Gene, 1995 May 26, 158(1), 61 - 6 A point mutation in a murine immunoglobulin V-region strongly influences the antibody yield in Escherichia coli; Duenas M et al.; Recombinant DNA technology has made it possible to produce specific Fab and scFv antibody (Ab) fragments in prokaryotic host cells . Using vectors designed for periplasmic expression of encoded Ab fragments, we have been studying how the sequence and genetic localization of the light chain (L-chain) variable region gene of a mouse Ab (CB-Nm.1) determined the level of Ab production . The variable region was shown to belong to the V kappa V family and contained a previously unreported Ile72 . Nine different Ab constructions were tested in monocistronic (scFv) or dicistronic (Fab) operons for their ability to affect the synthesis level of the L-chain . When the gene coding for the L-chain was located downstream from the Fd fragment gene, the substitution of codons encoding Ile by a codon encoding Thr was found to be crucial for any expression of the L-chain fragment . This was, however, not accompanied by an increase in L-chain-specific mRNA, neither was there any change in the size of the mRNA . The fact that the unmutated L-chain protein was produced from cells transformed with certain other constructions indicated that the protein as such was not incompatible with the prokaryotic environment . Together, this suggested that the translation process was involved in the restricted production of the L-chain . Thus, surprisingly small substitutions significantly affected the expression level, a fact that will have important implications on the library size expressed in prokaryotic hosts, including phage-displayed Ab libraries. Gene, 1995 May 26, 158(1), 107 - 11 Cloning and characterization of the lipase operon from Mycoplasma mycoides subspecies mycoides LC; Rawadi G et al.; Lipases, serine esterase enzymes, play an essential role in the mycoplasmal nutritional requirement for long-chain fatty acids . Although the lipase(s) activity in different mycoplasma species has been investigated, the molecular biology of the corresponding genes has not been studied . Using a single-primer PCR technique combined to more classical cloning systems, an operon containing three open reading frames (ORF), each of which could encode a lipase protein of 264, 264 or 269 amino acids (aa), was identified from Mycoplasma mycoides subsp . mycoides LC . Analysis of aa sequences of the encoded polypeptides showed that they display high aa similarity between each other (65-79%) and 28-31% identity to other prokaryotic lipases . Moreover, a lipase-esterase activity could be detected when the mycoplasmal lipase-encoding genes were expressed in a strong opal-suppressor-bearing Escherichia coli strain. Science, 1995 May 26, 268(5214), 1179 - 83 Inducible gene expression in trypanosomes mediated by a prokaryotic repressor; Wirtz E et al.; An inducible expression system was developed for the protozoan parasite Trypanosoma brucei . Transgenic trypanosomes expressing the tetracycline repressor of Escherichia coli exhibited inducer (tetracycline)-dependent expression of chromosomally integrated reporter genes under the control of a procyclic acidic repetitive protein (PARP) promoter bearing a tet operator . Reporter expression could be controlled over a range of four orders of magnitude in response to tetracycline concentration, a degree of regulation that exceeds those exhibited by other eukaryotic repression-based systems . The tet repressor-controlled PARP promoter should be a valuable tool for the study of trypanosome biochemistry, pathogenicity, and cell and molecular biology. J Biol Chem, 1995 May 26, 270(21), 12926 - 32 Cloning and expression analysis of a novel human serine hydrolase with sequence similarity to prokaryotic enzymes involved in the degradation of aromatic compounds; Puente XS et al.; A full-length cDNA coding for a novel human serine hydrolase has been cloned from a breast carcinoma cDNA library . Nucleotide sequence analysis has shown that the isolated cDNA contains an open reading frame coding for a polypeptide of 274 amino acids and a complete Alu repetitive sequence within its 3'-untranslated region . The predicted amino acid sequence contains the Gly-X-Ser-X-Gly motif characteristic of serine hydrolases and displays extensive similarity to several prokaryotic hydrolases involved in the degradation of aromatic compounds . The highest degree of identities was detected with four serine hydrolases encoded by the bphD genes of different strains of Pseudomonas with the ability to degrade biphenyl derivatives . On the basis of these sequence similarities, this novel human enzyme has been tentatively called Biphenyl hydrolase-related protein (Bph-rp) . The Bph-rp cDNA was expressed in Escherichia coli, and after purification, the recombinant protein was able to degrade p-nitrophenylbutyrate, a water-soluble substrate commonly used for assaying serine hydrolases . This hydrolytic activity was abolished by diisopropyl fluorophosphate, a covalent inhibitor of serine hydrolases, providing additional evidence that the isolated cDNA encodes a member of this protein superfamily . Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues revealed that Bph-rp is mainly expressed in liver and kidney, which was also confirmed at the protein level by Western blot analysis with antibodies raised against purified recombinant Bph-rp . According to structural characteristics, hydrolytic activity and tissue distribution of Bph-rp, a potential role of this enzyme in detoxification processes is proposed. Gene, 1995 May 19, 157(1-2), 125 - 6 Studies on the function of conserved sequence motifs in the T4 Dam-{N6-adenine} and EcoRII {C5-cytosine} DNA methyltransferases; Kossykh VG et al.; We used site-directed oligodeoxyribonucleotide-mediated mutagenesis and kinetic studies with purified wild-type (wt) and mutant proteins to evaluate the role of the conserved sequence motifs in two prokaryotic DNA MTases . We suggest that: (i) the main role of Pro in the M.EcoRII PC-motif is to restrict the conformational freedom of Cys and orient it in a manner essential for catalysis; (ii) in both M.EcoRII and T4 Dam the FXGXG-motif positions AdoMet with respect to the catalytic site; (iii) the DPPY-motif in T4 Dam (region IV) is important for AdoMet-binding and may be part of the binding site; and (iv) the RXNXKXXFXXPFK-motif in T4 Dam (region III) is part of the DNA binding/recognition domain. Structure, 1995 May 15, 3(5), 491 - 502 Crystal structure of catalase HPII from Escherichia coli; Bravo J et al.; BACKGROUND: Catalase is a ubiquitous enzyme present in both the prokaryotic and eukaryotic cells of aerobic organisms . It serves, in part, to protect the cell from the toxic effects of small peroxides . Escherichia coli produces two catalases, HPI and HPII, that are quite distinct from other catalases in physical structure and catalytic properties . HPII, studied in this work, is encoded by the katE gene, and has been characterized as an oligomeric, monofunctional catalase containing one cis-heme d prosthetic group per subunit of 753 residues . RESULTS: The crystal structure of catalase HPII from E . coli has been determined to 2.8 A resolution . The asymmetric unit of the crystal contains a whole molecule, which is a tetramer with accurate 222 point group symmetry . In the model built, that includes residues 27-753 and one heme group per monomer, strict non-crystallographic symmetry has been maintained . The crystallographic agreement R-factor is 20.1% for 58,477 reflections in the resolution shell 8.0-2.8 A . CONCLUSIONS: Despite differences in size and chemical properties, which were suggestive of a unique catalase, the deduced structure of HPII is related to the structure of catalase from Penicillium vitale, whose sequence is not yet known . In particular, both molecules have an additional C-terminal domain that is absent in the bovine catalase . This extra domain contains a Rossmann fold but no bound nucleotides have been detected, and its physiological role is unknown . In HPII, the heme group is modified to a heme d and inverted with respect to the orientation determined in all previously reported heme catalases . HPII is the largest catalase for which the structure has been determined to almost atomic resolution. Nucleic Acids Res, 1995 May 11, 23(9), 1487 - 94 Prokaryotic ribosomes recode the HIV-1 gag-pol-1 frameshift sequence by an E/P site post-translocation simultaneous slippage mechanism; Horsfield JA et al.; The mechanism favoured for -1 frameshifting at typical retroviral sites is a pre-translocation simultaneous slippage model . An alternative post-translocation mechanism would also generate the same protein sequence across the frameshift site and therefore in this study the strategic placement of a stop codon has been used to distinguish between the two mechanisms . A 26 base pair frameshift sequence from the HIV-1 gag-pol overlap has been modified to include a stop codon immediately 3' to the heptanucleotide frameshift signal, where it often occurs naturally in retroviral recoding sites . Stop codons at the 3'-end of the heptanucleotide sequence decreased the frame-shifting efficiency on prokaryote ribosomes and the recording event was further depressed when the levels of the release factors in vivo were increased . In the presence of elevated levels of a defective release factor 2, frameshifting efficiency in vivo was increased in the constructs containing the stop codons recognized specifically by that release factor . These results are consistent with the last six nucleotides of the heptanucleotide slippery sequence occupying the ribosomal E and P sites, rather than the P and A sites, with the next codon occupying the A site and therefore with a post-translocation rather than a pre-translocation -1 slippage model. Virology, 1995 May 10, 209(1), 122 - 35 Sequence motifs in the replicator protein of parvovirus MVM essential for nicking and covalent attachment to the viral origin: identification of the linking tyrosine; Nuesch JP et al.; Parvoviral DNA replication has many features in common with prokaryotic rolling circle replication (RCR), including the pivotal role of an initiator protein which introduces a site-specific, single strand nick into a duplex origin sequence . In this process, the protein becomes covalently attached to the new 5' end of the DNA, while making available a 3' hydroxyl to prime de novo synthesis . Sequence comparisons of prokaryotic RCR initiators has revealed a set of three common motifs, two of which, a putative metal coordination site and a downstream active-site tyrosine motif, could be tentatively identified in parvoviral replicator proteins . We have introduced mutations into the NS1 gene of the murine parvovirus minute virus of mice (MVM), in the putative metal coordination site at H129, and into the three candidate tyrosine motifs at Y188, Y197, and Y210 . Histidine-tagged mutant proteins were expressed in HeLa cells from recombinant vaccinia virus vectors and partially purified . None of the mutant proteins were able to initiate replication of origin-containing plasmids in vitro, and each showed impaired site-specific binding to the viral origin, with Y188 and Y197 being most severely defective . If this deficiency was minimized using low salt conditions, however, Y188 and Y197 mutant proteins were able to nick and become covalently attached to origin DNA, whereas Y210 and H129 mutant proteins were not, suggesting that the latter residues are part of the catalytic site of the NS1 nickase . Transfer of {32P}phosphate from substrate DNA to NS1, followed by cyanogen bromide cleavage of the complex, gave the single, labeled peptide consistent with Y210 being the linking tyrosine. Biochemistry, 1995 May 9, 34(18), 6183 - 7 Prokaryotic translation initiation factor IF3 is an elongated protein consisting of two crystallizable domains; Kycia JH et al.; We show that translation initiation factor IF3 can be split into two fragments of nearly equal size by the Escherichia coli outer membrane protease omptin . Circular dichroism and small-angle neutron scattering show that the two fragments are structured as domains . Each domain is relatively compact, and they are separated by about 45 A in intact IF3 . Thus IF3 is an elongated protein that consists of two well-separated domains . We suggest that these two domains are involved in ribosome binding across the cleft of the 30S ribosome . We also report the crystallization of each domain of IF3. J Biol Chem, 1995 May 5, 270(18), 10571 - 5 Reversal by GroES of the GroEL preference from hydrophobic amino acids toward hydrophilic amino acids; de Crouy-Chanel A et al.; The chaperones GroEL/hsp60 are present in all prokaryotes and in mitochondria and chloroplasts of eukaryotic cells . They are involved in protein folding, protein targeting to membranes, protein renaturation, and control of protein-protein interactions . They interact with many polypeptides in an ATP-dependent manner and possess a peptide-dependent ATPase activity . GroEL/hsp60 cooperates with GroES/hsp10, and the productive folding of proteins by GroEL generally requires GroES, which appears to regulate the binding and release of substrate proteins by GroEL . In a recent study, we have shown that GroEL interacts preferentially with the side chains of hydrophobic amino acids (Ile, Phe, Val, Leu, and Trp) and more weakly with several polar or charged amino acids, including the strongest alpha-helix and beta-sheet formers (Glu, Gln, His, Thr, and Tyr) . In this study, we show that GroES reduces the specificity of GroEL for hydrophobic amino acids and increases its specificity for hydrophilic ones . This shift by GroES of the GroEL specificity from hydrophobic amino acids toward hydrophilic ones might be of importance for its function in protein folding. J Biol Chem, 1995 May 5, 270(18), 10412 - 9 The dissociation of ATP from hsp70 of Saccharomyces cerevisiae is stimulated by both Ydj1p and peptide substrates; Ziegelhoffer T et al.; hsp70 proteins of both eukaryotes and prokaryotes possess both ATPase and peptide binding activities . These two activities are crucial for the chaperone activity of hsp70 proteins . The activity of DnaK, the primary hsp70 of Escherichia coli, is modulated by the GrpE and DnaJ proteins . In the yeast Saccharomyces cerevisiae, the predominant cytosolic hsp70, Ssa1p, interacts with a DnaJ homologue, Ydj1p . In order to better understand the function of the Ssa1p/Ydj1p chaperone, the effects of polypeptide substrates and Ydj1p on Ssa1p ATPase activity were assessed using a combination of steady-state kinetic analysis and single turnover substrate hydrolysis experiments . Polypeptide substrates and Ydj1p both serve to stimulate ATPase activity of Ssa1p . The two types of effector are biochemically distinct, each conferring a characteristic K+ dependence on Ssa1p ATPase activity . However, in single turnover ATP hydrolysis experiments, both polypeptide substrates and Ydj1p destabilized the ATP.Ssa1p complex through a combination of accelerated hydrolysis of bound ATP and accelerated release of ATP from Ssa1p . The acceleration of ATP release by Ydj1p is a previously unidentified function of a DnaJ homologue . In the case of Ydj1p-stimulated Ssa1p, steady-state ATPase activity is increased less than 2-fold at physiological K+ concentrations, despite a 15-fold increase in the hydrolysis of bound ATP . The primary effect of Ydj1p appears to be to disfavor an ATP form of Ssa1p . On the other hand, peptide stimulation of Ssa1p ATPase activity was enhanced at physiological K+ concentrations, supporting the idea that cycles of ATP hydrolysis play an important role in the interaction of hsp70 with polypeptide substrates . The enhanced ATP dissociation caused by both polypeptide substrates and Ydj1p may play a role in the regulation of Ssa1p chaperone activity by altering the relative abundance of ATP-and ADP-bound forms. Science, 1995 May 5, 268(5211), 667 - 75 The ethylene signal transduction pathway in plants; Ecker JR; Ethylene (C2H4), the chemically simplest plant hormone, is among the best-characterized plant growth regulators . It participates in a variety of stress responses and developmental processes . Genetic studies in Arabidopsis have defined a number of genes in the ethylene signal transduction pathway . Isolation of two of these genes has revealed that plants sense this gas through a combination of proteins that resemble both prokaryotic and eukaryotic signaling proteins . Ethylene signaling components are likely conserved for responses as diverse as cell elongation, cell fate patterning in the root epidermis, and fruit ripening . Genetic manipulation of these genes will provide agriculture with new tools to prevent or modify ethylene responses in a variety of plants. Biochem Cell Biol, 1995 May-Jun, 73(5-6), 223 - 34 Membrane permeation and intracellular trafficking of long chain fatty acids: insights from Escherichia coli and 3T3-L1 adipocytes; Mangroo D et al.; Long chain fatty acids are important substrates for energy production and lipid synthesis in prokaryotes and eukaryotes . Their cellular uptake represents an important first step leading to metabolism . This step is induced in Escherichia coli by growth in medium containing long chain fatty acids and in murine 3T3-L1 cells during differentiation to adipocytes . Consequently, these have been used extensively as model systems to study the cellular uptake of long chain fatty acids . Here, we present an overview of our current understanding of long chain fatty acid uptake in these cells . It consists of several distinct steps, mediated by a combination of biochemical and physico-chemical processes, and is driven by conversion of long chain fatty acids to acyl-CoA by acyl-CoA synthetase . An understanding of long chain fatty acid uptake may provide valuable insights into the roles of fatty acids in the regulation of cell signalling cascades, in the regulation of a variety of metabolic and transport processes, and in a variety of mammalian pathogenic conditions such as obesity and diabetes. J Bacteriol, 1995 May, 177(9), 2490 - 6 Active contribution of two domains to cooperative DNA binding of the enhancer-binding protein nitrogen regulator I (NtrC) of Escherichia coli: stimulation by phosphorylation and the binding of ATP; Chen P et al.; Activation by the prokaryotic activator nitrogen regulator I (NRI, or NtrC) of Escherichia coli requires an interaction between two NRI dimers . ATP-dependent phosphorylation stimulates this tetramerization, which can be detected as cooperative binding to DNA . A polypeptide containing only the DNA-binding carboxyl-terminal domain has been previously shown to bind noncooperatively to DNA . Our primary purpose was to determine whether the highly conserved N-terminal domain or the ATP-binding central domain is required for cooperative DNA binding . Because ATP was present in the experiments that showed that phosphorylation enhances cooperative bindings, it is possible that ATP and not phosphorylation stimulated cooperative binding . Our secondary purpose was to separately assess the effects of ATP and phosphorylation on cooperative binding . We showed that a variant with a deletion of the central domain, NRI-(delta 143-398), binds cooperatively as well as unphosphorylated wild-type NRI, implying that the N-terminal domain mediates phosphorylation-independent cooperative binding . Phosphorylation of NRI-(delta 143-398) did not further stimulate this binding, suggesting that the ATP-binding central domain may be required for the phosphorylation-dependent enhancement . Cooperative binding was enhanced by either acetyl-phosphate-dependent (i.e., ATP-independent) phosphorylation of NRI or the specific binding of ATP to the central domain . Their effects were not additive, a finding which is consistent with the interpretation that each promotes a similar dimer-dimer interaction . We discuss these results within the context of the hypothesis that the highly conserved N-terminal domain mediates phosphorylation-independent cooperativity and the central domain is required for cooperativity stimulated by ATP binding or phosphorylation. J Bacteriol, 1995 May, 177(9), 2387 - 95 Envelope structure of four gliding filamentous cyanobacteria; Hoiczyk E et al.; The cell walls of four gliding filamentous Oscillatoriaceae species comprising three different genera were studied by freeze substitution, freeze fracturing, and negative staining . In all species, the multilayered gram-negative cell wall is covered with a complex external double layer . The first layer is a tetragonal crystalline S-layer anchored on the outer membrane . The second array is formed by parallel, helically arranged surface fibrils with diameters of 8 to 12 nm . These fibrils have a serrated appearance in cross sections . In all cases, the orientation of the surface fibrils correlates with the sense of revolution of the filaments during gliding, i.e., clockwise in both Phormidium strains and counterclockwise in Oscillatoria princeps and Lyngbya aeruginosa . The lack of longitudinal corrugations or contractions of the surface fibrils and the identical appearances of motile and nonmotile filaments suggest that this structure plays a passive screw thread role in gliding . It is hypothesized that the necessary propulsive force is generated by shear forces between the surface fibrils and the continuing flow of secreted extracellular slime . Furthermore, the so-called junctional pores seem to be the extrusion sites of the slime . In motile cells, these pores exhibit a different staining behavior than that seen in nonmotile ones . In the former, the channels of the pores are filled with electron-dense material, whereas in the latter, the channels appear comparatively empty, highly contrasting the peptidoglycan . Finally, the presence of regular surface structures in other gliding prokaryotes is considered an indication that comparable structures are general features of the cell walls of gliding microbes. Infect Immun, 1995 May, 63(5), 1767 - 76 A plasmid-encoded regulatory region activates chromosomal eaeA expression in enteropathogenic Escherichia coli; Gomez-Duarte OG et al.; Enteropathogenic Escherichia coli (EPEC) organisms produce a characteristic histopathology in intestinal epithelial cells called attaching and effacing lesions . The eaeA gene is associated with attaching and effacing lesions and encodes intimin, a 94-kDa outer membrane protein . A 60-MDa plasmid, pMAR2, is essential for full virulence of EPEC strain E2348/69 (O127:H6) . We have cloned sequences from pMAR2 that increase expression of the chromosomal eaeA gene as shown by increased alkaline phosphatase activity of an eaeA::TnphoA gene fusion, increased expression of the intimin protein, and increased production of eaeA mRNA . These sequences are called per for plasmid-encoded regulator . pMAR2-cured JPN15 containing cloned per sequences adheres to HEp-2 cells in greater numbers than JPN15 carrying the plasmid vector only . The cloned per sequences contain four open reading frames (ORFs) which have been designated perA through perD . Only perC can by itself activate expression of eaeA::TnphoA, although the levels of alkaline phosphatase activity seen with this ORF alone are considerably lower than those seen when all four ORFs are present . The molecular sizes of polypeptides predicted from perA, perB, perC, and perD ORFs are 24, 14.8, 10.5, and 9.4 kDa, respectively . The PerA predicted protein shares homology with members of the AraC family of bacterial regulators, but PerB, PerC, and PerD have no striking homology with previously described prokaryotic proteins . Our studies indicate that plasmid-encoded factors regulate the expression of eaeA and possibly genes encoding other outer membrane proteins and may be important for virulence of EPEC. Endocrinology, 1995 May, 136(5), 2074 - 81 Early detection of secretogranin-II (SgII) in the human fetal pituitary: immunocytochemical study using an antiserum raised against a human recombinant SgII; Vallet VS et al.; Secretogranin-II (SgII) is a protein contained within secretory granules of mainly gonadotrophs . The purpose of this study was to determine whether SgII immunoreactivity (SgII-IR) in the human fetal pituitary was temporally related to gonadotropin immunoreactivity . A specific antihuman SgII antiserum was thus required . A complementary DNA clone with an open reading frame for human (h) SgII was synthesized by reverse transcription-polymerase chain reaction from pituitary total RNA . This clone was used to obtain the SgII polypeptide (-9 to 152) as a fusion protein, in a heterologous expression prokaryotic system . Antisera against the fusion protein were raised in rabbits and checked for specificity and sensitivity through Western blotting . Human fetal pituitaries from week 6 of gestation onward were used for immunocytochemical studies . Consecutive semithin sections were treated with the specific antisera against hSgII, beta-endorphin, and hPRL and with monoclonal antibodies to hCG alpha, hLH, and hFSH . SgII immunoreactivity appeared at week 8 and was restricted to pituitary cells expressing beta-endorphin (100% colocalization) . At week 9, FSH-positive cells did not contain SgII . From week 10, gonadotrophs progressively exhibited SgII-IR, up to 50% of that in FSH-containing cells at week 26 . The granin was never found in PRL cells whatever the stage of development . The present data demonstrate that SgII-IR is detected very early in fetal life; however, the positive cells are not gonadotrophs, but corticotrophs . Within gonadotrophs, SgII appears subsequent to hormones . At birth, more than 90% of SgII-IR cells are represented by corticotrophs and gonadotrophs. Genetics, 1995 May, 140(1), 325 - 43 Intraspecific and interspecific variation in 5S RNA genes are decoupled in diploid wheat relatives; Kellogg EA et al.; 5S RNAs form part of the ribosome in most organisms . In some, e.g., prokaryotes and some fungi, the genes are part of the ribosomal operon, but in most eukaryotes they are in tandem arrays of hundreds to thousands of copies separate from the main ribosomal array . 5S RNA genes can be aligned across kingdoms . We were therefore surprised to find that, for 28 diploid species of the wheat tribe (Triticeae), nucleotide diversity within an array is up to 6.2% in the genes, not significantly different from that of the nontranscribed spacers . Rates of concerted evolution must therefore be insufficient to homogenize the entire array . Between species, there are significantly fewer fixed differences in the gene than would be expected, given the high within-species variation . In contrast, the amount of variation between species in the spacer is the same as or greater than that within individuals . This leads to a paradox . High variation within an individual suggests that there is little selection on any particular gene within an array . But conservation of the gene across species implies that polymorphisms are periodically eliminated at a rate approximately equal to or greater than that of speciation . Levels of intraspecific polymorphism and interspecific divergence are thus decoupled . This implies that selective mechanisms exist to eliminate mutations in the gene without also affecting the spacer. Biotechniques, 1995 May, 18(5), 878 - 85 Cloning of ligand-binding domains of bacterial receptors by phage display; Jacobsson K et al.; We present an application of the phage display technique which makes it possible, through affinity selection, to clone the part of a prokaryotic receptor gene that encodes the ligand-binding domain . A phage display library was constructed by insertion of randomly fragmented chromosomal DNA from Staphylococcus aureus strain 8325-4 into the phagemid vector pHEN1 . Domains of the genes encoding staphylococcal protein A and fibronectin binding proteins were isolated from the library by affinity panning of the phage against the immobilized ligands . Approximately 1%-10% of the eluted phage encoded polypeptides that specifically bound the respective ligand . Nucleotide sequences of the isolated clones were in agreement with earlier known sequences of domains encoding the IgG and fibronectin-binding proteins . In addition, a second, so far unknown, nucleotide sequence encoding an IgG-binding polypeptide was identified. J Clin Microbiol, 1995 May, 33(5), 1308 - 13 PCR-based study of conserved and variable DNA sequences of Tritrichomonas foetus isolates from Saskatchewan, Canada; Riley DE et al.; The protozoan parasite Tritrichomonas foetus causes infertility and spontaneous abortion in cattle . In Saskatchewan, Canada, the culture prevalence of trichomonads was 65 of 1,048 (6%) among 1,048 bulls tested within a 1-year period ending in April 1994 . Saskatchewan was previously thought to be free of the parasite . To confirm the culture results, possible T . foetus DNA presence was determined by the PCR . All of the 16 culture-positive isolates tested were PCR positive by a single-band test, but one PCR product was weak . DNA fingerprinting by both T17 PCR and randomly amplified polymorphic DNA PCR revealed genetic variation or polymorphism among the T . foetus isolates . T17 PCR also revealed conserved loci that distinguished these T . foetus isolates from Trichomonas vaginalis, from a variety of other protozoa, and from prokaryotes . TCO-1 PCR, a PCR test designed to sample DNA sequence homologous to the 5' flank of a highly conserved cell division control gene, detected genetic polymorphism at low stringency and a conserved, single locus at higher stringency . These findings suggested that T . foetus isolates exhibit both conserved genetic loci and polymorphic loci detectable by independent PCR methods . Both conserved and polymorphic genetic loci may prove useful for improved clinical diagnosis of T . foetus . The polymorphic loci detected by PCR suggested either a long history of infection or multiple lines of T . foetus infection in Saskatchewan . Polymorphic loci detected by PCR may provide data for epidemiologic studies of T . foetus. Z Naturforsch {C}, 1995 May-Jun, 50(5-6), 380 - 90 The role of phosphatidylglycerol as a functional effector and membrane anchor of the D1-core peptide from photosystem II-particles of the cyanobacterium Oscillatoria chalybea; Kruse O et al.; The intrinsic polypeptide D1, isolated from photosystem (PS) II-particles of the cyanobacterium Oscillatoria chalybea, was obtained by electroelution and fractionated extraction with organic solvents . Purification was demonstrated by Western blotting and amino acid sequencing . By carrying out D1-immunization in rabbits a polyclonal monospecific D1-antiserum was obtained . For the qualitative characterization of D1 as a lipid-binding peptide, the effect of the lipids phosphatidylglycerol (PG), monogalactosyldiacylglyceride (MGDG) and phosphatidylcholine (PC) on PSII-oxygen evolution was analysed in reconstitution experiments . In these experiments purified photosystem II (PSII)-particle preparations were treated with the enzyme phospholipase A2 and supplemented with lipid emulsions . We were able to show that the inhibition of electron transport, as the consequence of this lipase treatment, was only relieved, if phosphatidylglycerol was added to the preparation . A model was proposed, in which phosphatidylglycerol is a functional effector for the optimal conformation of D1 in the PSII core complex . Phosphatidylglycerol molecules are unusually tightly bound to the D1 peptide by hydrophobic interactions . A covalent binding seems not probable . The localisation of phosphatidylglycerol binding sites was found by trypsin treatment of D1 and analysis of the obtained oligopeptides with HPLC and immunoblotting . The binding sites could be confined to the hydrophobic amino acid section between arginine 27 and arginine 225, which is known to be the membrane anchor of D1 . This has led us to the conclusion that the phospholipid phosphatidylglycerol plays an important role for anchoring the D1-peptide and for its orientation in the thylakoid membrane . Phosphatidylglycerol with its high amount of palmitic acid has in prokaryotic cyanobacteria apparently a role in stabilization and orientation . The high turn-over of D1 and the spatial separation of the synthesis- and incorporation-site in the membrane, developed during evolution in eukaryotic organisms, might have changed the requirement on the mobility and the orientation of D1 in photosynthetic membranes. Biochem Mol Biol Int, 1995 May, 35(6), 1223 - 31 beta-Glucosidase families revealed by computer analysis of protein sequences; Rojas A et al.; This computational study is a summary of how cloned beta-glucosidase subfamilies are organized . Computations were carried out using General Computer Group, Inc . (GCG) package programs . Twenty-two beta-glucosidases belonging to either cellulolytic or non-cellulolytic organisms were identified . The multialignment of a whole beta-glucosidase family is shown . Two sub-families, A and B, were clearly seen to exist . Sub-family A is further subdivided into sub-families A1 and A2 . A1 includes vegetal beta-glucosidases and A2 includes prokaryotic enzymes . Sub-family B has three new sub-families, B1, B2, and B3 . The enzymes in B2 are of yeast and/or fungi . Aspartic (D), glutamic (E) and histidine (H) residues, which are thought to be a part of the mechanism of the enzymatic hydrolysis are conserved . The well conserved amino acid sequences of the sub-family A are ITENGA; QUIEGA; HVD; and NEP . The well conserved amino acid sequences of the sub-family B are: SDW; and YN(R,K)(V,L)N. Mol Microbiol, 1995 May, 16(4), 719 - 31 A family of modular type mannuronan C-5-epimerase genes controls alginate structure in Azotobacter vinelandii; Ertesvag H et al.; The L-guluronic acid residues in the Azotobacter vinelandii polysaccharide alginate originate from a post-polymerization reaction catalysed by the enzyme mannuronan C-5-epimerase (ME) . We have previously reported the cloning and expression of an A . vinelandii gene encoding this enzyme, and we show here that the organism encodes at least four other ME genes originating from a common ancestor gene by a complex rearrangement process . The biological function of the corresponding enzymes is probably to catalyse the formation of alginates with a variety of physical properties . This model may explain the origin of the structural variability found in alginates isolated both from prokaryotic and eukaryotic organisms . The A . vinelandii enzymes may also potentially be useful for certain medical and biotechnological applications of this commercially important polysaccharide. Mol Microbiol, 1995 May, 16(4), 699 - 708 Mutational analysis of the putative nucleic acid-binding surface of the cold-shock domain, CspB, revealed an essential role of aromatic and basic residues in binding of single-stranded DNA containing the Y-box motif; Schroder K et al.; The major cold-shock protein of Bacillus subtilis, CspB, is a member of a protein family widespread among prokaryotes and eukaryotes that share the highly conserved cold-shock domain (CSD) . The CSD domain is involved in transcriptional and translational regulation and was shown to bind the Y-box motif, a cis-element that contains the core sequence ATTGG, with high affinity . The three-dimensional structure of CspB, a prototype of this protein family, revealed that this hydrophilic CSD domain creates a surface rich in aromatic and basic amino acids that may act as the nucleic acid-binding site . We have analysed the potential role of conserved aromatic and basic residues in nucleic acid binding by site-directed mutagenesis . In gel retardation and ultraviolet cross-linking experiments, the ability of CspB mutants to bind single-stranded oligonucleotides (ssDNA) that contain the Y-box motif was investigated . Single substitutions of three highly conserved phenylalanine residues (Phe-15, Phe-17, Phe-27) by alanine and substitution of one histidine (His-29) by glutamine, all located within the putative RNA-binding sites RNP-1 and RNP-2, abolished the nucleic acid-binding activity of CspB . Conservative substitutions of Phe-15 to tyrosine (F15Y) showed a small increase in binding affinity, whereas separate replacement of Phe-17 and Phe-27 by tyrosine caused a reduction in binding activity . These and other substitutions including the conserved basic residues Lys-7, Lys-13 and Arg-56 as well as the aromatic residues Trp-8 and Phe-30 strongly suggest that CspB uses the side-chains of these amino acids for specific interaction with nucleic acids . Ultraviolet cross-linking experiments for CspB mutants with ssDNA supported the idea of specific CspB/nucleic acid interaction and indicated an essential role for the aromatic and basic residues in this binding . In addition, two-dimensional nuclear magnetic resonance studies with F17A, K13Q, F15Y and F27Y revealed that the mutants have the same overall structure as the wild-type CspB protein. Mol Microbiol, 1995 May, 16(4), 625 - 34 Tracheal colonization factor: a Bordetella pertussis secreted virulence determinant; Finn TM et al.; We report here the identification of a virulence-associated factor, Tcf, (tracheal colonization factor), produced by strains of Bordetella pertussis but not Bordetella parapertussis or Bordetella bronchiseptica . This protein is encoded by the tcfA gene . When a strain of B . pertussis 18323 lacking this protein is used to infect mice with an aerosol challenge, the number of bacteria isolated from the tracheas is decreased 10-fold when compared with the parent 18323 . The derived amino acid sequence of tcfA predicts a 68 kDa RGD-containing, proline-rich protein, which after cleavage of a typical prokaryotic signal sequence would be 64 kDa . Amino acid sequence analysis demonstrates that the C-terminal 30 kDa of this protein shows 50% identity to the 30 kDa C-terminus of another Bordetella protein, the pertactin precursor . The N-terminal 34 kDa region contains the three amino-acid motif RGD and is 16.5% proline . Coupled in vitro transcription and translation analysis indicates that the tcfA gene product migrates as two bands of approximately 90 kDa . A fusion protein of the N-terminal, 34 kDa portion of Tcf to maltose-binding protein migrates, on SDS-PAGE, 30 kDa higher than expected from the combined molecular weights . Polyclonal antisera raised against the unique N-terminal portion of Tcf recognizes 90 kDa and 60 kDa bands in immunoblots of whole-cell lysates of strains of B . pertussis; it does not recognize any protein in whole-cell lysates of B . bronchiseptica or B . parapertussis . Supernatants of cultures of B . pertussis 18323 contain the 60 kDa form of the protein . Southern blot analysis of chromosomal DNA from strains of B . bronchiseptica and B . parapertussis, using a probe derived from tcfA, shows strong hybridization only to B . pertussis DNA . Thus, Tcf appears to be a unique virulence factor of B . pertussis. J Chromatogr A, 1995 Apr 28, 698(1-2), 203 - 24 Protein mutations revealed by two-dimensional electrophoresis; Cash P; High-resolution two-dimensional electrophoresis (2DE) can resolve many hundreds of proteins present in complex mixtures depending on the method of detection . These proteins can be characterised qualitatively, with respect to their electrophoretic mobilities (i.e . charge and apparent molecular mass) and quantitatively, using densitometry, to determine their amounts . There has been a widespread application of 2DE in the analysis and characterisation of protein mutations for a range of organisms . This review presents examples of the use of 2DE to study naturally occurring protein mutations and polymorphisms as well as the characterisation of induced protein mutations in prokaryotes and eukaryotes . Examples are presented to illustrate the use of 2DE to detect mutations affecting the electrophoretic mobility and biosynthesis of individual proteins as well as mutations leading to global alterations in cellular protein synthesis . The advantages and disadvantages of 2DE in the detection of protein mutations are discussed. Chem Biol Interact, 1995 Apr 28, 96(1), 17 - 32 The molecular biology of the flavin-containing monooxygenases of man; Phillips IR et al.; cDNA clones encoding five distinct members of the FMO family of man (FMOs 1, 2, 3, 4 and 5) were isolated by a combination of library screening and reverse transcription-polymerase chain reaction techniques . The deduced amino acid sequences of the human FMOs have 82-87% identity with their known orthologues in other mammal but only 51-57% similarity to each other . The hydropathy profiles of the proteins are very similar . From the calculated rate of evolution of FMOs (a 1% change in sequence per 6 million years) it would appear that individual members of the FMO gene family arose by duplication of a common ancestral gene some 250-300 million years ago . Each of the FMO genes was mapped by the polymerase chain reaction to the long arm of human chromosome 1 . The localization of the FMO1 gene was further refined to 1q23-q25 by in situ hybridization of human metaphase chromosomes . RNase protection assays demonstrated that in man each FMO gene displays a distinct developmental and tissue-specific pattern of expression . In the adult, FMO1 is expressed in kidney but not in liver, whereas in the foetus its mRNA is abundant in both organs . FMO3 expression is essentially restricted to the liver in the adult and the mRNA is either absent, or present in low amounts, in foetal tissues . FMO4 is expressed more constitutively . Human FMO1 and FMO3 cDNAs were functionally expressed in prokaryotic and eukaryotic cells . FMO1 and FMO3, expressed in either system, displayed product stereoselectivity in their catalysis of the N-oxidation of the pro-chiral tertiary amines, N-ethyl-N-methylaniline (EMA) and pargyline . Both enzymes were stereoselective with respect to the production of the (-)-S-enantiomer of EMA N-oxide . But in the case of pargyline, the enzymes displayed opposite stereoselectivity, FMO1 producing solely the (+)-enantiomer and FMO3 predominantly the (-)-enantiomer of the N-oxide. Nucleic Acids Res, 1995 Apr 25, 23(8), 1398 - 405 In vitro replication by prokaryotic and eukaryotic polymerases on DNA templates containing site-specific and stereospecific benzo{a}pyrene-7,8-dihydrodiol-9,10-epoxide adducts; Chary P et al.; DNA adducts of the environmental carcinogen benzo{a}pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) interact stereospecifically with prokaryotic and eukaryotic polymerases in vitro . Toward understanding the capacity to replicate past different diastereomers of BPDE at specific sites in DNA, six deoxyoligonucleotides, each 33 bases long, were constructed with stereochemically defined BPDE adducts on adenine N6 at position two of the human N-ras codon 61 . Four polymerases that were studied under single encounters with the template-primer complex terminated synthesis one base 3' to the lesion with all the adducted templates . When multiple encounters between polymerase and substrate were permitted, each of the polymerases analyzed revealed a unique pattern for a given adducted template . The general replication pattern was encompassed under two categories, reflecting the significance of the R and S configurations of C10 of the pyrenyl ring attached to the single-stranded DNA template . Furthermore, within each of these categories, every polymerase demonstrated distinct quantitative differences in product accumulation at a given site, for the various adducted templates . Among the polymerases utilized in this study, exonuclease-deficient Klenow fragment of polymerase I (exo- KF) exhibited the most efficient translesion synthesis resulting in approximately 16% full-length products with the modified templates bearing adducts with C10-S configuration . In contrast, chain elongation with bacteriophage T4 DNA polymerase bearing an active 3'-->5' exonucleolytic activity was most strongly inhibited by all six BPDE-adducted templates . Misincorporation of A opposite the adduct occurred in all the templates when polymerized with Sequenase, whereas exo- KF preferentially incorporated C opposite the C10-R BPDE adducts and A opposite the C10-S BPDE adducts. Arch Biochem Biophys, 1995 Apr 20, 318(2), 430 - 8 Active recombinant human cytosolic phospholipase A2 is expressed in Escherichia coli; Witmer MR et al.; The cDNA encoding human cytosolic phospholipase A2 (cPLA2) has been subcloned into a prokaryotic pET16b expression vector which also encodes an amino-terminal deca-histidine affinity tag to facilitate purification of the recombinant enzyme . Soluble, active fusion protein, designated His-cPLA2, has been obtained reproducibly from this expression system using the E . coli strain BL21 (DE3) . The protein has been purified to homogeneity in four steps and the mass confirmed by electrospray mass spectrometry . His-cPLA2 was characterized by kinetic analysis which demonstrated that the enzyme is similar to native cPLA2 in all respects investigated . Specifically, the enzyme binds to anionic vesicles containing substrate, and acts processively on these vesicles . Enzymatic activity is supported by the presence of Ca2+ and several other divalent metal ions, and is inhibited by several transition metal ions . Finally, the enzyme demonstrates lysophospholipase activity and exhibits a high selectivity for sn-2 arachidonyl esters . This prokaryotic expression system yields moderate amounts of unmodified recombinant His-cPLA2 and is advantageous for rapid production of protein and mutational analyses. Biochem Biophys Res Commun, 1995 Apr 17, 209(2), 417 - 25 Role of lipid peroxidation in electroporation-induced cell permeability; Maccarrone M et al.; Erythroleukemia K562 cells and lentil (Lens culinaris) root protoplasts have been subjected to pore-forming electric fields suitable for transfection experiments . Evidence is presented that the amount of hydroperoxides formed in cell membranes of both cell-types is a function of field strength applied . On the other hand, the electroporation-induced lipid peroxidation paralleled the enhancement of membrane permeability and was associated with greater membrane fluidity . The membrane hydroperoxides formed upon electric shock enhanced cell luminescence, and lipoxygenase activity appeared to be involved in the process . Electroporation of prokaryotic cells of Escherichia coli also enhanced light emission, which was higher in lipoxygenase-expressing clones. Gene, 1995 Apr 14, 156(1), 107 - 11 The 16S-23S rRNA intergenic spacer region of Bartonella (Rochalimaea) species is longer than usually described in other bacteria; Roux V et al.; We amplified by polymerase chain reaction (PCR) and sequenced using an automated laser fluorescent DNA sequencer (Pharmacia) the intergenic spacer region (ITS) between the 16S and 23S rRNAs of the four species of Rochalimaea which were recently renamed Bartonella sp . We obtained DNA fragments of 1211, 1262, 1258 and 1529 bp for the reference species of B . quintana, B . henselae, B . vinsonii and B . elizabethae, respectively . The ITS of the four species are longer than previously reported in prokaryotes and contained the genes encoding isoleucine-tRNA (tRNA(Ile)) and alanine-tRNA (tRNA(Ala)) . The sequences of the tRNA(Ala) genes are identical for the four Bartonella species, but the tRNA(Ile) gene sequence of B . quintana presents one mutation in comparison with the other species. J Biol Chem, 1995 Apr 14, 270(15), 8744 - 54 DNA looping by Saccharomyces cerevisiae high mobility group proteins NHP6A/B . Consequences for nucleoprotein complex assembly and chromatin condensation; Paull TT et al.; The formation of higher order protein.DNA structures often requires bending of DNA strands between specific sites, a process that can be facilitated by the action of nonspecific DNA-binding proteins which serve as assembly factors . A model for this activity is the formation of the invertasome, an intermediate structure created in the Hin-mediated site-specific DNA inversion reaction, which is stimulated by the prokaryotic nucleoid-associated protein HU . Previously, we have shown that the mammalian HMG1/2 proteins substitute for HU in this system and display efficient DNA wrapping activity in vitro . In the present work, we isolate the primary sources of assembly factor activity in Saccharomyces cerevisiae, as measured by the ability to stimulate invertasome formation, and show that these are the previously identified NHP6A/B proteins . NHP6A/B have comparable or greater activity in DNA binding, bending, and supercoiling with respect to HU and HMG1 and appear to form more stable protein.DNA complexes . In addition, expression of NHP6A in mutant Escherichia coli cells lacking HU and Fis restores normal morphological appearance to these cells, specifically in nucleoid condensation and segregation . From these data we predict diverse architectural roles for NHP6A/B in manipulating chromosome structure and promoting the assembly of multicomponent protein.DNA complexes. J Biol Chem, 1995 Apr 14, 270(15), 8610 - 22 Genetic and molecular characterization of a gene encoding a wide specificity purine permease of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes; Diallinas G et al.; In Aspergillus nidulans, loss-of-function mutations in the uapA and azgA genes, encoding the major uric acid-xanthine and hypoxanthine-adenine-guanine permeases, respectively, result in impaired utilization of these purines as sole nitrogen sources . The residual growth of the mutant strains is due to the activity of a broad specificity purine permease . We have identified uapC, the gene coding for this third permease through the isolation of both gain-of-function and loss-of-function mutations . Uptake studies with wild-type and mutant strains confirmed the genetic analysis and showed that the UapC protein contributes 30% and 8-10% to uric acid and hypoxanthine transport rates, respectively . The uapC gene was cloned, its expression studied, its sequence and transcript map established, and the sequence of its putative product analyzed . uapC message accumulation is: (i) weakly induced by 2-thiouric acid; (ii) repressed by ammonium; (iii) dependent on functional uaY and areA regulatory gene products (mediating uric acid induction and nitrogen metabolite repression, respectively); (iv) increased by uapC gain-of-function mutations which specifically, but partially, suppress a leucine to valine mutation in the zinc finger of the protein coded by the areA gene . The putative uapC gene product is a highly hydrophobic protein of 580 amino acids (M(r) = 61,251) including 12-14 putative transmembrane segments . The UapC protein is highly similar (58% identity) to the UapA permease and significantly similar (23-34% identity) to a number of bacterial transporters . Comparisons of the sequences and hydropathy profiles of members of this novel family of transporters yield insights into their structure, functionally important residues, and possible evolutionary relationships. J Biol Chem, 1995 Apr 7, 270(14), 8249 - 56 Glycosylation of human truncated Fc epsilon RI alpha chain is necessary for efficient folding in the endoplasmic reticulum; Letourneur O et al.; The high affinity immunoglobulin E (IgE) receptor is an alpha beta gamma 2 tetrameric complex . The truncated extracellular segment (alpha t) of the heavily glycosylated alpha chain is sufficient for high affinity binding of IgE . Here we have expressed various alpha t mutants in eukaryotic and prokaryotic cells to analyze the role of glycosylation in the folding, stability, and secretion of alpha t . All seven N-linked glycosylation sites in alpha t are glycosylated and their mutations have an additive effect on the folding and secretion of alpha t . Mutation of the seven N-glycosylation sites (delta 1-7 alpha t) induces misfolding and retention of alpha t in the endoplasmic reticulum . Similarly, tunicamycin treatment reduces substantially the folding efficiency of wild-type alpha t . In contrast, no difference in folding efficiency is detected between wild-type alpha t and delta 1-7 alpha t expressed in Escherichia coli . In addition, maturation of N-linked oligosaccharides and addition of O-linked carbohydrates are not required for either the transport or the IgE-binding function of alpha t . Furthermore, complete enzymatic deglycosylation does not affect the stability and the IgE-binding capacity of alpha t . Therefore, glycosylation is not intrinsically necessary for proper folding of alpha t but is required for folding in the endoplasmic reticulum . Our data are compatible with the concept that specific interactions between N-linked oligosaccharides and the folding machinery of the endoplasmic reticulum are necessary for efficient folding of alpha t in eukaryotic cells. J Biol Chem, 1995 Apr 7, 270(14), 7876 - 81 Recombinant human eosinophil cationic protein . Ribonuclease activity is not essential for cytotoxicity; Rosenberg HF; Eosinophil cationic protein (ECP) is a toxin secreted by activated human eosinophils that has anti-parasitic, antibacterial, and neurotoxic activities; ECP also has ribonuclease activity and structural homology to other mammalian ribonucleases . To determine the relationship between the ribonuclease activity and cytotoxicity of ECP, a method for producing recombinant ECP (rECP) in a prokaryotic expression system was devised . Periplasmic isolates from induced bacterial transfectants contained enzymatically active rECP; micromolar concentrations of rECP were shown to be toxic for Staphylococcus aureus (strain 502A) . In contrast, recombinant eosinophil-derived neurotoxin, with 67% amino acid sequence identity to ECP, had little to no toxicity for S . aureus; these findings are analogous to those obtained with purified, granule-derived ECP and eosinophil-derived neurotoxin . Two single base pair mutations were introduced into the coding sequence of rECP (Lys38 to Arg and His128 to Asp) to convert ribonuclease active-site residues into non-functional counterparts . These mutations eliminated the ribonuclease activity of rECP but had no discernible effect on the antibacterial activity of this protein, demonstrating that ribonuclease activity and cytotoxicity are, in this case, independent functions of ECP. Biochemistry, 1995 Apr 4, 34(13), 4393 - 401 Molecular basis for prokaryotic specificity of magainin-induced lysis; Tytler EM et al.; Magainins and mastoparans are examples of peptide antibiotics and peptide venoms, respectively . They have been grouped together as class L amphipathic helixes {Segrest, J.P., et al . (1990) Proteins 8, 103-117} because of similarities in the distribution of Lys residues along the polar face of the helix . Class L venoms lyse both eukaryotic and prokaryotic cells whereas class L antibiotics specifically lyse bacteria . The structural basis for the specificity of class L antibiotics is not well understood . Sequence analysis showed that class L antibiotics have a Glu residue on the nonpolar face of the amphipathic helix; this is absent from class L venoms . We synthesized three model class L peptides with or without Glu on the nonpolar face: 18LMG (LGSIWKFIKAFVGGIKKF), {E14}18LMG and {G5,E14}18LMG . Hemolysis, bacteriolysis, and bacteriostasis studies using these peptides showed that the specificity of lysis is due to both the presence of a Glu residue on the nonpolar face of the helix and the bulk of the nonpolar face . Studies using large unilamellar phospholipid vesicles showed that the inclusion of cholesterol greatly inhibited leakage by the two Glu-containing peptides . These results cannot be attributed to changes in the phase behavior of the lipids caused by the inclusion of cholesterol or to differences in the secondary structure of the peptides . These results suggest that eukaryotic cells are resistant to lysis by magainins because of peptide-cholesterol interactions in their membranes that inhibit the formation of peptide structures capable of lysis, perhaps by hydrogen bonding between Glu and cholesterol . Bacterial membranes, lacking cholesterol, are susceptible to lysis by magainins. Mol Cell Biol, 1995 Apr, 15(4), 2231 - 44 Identification and characterization of a sequence motif involved in nonsense-mediated mRNA decay; Zhang S et al.; In both prokaryotes and eukaryotes, nonsense mutations in a gene can enhance the decay rate or reduce the abundance of the mRNA transcribed from that gene, and we call this process nonsense-mediated mRNA decay . We have been investigating the cis-acting sequences involved in this decay pathway . Previous experiments have demonstrated that, in addition to a nonsense codon, specific sequences 3' of a nonsense mutation, which have been defined as downstream elements, are required for mRNA destabilization . The results presented here identify a sequence motif (TGYYGATGYYYYY, where Y stands for either T or C) that can predict regions in genes that, when positioned 3' of a nonsense codon, promote rapid decay of its mRNA . Sequences harboring two copies of the motif from five regions in the PGK1, ADE3, and HIS4 genes were able to function as downstream elements . In addition, four copies of this motif can function as an independent downstream element . The sequences flanking the motif played a more significant role in modulating its activity when fewer copies of the sequence motif were present . Our results indicate the sequences 5' of the motif can modulate its activity by maintaining a certain distance between the sequence motif and the termination codon . We also suggest that the sequences 3' of the motif modulate the activity of the downstream element by forming RNA secondary structures . Consistent with this view, a stem-loop structure positioned 3' of the sequence motif can enhance the activity of the downstream element . This sequence motif is one of the few elements that have been identified that can predict regions in genes that can be involved in mRNA turnover . The role of these sequences in mRNA decay is discussed. J Mol Evol, 1995 Apr, 40(4), 428 - 42 Phylogenetic relationship of the green alga Nanochlorum eukaryotum deduced from its chloroplast rRNA sequences; Schreiner M et al.; The marine green coccoidal alga Nanochlorum eukaryotum (N.e.) is of small size with an average diameter of 1.5 microns . It is characterized by primitive-appearing biochemical and morphological properties, which are considerably different from those of other green algae . Thus, it has been proposed that N.e . may be an early developed algal form . To prove this hypothesis, DNA of N.e . was isolated by a phenol extraction procedure, and the chloroplast DNA separated by preparative CsCl density-gradient centrifugation . The kinetic complexity of the nuclear and of the chloroplast DNA was evaluated by reassociation kinetics to 3 x 10(7) bp and 9 x 10(4) bp, respectively . Several chloroplast genes, including the rRNA genes, were cloned on distinct fragments . The order of the rRNA genes corresponds to the common prokaryotic pattern . The 16S rRNA gene comprises 1,548 bases and is separated from the 23S rRNA gene with its 2,920 bases by a short spacer of 460 bases, which also includes the tRNA(Ile) and tRNA(Ala) genes . The 5S rRNA gene has not been found; it must start further than 500 bases downstream from the 3'-end of the 23S rRNA gene . From the chloroplast rRNA sequences, we have deduced secondary structures of the 16S and 23S rRNAs, which are in agreement with standard models . The rRNA sequences were aligned with corresponding chloroplast sequences; phylogenetic relationships were calculated by several methods . From these calculations, we conclude that N.e . is most closely related to Chlorella vulgaris . Therefore, N.e . does not represent an early developed algal species; the primitive-appearing morphological and biochemical characteristics of N.e . must rather be explained by secondary losses. Mol Microbiol, 1995 Apr, 16(1), 87 - 96 Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB; Koronakis E et al.; The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding cassette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide . We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro . Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete loss of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change . Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro . The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity. Curr Biol, 1995 Apr 1, 5(4), 334 - 7 DNA replication . A familiar ring to DNA polymerase processivity; Wyman C et al.; Structural similarity reveals that prokaryotic and eukaryotic DNA polymerases share a mechanism for processivity--but the conservation of additional chromosomal replication mechanisms remains to be determined. Comput Appl Biosci, 1995 Apr, 11(2), 195 - 9 Middle-range clustering of nucleotides in genomes; Mrazek J et al.; We propose a novel, transparent and very simple algorithm to analyze middle-range correlations in genomic nucleotide sequences . Analysis by this algorithm of the EMBL Nucleotide Sequence Database demonstrates that all four nucleotides cluster in the genomic nucleotide sequences of eukaryotes on the scale of several hundred base pairs . In prokaryotes, the clustering is weak but still evident . The non-dominant three bases are deficient in the clusters, while A is the most deficient nucleotide in the clusters of C, and vice versa, and G is the most deficient nucleotide in the clusters of T, and vice versa . The algorithm also detects CG islands, extending over 1 kb, in vertebrate sequences . In plants, the CG islands are shown to be much smaller, if they exist at all . A clustering tendency is also exhibited by the TA doublet . Other doublets do not cluster . We observe no strong correlation between nucleotides separated in genomes by > 1 kb. Protein Expr Purif, 1995 Apr, 6(2), 132 - 40 Comparison of the expression of native and mutant bovine annexin IV in Escherichia coli using four different expression systems; Nelson MR et al.; Bovine annexin IV, a Ca(2+)-dependent, membrane-binding protein, was expressed in E . coli using four different prokaryotic expression vector systems . An annexin IV cDNA was mutated in the 5' noncoding region to introduce an NcoI restriction site at the translation initiation site . The coding sequence was then excised and ligated into the expression vectors: pKK233-2 (which uses a hybrid trc promoter), pFOG405 (which uses the alkaline phosphatase promoter and generates a fusion protein with the alkaline phosphatase signal sequence that targets the protein for secretion), pOTSNco12 (which provides temperature-sensitive expression from the lambda phage promoter), and pET11d (which uses the T7lac promoter and a protease-deficient host) . Expression of wild type and mutant annexin IV in the various systems was compared . Differences in level of expression, formation of inclusion bodies, and yield of purified protein were observed . The pET11d system was found to be the most effective expression system for annexin IV and various annexin IV mutant constructs, providing the highest yield of functional protein from the soluble fraction of cell lysates . Bovine chromaffin granule binding and aggregating activities of recombinant annexin IV were found to be virtually indistinguishable from those of bovine annexin IV isolated from liver tissue . Truncation constructs containing one, two, or three of the four conserved 70-amino-acid domains of native annexin IV were successfully created and expressed in E . coli, but the recombinant proteins were generally insoluble . pET11d annexin constructs containing point mutations in residues involved in binding calcium produced soluble protein at levels comparable to those of constructs expressing wild type protein. J Bacteriol, 1995 Apr, 177(8), 2143 - 50 Genes encoded on a cyanobacterial plasmid are transcriptionally regulated by sulfur availability and CysR; Nicholson ML et al.; A cyanobacterial sulfur-regulated gene (cysR), which encodes a protein with similarity to the Crp family of prokaryotic regulatory proteins, has recently been isolated and characterized . Polyacrylamide gel electrophoresis of periplasmic protein extracts reveals that a cysR mutant fails to synthesize a 36-kDa polypeptide that is normally induced in wild-type cells that have been grown under sulfur-deficient conditions . The amino-terminal sequence of this protein was obtained, and a synthetic oligonucleotide was used to isolated a clone containing a 1.9-kb NruI-KpnI fragment from a Synechococcus sp . strain PCC 7942 genomic library . RNA blot analysis indicates that this fragment encodes a transcript that is detectable in wild-type but not cysR mutant cells that have been starved for sulfur . DNA blot analysis revealed that the 1.9-kb NruI-KpnI fragment is contained within the Ba4 BamHI fragment of the endogenous 50-kb plasmid pANL . RNA blot studies indicate that the accumulation of a large number of pANL transcripts is regulated by sulfur levels and CysR . DNA sequence analysis confirmed that the gene encoding the sulfur-regulated 36-kDa periplasmic protein is encoded on the Ba4 fragment of pANL . The sequence of the 36-kDa protein displays sequence similarity to the enzyme catalase, and two downstream proteins exhibit 25 and 62% identity to a subunit of a P-type ATPase complex involved in Mg2+ transport and a chromate resistance determinant, respectively . Surprisingly, a strain in which the putative chromate resistance gene was interrupted by a drug resistance marker exhibited increased resistance to chromate when grown in media containing low sulfate concentrations . The possible role of this protein in the acclimation of cyanobacteria to conditions of low sulfur availability is discussed. Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2627 - 31 Two helices plus a linker: a small model substrate for eukaryotic RNase P; Carrara G et al.; Using precursor tRNA molecules to study RNA-protein interactions, we have identified an RNA motif recognized by eukaryotic RNase P (EC 3.1.26.5) . Analysis of circularly permuted precursors indicates that interruptions in the sugar-phosphate backbone are not tolerated in the acceptor stem, in the T stem-loop, or between residues A-9 and G-10 . Prokaryotic RNase P will function with a minihelix consisting of the acceptor stem connected directly to the T stem-loop . Eukaryotic RNase P cannot use such a minimal substrate unless a linker sequence is added in the gap where the D stem and anticodon stem-loop were deleted. J Biol Chem, 1995 Mar 24, 270(12), 6901 - 7 The role of cytochrome c-550 as studied through reverse genetics and mutant characterization in Synechocystis sp . PCC 6803; Shen JR et al.; The gene coding for cytochrome c-550 in Synechocystis sp . PCC 6803 was cloned based on the N-terminal sequence of the mature polypeptide . Using the most probable translation start codon, the gene is expected to code for 160 amino acid residues . This includes a cleavable N-terminal leader sequence of 25 residues . This leader sequence has an Arg-Asn-Arg sequence immediately before the cleavage site; this is characteristic for transit peptides in prokaryotes . Comparison of this sequence with the leader sequence of the photosystem II-associated extrinsic 33-kDa protein from the same cyanobacterium showed an identity of 13 out of 25 residues . These results suggest that after synthesis of the apoprotein, cytochrome c-550 is transported into the thylakoid lumen . Using the cloned gene, insertion and deletion mutants of Synechocystis sp . PCC 6803 were constructed . In the absence of cytochrome c-550, both mutants were capable of photoautotrophic growth but at a significantly reduced rate . Atrazine bindng and Western blot analysis showed that these mutants on a per-chlorophyll basis contained 53-67% of the amount of photosystem II as compared with wild type . The photosystem II-specific oxygen-evolving activity at saturating light intensity was reduced to about 40% of that in the wild type strain . Taken together, these results indicate that the cytochrome c-550 is transported into the thylakoid lumen and contributes to optimal functional stability of photosystem II in cyanobacteria . This supports our biochemical evidence that cytochrome c-550 is associated with the lumenal side of photosystem II as one of the extrinsic proteins enhancing oxygen evolution (Shen, J.-R., Ikeuchi, M., and Inoue, Y . (1992) FEBS Lett . 301, 145-149; Shen, J.-R., and Inoue, Y . (1993) Biochemistry 32, 1825-1832) . Based on these results, the gene for cytochrome c-550 was named psbV . The possible evolutionary relationship among extrinsic proteins of the photosystem II donor side is discussed. Gene, 1995 Mar 21, 155(1), 35 - 43 Ribozymes expressed within the loop of a natural antisense RNA form functional transcription terminators; Deshler JO et al.; As an initial step towards developing a widely applicable system for expressing small ribozymes (Rz) in various cell types using T7 RNA polymerase, we have replaced the loop domain of a natural prokaryotic antisense RNA (RNAout from Tn10), with hammerhead (Hh) Rz . RNAout was chosen, because the stem of its secondary structure gives it an unusually long half life in Escherichia coli which should also confer in vivo stability to small RNA sequences expressed within its loop domain . In order to define the 3' end of the Rz-RNAout chimeric RNAs, a poly(U) tract was inserted just 3' of the RNAout stem . Molecular analysis indicates that these RNAs function both as transcription terminators and Rz . In addition, the RNAs are stable in E . coli and can be expressed in mammalian cells . These results show that certain characteristics of a naturally evolved RNA, RNAout in this case, can be used to provide additional functions to short RNAs containing Hh Rz without disrupting the enzymatic activity of the Rz. Biochemistry, 1995 Mar 21, 34(11), 3686 - 93 Prokaryotic expression of the heme- and flavin-binding domains of rat neuronal nitric oxide synthase as distinct polypeptides: identification of the heme-binding proximal thiolate ligand as cysteine-415; McMillan K et al.; The heme- and flavin-binding domains of constitutive rat neuronal nitric oxide synthase (NOS) were expressed in Escherichia coli as distinct polypeptides with properties characteristic of the intact enzyme . The amino-terminal heme-binding domain (residues 1-714) was expressed using the expression vector pCW . The denatured molecular mass of the expressed protein was 80 kDa, and the protein was shown to be immunoreactive to rabbit anti-NOS IgG . The NOS hemoprotein exhibited a ferrous-carbon monoxide difference spectrum with a wavelength maximum at 445 nm . Spectral perturbation with L-arginine and BH4 elicited a type I difference spectrum, confirming the presence of binding sites for these molecules within the N-terminal NOS polypeptide . Site-directed mutagenesis was applied to the putative axial heme ligand, cysteine-415, generating the histidine mutant, which confirmed the identity of the proximal ligand . NOS flavoproteins, with (C1, residues 715-1429) and without (C2, residues 749-1429) an amino-terminal calmodulin-binding motif, were expressed using the vector pPROK-1 . The C1 and C2 flavoproteins were immunoreactive to anti-NOS IgG and were sized at approximately 80 kDa . Both of the purified flavoproteins exhibited optical absorbance properties typical of a flavin prosthetic group, with wavelength maxima at 380 and 450 nm, and were competent in NADPH-dependent electron transfer to cytochrome c, with observed rates of approximately 2-4 mumol/min/mg . The bacterial expression of the NO synthase heme-binding oxygenase and flavoprotein oxidoreductase domains as isolated proteins with specific properties of the intact enzyme represents an important development in structure-function studies of this complex enzyme. J Biol Chem, 1995 Mar 17, 270(11), 6062 - 70 Identification of a non-mitochondrial phosphatidylserine decarboxylase activity (PSD2) in the yeast Saccharomyces cerevisiae; Trotter PJ et al.; Phosphatidylserine decarboxylase (PSD1) plays a central role in the biosynthesis of aminophospholipids in both prokaryotes and eukaryotes by catalyzing the synthesis of phosphatidylethanolamine . Recent reports (Trotter, P . J., Pedretti, J., and Voelker, D . R . (1993) J . Biol . Chem . 268, 21416-21424; Clancey, C . J., Chang, S.-C., and Dowhan, W . (1993) J . Biol . Chem . 268, 24580-24590) described the cloning of a yeast structural gene for this enzyme (PSD1) and the creation of the null allele . Based on the phenotype of strains containing a null allele for PSD1 (psd1-delta 1::TRP1) it was hypothesized that yeast have a second phosphatidylserine decarboxylase . The present studies demonstrate the presence of a second enzyme activity (denoted PSD2), which, depending on the method of evaluation, accounts for 4-12% of the total cellular phosphatidylserine decarboxylase activity found in wild type . Recessive mutations resulting in loss of this enzyme activity (denoted psd2) in cells containing the psd1-delta 1::TRP1 null allele also result in ethanolamine auxotrophy . When incubated with {3H}serine these double mutants accumulate label in phosphatidylserine, while very little (< 5%) is converted to phosphatidylethanolamine . In addition, these mutants have a approximately 70% decrease in the amount of total phosphatidylethanolamine even when grown in the presence of exogenous ethanolamine . Strains containing psd1 or psd2 mutations were utilized for the subcellular localization of the PSD2 enzyme activity . Unlike the PSD1 activity, the PSD2 enzyme activity does not localize to the mitochondria, but to a low density subcellular compartment with fractionation properties similar to both vacuoles and Golgi. Biochem Biophys Res Commun, 1995 Mar 17, 208(2), 697 - 703 Identification of a 40-kDa human protein that cross-reacts with prokaryotic hsp60/chaperonins; Rosenberg HF et al.; Cross-reactivity between the immunodominant bacterial hsp60 proteins and heterologous human target proteins has led to numerous hypotheses on the role of hsp60 in the pathogenesis of autoimmune disease . In this work, we describe a novel 40-kDa human protein that cross-reacts with bacterial hsp60 proteins . CCP40 (chaperone cross-reacting protein, 40-kDa) was identified in extracts from HL-60 (human promyelocytic leukemia) cells on Western blots probed with A57-E4, a monoclonal antibody specifying a linear polypeptide epitope common among the bacterial hsp60 proteins (Yuan et . al . (1992) Inf . Immun . 60, 2288-2296) . CCP40 was detected in other human hematopoietic cell lines, but could not be detected in mature peripheral blood leukocytes . CCP40 was also expressed in human CD34+ peripheral blood progenitor cells, disappearing with cytokine-induced cellular maturation. Nature, 1995 Mar 16, 374(6519), 227 - 32 The major evolutionary transitions; Szathmary E et al.; There is no theoretical reason to expect evolutionary lineages to increase in complexity with time, and no empirical evidence that they do so . Nevertheless, eukaryotic cells are more complex than prokaryotic ones, animals and plants are more complex than protists, and so on . This increase in complexity may have been achieved as a result of a series of major evolutionary transitions . These involved changes in the way information is stored and transmitted. Eur J Biochem, 1995 Mar 15, 228(3), 616 - 24 DNA interactions of antitumor platinum(IV) complexes; Novakova O et al.; Modifications of natural DNA and synthetic double-stranded oligodeoxyribonucleotides by cis-diamminedichloro-trans-dihydroxyplatinum(IV) (oxoplatin) were studied by means of ELISA, Maxam-Gilbert footprinting techniques, HPLC of enzymically digested DNA, and transcription assay . It was found that oxoplatin can bind DNA directly without addition of a reducing agent . In addition, the antibodies elicited against DNA modified by cisplatin were not competitively inhibited by DNA modified by oxoplatin . However, DNA containing the adducts of oxoplatin became a strong inhibitor of these antibodies, if it was subsequently treated with ascorbic acid, which is a reducing agent . These results were interpreted to mean that oxoplatin can form DNA adducts containing the platinum moiety in the quadrivalent state . The direct irreversible binding of the platinum(IV) drug is, however, slow as compared to the reaction of its platinum(II) counterpart . It was also found that oxoplatin preferentially binds to guanine residues and can form DNA intrastrand and interstrand cross-links containing platinum(IV) . The DNA adducts containing platinum(IV) can inhibit in vitro transcription by a prokaryotic DNA-dependent RNA polymerase . We find that the platinum(IV) complex binds to DNA at similar sites as its platinum(II) counterpart . On the other hand, the DNA adducts containing the platinum(II) or platinum(IV) analogues differ in the number of ligands and the formal charge on their platinum center . We suggest that these differences could be responsible for distinct conformational features and stability of DNA modified by platinum(II) or platinum(IV) complexes. J Biol Chem, 1995 Mar 10, 270(10), 5412 - 7 Cloning and characterization of a dihydrolipoamide acetyltransferase (E2) subunit of the pyruvate dehydrogenase complex from Arabidopsis thaliana; Guan Y et al.; A cDNA encoding a dihydrolipoamide acetyltransferase (E2) subunit of the pyruvate dehydrogenase complex has been isolated from Arabidopsis thaliana . A cell culture cDNA expression library was screened with a monoclonal antibody (JIM 63) raised against nuclear matrix proteins, and four clones were isolated . One of these was 2175 base pairs in length, and it contained an open reading frame with an amino acid sequence and domain structure with strong similarity to the E2s of other eukaryotic and prokaryotic organisms . The organization and number of functional domains within the Arabidopsis protein are identical to those of the human E2, although the amino acid sequences within these domains are equally similar to those of the yeast and human proteins . The predicted amino acid sequence reveals the presence of a putative amino-terminal leader sequence with characteristics similar to those of other proteins, which are targeted to the plant mitochondrial matrix . The cross-reactivities of plant mitochondrial matrix proteins with JIM 63 and antibodies raised against the E2 and protein X components of eukaryotic pyruvate dehydrogenase complexes are consistent with the clone encoding a mitochondrial form of E2 and not the smaller protein X . The E2 mRNA of 2.2 kilobases was expressed in a range of Arabidopsis and Brassica napus tissues. J Biol Chem, 1995 Mar 3, 270(9), 4438 - 50 The role of Barbie box sequences as cis-acting elements involved in the barbiturate-mediated induction of cytochromes P450BM-1 and P450BM-3 in Bacillus megaterium; Liang Q et al.; In a previous publication (He, J.-S., and Fulco, A . J . (1991) J . Biol . Chem . 266, 7864-7869), we reported that a 15-17-base pair DNA sequence (designated a Barbie box element) in the 5'-regulatory regions of cytochrome P450BM-1 and P450BM-3 genes from Bacillus megaterium was recognized by a barbiturate-regulated protein . It is now recognized that essentially all eukaryotic and prokaryotic genes whose 5'-flanking regions are known and that encode barbiturate-inducible proteins contain the Barbie box element . A 4-base pair sequence (AAAG) is found in the same relative position in all Barbie box elements . In B . megaterium, mutation of the Barbie box located in the P450BM-1 gene leads to the constitutive synthesis of cytochrome P450BM-1 and a 10-fold increase of expression of Bm1P1, a small gene located upstream of the P450BM-1 gene, that encodes a putative regulatory protein . Mutation of the P450BM-3 Barbie box significantly increased the expression of both P450BM-3 and Bm3P1 (another small gene located upstream of the P450BM-3 gene that encodes a second putative regulatory protein) in response to pentobarbital induction but left the basal levels unaffected . In gel mobility shift assays, Bm3R1, a repressor of the P450BM-3 gene, was found to specifically interact with the Barbie box sequences of the B . megaterium P450 genes . Mutated Barbie boxes showed a decreased binding affinity for Bm3R1 compared to their wild type (unmutated) counterparts . Barbie box sequences were also shown to specifically interact with putative positive regulatory factors of B . megaterium cells . These putative positive factors were induced by pentobarbital and were also present at high levels during late stationary phase of B . megaterium cell cultures grown in the absence of barbiturates . The mutated Barbie box sequences had greater binding affinity for these positive factors than did unmutated Barbie box sequences . DNase I footprinting analysis of the 5'-flanking region of the P450BM-1 gene revealed that these positive factors protected a segment of DNA covering a portion of the Barbie box sequence and a small flanking region . Similar footprinting experiments with the 5'-flanking region of the P450BM-3 gene failed, however, to unambiguously reveal protected sequences in the Barbie box region . The evidence suggests that the positive factors and Bm3R1 compete with each other for binding to the Barbie box region, especially in the 5'-flanking region of the P450BM-1 gene, and for putative roles in the regulation of transcription from the B . megaterium P450 genes.(ABSTRACT TRUNCATED AT 400 WORDS) J Clin Invest, 1995 Mar, 95(3), 1193 - 8 Cross-resistance between cisplatin, antimony potassium tartrate, and arsenite in human tumor cells; Naredi P et al.; Cross-resistance between cisplatin (DDP) and metalloid salts in human cells was sought on the basis that mechanisms that mediate metalloid salt cross-resistance in prokaryotes are evolutionarily conserved . Two ovarian and two head and neck carcinoma cell lines selected for DDP resistance were found to be cross-resistant to antimony potassium tartrate, which contains trivalent antimony . The DDP-resistant variant 2008/A was also cross-resistant to arsenite but not to stibogluconate, which contains pentavalent antimony . A variant selected for resistance to antimony potassium tartrate was cross-resistant to DDP and arsenite . Resistance to antimony potassium tartrate and arsenite was of a similar magnitude (3-7-fold), whereas the level of resistance to DDP was greater (17-fold), irrespective of whether the cells were selected by exposure to DDP or to antimony potassium tartrate . In the resistant sublines, uptake of {3H}-dichloro(ethylenediamine) platinum(II) was reduced to 41-52% of control, and a similar deficit was observed in the accumulation of arsenite . We conclude that DDP, antimony potassium tartrate, and arsenite all share a common mechanism of resistance in human cells and that this is due in part to an accumulation defect. Lipids, 1995 Mar, 30(3), 231 - 4 Identification of the active site of vertebrate oxidosqualene cyclase; Abe I et al.; Active site mapping of rat liver oxidosqualene cyclase (OSC), a 78 kDa membrane-bound enzyme, was carried out using the mechanism-based irreversible inhibitor, {3H}29-methylidene-2,3-oxidosqualene . The amino acid sequence of the radiolabeled CNBr peptide fragment showed unexpectedly high similarity to the yeast OSC, plant OSC, and bacterial squalene cyclases . Further, radio analysis established that the two adjacent Asp residues in the highly conserved region (Asp-Asp-Thr-Ala-Glu-Ala, or DDTAEA) were equally labeled by the irreversible inhibitor . This result provided the first information on the structural details of the active site of OSC, and showed for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases . Interestingly, the covalently-modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of other terpene cyclases and prenyl transferases . The Asp-rich motif may act as a point charge to stabilize incipient cationic charge. Cell Mol Biol (Noisy-le-grand), 1995 Mar, 41(2), 279 - 87 A new procedure for the preparation of highly active melonin from the dry seeds of Cucumis melo L; Rojo MA et al.; Melon (Cucumis melo L.) dry seeds contain melonin, a protein that strongly inhibits ribosomes from different prokaryotic and eukaryotic sources including those from melon . The protein was purified by a new method to yield highly active and stable protein preparations that involves chromatography through S-Sepharose Fast Flow, CM-Sepharose, Superdex 75 and Mono-S . Melonin shows important functional properties: 1) its inhibitory effects on translation were irreversible; 2) it is a single unglycosylated polypeptide chain with an apparent M(r) of 22000; 3) it degrades RNA in a dose-dependent way without affecting DNA . In the light of present results melonin can be considered as a new plant RNase of unusual properties. AIDS Res Hum Retroviruses, 1995 Mar, 11(3), 405 - 8 Polyclonal rabbit antisera that detect the Vpr protein of SIVSM and SIVMAC on immunoblots of purified virions; Newman MA et al.; Antisera suitable for detection of SIVSM or SIVMAC Vpr proteins on Western blots of purified virions are currently not available . We have expressed the Vpr protein of SIVSMPBj1.9 in a gst-based prokaryotic expression system and used it to raise polyclonal antisera in rabbits . Two immune sera were obtained that specifically recognized both cell- and virion-associated Vpr protein on immunoblots of three different SIV isolates (SIVSMPBj1.9, SIVMACBK28, and SIVMAC239) . Because Vpr is believed to play an important role in HIV/SIV replication and pathogenesis, these reagents will allow the extension of functional analyses of this protein to a broader spectrum of viruses . Both antisera and the gst-Vpr expression plasmid have been submitted to the NIAID AIDS Research and Reagent Program and are available to interested investigators. Plant Cell Physiol, 1995 Mar, 36(2), 207 - 13 Structure, function and regulation of the nitrate transport system of the cyanobacterium Synechococcus sp . PCC7942; Omata T; The active nitrate transport system of the cyanobacterium Synechococcus sp . PCC7942 is encoded by the four genes nrtA, nrtB, nrtC and nrtD . It is essential for the growth of the cyanobacterium at physiological concentrations of nitrate and has been shown to be involved in the active transport of nitrite as well . The deduced amino acid sequences of the NrtB, NrtC and NrtD proteins indicate that the transporter is a member of the ABC (ATP-binding cassette) superfamily of active transporters . Among the prokaryotic ABC transporters, the cyanobacterial nitrate/nitrite transporter is unique in having a membrane-bound protein NrtA and an NrtA-like extra domain linked to one of the ATP-binding subunits (C-terminal domain of NrtC) . Molecular biological, biochemical and physiological studies suggest that NrtA is the substrate-binding protein required for the transport of nitrate/nitrite and that the C-terminal domain of NrtC has a regulatory role . Comparison of the structures of nitrate transporters from eukaryotic and prokaryotic, photosynthetic and non-photosynthetic organisms indicate that the nrt nitrate/nitrite transporter represents a prokaryotic nitrate transporter distinct from the nitrate transporters of eukaryotes. Plant Mol Biol, 1995 Mar, 27(6), 1189 - 96 Characterization of the single psbA gene of Prochlorococcus marinus CCMP 1375 (Prochlorophyta); Hess WR et al.; DNA sequence, copy number, expression and phylogenetic relevance of the psbA gene from the abundant marine prokaryote P . marinus CCMP 1375 was analyzed . The 7 amino acids near the C-terminus missing in higher plant and in Prochlorothrix hollandica D1 proteins are present in the derived amino acid sequence . P . marinus contains only a single psbA gene . Thus, this organism lacks the ability to adapt its photosystem II by replacement of one type of D1 by another, as several cyanobacteria do . Phylogenetic trees suggested the D1-1 iso-form from Synechococcus PCC 7942 as the next related D1 protein and place P . marinus separately from Prochlorothrix hollandica among the cyanobacteria. Curr Microbiol, 1995 Mar, 30(3), 133 - 6 Buchnera aphidicola (aphid-endosymbiont) glyceraldehyde-3-phosphate dehydrogenase: molecular cloning and sequence analysis; Kolibachuk D et al.; Buchnera aphidicola is an endosymbiont of the aphid Schizaphis graminum . A 3.9-kb B . aphidicola DNA fragment was sequenced and found to contain two open reading frames (ORFs) . The deduced amino acid sequence of one of the ORFs had an 85% identity to Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (Gap) . Both of these proteins have a higher similarity to eukaryotic than to prokaryotic Gaps . The second ORF could not be readily identified . The sequence of the putative product indicated that it was a member of the family of ATP-binding, membrane-associated proteins . The highest amino acid identity (36%) was with E . coli FtsE, a protein involved in cell division. Analyst, 1995 Mar, 120(3), 693 - 7 Iron species in iron homeostasis and toxicity; Crichton RR et al.; Iron homeostasis in prokaryotic cells appears to be regulated essentially at the level of the genome by the Fur protein . When iron is in short supply the uptake and assimilation pathways are de-repressed and siderophores are synthesized together with the outer, inner (plasma) membrane, periplasmic and cytosolic components necessary for the uptake of ferri-siderophores . When iron is no longer limiting the Fe2+ the Fur complex acts as a transcriptional repressor, and shuts down the synthesis of all the components of iron assimilation . In euykarotic cells, iron homeostasis is dependent upon the iron regulatory factor (IRF), a cytoplasmic protein that can bind to specific stem loops, iron responsive elements (IREs) on the messenger ribonucleic acid molecules (mRNAs) of proteins involved in iron storage (ferritin), utilization (erythroid delta-aminolaevulinate synthase, AIS), and uptake (transferrin receptor) . During ion depletion the IRE is in a high affinity form, which, by binding strongly to the corresponding mRNAs, down regulates iron storage and utilization, while up-regulating transferrin receptor expression . When the cells are iron replete, IRF binding to IREs is weak, allowing transferrin receptor mRNA to be degraded . In this paper it is shown that in physiological conditions of iron overload and depletion, IRF functions in vivo in the manner already described for in vitro models . The nature and the speciation of the various iron species within the low molecular weight pool of eukaryotic cells remains unclear. Mol Immunol, 1995 Mar, 32(4), 267 - 75 Class I MHC alpha 3 domain can function as an independent structural unit to bind CD8 alpha; Fayen J et al.; Functional interactions between CD8-dependent cytotoxic T cells and their targets require physical contact between CD8 and a non-polymorphic determinant on the alpha 3 domain of the class I MHC molecule . We developed a cell-free assay to directly monitor this molecular interaction, specifically excluding the participation of other cellular proteins and lipids . This assay employed a soluble CD8 derivative and a plate-bound HLA-A2.1 derivative, alpha 3/MalE, in which the alpha 3 domain has been expressed independently of its neighboring polypeptide domains on the native class I MHC molecule and beta 2-microglobulin (beta 2-m) . These proteins were produced using eukaryotic and prokaryotic expression systems, respectively . Our data demonstrated specific, saturable binding between soluble CD8 alpha (sCD8 alpha) and alpha 3/MalE, and the Kd of this interaction was determined to be 4.5 x 10(-7) M . Monoclonal antibodies (mAb) directed against either CD8 or the alpha 3 domain of class I MHC inhibited binding; mAb directed against other sites on class I MHC and beta 2-m did not . Our data suggest that the interaction between CD8 alpha and the alpha 3 domain of class I MHC does not require the participation of neighboring class I sequences or beta 2-m. J Mol Evol, 1995 Mar, 40(3), 337 - 42 Segmented structure of protein sequences and early evolution of genome by combinatorial fusion of DNA elements; Trifonov EN; A theory of an early stage of genome evolution by combinatorial fusion of circular DNA units is suggested, based on protein sequence "fossil" evidence . The evidence includes preference of protein sequence lengths for certain sizes--multiples of 123 aa for eukaryotes and multiples of 152 aa for prokaryotes . At the DNA level these sizes correspond to 350-450 base pairs--the known optimal range for DNA ring closure . The methionine residues repeatedly appear along the sequences with the same period of about 120 aa (in eukaryotes), presumably marking the sites of insertion of the early genes--rings of protein-coding DNA . No torsional constraint in this DNA results in very sharp estimate of the helical periodicity of the early DNA, indistinguishable from the experimental mean value for extant DNA . According to the combinatorial fusion theory, based on the above evidence, in the pregenomic, prerecombinational stage the genes and the noncoding sequences existed in form of autonomously replicating DNA rings of close to standard size, randomly segregating between dividing cells, like modern plasmids do . In the recombinational early genomic stage the rings started to fuse, forming larger DNA molecules consisting of several unit genes connected in various combinations and forming long protein-coding sequences (combinatorial fusion) . This process, which involved, perhaps, noncoding sequences as well, eventually resulted in the formation of large genomes . The dispersed circular DNA--or, rather, evolutionarily advanced derivatives thereof--may still exist in the form of various mobile DNA elements. J Mol Evol, 1995 Mar, 40(3), 238 - 48 The expanding small heat-shock protein family, and structure predictions of the conserved "alpha-crystallin domain"; Caspers GJ et al.; The ever-increasing number of proteins identified as belonging to the family of small heat-shock proteins (shsps) and alpha-crystallins enables us to reassess the phylogeny of this ubiquitous protein family . While the prokaryotic and fungal representatives are not properly resolved, most of the plant and animal shsps and related proteins are clearly grouped in distinct clades, reflecting a history of repeated gene duplications . The members of the shsp family are characterized by the presence of a conserved homologous "alpha-crystallin domain," which sometimes is present in duplicate . Predictions are made of secondary structure and solvent accessibility of this domain, which together with hydropathy profiles and intron positions support the presence of two similar hydrophobic beta-sheet-rich motifs, connected by a hydrophilic alpha-helical region . Together with an overview of the newly characterized members of the shsp family, these data help to define this family as being involved as stable structural proteins and as molecular chaperones during normal development and induced under pathological and stressful conditions. Genes Dev, 1995 Mar 1, 9(5), 587 - 99 SMC2, a Saccharomyces cerevisiae gene essential for chromosome segregation and condensation, defines a subgroup within the SMC family; Strunnikov AV et al.; We characterized the SMC2 (structural maintenance of chromosomes) gene that encodes a new Saccharomyces cerevisiae member of the growing family of SMC proteins . This family of evolutionary conserved proteins was introduced with identification of SMC1, a gene essential for chromosome segregation in budding yeast . The analysis of the putative structure of the Smc2 protein (Smc2p) suggests that it defines a distinct subgroup within the SMC family . This subgroup includes the ScII, XCAPE, and cut14 proteins characterized concurrently . Smc2p is a nuclear, 135-kD protein that is essential for vegetative growth . The temperature-sensitive mutation, smc2-6, confers a defect in chromosome segregation and causes partial chromosome decondensation in cells arrested in mitosis . The Smc2p molecules are able to form complexes in vivo both with Smc1p and with themselves, suggesting that they can assemble into a multimeric structure . In this study we present the first evidence that two proteins belonging to two different subgroups within the SMC family carry nonredundant biological functions . Based on genetic, biochemical, and evolutionary data we propose that the SMC family is a group of prokaryotic and eukaryotic chromosomal proteins that are likely to be one of the key components in establishing the ordered structure of chromosomes. J Steroid Biochem Mol Biol, 1995 Mar, 52(3), 209 - 18 The rat 17 alpha-hydroxylase-17,20-desmolase (CYP17) active site: computerized homology modeling and site directed mutagenesis; Buczko E et al.; A homology model of the rat 17 alpha-hydroxylase-17,20 desmolase (CYP17) steroid binding domain was derived from the alpha/beta F supersecondary structural element of the 3 alpha/20 beta hydroxysteroid dehydrogenase (HSD) of Streptomyces hydrogenans that constitutes a major segment of the C19 steroid binding cavity . A CYP17 arginine-rich domain, including Arg346, Arg361 and Arg363, that has previously been shown to be important to CYP17 catalytic activity, is conserved in this HSD structural element between two HSD domains known to be important to C19 steroid binding . These two HSD motifs, in addition to a C-terminal domain at the apex of the steroid binding cavity, are also present in similar though not identical forms in the rat CYP17 sequence . The model was evaluated in terms of both hydroxylase/lyase activity and stability of CYP17 mutant proteins (Tyr334Phe, Phe343Ile, Arg357Ala, Arg361Ala, Asp345Ala), and further tested with mutagenesis of Glu353, Glu358, and Tyr431 . Those amino acids located at folding junctions in the model steroid binding domain (Glu358, Arg361, and Tyr431) are each individually required to prevent degradation of the nascent protein, as well as for basic hydroxylase/lyase activity . Genomic analysis of the rat CYP17 gene reveals that this domain is contained in exon 6, and a correlation exists between the length of exon 6 and the boundaries of the HSD supersecondary element . These studies demonstrate that exon 6 of the rat CYP17 is essential for CYP17 activity, and may be structurally related to the NAD-linked prokaryote alpha/beta F supersecondary element. Vet Parasitol, 1995 Mar, 57(1-3), 43 - 9 Recent developments in the molecular biology of anaplasmosis; Barbet AF; Recent applications of DNA analysis, cloning, sequencing and expression technology have resulted in significant advances in our understanding of the hemoparasite Anaplasma marginale . Analysis of 16S ribosomal RNA has confirmed a phylogenetic position close to Ehrlichia sp . and Cowdria ruminantium . Intact genomic DNA of A . marginale digested with SfiI separates into bands from 14 to 170 kbp on pulse-field gels, with a total genome size of 1200-1260 kbp and G + C content of 56 mol% . Major surface proteins (MSP1-MSP5) have been identified and DNA coding sequences are available for most of these . These data have revealed that MSPs may be quite polymorphic between different geographic isolates, may be encoded by multi-gene families, and have some similar features to other prokaryotes including signal peptidase cleavage sites and gene regulatory sequences . Homologies have been detected between MSPs and immunodominant proteins of Cowdria ruminantium . Several MSPs have been expressed to high level and purified from recombinant Escherichia coli . MSP 1, 2 and 4 have potential for the development of vaccines and MSP3 and 5 for improved diagnostic assays. Biofizika, 1995 Mar-Apr, 40(2), 296 - 316 {The role of disulfide bridges of the residual protein in chromosomal DNA organization}; Struchkov VA et al.; The review of literature data and our investigation on the role of disulfide bridges of residual protein (RP) in structural organization of chromosomal DNA is presented . It was studied the action of several S-S cleaving agents (2-mercaptoethanol, dithiothreitol, NaBH4, glutathione reductase) on native DNA-RP complexes, isolating from the different eukaryotic and prokaryotic cells . It was shown, that the thiols result the fragmentation of DNA-RP complex in double-strand subunits of several size (5 x 10(5), (18-20) x 10(6), 70 x 10(6) Da) on dependence of the incubate condition (concentration of thiols, pH, time) . It was observed, that specific S-S bonds (thiol-sensitivity at neutral or acid conditions, glutathione reductase-sensitivity) are present in DNA-RP complexes, which may control of different structural levels of DNA into chromosome (gen-transcription-replicon-domain) . The possible quasisubunit structure of chromosomal DNA with participation of polypeptide S-S bonds and complementary "sticky" ends of subunits is discussed. Mol Biol (Mosk), 1995 Mar-Apr, 29(2), 258 - 80 {Reverse transcriptase and its biological role}; Ivanov VA et al.; Elements with open-reading translation frames homologous to retroviral RNA-dependent DNA polymerases (reverse transcriptases) were found in eukaryotic cell genomes . Expression of endogenous reverse transcriptases in prokaryotic and eukaryotic cells is confirmed directly and indirectly . Evolution and function of reverse transcriptases are analyzed, and their phylogenetic and ontogenetic role is evaluated. J Med Virol, 1995 Mar, 45(3), 253 - 8 Prokaryotic expression and analysis of the antibody response to a Newcastle isolate of the core gene of hepatitis C; Milton ID et al.; The full length hepatitis C virus (HCV) core gene was isolated from a Newcastle strain and expressed in E . coli . A truncated HCV core gene which lacks the hydrophobic carboxyl-terminal sequence was also expressed . The truncated HCV core was expressed at higher levels with fewer cleavage products . Antibody reactivity to the recombinant HCV core antigen was analysed by ELISA and Western blotting in 60 HCV antibody-positive patients with a broad spectrum of liver disease . There was no significant difference between the presence of IgG to recombinant HCV core and reactivity to the core antigen in the RIBA-2 test . There was also no significant difference between the presence of IgG to recombinant core and diagnostic PCR as a marker for active liver inflammation. Curr Microbiol, 1995 Mar, 30(3), 149 - 53 Detection of mip-like sequences and Mip-related proteins within the family Rickettsiaceae; Cianciotto NP et al.; The Mip surface protein, a prokaryotic analog of the FK506-binding proteins, enhances the ability of Legionella pneumophila to infect macrophages and protozoa . Using mip-specific probes and low-stringency Southern hybridizations, we have detected DNA sequences homologous to mip within Coxiella burnetii and Rochalimaea quintana . Using specific anti-Mip antisera and immunoblot analysis, we also detected Mip-related proteins within these bacteria as well as within Rickettsia and Ehrlichia species . These data suggest that Mip-related proteins have broad significance for host-parasite interactions . However, they also indicate that care must be exercised when using mip probes or anti-Mip antibodies for the detection of Legionella organisms in water or clinical samples. Proc Natl Acad Sci U S A, 1995 Feb 28, 92(5), 1604 - 8 Possible proton relay pathways in cytochrome c oxidase; Fetter JR et al.; As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase (EC 1.9.3.1) catalyzes the reduction of oxygen to water and generates a proton gradient . To test for proton pathways through the oxidase, site-directed mutagenesis was applied to subunit I of the Rhodobacter sphaeroides enzyme . Mutants were characterized in three highly conserved regions of the peptide, comprising possible proton loading, unloading, and transfer sites: an interior loop between helices II and III (Asp132Asn/Ala), an exterior loop between helices IX and X (His411Ala, Asp412Asn, Thr413Asn, Tyr414Phe), and the predicted transmembrane helix VIII (Thr352Ala, Pro358Ala, Thr359Ala, Lys362Met) . Most of the mutants had lower activity than wild type, but only mutants at residue 132 lost proton pumping while retaining electron transfer activity . Although electron transfer was substantially inhibited, no major structural alteration appears to have occurred in D132 mutants, since resonance Raman and visible absorbance spectra were normal . However, lower CO binding (70-85% of wild type) suggests some minor change to the binuclear center . In addition, the activity of the reconstituted Asp132 mutants was inhibited rather than stimulated by ionophores or uncoupler . The inhibition was not observed with the purified enzyme and a direct pH effect was ruled out, suggesting an altered response to the electrical or pH gradient . The results support an important role for the conserved II-III loop in the proton pumping process and are consistent with the possibility of involvement of residues in helix VIII and the IX-X loop. Nucleic Acids Res, 1995 Feb 25, 23(4), 689 - 95 An overabundance of long oligopurine tracts occurs in the genome of simple and complex eukaryotes; Behe MJ; A search of sequence information in the GenBank files shows that tracts of 15-30 contiguous purines are greatly overrepresented in all eukaryotic species examined, ranging from yeast to human . Such an overabundance does not occur in prokaryotic sequences . The large increase in the number of oligopurine tracts cannot be explained as a simple consequence of base composition, nearest-neighbor frequencies, or the occurrence of an overabundance of oligoadenosine tracts . Oligopurine sequences have previously been shown to be versatile structural elements in DNA, capable of occuring in several alternate conformations . Thus the bias toward long oligopurine tracts in eukaryotic DNA may reflect the usefulness of these structurally versatile sequences in cell function. Nucleic Acids Res, 1995 Feb 25, 23(4), 595 - 8 Cloning and characterization of the hrpA gene in the terC region of Escherichia coli that is highly similar to the DEAH family RNA helicase genes of Saccharomyces cerevisiae; Moriya H et al.; During the course of systematic nucleotide sequence analysis of the terC region of E.coli K-12 by using the ordered lambda phage clones, we found the presence of a gene, termed hrpA, that showed a high degree of sequence similarity to the PRP2, PRP16 and PRP22 genes of Saccharomyces cerevisiae . The products of these yeast genes are known to play their roles in mRNA splicing, and belong to a group of proteins collectively called the DEAH family . The hrpA gene is the first example of a DEAH family gene in prokaryotes . The N-terminal region of the protein it encodes contains conserved sequence stretches characteristic of an RNA helicase . Its molecular mass is calculated to be 146 kDa . Previously, a 135 kDa protein was identified by Moir et al . {J . Bacteriol . (1992) 174, 2102-2110} in this region which is most likely identical to that encoded by hrpA . The C-terminal region of the hrpA gene product seems to contain an RNA binding motif weakly resembling that of ribosomal protein S1 of E.coli . Disruption of the hrpA gene suggested that it is not essential for the growth of E.coli. Nucleic Acids Res, 1995 Feb 25, 23(4), 580 - 7 Bulged-out nucleotides in an antisense RNA are required for rapid target RNA binding in vitro and inhibition in vivo; Hjalt TA et al.; Naturally occurring antisense RNAs in prokaryotes are generally short, highly structured and untranslated . Stem-loops are always present, and loop regions serve as primary recognition structures in most cases . Single-stranded tails or internal unstructured regions are required for initiation of stable pairing between antisense and target RNA . Most antisense RNAs contain bulged-out nucleotides or small internal loops in upper stem regions . Here we investigated the role of the bulged-out nucleotides of CopA (the copy number regulator of plasmid R1) in determining the binding properties of this antisense RNA to its target in vitro and the efficiency of a translational inhibition in vivo . The introduction of perfect helicity in the region of the two bulges in CopA decreased pairing rate constants by up to 180-fold, increased equilibrium dissociation constants of the 'kissing intermediate' up to 14-fold, and severely impaired inhibition of repA expression . A previously described loop size mutant of CopA showed decreased pairing rates, but, in contrast to the bulge-less mutant CopAs, shows a decreased dissociation constant of the 'kissing complex' . We conclude that removal of the specific bulges/internal loops within the stem-loop II of CopA impairs the inhibitor, and that creation of an internal loop at a different position does not restore activity, emphasizing the optimal folding of wild-type CopA . The accompanying paper shows that an additional function of bulges can be protection from RNase III cleavage. J Biol Chem, 1995 Feb 24, 270(8), 3611 - 8 Identification of the potential active site of the signal peptidase SipS of Bacillus subtilis . Structural and functional similarities with LexA-like proteases; van Dijl JM et al.; Signal peptidases remove signal peptides from secretory proteins . By comparing the type I signal peptidase, SipS, of Bacillus subtilis with signal peptidases from prokaryotes, mitochondria, and the endoplasmic reticular membrane, patterns of conserved amino acids were discovered . The conserved residues of SipS were altered by site-directed mutagenesis . Replacement of methionine 44 by alanine yielded an enzyme with increased activity . Two residues (aspartic acid 146 and arginine 84) appeared to be conformational determinants; three other residues (serine 43, lysine 83, and aspartic acid 153) were critical for activity . Comparison of SipS with other proteases requiring serine, lysine, or aspartic acid residues in catalysis revealed sequence similarity between the region of SipS around serine 43 and lysine 83 and the active-site region of LexA-like proteases . Furthermore, self-cleavage sites of LexA-like proteases closely resembled signal peptidase cleavage sites . Together with the finding that serine and lysine residues are critical for activity of the signal peptidase of Escherichia coli (Tschantz, W.R., Sung, M., Delgado-Partin, V.M., and Dalbey, R.E . (1993) J . Biol . Chem . 268, 27349-27354), our data indicate that type I signal peptidases and LexA-like proteases are structurally and functionally related serine proteases . A model envisaging a catalytic serine-lysine dyad in prokaryotic type I signal peptidases is proposed to accommodate our observations. J Mol Biol, 1995 Feb 17, 246(2), 273 - 83 Alignment/phylogeny of the papain superfamily of cysteine proteases; Berti PJ et al.; An alignment/phylogeny of the papain superfamily of cysteine proteases was created using an initial structure-based alignment followed by successive iterations of sequence alignment and phylogenetic inference . The iterative approach resulted in significant improvements in the alignment/phylogeny . There were three groups of cysteine proteases that were distantly related and which could be aligned against each other only in the active site regions: the papain group, which included such stereotypical cysteine proteases as cathepsins B, C, H, L and S; and the bleomycin hydrolase and calpain groups . There was one bacterial sequence in each of the bleomycin hydrolase and calpain groups . The former probably arose by lateral gene transfer, the latter possibly by direct evolution from an ancestral protease predating the eukaryote/prokaryote divergence . The phylogeny of the papain group indicated that many families diverged almost simultaneously early during eukaryotic evolution . In mammals there are at least 12 distinct families of cysteine proteases, possibly many more, including at least two as yet uncharacterized enzymes. Oncogene, 1995 Feb 16, 10(4), 757 - 63 The product of the NF2 tumour suppressor gene localizes near the plasma membrane and is highly expressed in muscle cells; den Bakker MA et al.; Neurofibromatosis type 2 (NF2) is a disease resulting in the formation of schwannomas of the eighth cranial nerve, and other central nervous system tumours . A tumour suppressor gene has been found to be responsible for this disorder . The 595 amino acid NF2 protein shows a great deal of homology to a superfamily of membrane organizing proteins . To generate antibodies against the NF2 protein four synthetic peptides (SP) were injected in rabbits . COS cells transfected with an NF2 cDNA construct in an expression vector were used for immunocytochemical staining experiments; lysates of transfected COS cells were used for Western blotting experiments, as were lysates of E . coli cultures transformed with an NF2 cDNA construct subcloned in a prokaryotic expression vector . In western blots all sera detected a band indicating the appropriate molecular weight in lysates of transfected COS cells and E . coli . Immunocytochemical staining experiments indicate that the NF2 protein localizes in or near the cell membrane . Immunohistochemical staining of human tissue sections demonstrated the presence of the NF2 protein in muscle-, and Sch