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