expressed neuronal NO synthase (nNOS) is responsible for a large part of NO production, yet its regulation is poorly understood . Recent reports of two alternative spliceforms of nNOS in the mouse and in man have raised the possibility of spatial and temporal modulation of expression . This study demonstrates the existence of at least three transcripts of the rat nNOS gene designated nNOSa, nNOSb, and nNOSc, respectively, with distinct 5' untranslated first exons that arise from alternative splicing to a common second exon . Expression of the alternative transcripts occurs with a high degree of tissue and developmental specificity, as demonstrated by RNase protection assays on multiple tissues from both fetal and adult rats . Furthermore, terminal differentiation of rat pheochromocytoma-derived PC12 cells into neurons is associated with induction of nNOSa, suggesting, likewise, development- and tissue-specific transcriptional control of nNOS isoform expression . Physical mapping using a rat yeast artificial chromosome clone shows that the alternatively spliced first exons 1a, 1b, and 1c are separated by at least 15-60 kb from the downstream coding sequence, with exons 1b and 1c being positioned within 200 bp of each other . These findings provide evidence that the biological activity of nNOS is tightly and specifically regulated by a complex pattern of alternative splicing, indicating that the notion of constitutive expression of this isoform needs to be revised.

Neuron, 1997 Aug, 19(2), 239 - 49
Aberrant splicing of a mouse disabled homolog, mdab1, in the scrambler mouse; Ware ML et al.; Although accurate long-distance neuronal migration is a cardinal feature of cerebral cortical development, little is known about control of this migration . The scrambler (scm) mouse shows abnormal cortical lamination that is indistinguishable from reeler . Genetic and physical mapping of scm identified yeast artificial chromosomes containing an exon of mdab1, a homolog of Drosophila disabled, which encodes a phosphoprotein that binds nonreceptor tyrosine kinases . mdab1 transcripts showed abnormal splicing in scm homozygotes, with 1.5 kb of intracisternal A particle retrotransposon sequence inserted into the mdab1 coding region in antisense orientation, producing a mutated and truncated predicted protein . Therefore, mdab1 is most likely the scm gene, thus implicating nonreceptor tyrosine kinases in neuronal migration and lamination in developing cerebral cortex.

Plant Mol Biol, 1997 Sep, 35(1-2), 187 - 95
Towards map-based cloning of the barley stem rust resistance genes Rpg1 and rpg4 using rice as an intergenomic cloning vehicle; Kilian A et al.; The barley stem rust resistance genes Rpg1 and rpg4 were mapped in barley on chromosomes 1P and 7M, respectively and the syntenous rice chromosomes identified as 6P and 3P by mapping common probes in barley and rice . Rice yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and cosmid clones were used to isolate probes mapping to the barley Rpg1 region . The rice BAC isolated with the pM13 probe was a particularly excellent source of probes . A high-resolution map of the Rpg1 region was established with 1400 gametes yielding a map density of 3.6 markers per 0.1 cM . A detailed physical map was established for the rice BAC fragment containing the Rpg1-flanking markers pM13 and B24 . This fragment covers a barley genetic distance of 0.6 cM and a rice DNA physical distance of ca . 70 kb . The distribution of barley cross-overs in relation to the rice DNA physical distances was extremely uneven . The barley genetic distance between the pM13 marker and Rpg1 was 0.1 cM per ca . 55 kb, while on the proximal side it was 0.5 cm per ca . 15 kb . Three probes from the distal end of the pM13 BAC mapped 3.0 cm proximal of Rpg1 and out of synteny with rice . These experiments confirm the validity of using large insert rice clones as probe sources to saturate small barley (and other large genome cereals) genome regions with markers . They also establish a note of caution that even in regions of high microsynteny, there may be small DNA fragments that have transposed and are no longer in syntenous positions.

Plant Mol Biol, 1997 Sep, 35(1-2), 115 - 27
Physical mapping of the rice genome with BACs; Zhang HB et al.; The development of genetics in this century has been catapulted forward by several milestones: rediscovery of Mendel's laws, determination of DNA as the genetic material, discovery of the double-helix structure of DNA and its implications for genetic behavior, and most recently, analysis of restriction fragment length polymorphisms (RFLPs) . Each of these milestones has generated a huge wave of progress in genetics . Consequently, our understanding of organismal genetics now extends from phenotypes to their molecular genetic basis . It is now clear that the next wave of progress in genetics will hinge on genome molecular physical mapping, since a genome physical map will provide an invaluable, readily accessible system for many detailed genetic studies and isolation of many genes of economic or biological importance . Recent development of large-DNA fragment (> 100 kb) manipulation and cloning technologies, such as pulsed-field gel electrophoresis (PFGE), and yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) cloning, has provided the powerful tools needed to generate molecular physical maps for higher-organism genomes . This chapter will discuss (1) an ideal physical map of plant genome and its applications in plant genetic and biological studies, (2) reviews on physical mapping of the genomes of Caenorhabditis elegans, Arabidopsis thaliana, and man, (3) large-insert DNA libraries: cosmid, YAC and BAC, and genome physical mapping, (4) physical mapping of the rice genome with BACs, and (5) perspectives on the physical mapping of the rice genome with BACs.

Plant Mol Biol, 1997 Sep, 35(1-2), 101 - 13
Physical mapping of the rice genome with YAC clones; Kurata N et al.; Construction of a rice physical map covered by YAC clones which have been arranged over half of the genome length is presented here . A total of 1285 RFLP and RAPD markers almost evenly distributed on the rice genetic map could select 2974 YAC clones and 2443 clones of them were located on their original positions . Rice YACs carrying 350 kb average insert fragments of 2443 clones could cover 222 megabase length of the rice genome, corresponding to 52% of the whole genome size (4.3 Mb) . Chromosome landing with many YAC clones on the high-density genetic map loci efficiently integrated the genetic map with a physical map . This is the first step to generate a comprehensive genome map of rice . An integrated genome map should be an indispensable tool to figure out genome structure as well as to clone trait genes by map-based cloning.

Genes Chromosomes Cancer, 1997 Sep, 20(1), 73 - 81
Cytogenetic, fluorescence in situ hybridisation, and clinical evaluation of translocations with concomitant deletion at 13q14 in chronic lymphocytic leukaemia; Gardiner AC et al.; Deletions and translocations of 13q14 are the most frequent structural chromosome abnormalities found in chronic lymphocytic leukaemia (CLL) . We have identified 13q14 translocations in the blood of 30 of 450 (6.6%) CLL patients by conventional cytogenetics, using tetradecanoyl phorbol 12-myristate 13-acetate (TPA) as a mitogen . The translocations are characterised by multiple partner chromosomes and a high incidence, 6 of 30 cases, of complex rearrangements . Seven cases were also studied by fluorescence in situ hybridisation (FISH) using four previously ordered YACs, to define the breakpoints further . Deletions with varying proximal and distal breakpoints were found in six cases . Two of the cases had deletions of the cytogenetically normal chromosome 13 at q14, and in one case the 13q14 translocation was a secondary genetic event . No difference in the clinical features between the patients with 13q14 translocation and 54 patients with 13q14 deletions or four patients with both a translocation and a deletion was observed . These data suggest that the genetic consequence of 13q14 translocations in CLL is the loss of a tumour suppressor gene.

Fungal Genet Biol, 1997 Jun, 21(3), 364 - 72
Multicellular ascomycetous fungal genomes contain more than 8000 genes; Kupfer DM et al.; Fungi comprise a large monophyletic group of uni- and multicellular eukaryotic organisms in which many species are of economic or medical importance . Fungal genomes are variable in size (13-42 Mb), and multicellular species support true spatial and temporal cell-type-specific regulation of gene expression . In a 38.8-kb Aspergillus nidulans contiguous genomic DNA region, a transposable element and 12 potential genes were identified, 7 similar to genes in other organisms . This observation is consistent with the prediction that multicellular ascomycetous fungi harbor 8000-9000 genes in a 36-Mb average genome . Thus, the genomic DNA sequence of filamentous fungi will provide substantial amounts of genetic and functional information that is not available in yeast, for the human and other metazoan minimal gene complement.

Fungal Genet Biol, 1997 Jun, 21(3), 302 - 7
The design of pooling experiments for screening a clone map; Balding DJ et al.; We consider nonadaptive pooling designs for unique-sequence screening of a 1530-clone map of Aspergillus nidulans . The map has the properties that the clones are, with possibly a few exceptions, ordered and no more than 2 of them cover any point on the genome . We propose two subdesigns of the Steiner system S(3, 5, 65), one with 65 pools and approximately 118 clones per pool, the other with 54 pools and about 142 clones per pool . Each design allows 1 or 2 positive clones to be detected, even in the presence of substantial experimental error rates . More efficient designs are possible if the overlap information in the map is exploited, if there is no constraint on the number of clones in a pool, and if no error tolerance is required . An information theory lower bound requires at least 12 pools to satisfy these minimal criteria, and an "interleaved binary" design can be constructed on 20 pools, with about 380 clones per pool . However, the designs with more pools have important properties of robustness to various possible errors and general applicability to a wider class of pooling experiments.

Nucleic Acids Res, 1997 Sep 15, 25(18), 3665 - 71
FLP-mediated DNA mobilization to specific target sites in Drosophila chromosomes; Golic MM et al.; The ability to place a series of gene constructs at a specific site in the genome opens new possibilities for the experimental examination of gene expression and chromosomal position effects . We report that the FLP- FRT site-specific recombination system of the yeast 2mu plasmid can be used to integrate DNA at a chromosomal FRT target site in Drosophila . The technique we used was to first integrate an FRT- flanked gene by standard P element-mediated transformation . FLP was then used to excise the FRT- flanked donor DNA and screen for FLP-mediated re-integration at an FRT target at a different chromosome location . Such events were recovered from up to 5% of the crosses used to screen for mobilization and are easily detectable by altered linkage of a white reporter gene or by the generation of a white + gene upon integration.

Science, 1997 Sep 12, 277(5332), 1672 - 6
Proteolysis and DNA replication: the CDC34 requirement in the Xenopus egg cell cycle; Yew PR et al.; The cell division cycle gene, CDC34, is required for ubiquitin-mediated degradation of G1 regulators and cell cycle progression through the transition from G1 to S phase in budding yeast . A CDC34 requirement for S phase onset in higher eukaryotes has not been established . Studies of the simple embryonic cell cycle of Xenopus laevis eggs demonstrated that Cdc34p in a large molecular size complex was required in the initiation of DNA replication . Cdc34p appears to regulate the initiation function of Cdk2-cyclin E, perhaps through the degradation of the Xenopus cdk inhibitor, Xic1.

Am J Med Genet, 1997 Sep 5, 71(4), 420 - 5
Growth hormone insufficiency associated with haploinsufficiency at 18q23; Cody JD et al.; Growth hormone insufficiency is a common cause of growth failure in children with the 18q- syndrome . Individuals with this syndrome have a deletion as large as 36 Mb from the long arm of chromosome 18 . We have evaluated 33 children with this syndrome for growth hormone production and have identified a region of approximately 2 Mb, which is deleted in every growth hormone insufficient patient . Two genes contained in this region, myelin basic protein, and the galanin receptor, are candidate genes for the growth hormone insufficiency phenotype.

Science, 1997 Sep 5, 277(5331), 1518 - 23
Dynamic molecular combing: stretching the whole human genome for high-resolution studies; Michalet X et al.; DNA in amounts representative of hundreds of eukaryotic genomes was extended on silanized surfaces by dynamic molecular combing . The precise measurement of hybridized DNA probes was achieved directly without requiring normalization . This approach was validated with the high-resolution mapping of cosmid contigs on a yeast artificial chromosome (YAC) within yeast genomic DNA . It was extended to human genomic DNA for precise measurements ranging from 7 to 150 kilobases, of gaps within a contig, and of microdeletions in the tuberous sclerosis 2 gene on patients' DNA . The simplicity, reproducibility, and precision of this approach makes it a powerful tool for a variety of genomic studies.

Science, 1997 Sep 5, 277(5331), 1501 - 5
Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216; Peng CY et al.; Human Cdc25C is a dual-specificity protein phosphatase that controls entry into mitosis by dephosphorylating the protein kinase Cdc2 . Throughout interphase, but not in mitosis, Cdc25C was phosphorylated on serine-216 and bound to members of the highly conserved and ubiquitously expressed family of 14-3-3 proteins . A mutation preventing phosphorylation of serine-216 abrogated 14-3-3 binding . Conditional overexpression of this mutant perturbed mitotic timing and allowed cells to escape the G2 checkpoint arrest induced by either unreplicated DNA or radiation-induced damage . Chk1, a fission yeast kinase involved in the DNA damage checkpoint response, phosphorylated Cdc25C in vitro on serine-216 . These results indicate that serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify Cdc25C as a potential target of checkpoint control in human cells.

Science, 1997 Sep 5, 277(5331), 1495 - 7
Cdc25 mitotic inducer targeted by chk1 DNA damage checkpoint kinase; Furnari B et al.; Arrest of the cell cycle at the G2 checkpoint, induced by DNA damage, requires inhibitory phosphorylation of the kinase Cdc2 in both fission yeast and human cells . The kinase Wee1 and the phosphatase Cdc25, which regulate Cdc2 phosphorylation, were evaluated as targets of Chk1, a kinase essential for the checkpoint . Fission yeast cdc2-3w Deltacdc25 cells, which express activated Cdc2 and lack Cdc25, were responsive to Wee1 but insensitive to Chk1 and irradiation . Expression of large amounts of Chk1 produced the same phenotype as did loss of the cdc25 gene in cdc2-3w cells . Cdc25 associated with Chk1 in vivo and was phosphorylated when copurified in Chk1 complexes . These findings identify Cdc25, but not Wee1, as a target of the DNA damage checkpoint.

Nature, 1997 Sep 4, 389(6646), 85 - 9
Smad4 and FAST-1 in the assembly of activin-responsive factor; Chen X et al.; Members of the TGF-beta superfamily of signalling molecules work by activating transmembrane receptors with phosphorylating activity (serine-threonine kinase receptors); these in turn phosphorylate and activate SMADs, a class of signal transducers . Activins are growth factors that act primarily through Smad2, possibly in partnership with Smad4, which forms heteromeric complexes with different ligand-specific SMADs after activation . In frog embryos, Smad2 participates in an activin-responsive factor (ARF), which then binds to a promoter element of the Mix.2 gene . The principal DNA-binding component of ARF is FAST-1, a transcription factor with a novel winged-helix structure . We now report that Smad4 is present in ARF, and that FAST-1, Smad4 and Smad2 co-immunoprecipitate in a ligand-regulated fashion . We have mapped the site of interaction between FAST-1 and Smad2/Smad4 to a novel carboxy-terminal domain of FAST-1, and find that overexpression of this domain specifically inhibits activin signalling . In a yeast two-hybrid assay, the FAST-1 carboxy terminus interacts with Smad2 but not Smad4 . Deletion mutants of the FAST-1 carboxy terminus that still participate in ligand-regulated Smad2 binding no longer associated with Smad4 or ARF . These results indicate that Smad4 stabilizes a ligand-stimulated Smad2-FAST-1 complex as an active DNA-binding factor.

Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9973 - 8
F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF); Jiang CJ et al.; Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins . They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing . In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin . One mutant, zmadf3-1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3 . A second mutant, zmadf3-2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished . The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively . Using the tertiary structure of destrin, yeast cofilin, and Acanthamoeba actophorin, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding . We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long alpha-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding . The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 A, respectively, with corresponding bond angles of 99.5 degrees and 113 degrees, close to the optimum for a strong hydrogen bond.

Cancer Res, 1997 Sep 1, 57(17), 3708 - 16
Identification of a new P-glycoprotein-like ATP-binding cassette transporter gene that is overexpressed during hepatocarcinogenesis; Furuya KN et al.; The liver is remarkably insensitive to a variety of cytotoxins and expresses a number of known drug resistance genes . To isolate new P-glycoprotein (Pgp)-related genes, we screened a normal rat liver cDNA library at low stringency with a MDR1 cDNA fragment containing the P-loop and ATP binding site . We isolated a novel cDNA closely related to the Pgps that is dramatically increased in hepatic neoplasia and refer to it as P-glycoprotein-related protein (PRP) . The predicted protein shows PRP to be a member of the ATP-Binding Cassette (ABC) family of proteins, and a multisequence comparison of the nucleotide binding domain and the ABC family signature sequences reveals that PRP sequences are highly conserved with the greatest similarity to the yeast heavy metal transporter encoded by hmtl . However, the hydropathy plot analysis suggests that PRP does not have any prominent membrane-spanning domains and thus is not typical of ABC transporters . The PRP transcript is detected in many normal tissues . In the H35 hepatoma cell line, PRP was overexpressed compared to normal liver . Southern blot analysis of DNA from the H35 rat hepatoma cells reveals that the PRP gene was amplified compared to normal liver . The orotic acid model of hepatocarcinogenesis was used to determine if during stepwise progression to liver cancer, PRP changed with hepatocarcinogenesis . At the hyperplastic nodule stage, PRP expression was increased over its expression in normal surrounding liver . More dramatic increases in PRP expression were found in frank hepatic carcinomas . Cumulatively, these studies are the first to link a novel ABC family member to the hepatic neoplastic process, a role that may be recapitulated in other cells, considering the ubiquitous expression of PRP.

Nat Genet, 1997 Sep, 17(1), 119 - 21
Reverse genetics by chemical mutagenesis in Caenorhabditis elegans; Jansen G et al.; Traditional reverse genetics on yeast, mice and other organisms uses homologous recombination with transgenic DNA to interrupt a target gene . Here we report that target-selected gene inactivation can be be achieved in Caenorhabditis elegans with the use of chemical mutagens . We use PCR to selectively visualize deletions in genes of interest; the method is sensitive enough to permit detection of a single mutant among more than 15,000 wild types . A permanent frozen mutant collection of more than a million mutagenized animals has been established, and deletion mutants of several G-protein genes were isolated from it . The approach is suitable to be scaled up for systematic inactivation of all 17,000 C . elegans genes . Because it requires no transgenesis or cell culturing, it may also be applicable to small organisms usually considered to be outside the realm of reverse genetics (for example, other nematodes and insects) . Any sequenced gene in any organism that can be handled in very large numbers can possibly be targeted in this way.

Nat Genet, 1997 Sep, 17(1), 96 - 9
Clustering of missense mutations in the ataxia-telangiectasia gene in a sporadic T-cell leukaemia; Vorechovsky I et al.; Ataxia-telangiectasia (A-T) is a recessive multi-system disorder caused by mutations in the ATM gene at 11q22-q23 (ref . 3) . The risk of cancer, especially lymphoid neoplasias, is substantially elevated in A-T patients and has long been associated with chromosomal instability . By analysing tumour DNA from patients with sporadic T-cell prolymphocytic leukaemia (T-PLL), a rare clonal malignancy with similarities to a mature T-cell leukaemia seen in A-T, we demonstrate a high frequency of ATM mutations in T-PLL . In marked contrast to the ATM mutation pattern in A-T, the most frequent nucleotide changes in this leukaemia were missense mutations . These clustered in the region corresponding to the kinase domain, which is highly conserved in ATM-related proteins in mouse, yeast and Drosophila . The resulting amino-acid substitutions are predicted to interfere with ATP binding or substrate recognition . Two of seventeen mutated T-PLL samples had a previously reported A-T allele . In contrast, no mutations were detected in the p53 gene, suggesting that this tumour suppressor is not frequently altered in this leukaemia . Occasional missense mutations in ATM were also found in tumour DNA from patients with B-cell non-Hodgkin's lymphomas (B-NHL) and a B-NHL cell line . The evidence of a significant proportion of loss-of-function mutations and a complete absence of the normal copy of ATM in the majority of mutated tumours establishes somatic inactivation of this gene in the pathogenesis of sporadic T-PLL and suggests that ATM acts as a tumour suppressor . As constitutional DNA was not available, a putative hereditary predisposition to T-PLL will require further investigation.

Nat Genet, 1997 Sep, 17(1), 65 - 70
Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion; David G et al.; The gene for spinocerebellar ataxia 7 (SCA7) has been mapped to chromosome 3p12-13 . By positional cloning, we have identified a new gene of unknown function containing a CAG repeat that is expanded in SCA7 patients . On mutated alleles, CAG repeat size is highly variable, ranging from 38 to 130 repeats, whereas on normal alleles it ranges from 7 to 17 repeats . Gonadal instability in SCA7 is greater than that observed in any of the seven known neuro-degenerative diseases caused by translated CAG repeat expansions, and is markedly associated with paternal transmissions . SCA7 is the first such disorder in which the degenerative process also affects the retina.

Nat Genet, 1997 Sep, 17(1), 25 - 31
A candidate gene for familial Mediterranean fever . The French FMF Consortium; Huntingtin-associated protein 1 (HAP1) binds to a Trio-like polypeptide et al.; The Department of Psychiatry, The Johns Hopkins School of Medicine, Baltimore, MD 21205-2196, USAHuntington's disease (HD) occurs when the widely expressed protein huntingtin contains an expanded glutamine repeat . The selective degeneration and neuronal morphologic abnormalities of HD may involve interactions with proteins that bind to huntingtin, such as HAP1 . The biological significance of this interaction is unclear because neither HAP1 nor huntingtin have significant homology to known proteins . Therefore, we sought to identify HAP1-binding proteins . Using the yeast two-hybrid system, we isolated a rat cDNA encoding part of a protein that interacts with HAP1, and we confirmed the specificity of this interaction using an in vitro protein-binding assay . We called the protein Duo because it is closely related to the human protein Trio but is shorter . Northern blot analysis indicates brain-specific expression of Duo . Human Duo contains a guanine nucleotide exchange factor (GEF) domain that is likely to be rac1-specific, a pleckstrin homology (PH) domain and spectrin-like repeat units . These data support the hypothesis that huntingtin is involved in vesicle trafficking and cytoskeletal functions, and raise the possibility of a role for huntingtin in the regulation of a ras-related signaling pathway.

J Clin Endocrinol Metab, 1997 Sep, 82(9), 3047 - 53
Complementary deoxyribonucleic acid cloning and characterization of a putative human axonemal dynein light chain gene; Kastury K et al.; Immotile Cilia Syndrome (ICS) is characterized by recurrent sinus and lung infections, bronchiectasis, and sperm immotility . Nasal cilia and sperm tails in patients with ICS exhibit a variety of ultrastructural defects, often including shortening or absence of the inner dynein arms . Immotile mutant strains of Chlamydomonas, a biflagellated algae, have ultrastructural defects similar to those seen in patients with this clinical disorder . Furthermore, splice-site mutations in the Chlamydomonas inner dynein arm gene (p28) are associated with impaired flagellar motility . We therefore hypothesized that the human homologue of the Clamydomonas dynein p28 gene would be an attractive candidate gene for patients with ICS . Accordingly, we cloned the full length complementary DNA (cDNA) and genomic clone by screening of appropriate libraries and databases, using the protein sequence of the Chlamydomonas p28 gene . The human homologue is encoded by a 921 bp transcript (accession no . AF006386) with an open reading frame of 257 amino acids . Using somatic cell and radiation hybrid panels, the hp28 gene was mapped to human chromosome 1p35.1 . The hp28 cDNA probe hybridizes to sequences in all species on a zoo blot containing genomic DNA from yeast to human . Northern blot analysis reveals two hp28 gene transcripts, 0.9 and 2.5 kb, in many tissues . The 0.9 kb transcript is expressed at a 20-fold higher level than the 2.5-kb transcript in the testis . The entire gene is included in a 20-kb EcoRI genomic fragment and has 7 exons and 6 introns . Cloning of the hp28 cDNA and mapping of the intron-exon junctions should now make it possible to test whether a subset of ICS is a consequence of mutations in the human axonemal dynein light chain gene hp28.

Mol Biochem Parasitol, 1997 Sep, 88(1-2), 151 - 62
A Plasmodium falciparum homologue of the ATPase subunit of a multi-protein complex involved in chromatin remodelling for transcription; Ji DD et al.; A Plasmodium falciparum homologue of one of the components of a chromatin-remodelling complex which controls binding of transcription factors to nucleosome core particles has been cloned and characterised . The gene encodes 1422 amino acids with an estimated molecular mass of 167 kDa . The protein, SNF2L, shares 60% amino acid identity in its conserved DNA-dependent ATPase domain with yeast transcription factors originally identified by characterising mating type switch mutants . It also contains sequences related to the so-called SWI3, ADA2, N-CoR and TFIIIB B" or SANT DNA binding domains which are characteristic of these transcriptional activation factors . The SNF2L gene has two short introns in the 3' region of the coding sequence of the gene and is transcribed into a single approximately 6.5 kb messenger RNA species which is present throughout the asexual stages of the cell cycle . Southern blotting and pulsed field gel electrophoresis experiments show that SNF2L is a single copy gene . located on P . falciparum chromosome 11.

Hum Genet, 1997 Sep, 100(3-4), 481 - 5
Mapping of the gene encoding the B56 beta subunit of protein phosphatase 2A (PPP2R5B) to a 0.5-Mb region of chromosome 11q13 and its exclusion as a candidate gene for multiple endocrine neoplasia type 1 (MEN1); Forbes SA et al.; The multiple endocrine neoplasia type 1 (MEN1) locus has been previously localised to 11q13 by combined tumour deletion mapping and recombination studies, and a 0.5-Mb region, flanked by PYGM and D11S449, has been defined . In the course of constructing a conting, we have identified the location of the gene encoding the B56 beta subunit of protein phosphatase 2A (PP2A), which is involved in cell signal transduction pathways and thus represents a candidate gene for MEN1 . We have searched for mutations in the PP2A-B56 beta coding region, together with the 5' and 3' untranslated regions in six MEN1 patients . DNA sequence abnormalities were not identified and thus the PP2A-B56 beta gene is excluded as the candidate gene for MEN1 . However, our precise localisation of PP2A-B56 beta to this region of 11q13 may help in elucidating the basis for other disease genes mapping to this generich region.

Hum Genet, 1997 Sep, 100(3-4), 477 - 80
Mapping of a novel SH3 domain protein and two proteins of unknown function to human chromosome 21; Katsanis N et al.; Down syndrome, caused by trisomy of human chromosome 21 (HSA21), is the most common autosomal form of mental retardation . To understand the aetiology of the syndrome we need to identify the genes involved . We have utilised the information generated by the various EST sequencing projects to enrich the transcription map of chromosome 21 . Here we report the mapping of SH3P17 to 21q22.1 and the localisation of two genes previously mapped to HSA21 by Nagase and colleagues, KIAA0136 and KIAA0179 to 21q22.2 and 21q22.3, respectively . SH3P17 has unknown function but contains four SH3 domains . KIAA0136 shows no homology to a yeast open reading frame . Further investigation of these three genes will add to our functional understanding of HSA21.

Hum Genet, 1997 Sep, 100(3-4), 334 - 8
A de nevo complex t(7;13;8) translocation with a deletion in the TRPS gene region; Brandt CA et al.; Molecular cytogenetic analyses have resolved the pathogenetic aberration of an 8-year-old girl with tricho-rhino-phalangeal syndrome type I (TRPS I), normal intelligence, and a karyotype originally described as 46,XX,t(8;13)(q24;q21) . R- and Q-banding and high resolution R-banding analyses have also disclosed a seemingly mosaic abnormality of the distal short arm of chromosome 7 but have not fully characterized this abnormality . Combined primed in situ labelling and chromosome painting, and three-colour chromosome painting have revealed a complex, apparently balanced translocation t(7;13;8) . Fluorescence in situ hybridization with yeast artificial chromosome and cosmid clones from 8q24.1 has shown an interstitial deletion of at least 3 Mb covering most of the TRPS I critical region.

Mamm Genome, 1997 Sep, 8(9), 668 - 72
Cloning and mapping of a human and mouse gene with homology to ecto-ATPase genes; Chadwick BP et al.; The human CD39-like-1 gene (CD39L1) was isolated from a selected cDNA library enriched for transcripts from regions of human Chromosome (Chr) 9q . Database searches with sequences of one group of clones from the selected cDNA library showed strong amino acid homology to the lymphoid cell activation antigen CD39, an ecto-apyrase gene from human and mouse . The full-length sequence for CD39L1 identified a putative 472 amino acid protein with greater than 60% identity with the chicken muscle ecto-ATPase protein, as well as homology to a number of other known ecto-ATPases and ecto-apyrases from rat, garden pea, yeast, and Toxoplasma gondii . A high level of amino acid identity suggests that CD39L1 is closely related to the chicken muscle ecto-ATPase . The presence of the human ABC2 gene on an overlapping cosmid and hybridizations to somatic cell mapping panels suggest that CD39L1 maps to human Chr 9q34 . A mouse homolog was isolated (showing greater than 78% nucleotide sequence identity) and mapped by FISH to mouse Chr 2, the syntenic region of human 9q34 . The genomic structure of CD39L1 reveals 9 exons covering less than 7 kb.

Immunogenetics, 1997, 46(5), 383 - 95
Cloning, structural analysis, and mapping of the B30 and B7 multigenic families to the major histocompatibility complex (MHC) and other chromosomal regions; Henry J et al.; We present the cloning, structural analysis, and mapping of new members belonging to two multigenic families, the B30-RING finger family and the B7.1-B7.2 family, as well as two genes derived by exon shuffling from members of these families . Eight new members were found and three of them map to the human major histocompatibilitiy complex (MHC) region . Phylogenic and physical mapping analysis allowed us to decipher the evolutionary story of these two multigenic families and to shed light on the evolution of the MHC region . We also show that a deductive analysis can be used to predict the existence of a given gene.

Mol Cell Biol, 1997 Sep, 17(9), 5117 - 26
Luman, a new member of the CREB/ATF family, binds to herpes simplex virus VP16-associated host cellular factor; Lu R et al.; The human host cell factor (HCF) is expressed in a variety of adult and fetal tissues, and its gene is conserved in animals as diverse as mammals and insects . However, its only known function is to stabilize the herpes simplex virus virion transactivator VP16 in a complex with the cellular POU domain protein Oct-1 and cis-acting regulatory elements in promoters of immediate-early viral genes . To identify a cellular function for HCF, we used the yeast two-hybrid system to identify a cellular ligand for HCF . This protein, Luman, appears to be a cyclic AMP response element (CRE)-binding protein/activating transcription factor 1 protein of the basic leucine zipper superfamily . It binds CREs in vitro and activates CRE-containing promoters when transfected into COS7 cells . This activation of transcription was synergistically enhanced by the presence of CCAAT/enhancer-binding protein elements and inhibited by AP-1 elements in the promoter . In addition to a basic DNA binding domain, Luman possesses an unusually long leucine zipper and an acidic amino-terminal activation domain . These features in Luman are also present in what appear to be homologs in the mouse, Drosophila melanogaster, and Caenorhabditis elegans . Luman and VP16 appear to have similar mechanisms for binding HCF, as in vitro each competitively inhibited the binding of the other to HCF . In transfected cells, however, while VP16 strongly inhibited the ability of GAL-Luman to activate transcription from a GAL4 upstream activation sequence-containing promoter, Luman was unable to inhibit the activity of GAL-VP16 . Luman appears to be a ubiquitous transcription factor, and its mRNA was detected in all human adult and fetal tissues examined . The possible role of HCF in regulating the function of this ubiquitous transcription factor is discussed.

Mol Cell Biol, 1997 Sep, 17(9), 5087 - 96
Ran-binding protein 5 (RanBP5) is related to the nuclear transport factor importin-beta but interacts differently with RanBP1; Deane R et al.; We report the identification and characterization of a novel 124-kDa Ran binding protein, RanBP5 . This protein is related to importin-beta, the key mediator of nuclear localization signal (NLS)-dependent nuclear transport . RanBP5 was identified by two independent methods: it was isolated from HeLa cells by using its interaction with RanGTP in an overlay assay to monitor enrichment, and it was also found by the yeast two-hybrid selection method with RanBP1 as bait . RanBP5 binds to RanBP1 as part of a trimeric RanBP1-Ran-RanBP5 complex . Like importin-beta, RanBP5 strongly binds the GTP-bound form of Ran, stabilizing it against both intrinsic and RanGAP1-induced GTP hydrolysis and also against nucleotide exchange . The GAP resistance of the RanBP5-RanGTP complex can be relieved by RanBP1, which might reflect an in vivo role for RanBP1 . RanBP5 is a predominantly cytoplasmic protein that can bind to nuclear pore complexes . We propose that RanBP5 is a mediator of a nucleocytoplasmic transport pathway that is distinct from the importin-alpha-dependent import of proteins with a classical NLS.

Mol Cell Biol, 1997 Sep, 17(9), 5033 - 43
Identification of mouse histone deacetylase 1 as a growth factor-inducible gene; Bartl S et al.; Reversible acetylation of core histones plays an important role in transcriptional regulation, cell cycle progression, and developmental events . The acetylation state of histones is controlled by the activities of acetylating and deacetylating enzymes . By using differential mRNA display, we have identified a mouse histone deacetylase gene, HD1, as an interleukin-2-inducible gene in murine T cells . Sequence alignments revealed that murine HD1 is highly homologous to the yeast RPD3 pleiotropic transcriptional regulator . Indirect immunofluorescence microscopy proved that mouse HD1 is a nuclear protein . When expressed in yeast, murine HD1 was also detected in the nucleus, although it failed to complement the rpd3delta deletion phenotype . HD1 mRNA expression was low in G0 mouse cells but increased when the cells crossed the G1/S boundary after growth stimulation . Immunoprecipitation experiments and functional in vitro assays showed that HD1 protein is associated with histone deacetylase activity . Both HD1 protein levels and total histone deacetylase activity increased upon interleukin-2 stimulation of resting B6.1 cells . When coexpressed with a luciferase reporter construct, HD1 acted as a negative regulator of the Rous sarcoma virus enhancer/promoter . HD1 overexpression in stably transfected Swiss 3T3 cells caused a severe delay during the G2/M phases of the cell cycle . Our results indicate that balanced histone acetylation/deacetylation is crucial for normal cell cycle progression of mammalian cells.

J Virol, 1997 Sep, 71(9), 6887 - 97
Interferon-independent and -induced regulation of Epstein-Barr virus EBNA-1 gene transcription in Burkitt lymphoma; Nonkwelo C et al.; Replication of the Epstein-Barr virus (EBV) genome within latently infected cells is dependent on the EBV EBNA-1 protein . The objective of this study was to identify transcriptional regulatory proteins that mediate EBNA-1 expression via the viral promoter Qp, which is active in EBV-associated tumors such as Burkitt lymphoma and nasopharyngeal carcinoma . Results of a yeast one-hybrid screen suggested that a subset of the interferon regulatory factor (IRF) family may regulate EBNA-1 transcription by targeting an essential cis-regulatory element of Qp, QRE-2 . Further investigation indicated that the transcriptional activator IRF-1 and the closely related IRF-2, a repressor of interferon-induced gene expression, are both capable of activating Qp . However, the major QRE-2-specific binding activity detected within extracts of Burkitt lymphoma cells was attributed to IRF-2, suggesting that interferon-independent activation of Qp is largely mediated by IRF-2 in these cells . We observed no effect of gamma interferon on Qp activity in transfection assays, whereas we observed a moderate but significant repression of Qp activity in response to alpha interferon, possibly mediated by either the interferon consensus sequence binding protein or IRF-7, a novel alpha interferon-inducible factor identified in this study . Since expression of IRF-1 and IRF-2 is increased in response to interferons, the Qp activity observed in the presence of interferon likely represented an equilibrium between IRF factors that activate and those that repress gene expression in response to interferon . Thus, by usurping both IRF-1 and its transcriptional antagonist IRF-2 to activate Qp, EBV has evolved not only a mechanism to constitutively express EBNA-1 but also one which may sustain EBNA-1 expression in the face of the antiviral effects of interferon.

J Cell Biochem, 1997 Sep 1, 66(3), 277 - 85
Roles of p300, pocket proteins, and hTBP in E1A-mediated transcriptional regulation and inhibition of p53 transactivation activity; Sang N et al.; The conserved region 1 and the extreme N-terminus of adenoviral oncoprotein E1A are essential for transforming activity . They also play roles in the interaction of E1A with p300/CBP and pRb and are involved in both transactivation and repression of host gene expression . It was reported recently that p53-mediated transactivation is specifically repressed by E1A and that p53-induced apoptosis can be protected by pRb . In this report, we investigated the roles of pRb and p300 in the N-terminus of E1A-mediated transcriptional regulation . We demonstrate here that p300 and pRb have no effect on DBD.1-70 transactivation and that overexpression of p300 or pRb failed to relieve the repression by E1A . Repression of p53 transactivation requires both the extreme amino terminus and CR1 but not CR2 . This repressive activity of E1A specifically correlates with E1A's ability to bind p300 and TBP . On the other hand, E1A inhibited the transactivation activity of a fusion construct containing the DNA binding domain of yeast Gal4 and the transactivation domain of p53 . When p53 was contransfected with E1A, similar inhibition was found in Saos-2 cells that lack endogenous pRb and p53 activity . Introduction of pRb into Saos-2 cells did not affect p53 transcription activity . E1A-mediated repression can be relieved be overexpression of either p300, hTBP, or-TFIIB but cannot be released by overexpression of pocket proteins . Our data suggest that p300/CBP and TBP but not the pocket proteins, pRb, p107, and pRb2/p130 are functional targets of E1A in transcriptional regulation and that p53 transactivation requires the function of the p300/TBP/TFIIB complex, thus delineating a new pathway by which E1A may exert its transforming activity.

J Biol Chem, 1997 Aug 29, 272(35), 22243 - 7
Specific interactions of the autoantigen L7 with multi-zinc finger protein ZNF7 and ribosomal protein S7; Witte S et al.; The eucaryotic protein L7, which associates with the large subunit of ribosomes, has been shown to be a major autoantigen in systemic autoimmune arthritis . The N terminus carries a sequence motif that is similar to the leucine zipper domain of eucaryotic transcription factors . This domain promotes the homodimerization of protein L7 through alpha-helical coiled-coil formation and binds to distinct mRNAs, thereby inhibiting their cell-free translation . Using a yeast two-hybrid selection, we have identified from a Jurkat T lymphoma cDNA library ribosomal protein S7 and the multi-zinc finger protein ZNF7 as proteins that interact with protein L7 . A fragment of L7 carrying the leucine zipper-like domain is fully sufficient to mediate these interactions . Their potential biological significance is indicated by low apparent dissociation constants of S7-L7 (15 x 10(-9) M) and, respectively, ZNF7-L7 (2 x 10(-9) M) complexes and co-immunoprecipitation of proteins S7, ZNF7, and L7 from a cell lysate with an anti-L7 antibody . We also show that ZNF7-like L7 and S7 can exist in a ribosome-bound form . This study provides further evidence suggesting that L7 is involved in translational regulation through interactions with components of the translational apparatus.

J Biol Chem, 1997 Aug 29, 272(35), 22227 - 35
Sex-lethal interactions with protein and RNA . Roles of glycine-rich and RNA binding domains; Wang J et al.; Sex-lethal (Sxl) is an RNA-binding protein, containing two conserved RNA binding domains (RBDs) and a glycine-rich region, which functions as a regulator of alternative splicing in Drosophila sex determination . Previous work demonstrated that Sxl monomers interact cooperatively upon binding to target RNAs and that the cooperativity depends on the glycine-rich N terminus . Here we use band shift experiments to show that RNA binding patterns are altered when Sxl is combined with other proteins having similar glycine-rich domains, including mammalian heterogeneous nuclear (hn) RNP L and Drosophila Hrb87F (an hnRNP A/B homolog) . Direct involvement of the Sxl glycine-rich region in protein interactions was verified by Far-Western analysis . Two interaction domains, the Sxl N terminus and the Sxl first RNA binding domain, were suggested by the yeast two-hybrid assay . In a systematic examination of the RNA binding properties of Sxl domains, it was found that the Sxl termini as well as the RBDs influence RNA binding specificity . Finally, selection of the Sxl optimal binding site (SELEX) confirms the importance of U-runs in the Sxl binding site and suggests a second type of non-U-run target that may be associated with RNA secondary structure.

J Cell Biol, 1997 Aug 25, 138(4), 927 - 38
Rho- and rac-dependent assembly of focal adhesion complexes and actin filaments in permeabilized fibroblasts: an essential role for ezrin/radixin/moesin proteins; Mackay DJ et al.; The small GTPases Rho and Rac regulate actin filament assembly and the formation of integrin adhesion complexes to produce stress fibers and lamellipodia, respectively, in mammalian cells . Although numerous candidate effectors that might mediate these responses have been identified using the yeast two-hybrid and affinity purification techniques, their cellular roles remain unclear . We now describe a biological assay that allows components of the Rho and Rac signaling pathways to be identified . Permeabilization of serum-starved Swiss 3T3 cells with digitonin in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) induces both actin filament and focal adhesion complex assembly through activation of endogenous Rho and Rac . These responses are lost when GTPgammaS is added 6 min after permeabilization, but can be reconstituted using concentrated cytosolic extracts . We have achieved a 10,000-fold purification of the activity present in pig brain cytosol and protein sequence analysis shows it to contain moesin . Using recombinant proteins, we show that moesin and its close relatives ezrin and radixin can reconstitute stress fiber assembly, cortical actin polymerization and focal complex formation in response to activation of Rho and Rac.

Nature, 1997 Aug 21, 388(6644), 798 - 801
DNA renaturation activity of the SMC complex implicated in chromosome condensation; Sutani T et al.; Chromosome condensation occurs in mitosis before the separation of sister chromatids, and requires DNA topoisomerase II and a group of proteins called SMCs . The resulting condensed chromosomes in metaphase have a complex hierarchical structure . SMCs, the components of condensed chromosomes, are also required for the separation of sister chromatids and gene dosage compensation, and are found in a range of organisms from yeasts to mammals . However, the mechanisms by which the SMCs contribute to chromosome condensation are unknown . We have studied chromosomes in fission-yeast SMC mutants cut3-477 and cut14-208, which remain largely non-condensed during mitosis at the restrictive temperature (36 degrees C) . To test their role in DNA condensation, we isolated the proteins Cut3 and Cut14 as an oligomeric complex, and tested their interactions with isolated DNA . The complex efficiently promoted the DNA renaturation reactions (the winding up of single-strand DNAs into double helical DNA) as much as approximately 70-fold more efficiently than RecA, which is a bacterial protein with similar activity . The activity of the mutant complex was heat sensitive . As DNA winding by renaturation is a potential cause of supercoiling, the SMC complex may be implicated in promoting the higher-order DNA coiling found in condensed chromosomes.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9249 - 54
Screening for imprinted genes by allelic message display: identification of a paternally expressed gene impact on mouse chromosome 18; Hagiwara Y et al.; A systematic screen termed the allelic message display (AMD) was developed for the hunting of imprinted genes . In AMD, differential display PCR is adopted to image allelic expression status of multiple polymorphic transcripts in two parental mouse strains, reciprocal F1 hybrids and pooled backcross progenies . From the displayed patterns, paternally and maternally expressed transcripts can be unequivocally identified . The effectiveness of AMD screening was clearly demonstrated by the identification of a paternally expressed gene Impact on mouse chromosome 18, the predicted product of which belongs to the YCR59c/yigZ hypothetical protein family composed of yeast and bacterial proteins with currently unknown function . In contrast with previous screening methods necessitating positional cloning efforts or generation of parthenogenetic embryos, this approach requires nothing particular but appropriately crossed mice and can be readily applied to any tissues at various developmental stages . Hence, AMD would considerably accelerate the identification of imprinted genes playing pivotal roles in mammalian development and the pathogenesis of various diseases.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9238 - 43
The mouse pale ear (ep) mutation is the homologue of human Hermansky-Pudlak syndrome; Gardner JM et al.; The recessive mutation at the pale ear (ep) locus on mouse chromosome 19 was found to be the homologue of human Hermansky-Pudlak syndrome (HPS) . A positional cloning strategy using yeast artificial chromosomes spanning the HPS locus was used to identify the HPS gene and its murine counterpart . These genes and their predicted proteins are highly conserved at the nucleotide and amino acid levels . Sequence analysis of the mutant ep gene revealed the insertion of an intracisternal A particle element in a protein-coding 3' exon . Here we demonstrate that mice with the ep mutation exhibit abnormalities similar to human HPS patients in melanosomes and platelet-dense granules . These results establish an animal model of HPS and will facilitate biochemical and molecular analyses of the functions of this protein in the membranes of specialized intracellular organelles.

Biochemistry, 1997 Aug 19, 36(33), 10178 - 84
Kinetics of protoporphyrinogen oxidase inhibition by diphenyleneiodonium derivatives; Arnould S et al.; Protoporphyrinogen oxidase, the last enzyme of the common branch of the heme and chlorophyll pathways in plants, is the molecular target of diphenyl ether-type herbicides . These compounds inhibit the enzyme competitively with respect to the tetrapyrrole substrate, protoporphyrinogen IX . We used the flavinic nature of protoporphyrinogen oxidase to investigate the reactivity of the enzyme toward the 2,2'-diphenyleneiodonium cation, a known inhibitor of several flavoproteins . Diphenyleneiodonium inhibited the membrane-bound yeast protoporphyrinogen oxidase competitively with molecular oxygen . The typical slow-binding kinetics suggested that the enzyme with a reduced flavin rapidly combined with the inhibitor to form an initial complex which then slowly isomerized to a modified enzyme-inhibitor complex (Ki = 6.75 x 10(-8) M, Ki* = 4.1 x 10(-9) M) . This inhibition was strongly pH-dependent and was maximal at pH 8 . Substituted diphenyleneiodoniums were synthesized and shown to be even better inhibitors than 2,2'-diphenyleneiodonium: Ki = 4.4 x 10(-8) M and Ki* = 1.3 x 10(-9) M for 4-methyl-2,2'-diphenyleneiodonium, Ki = 2.2 x 10(-8) M and Ki * = 1.1 x 10(-9) M for 6-methyl-2,2'-diphenyleneiodonium, and Ki = 6.4 x 10(-9) M and Ki* = 1.2 x 10(-1)2 M for 4-nitro-2,2'-diphenyleneiodonium . The 4-nitro-2,2'-diphenyleneiodonium was a quasi irreversible inhibitor (k5/k6 > 5000) . Diphenyleneiodoniums are a new class of protoporphyrinogen oxidase inhibitors that act via a mechanism very different from that of diphenyl ether-type herbicides and appear to be promising tools for studies on the structure-function relationships of this agronomically important enzyme.

Biochem Biophys Res Commun, 1997 Aug 18, 237(2), 245 - 50
Interaction of DA41, a DAN-binding protein, with the epidermal growth factor-like protein, S(1-5); Ozaki T et al.; Recently, we have identified a new protein (DA41) which can associate with candidate tumor-suppressor DAN protein . In the present study, we have searched for DA41-interacting protein(s), using a yeast two-hybrid system . An adult rat lung cDNA library was screened by using a truncated form of DA41 (1-308) which lacks a DAN-binding region as bait . One of the positive clones, T16, contained a cDNA sequence of 1934 nucleotides with a single open reading frame of 493 amino acids . A data base search revealed that T16 exhibited a strong sequence similarity to the human epidermal growth factor (EGF)-like protein, S(1-5) . The region encoding amino acids 155-232 of DA41 was identified for the interaction with T16 . Since DAN and S(1-5) proteins are known to suppress and stimulate DNA synthesis, respectively, it is possible that functional interaction of DAN with S(1-5) through DA41 might play an important role(s) in the regulation of cell growth.

Genomics, 1997 Aug 15, 44(1), 110 - 7
Physical mapping of potassium channel gene clusters on mouse chromosomes three and six; Street VA et al.; Mammalian voltage-gated K channel genes have been divided into four subfamilies (Shaker, Shab, Shal, and Shaw) based on their sequence identity and similarity to related genes in Drosophila . Genetic mapping of the voltage-gated K channel genes has shown that similar multigene clusters exist on mouse Chr 3 and 6 and suggests that the clusters may have arisen through chromosomal duplication . In this report, YAC-based physical maps of the clustered mouse Shaker-like K channel genes have been constructed using restriction endonuclease and yeast chromosome fragmentation approaches . These data define the physical spacing as 5'-Kcna3-(60 kb)-Kcna2-(90 kb)-Kcna8-3' on Chr 3, and as 5'-Kcna6-(80 kb)-Kcna1-(110 kb)-Kcna5-3' on Chr 6, with all genes oriented in a head-to-tail manner within their respective clusters . These detailed physical maps of both K channel gene clusters provide additional support for the idea of an ancient genome tetraploidization event.

Genomics, 1997 Aug 15, 44(1), 61 - 70
AFLP-based fine mapping of the Mlo gene to a 30-kb DNA segment of the barley genome; Simons G et al.; Resistance of barley (Hordeum vulgare) to the powdery mildew fungus Erysiphe graminis f.sp . hordei is conferred by several dominant genes, but also by recessive alleles of the Mlo locus mapping on the long arm of chromosome 4 . In addition, this single-factor-mediated resistance is active against all known physiological races of the parasite . Thus the mechanism underlying mlo-mediated resistance should differ substantially from that mediated by the dominant genes . A positional cloning strategy to isolate the Mlo gene from the barley genome, the size of which is almost double the size of the human genome, has been designed . The AFLP technique was employed to identify markers tightly linked to the Mlo locus and to produce a local high-resolution genetic map . The use of this high-volume marker technology allowed the rapid screening of approximately 250,000 loci for linkage to Mlo . A large number of Mlo-linked AFLP markers were identified, one of which cosegregated with Mlo on the basis of more than 4000 meiotic events . A four-genome-equivalent barley YAC library (average insert size 480 kb) was constructed and screened with this cosegregating marker . Four YACs containing this marker were isolated and subsequent characterization by AFLP-based physical mapping allowed the physical delimitation of the Mlo locus to a DNA segment of 30 kb.

Genomics, 1997 Aug 15, 44(1), 45 - 51
Cloning, chromosomal localization, and interspecies interaction of mouse DNA polymerase delta small subunit (PolD2); Hindges R et al.; DNA polymerase delta core is a heterodimeric enzyme with a catalytic subunit of 125 kDa and a second subunit of 50 kDa with an as yet unknown function . It is an essential enzyme for DNA replication and DNA repair . We cloned the full-length cDNA encoding the DNA polymerase delta small subunit from mouse cells . The sequence of the predicted polypeptide of 51,336 Da is, like the catalytic subunit, highly conserved not only among mammals (93% identity and 96% similarity), but also between yeast and mammals (34% identity and 57% similarity) . Fluorescence in situ hybridization experiments indicated that the gene for the small DNA polymerase delta of mouse is localized on chromosome 11, band A2 . By using the yeast two-hybrid system we found that the mouse 125-kDa DNA polymerase catalytic subunit is able to interact with the 50-kDa subunit of the human enzyme, suggesting an in vivo interspecies interaction between the two subunits of DNA polymerase delta.

Genomics, 1997 Aug 15, 44(1), 22 - 34
NVL: a new member of the AAA family of ATPases localized to the nucleus; Germain-Lee EL et al.; We report the cloning of NVL, a newly recognized human gene that encodes an approximately 110-kDa nuclear protein designated NVLp (nuclear VCP-like protein), which is a member of a rapidly growing family of ATP-binding proteins recently denoted the AAA family (ATPases associated with diverse cellular activities) (W . H . Kunau et al., 1993, Biochimie 75:209-224) . NVL was isolated by degenerate PCR using oligonucleotides corresponding to the yeast PEX1 gene, which is necessary for peroxisomal biogenesis . Two cDNAs, designated NVL.1 and NVL.2, may represent alternatively spliced forms of a single gene that maps to chromosome 1q41-q42.2 . NVL has greatest similarity to the VCP subfamily of AAA proteins, is widely expressed, and encodes a nuclear protein with two highly similar ATP-binding domains . We speculate that NVLp is involved in an ATP-dependent nuclear process.

Cancer Res, 1997 Aug 15, 57(16), 3548 - 53
Isolation of a candidate gene, CAB1, for cholesterol transport to mitochondria from the c-ERBB-2 amplicon by a modified cDNA selection method; Akiyama N et al.; An improved cDNA selection method was established to isolate expressed genes efficiently from an amplified chromosome region in human cancer . Biotinylated yeast artificial chromosome DNA containing c-ERBB-2 was hybridized in solution with PCR-amplifiable cDNAs of an esophageal cancer cell line bearing the c-ERBB-2 amplification . After capturing the hybrids on avidin-coated magnetic beads, the cDNAs were amplified by PCR . Four new genes (A39, C51, CAB1, and GRB-7) coamplified with c-ERBB-2 were isolated from the enriched cDNA library . CAB1, GRB-7, and c-ERBB-2 were overexpressed in gastric and esophageal cancer cells in correspondence with the amplification . The deduced amino acid sequence of the CAB1 gene had significant homology to the recently discovered steroidogenic acute regulatory protein, StAR, which plays an essential role in cholesterol transport to mitochondria . It was established that multiple overexpressed genes are frequently present in a single amplicon.

J Biol Chem, 1997 Aug 15, 272(33), 20805 - 10
Phosphorylation-dependent regulation of N-methyl-D-aspartate receptors by calmodulin; Hisatsune C et al.; The N-methyl-D-aspartate (NMDA) receptor plays important roles in synaptic plasticity and brain development . The NMDA receptor subunits have large intracellular domains in the COOH-terminal region that may interact with signal-transducing proteins . By using the yeast two-hybrid system, we found that calmodulin interacts with the COOH terminus of the NR1 subunit and inactivates the channels in a Ca2+-dependent manner . Here we show that protein kinase C (PKC)-mediated phosphorylation on serine residues of NR1 decreases its affinity for calmodulin . This suggests that PKC-mediated phosphorylation of NR1 prevents calmodulin from binding to the NR1 subunit and thereby inhibits the inactivation of NMDA receptors by calmodulin . In addition, we show that stimulation of metabotropic glutamate receptor 1alpha, which potentiates NMDA channels through PKC, decreases the ability of NR1 to bind to calmodulin . Thus, our data provide clues to understanding the basis of cross-talk between two types of receptors, metabotropic glutamate receptors and the NR1 subunit, in NMDA channel potentiation.

J Biol Chem, 1997 Aug 15, 272(33), 20730 - 5
Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors Tom20, Tom22, and Tom70; Brix J et al.; The preprotein translocase of the outer mitochondrial membrane (Tom) is a multi-subunit complex required for specific recognition and membrane translocation of nuclear-encoded preproteins . We have expressed and purified the cytosolic domains of three postulated import receptors, Tom20, Tom22, and Tom70 . Each receptor domain is able to bind mitochondrial preproteins but with different specificity . Tom20 binds both preproteins with N-terminal presequences and preproteins with internal targeting signals; the binding is enhanced by the addition of salt . Tom22 selectively recognizes presequence-carrying preproteins in a salt-sensitive manner . Tom70 preferentially binds preproteins with internal targeting information . A chemically synthesized presequence peptide competes with preproteins for binding to Tom20 and Tom22 but not to Tom70 . We conclude that each of the three import receptors binds preproteins independently and by a different mechanism . Both Tom20 and Tom22 function as presequence receptors.

J Biol Chem, 1997 Aug 15, 272(33), 20538 - 44
Hrs, a tyrosine kinase substrate with a conserved double zinc finger domain, is localized to the cytoplasmic surface of early endosomes; Komada M et al.; Hrs is a 115-kDa double zinc finger protein that is rapidly tyrosine phosphorylated in growth factor-stimulated cells . However, its function remains unknown . Here we show that Hrs is localized to early endosomes . Intracellular localization of endogenous Hrs and exogenously expressed Hrs tagged with the hemagglutinin epitope was examined by immunofluorescence staining using anti-Hrs and anti-hemagglutinin epitope antibodies, respectively . Hrs was detected in vesicular structures and was colocalized with the transferrin receptor, a marker for early endosomes, but only partially with CD63, a marker for late endosomes . A zinc finger domain deletion mutant of Hrs was also colocalized with the transferrin receptor, suggesting that the zinc finger domain is not required for its correct localization . Immunoelectron microscopy showed that Hrs was localized to the cytoplasmic surface of these structures . By subcellular fractionation, Hrs was recovered both in the cytoplasmic and membrane fractions . The membrane-associated Hrs was extracted from the membrane by alkali treatment, suggesting that it is peripherally associated with early endosomes . These results, together with our finding that Hrs is homologous to Vps27p, a protein essential for protein traffic through a prevacuolar compartment in yeast, suggest that Hrs is involved in vesicular transport through early endosomes.

J Biol Chem, 1997 Aug 15, 272(33), 20332 - 5
The actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells; Lamaze C et al.; Actin filament organization is essential for endocytosis in yeast . In contrast, the actin-depolymerizing agent cytochalasin D has yielded ambiguous results as to a role for actin in receptor-mediated endocytosis in mammalian cells . We have therefore re-examined this issue using highly specific reagents known to sequester actin monomers . Two of these reagents, thymosin beta4 and DNase I, potently inhibited the sequestration of transferrin receptors into coated pits as measured in a cell-free system using perforated A431 cells . At low concentrations, thymosin beta4 but not DNase I was stimulatory . Importantly, the effects of both reagents were specifically neutralized by the addition of actin monomers . A role for the actin cytoskeleton was also detected in intact cells where latrunculin A, a drug that sequesters actin monomers, inhibited receptor-mediated endocytosis . Biochemical and morphological analyses suggest that these reagents inhibit later events in coated vesicle budding . These results provide new evidence that the actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells.

J Biol Chem, 1997 Aug 8, 272(32), 20162 - 6
GS15, a 15-kilodalton Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) homologous to rbet1; Xu Y et al.; Bet1p plays an essential role in vesicular transport from the endoplasmic reticulum (ER) to the Golgi in yeast, and it functions as a vesicle soluble N-ethylmaleimide-sensitive factor protein receptor (v-SNARE) . A mammalian protein related to Bet1p has been reported previously and was referred to as rbet1 . We have now identified a new mammalian protein that is homologous to rbet1 (28% amino acid identity) . mRNA for this rbet1 homologue is widely expressed in rat tissues . Affinity-purified polyclonal antibodies raised against recombinant protein specifically recognized a 15-kilodalton integral membrane protein highly enriched in Golgi membranes . Indirect immunofluorescence microscopy revealed that this protein is specifically associated with the Golgi apparatus in diverse cell types . Biochemical characterization established that this protein behaves like a SNARE and was named GS15 (Golgi SNARE with a size of 15 kilodaltons) . These properties raise the possibility that GS15 is a novel SNARE mediating a yet to be defined transport event associated with the Golgi apparatus.

J Biol Chem, 1997 Aug 8, 272(32), 19758 - 62
Splicing of intron-containing tRNATrp by the archaeon Haloferax volcanii occurs independent of mature tRNA structure; Armbruster DW et al.; We have investigated the requirements for mature tRNA structure in the in vivo splicing of the Haloferax volcanii, intron-containing tRNATrp RNA . A partial tRNATrp gene, which contained only the anticodon stem-loop region of the mature tRNA, was fused to a carrier yeast tRNA gene for expression in H . volcanii . Transcripts from this hybrid gene were found to be processed by endonuclease and ligase at the tRNATrp exon-intron boundaries . These results verify that the substrate recognition properties of the halobacterial endonuclease observed in vitro reflect the properties of this enzyme in vivo, namely that mature tRNA structure is not essential for recognition by the endonuclease . The independence of these reactions on mature tRNA provides further support for a relationship between archaeal tRNA and rRNA intron-processing systems and highlight a difference in the substrate recognition properties between the archaeal and eucaryal processing systems . The significance of these differences is discussed in light of the observation that the tRNA endonucleases of these organisms are related.

J Biol Chem, 1997 Aug 8, 272(32), 19672 - 81
In vitro characterization of the novel proprotein convertase PC7; Munzer JS et al.; Biochemical and enzymatic characterization of the novel proprotein convertase rat PC7 (rPC7) was carried out using vaccinia virus recombinants overexpressed in mammalian BSC40 cells . Pro-PC7 is synthesized as a glycosylated zymogen (101 kDa) and processed into mature rPC7 (89 kDa) in the endoplasmic reticulum . No endogenously produced soluble forms of this membrane-anchored protein were detected . A deletion mutant (65 kDa), truncated well beyond the expected C-terminal boundary of the P-domain, produced soluble rPC7 in the culture medium . Enzymatic activity assays of rPC7 using fluorogenic peptidyl substrates indicated that the pH optimum, Ca2+ dependence, and cleavage specificity of this enzyme are largely similar to those of furin . However, with some substrates, cleavage specificity more closely resembled that of yeast kexin, suggesting differential processing of proprotein substrates by this novel convertase . We examined the rPC7- and human furin-mediated cleavage of synthetic peptides containing the processing sites of three proteins known to colocalize in situ with rPC7 . Whereas both enzymes correctly processed the pro-parathyroid hormone tridecapeptide and the pro-PC4 heptadecapeptide, neither enzyme cleaved a pro-epidermal growth factor hexadecapeptide . Thus, this study establishes that rPC7 is an enzymatically functional subtilisin/kexin-like serine proteinase with a cleavage specificity resembling that of hfurin . In addition, we have demonstrated that rPC7 can correctly process peptide precursors that contain the processing sites of at least two potential physiological substrates.

Science, 1997 Aug 8, 277(5327), 805 - 8
Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34; van Slegtenhorst M et al.; Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the widespread development of distinctive tumors termed hamartomas . TSC-determining loci have been mapped to chromosomes 9q34 (TSC1) and 16p13 (TSC2) . The TSC1 gene was identified from a 900-kilobase region containing at least 30 genes . The 8.6-kilobase TSC1 transcript is widely expressed and encodes a protein of 130 kilodaltons (hamartin) that has homology to a putative yeast protein of unknown function . Thirty-two distinct mutations were identified in TSC1, 30 of which were truncating, and a single mutation (2105delAAAG) was seen in six apparently unrelated patients . In one of these six, a somatic mutation in the wild-type allele was found in a TSC-associated renal carcinoma, which suggests that hamartin acts as a tumor suppressor.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8658 - 63
Xeroderma pigmentosum and trichothiodystrophy are associated with different mutations in the XPD (ERCC2) repair/transcription gene; Taylor EM et al.; The xeroderma pigmentosum group D (XPD) protein has a dual function, both in nucleotide excision repair of DNA damage and in basal transcription . Mutations in the XPD gene can result in three distinct clinical phenotypes, XP, trichothiodystrophy (TTD), and XP with Cockayne syndrome . To determine if the clinical phenotypes of XP and TTD can be attributed to the sites of the mutations, we have identified the mutations in a large group of TTD and XP-D patients . Most sites of mutations differed between XP and TTD, but there are three sites at which the same mutation is found in XP and TTD patients . Since the corresponding patients were all compound heterozygotes with different mutations in the two alleles, the alleles were tested separately in a yeast complementation assay . The mutations which are found in both XP and TTD patients behaved as null alleles, suggesting that the disease phenotype was determined by the other allele . If we eliminate the null mutations, the remaining mutagenic pattern is consistent with the site of the mutation determining the phenotype.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8405 - 10
Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation; Rossi F et al.; We present an approach for monitoring protein-protein interactions within intact eukaryotic cells, which should increase our understanding of the regulatory circuitry that controls the proliferation and differentiation of cells and how these processes go awry in disease states such as cancer . Chimeric proteins composed of proteins of interest fused to complementing beta-galactosidase (beta-gal) deletion mutants permit a novel analysis of protein complexes within cells . In this approach, the beta-gal activity resulting from the forced interaction of nonfunctional weakly complementing beta-gal peptides (Deltaalpha and Deltaomega) serves as a measure of the extent of interaction of the non-beta-gal portions of the chimeras . To test this application of lacZ intracistronic complementation, proteins that form a complex in the presence of rapamycin were used . These proteins, FRAP and FKBP12, were synthesized as fusion proteins with Deltaalpha and Deltaomega, respectively . Enzymatic beta-gal activity served to monitor the formation of the rapamycin-induced chimeric FRAP/FKBP12 protein complex in a time- and dose-dependent manner, as assessed by histochemical, biochemical, and fluorescence-activated cell sorting assays . This approach may prove to be a valuable adjunct to in vitro immunoprecipitation and crosslinking methods and in vivo yeast two-hybrid and fluorescence energy transfer systems . It may also allow a direct assessment of specific protein dimerization interactions in a biologically relevant context, localized in the cell compartments in which they occur, and in the milieu of competing proteins.

Regul Toxicol Pharmacol, 1997 Aug, 26(1), 96 - 101
Failure to Confirm Estrogenic Activity for Benzoic Acid and Clofibrate: Implications for Lists of Endocrine-Disrupting Agents
Ashby J, Lefevre PA, Odum J, Tinwell H, Kennedy SJ, Beresford N, Sumpter JP.
Earlier reports that benzoic acid is uterotrophic to the rat and mouse and that clofibrate is uterotrophic to the rat have not been confirmed . The studies reported here involved the use of a range of test protocols and dose levels, including the protocols/dose levels used by the original investigators . In addition, both chemicals were inactive in a human estrogen receptor (hERalpha) yeast estrogenicity assay . It is concluded that benzoic acid and clofibrate are not estrogenic in the assays used here . This conclusion has implications for the compilation of lists of endocrine-disrupting chemicals .

Regul Toxicol Pharmacol, 1997 Aug, 26(1), 60 - 8
Development of a Tier I Screening Battery for Detecting Endocrine-Active Compounds (EACs)
Cook JC, Kaplan AM, Davis LG, O'connor JC.
One of the components of our research program is development of a mode-of-action screening battery to detect several different types of endocrine-active compounds (EACs) . Our working hypothesis is that a comprehensive short-term in vivo/in vitro battery can be developed to identify endocrine toxicants using a collection of endpoints . The goals of this battery are that it be quick, cost effective, and predictive . The purpose of this battery is to identify potential EACs and to assess their potency in order to prioritize compounds for further study . Two in vivo screens (intact male and ovariectomized female rats) are being evaluated for their ability to detect several different types of endocrine activity . To validate this screen, 15 compounds with known endocrine activities are being used to evaluate a collection of different endpoints for their variability, stability over time, predictiveness, and dose dependency . These positive controls were chosen because they can modulate development, reproduction, or cancer . The advantage of an in vivo screen is that it utilizes a metabolically and physiologically intact system . The male in vivo battery will be used to assess several different types of endocrine activity, primarily by using a comprehensive hormonal battery . The female in vivo battery will be used to identify compounds which are either estrogenic/antiestrogenic or can alter the prolactin pathway . The in vitro portion of the screening battery consists of a yeast transactivation system (YTS) . The YTS is being evaluated for its ability to identify compounds which are agonists or antagonists to the estrogen, androgen, or progesterone receptors . The expression of mammalian receptors in yeast allows for assessment of steroid-dependent transcriptional activators . The value of this system is that it can be used as a routine screen for compounds that interact with steroid receptors . Alterations in ligand binding to these receptors can be correlated with alterations in development via masculinization of females and/or feminization of males, decreases in reproductive success, or modulation of cancer incidence from in vivo tests . The in vivo and in vitro screens are designed to be run in parallel with built-in redundancy in order to reduce the probability of false-negative/positive responses .

Mol Cell Biochem, 1997 Aug, 173(1-2), 113 - 9
Alcohol dehydrogenase-I from horse liver stimulates immunoglobulin production by human hybridoma and human peripheral blood lymphocytes; Sugahara T et al.; Immunoglobulin production stimulating activity of alcohol dehydrogenase {EC 1.1.1.1} was assessed . Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells producing human lung cancer specific monoclonal IgM . IgM production of HB4C5 cells was enhanced more than 6 fold by the addition of ADH-I at 400 microg/ml under serum-free condition . However, yeast derived ADHs, such as ADH-II and -III were ineffective to accelerate immunoglobulin production of the hybridoma line . These results imply that the immunoglobulin production stimulating effect of ADH-I is irrelevant to its enzymatic function, and defined as a novel feature of ADH-I . This enzyme also stimulated IgM and IgG production by human peripheral blood lymphocytes 2.9 fold and 1.4 fold, respectively . This fact suggests that ADH-I stimulates immunoglobulin production not only by specific hybridoma cell line, but also by non-specific immunoglobulin producers.

Biochem J, 1997 Aug 1, 325 ( Pt 3), 779 - 86
Nramp1 locus encodes a 65 kDa interferon-gamma-inducible protein in murine macrophages; Atkinson PG et al.; The murine Nramp1 (natural-resistance-associated macrophage protein) locus, formerly known as Ity/Lsh/Bcg, was isolated previously on the basis of chromosomal location, and as conferring natural resistance to infection against intracellular macrophage pathogens . The gene encodes a transporter molecule of unknown function . We have prepared polyclonal antisera against the C-terminal 35 amino acids of murine Nramp1 . This serum is reactive towards a 65 kDa protein, expressed in murine macrophage cells from resistant or susceptible mice stimulated with interferon-gamma and lipopolysaccharide, but not in non-macrophage cells . Evidence indicates that Nramp1 is localized in a subcellular membrane rather than at the cell surface . This evidence includes: the identification of conserved endocytic targeting motifs following inspection of human and murine Nramp sequences; the enrichment of Nramp1, following magnetic selection of phagolysosomal vesicles from activated macrophages that were allowed to phagocytose magnetic, IgG-coated beads; confocal microscopy . These studies place Nramp1 on a membrane in close proximity to obligate intracellular pathogens . A link between Nramp1 and divalent-cation transport is suggested by sequence similarity with yeast SMF1 . Evidence showing modulation of Nramp1 protein levels by iron chelation provides a direct link with Nramp1 function and divalent-cation metabolism.

Biochem J, 1997 Aug 1, 325 ( Pt 3), 741 - 9
A novel murine P-450 gene, Cyp4a14, is part of a cluster of Cyp4a and Cyp4b, but not of CYP4F, genes in mouse and humans; Heng YM et al.; Genomic clones for Cyp4a12 and a novel member of the murine Cyp4a gene family were isolated . The novel gene, designated Cyp4a14, has a GC rich sequence immediately 5' of the transcription start site, and is similar to the rat CYP4A2 and CYP4A3 genes . The Cyp4a14 gene spans approximately 13 kb, and contains 12 exons; sequence similarity to the rat CYP4A2 gene sequence falls off 300 bp upstream from the start site . In view of the known sex-specific expression of the rat CYP4A2 gene, the expression and inducibility of Cyp4a14 was examined . The gene was highly inducible in the liver when mice were treated with the peroxisome proliferator, methylclofenapate; induction levels were low in control animals and no sex differences in expression were observed . By contrast, the Cyp4a12 RNA was highly expressed in liver and kidney of control male mice but was expressed at very low levels in liver and kidney of female mice . Testosterone treatment increased the level of this RNA in female liver slightly, and to a greater extent in the kidney of female mice . In agreement with studies on the cognate RNA, expression of Cyp4a12 protein was male-specific in the liver of control mice and extremely high inducibility of Cyp4a10 protein, with no sex differences, was also demonstrated . In view of the overlapping patterns of inducibility of the three Cyp4a genes, we investigated whether the three genes were co-localized in the genome . Two overlapping yeast artificial chromosome (YAC) clones were isolated, and the three Cyp4a genes were shown to be present on a single YAC of 220 kb . The Cyp4a genes are adjacent to the Cyp4b1 gene, with Cyp4a12 most distant from Cyp4b1 . The clustering of these two gene subfamilies in the mouse was replicated in the human, where the CYPA411 and CYP4B1 genes were present in a single YAC clone of 440 kb . However, the human CYP4F2 gene was mapped to chromosome 19 . Phylogenetic analysis of the CYP4 gene families demonstrated that CYP4A and CYP4B are more closely related than CYP4F.

Genomics, 1997 Aug 1, 43(3), 387 - 9
Closely spaced tandem arrangement of AQP2, AQP5, and AQP6 genes in a 27-kilobase segment at chromosome locus 12q13; Ma T et al.; The aquaporins (AQPs) are a family of water-transporting proteins that facilitate osmotically driven water movement across cell plasma membranes . Among the seven human aquaporins cloned to date (AQPs 0-6), genes encoding the four most closely related aquaporins (AQP0, AQP2, AQP5, and AQP6) have been mapped to chromosome band 12q13, suggesting an aquaporin family gene cluster at this locus . To construct a physical map and identify novel aquaporin gene members on this cluster, a human CEPH B yeast artificial chromosome (YAC) library was screened by PCR using primers derived from exon 4 of AQP2 and AQP0 genes . A YAC clone with 200 kb of human DNA was isolated and analyzed . Primary pulsed-field gel electrophoresis and Southern blot analysis indicated the presence of AQP2, AQP5, and AQP6 genes, but not AQP0 . Restriction mapping and PCR analysis yielded a precise physical map in which the three aquaporin genes span only approximately 27 kb in the order, transcriptional orientation, and spacer length 5'-AQP2-5 kb spacer-AQP5-7 kb spacer-AQP6-3'.

Genomics, 1997 Aug 1, 43(3), 376 - 9
Identification and cloning of the human homolog (JAG1) of the rat Jagged1 gene from the Alagille syndrome critical region at 20p12; Oda T et al.; Notch proteins are a family of closely related transmembrane receptors proven to be instrumental in cell fate decisions . Recently, Notch ligands Delta and Jagged have been identified in Drosophila and rat, respectively . We have isolated the human homolog of the rat Jagged1 gene, JAG1, from a CpG island in a YAC clone covering the Alagille syndrome critical region at chromosome 20p12 (tel-SNAP-D20S186-cen) . Alagille syndrome is an autosomal dominant disorder characterized by neonatal jaundice, paucity of intrahepatic bile ducts, and abnormalities of the heart, skeleton, and eyes . The human Jagged1 (JAG1), therefore, appears to be a strong candidate gene for this disease . Here we describe the identification, full-length cDNA cloning, expression patterns, and precise physical location of this gene within the Alagille syndrome critical region.

Genomics, 1997 Aug 1, 43(3), 349 - 58
A 2-Mb YAC contig and physical map covering the chromosome 8q12 breakpoint cluster region in pleomorphic adenomas of the salivary glands; Kas K et al.; Pleomorphic adenomas are benign epithelial tumors originating from the major and minor salivary glands . Extensive cytogenetic studies have demonstrated that they frequently show chromosome abnormalities involving chromosome 8, with consistent breakpoints at 8q12 . In previous studies, we have shown that these breakpoints are located in a 9-cM interval between MOS/D8S285 and D8S260 . Here, we describe directional chromosome walking studies starting from D8S260 as well as D8S285 . Using the CEPH and ICRF YAC libraries, these studies resulted in the construction of two nonoverlapping YAC contigs of about 2 and 5 Mb, respectively . Initial fluorescence in situ hybridization (FISH) analysis suggested that the majority of 8q12 breakpoints clustered within the 2-Mb contig, which was mapped to the centromeric part of chromosome band 8q12 . This contig has at least double coverage and consists of 34 overlapping YAC clones . The localization of the YACs was confirmed by FISH analysis . On the basis of mapping data of landmarks with an average spacing of 65 kb as well as restriction enzyme analysis, a long-range physical map was established for the chromosome region spanned by the 2-Mb contig . The relative positions of various known genes and expressed sequence tags within this contig were also determined . Subsequent FISH analyses of pleomorphic adenomas using YACs as well as cosmids revealed that all but two of the 8q12 breakpoints in the primary tumors tested mapped within a 300-kb interval between the MOS proto-oncogene and STS EM156 . The target gene affected by the chromosome aberrations mapping within this interval was recently shown to be the PLAG1 gene, which encodes a novel zinc finger protein.

Genomics, 1997 Aug 1, 43(3), 285 - 97
Characterization of the C3 YAC contig from proximal mouse chromosome 17 and analysis of allelic expression of genes flanking the imprinted Igf2r gene; Schweifer N et al.; The imprinted mouse insulin-like growth factor type 2 receptor (Igf2r) maps to the middle of a gene-rich region in band A2 of mouse chromosome 17 . The t(Lub2) chromosome 17 variant contains a small deletion that removes at least seven genes including Igf2r . We have constructed a YAC contig spanning the entire t(Lub2) deletion and created a restriction map that covers 700 kb . The position, transcription orientation, and imprinted status of the genes immediately flanking Igf2r have been assessed . We show here that the Mas gene, which lies 65 kb upstream to Igf2r, contains a novel 5' exon and is not imprinted in adult tissues . We further show that the recently identified Lx1 gene lies immediately downstream and is also expressed from both parental alleles in adult tissues . The remaining genes in this region have previously been shown to be biallelically expressed.

Genome Res, 1997 Aug, 7(8), 787 - 801
A high-resolution physical and transcript map of the Cri du chat region of human chromosome 5p; Church DM et al.; A high-resolution physical and transcription map has been generated of a 3.5-Mb region of 5p15.2 that is associated with the Cri du chat (CDC) syndrome . Utilizing a variety of resources including a natural deletion panel, a chromosome specific radiation hybrid panel, and YAC, PAC, and BAC genomic clones we have ordered > 60 STSs within this region . Approximately 45% of these STSs were obtained from publicly available whole genome maps, thus allowing for integration of this map with currently available resources . Thirteen of these markers were ESTs . In addition, > 70 exon trapped products have been mapped on the natural deletion panel and bacterial clone resource . The combination of these resources has allowed for the identification of 17 transcripts within this region, all of which represent candidate genes for CDC . Further characterization of the genomic contig also revealed that this region of 5p15 contains a large number of repetitive elements.

Curr Opin Biotechnol, 1997 Aug, 8(4), 455 - 8
Production of human antibody repertoires in transgenic mice; Bruggemann M et al.; Transgenic mice have been created that carry human immunoglobulin heavy and light chain genes in germline configuration and that have the corresponding endogenous genes silenced . The transgenes are either minigene constructs or large, almost authentic, transloci on yeast artificial chromosomes and undergo B-cell-specific DNA rearrangement and hypermutation in the mouse lymphoid tissue . Monoclonal antibodies with good affinities for human antigens have been obtained after immunisation . These mice may be a future source of human antibodies for therapy.

Leukemia, 1997 Aug, 11(8), 1187 - 92
Smad5, a tumor suppressor candidate at 5q31.1, is hemizygously lost and not mutated in the retained allele in human leukemia cell line HL60; Zavadil J et al.; Deletions of the long arm of chromosome 5 with common overlapping segment 5q31.1 are among the most frequent cytogenetic aberrations in myelodysplastic syndromes and acute myeloid leukemias (MDS/AML) . We have constructed a YAC-based physical map of the 5q31.1 critical locus and localized the transcriptional transactivator Smad5 adjacent to loci showing consistent loss of heterozygosity in these disorders . Smad5 plays a key role along the bone morphogenetic protein-4 (BMP-4) inhibitory signalling pathway inducing embryonic hematopoiesis . Smad5 homologs Smad2 and DPC4 have recently been linked to human cancer . FISH analysis of AML-M2 cell line HL60 and of four MDS/AML patients revealed consistent hemizygous loss of the Smad5 locus . In HL60 cells, a translocation event within 5q31.1 associated with loss of adjacent material leads to disruption of the critical locus with partial retention of the 5q31.1 genomic sequences on a marker chromosome . RT-PCR sequencing analysis of the HL60 Smad5 remaining allele ruled out the functional inactivation of the gene analogous to that occurring in the Smad5 homologs DPC4 and Smad2 in cases of pancreatic and colorectal cancers . Mutational analysis of Smad5 in MDS/AML cases is in progress.

Curr Opin Cell Biol, 1997 Aug, 9(4), 505 - 12
SNAREs and NSF in targeted membrane fusion; Hay JC et al.; A major current issue in vesicle trafficking is whether NSF (N-ethylmaleimide-sensitive factor) and alpha-SNAP (alpha-soluble NSF attachment protein) are required prior to SNARE (SNAP receptor) complex formation to allow vesicle docking, or after docking at a step close to membrane fusion . Recent studies of yeast vacuolar fusion indicated that the requirement for ATP, NSF and alpha-SNAP could be completely satisfied prior to SNARE docking complex assembly; however, the universality of a predocking role for these factors remains to be established . The vacuolar fusion system has also been used to directly demonstrate a requirement for SNARE proteins on both fusing membranes, verifying a central postulate of current fusion models.

Hum Mol Genet, 1997 Aug, 6(8), 1295 - 304
Generation of novel human MHC class II mutant B-cell lines by integrating YAC DNA into a cell line homozygously deleted for the MHC class II region; Fabb SA et al.; The human B lymphoblastoid cell line (LCL) 721.174 sustains a large homozygous deletion in the major histocompatibility complex (MHC) class II region that results in an absence of DQ and DR molecules as well as a deficiency in the assembly and transport of class I molecules to the cell surface . The deleted genes include the transporters associated with antigen processing TAP1 and TAP2, DMA and DMB which are involved in editing class II bound peptides, and four genes whose roles in antigen processing are unclear; the low mass polypeptide genes LMP2 and LMP7, and DNA and DOB . To study this region we have integrated into 721.174 two overlapping yeast artificial chromosome (YAC) clones which cover the interval LMP2-DRA inclusive . Three clones (11.2A1.1, 4D1D10.1 and 4D1D10.2), containing complete copies of the transfected YAC, produced varying levels of mRNA from the LMP, TAP, DQ and DR genes and corresponding levels of LMP and TAP protein . Class I cell surface expression was restored in 11.2A1.1 and 4D1D10.1, as was DR expression in both 4D1D10 transfectants . These studies demonstrate the feasibility of introducing large groups of functional genes back into human lymphoblastoid cells sustaining deletions, with full restoration of biological function . The procedure could be exploited in order to restore all but one gene covered by the deletion, effectively producing a single gene defect . This could be used to introduce copies of genes engineered to contain mutations and to study cis regulatory elements at some distance from the chosen loci.

Hum Mol Genet, 1997 Aug, 6(8), 1241 - 50
Autism and multiple exostoses associated with an X;8 translocation occurring within the GRPR gene and 3' to the SDC2 gene; Ishikawa-Brush Y et al.; An X;8 translocation was identified in a 27-year-old female patient manifesting multiple exostoses and autism accompanied by mental retardation and epilepsy . Through molecular analysis using yeast artificial chromosomes (YACs) and cosmid clones, the translocation breakpoint was isolated and confirmed to be reciprocal within a 5'-GGCA-3' sequence found on both X and 8 chromosomes without gain or loss of a single nucleotide . The translocation breakpoint on the X chromosome occurred in the first intron of the gastrin-releasing peptide receptor (GRPR) gene and that on chromosome 8 occurred approximately 30 kb distal to the 3' end of the Syndecan-2 gene (SDC2), also known as human heparan sulfate proteoglycan or fibroglycan . The GRPR gene was shown to escape X-inactivation . A dosage effect of the GRPR and a position effect of the SDC2 gene may, however, contribute the phenotype observed in this patient since the orientation of these genes with respect to the translocation was incompatible with the formation of a fusion gene . Investigation of mutations in these two genes in unrelated patients with either autism or multiple exostoses as well as linkage and association studies is needed to validate them as candidate genes.

Genes Chromosomes Cancer, 1997 Aug, 19(4), 273 - 7
FISH analysis of translocations involving the short arm of chromosome 9 in lymphoid malignancies; Leblanc T et al.; Deletion of the short arm of chromosome 9 (9p), resulting in the loss of the p16INK4a/MTS1 gene, now called CDKN2, has been found to occur frequently in acute lymphoblastic leukemia, even in the absence of a microscopically visible deletion . In this study, we have used YAC probes encompassing the CDKN2 locus to analyze by fluorescence in situ hybridization patients with leukemia and lymphoma and translocations involving 9p in order to establish the CDKN2 status in relation to the karyotype . We found that, in leukemic cells exhibiting loss of heterozygosity at the CDKN2 locus, the deleted allele was from the cytogenetically normal chromosome 9, whereas the other allele was located on a rearranged chromosome . This finding suggests that CDKN2 gene loss is nonrandomly associated with 9p translocation in lymphoid proliferations . Genes Chromosom.

Genes Chromosomes Cancer, 1997 Aug, 19(4), 220 - 7
Normal FHIT transcripts in renal cell cancer- and lung cancer-derived cell lines, including a cell line with a homozygous deletion in the FRA3B region; van den Berg A et al.; The recently identified FHIT gene encompasses the FRA3B region and the breakpoint of a constitutive t(3;8) occurring in a family with hereditary renal cell cancer . Occurrence of aberrant transcripts in different types of tumours has led to the suggestion that FHIT might play a critical role in the development of various types of cancer . We have analyzed the gene and its transcripts in lung cancers and renal cell cancer-derived cell lines . A lung adenocarcinoma cell line, GLC-A2, appeared to have a homozygous deletion in intron 5 of FHIT . RT-PCR analysis revealed a normal-sized PCR product in all of the cell lines: Including GLC-A2 . A number of them had an additional aberrant product . Analysis of a great number of control cell lines and tissues showed that the majority of these also had aberrant PCR products in addition to a normal-sized PCR product . Different specimens of the same cell type showed variable additional RT-PCR products . Normal-sized PCR products had a sequence identical to the FHIT sequence . PCR products longer than normal had insertions of different sizes at different positions . With three exceptions, PCR products shorter than normal represented FHIT sequences missing one or more entire exons . Thus, the presence of aberrant transcripts is not cancer-specific . Conceivably, sequence responsible for the instability of the FRA3B region are being transcribed into FHIT pre-mRNA and may cause the abnormal splicing and processing of the transcripts.

RNA, 1997 Aug, 3(8), 893 - 904
Structure and aminoacylation capacities of tRNA transcripts containing deoxyribonucleotides; Aphasizhev R et al.; The contribution of the ribose 2'-hydroxyls to RNA structure and function has been analyzed, but still remains controversial . In this work, we report the use of a mutant T7 RNA polymerase as a tool in RNA studies, applied to the aspartate and methionine tRNA aminoacylation systems from yeast . Our approach consists of determining the effect of substituting natural ribonucleotides by deoxyribonucleotides in RNA and, thereby, defining the subset of important 2'-hydroxyl groups . We show that deoxyribose-containing RNA can be folded in a global conformation similar to that of natural RNA . Melting curves of tRNAs, obtained by temperature-gradient gel electrophoresis, indicate that in deoxyribo-containing molecules, the thermal stability of the tertiary network drops down, whereas the stability of the secondary structure remains unaltered . Nuclease footprinting reveals a significant increase in the accessibility of both single- and double-stranded regions . As to the functionality of the deoxyribose-containing tRNAs, their in vitro aminoacylation efficiency indicates striking differential effects depending upon the nature of the substituted ribonucleotides . Strongest decrease in charging occurs for yeast initiator tRNA(Met) transcripts containing dG or dC residues and for yeast tRNA(Asp) transcripts with dU or dG . In the aspartate system, the decreased aminoacylation capacities can be correlated with the substitution of the ribose moieties of U11 and G27, disrupting two hydrogen bond contacts with the synthetase . Altogether, this suggests that specific 2'-hydroxyl groups in tRNAs can act as determinants specifying aminoacylation identity.

Appl Environ Microbiol, 1997 Aug, 63(8), 3301 - 3
Interference of peptone and tyrosine with the lignin peroxidase assay; ten Have R et al.; The N-unregulated white rot fungus Bjerkandera sp . strain BOS55 was cultured in 1 liter of peptone-yeast extract medium to produce lignin peroxidase (LiP) . During the LiP assay, the oxidation of veratryl alcohol to veratraldehyde was inhibited due to tyrosine present in the peptone and the yeast extract.

Mol Biochem Parasitol, 1997 Aug, 87(2), 169 - 81
Plasmodium falciparum asparagine and aspartate rich protein 2 is an evolutionary conserved protein whose repeats identify a new family of parasite antigens; Barale JC et al.; We describe here a new Plasmodium falciparum antigen, asparagine and aspartate rich protein 2 (PfAARP2) of 150 kDa, which is encoded by a unique gene on chromosome 1 . PfAARP2 is first expressed 12 h post-invasion and accumulates in trophozoites and schizonts . Immunofluorescence studies indicate that PfAARP2 is translocated into the red blood cell cytoplasm . The central region of Pfaarp2 contains blocks of repetitions encoding asparagine and aspartate residues, which define a new family of related genes dispersed on different chromosomes, and two members of this family have also been identified . Interestingly, the non-repeated N- and C-termini of PfAARP2 display significant similarity to two yeast and human predicted proteins, and its possible function is discussed.

J Mol Biol, 1997 Aug 1, 270(5), 688 - 95
Tissue-specific expression and alpha-actinin binding properties of the Z-disc titin: implications for the nature of vertebrate Z-discs; Sorimachi H et al.; Titins are giant filamentous proteins which connect Z-discs and M-lines in the sarcomeres of vertebrate striated muscles . Comparison of the N-terminal region of titin (Z-disc region) from different skeletal and cardiac muscles reveals a 900-residue segment which is expressed in different length variants, dependent on tissue type . When searching for ligands of this differentially expressed domain by a yeast-two hybrid approach, we detected binding to alpha-actinin . The isolated alpha-actinin cDNAs were derived from the C-terminal region of the alpha-actinin isoform (alpha-actinin-2) encoded by the ACTN2 gene . Therefore, the two antiparallel subunits of an alpha-actinin-2 homodimer will attach to actin at their respective C termini, whereas they will bind to the Z-disc titin at their N termini . This may thus explain how alpha-actinins can cross-link antiparallel titin and thin filaments from opposing sarcomeres . The alpha-actinin-2 binding site of the Z-disc titin is located within a sequence of 45-residue repeats, referred to as Z-repeat region . Both the N-terminal and C-terminal Z-repeats have alpha-actinin binding properties and are expressed in all striated muscles . By contrast, the more central Z-repeats are expressed in slow and fast skeletal muscles, as well as embryonic and adult cardiac muscles, in different copy numbers . Such alternative splicing of the Z-disc titin appears to be important for the tissue and fibre type diversity of the Z-disc lattice.

Cancer Res, 1997 Aug 1, 57(15), 3288 - 93
Inhibition of nonsense-mediated messenger RNA decay in clinical samples facilitates detection of human MSH2 mutations with an in vivo fusion protein assay and conventional techniques; Andreutti-Zaugg C et al.; Germ-line mutations in the human MSH2 (hMSH2) gene account for about 40% of known defects in kindreds with hereditary nonpolyposis colon cancer . We describe a simple fusion protein assay for detection of hMSH2 nonsense mutations in yeast . Detection of nonsense mutations with this assay is severely compromised in many cases by nonsense-mediated mRNA decay, a physiological process that destabilizes the mutant RNA . Triggering of nonsense-mediated decay requires mRNA scanning by the ribosome to detect the stop codon . We show that treatment of cells with the translation inhibitor puromycin suppresses nonsense-mediated decay and facilitates the detection of nonsense mutations in clinical samples by cDNA sequencing, in vitro protein truncation tests, and the yeast fusion protein assay . Given the prevalence of chain-terminating mutations in human disease genes, puromycin treatment of blood samples should improve the signal-to-noise ratio and hence the sensitivity of many RNA-based diagnostic tests . Paradoxically, the yeast hMSH2::ADE2 fusion protein assay also detects some in-frame mutations, presumably through an effect on the folding of the fusion protein.

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