Prokaryotes

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 Schwann cells . These results support the hypothesis that the NF2 protein functions as a membrane organizing element.

Genes Dev, 1995 Feb 15, 9(4), 423 - 36
Identification and characterization of genes that are required for the accelerated degradation of mRNAs containing a premature translational termination codon; Cui Y et al.; In both prokaryotes and eukaryotes nonsense mutations in a gene can enhance the decay rate of the mRNA transcribed from the gene, a phenomenon described as nonsense-mediated mRNA decay . In yeast, the products of the UPF1 and UPF3 genes are required for this decay pathway, and in this report we focus on the identification and characterization of additional factors required for rapid decay of nonsense-containing mRNAs . We present evidence that the product of the UPF2 gene is a new factor involved in this decay pathway . Mutation of the UPF2 gene or deletion of it from the chromosome resulted in stabilization of nonsense-containing mRNAs, whereas the decay of wild-type transcripts was not affected . The UPF2 gene was isolated, and its transcript was characterized . Our results demonstrate that the UPF2 gene encodes a putative 126.7-kD protein with an acidic region at its carboxyl terminus (-D-E)n found in many nucleolar and transcriptional activator proteins . The UPF2 transcript is 3600 nucleotides in length and contains an intron near its 5' end . The UPF2 gene is dispensable for vegetative growth, but upf2 delta strains were found to be more sensitive to the translational elongation inhibitor cycloheximide than UPF2+ . A genetic analysis of other alleles proposed to be involved in nonsense-mediated mRNA decay revealed that the UPF2 gene is allelic to the previously identified sua1 allele, a suppressor of an out-of-frame ATG insertion shown previously to reduce translational initiation from the normal ATG of the CYC1 gene . In addition, we demonstrate that another suppressor of this cyc1 mutation, sua6, is allelic to upf3, a previously identified lesion involved in nonsense-mediated mRNA decay.

Gene, 1995 Feb 14, 153(2), 185 - 9
Hepatitis C virus core protein: synthesis, affinity purification and immunoreactivity with infected human sera; Khanna A et al.; The genomic region encoding the core (C) protein (amino acids 1-162) of hepatitis C virus (HCV) was expressed in Escherichia coli as a recombinant (re-) protein with the maltose-binding protein (MBP) using the prokaryotic expression vector pMAL-CR1 . The fusion protein (C::MBP) was identified as a approx . 62-kDa polypeptide by immunoblot analysis using antiserum to MBP and HCV-infected human sera . The size of C::MBP corresponded to the calculated combined molecular mass of the approx . 20-kDa HCV C protein and the approx . 42-kDa MBP . The approx . 62-kDa C::MBP was purified using amylose resin as a matrix in affinity chromatography, and showed specific reactivity with HCV-infected human sera . These results suggest that C::MBP may serve as a source of the core antigen for immunological studies on HCV infection.

J Theor Biol, 1995 Feb 7, 172(3), 279 - 91
Identification and simulation of shifted periodicities common to protein coding genes of eukaryotes, prokaryotes and viruses; Arques DG et al.; The distribution of nucleotides in protein coding genes is studied with autocorrelation functions . The autocorrelation function YRY(N)iYRY, analysing the occurrence probability of the i-motif YRY(N)iYRY (two motifs YRY separated by any i bases N, R = purine = Adenine or Guanine, Y = pyrimidine = Cytosine or Thymine, N = R or Y) in the protein coding genes of eukaryotes, prokaryotes and viruses, reveals the classical periodicity 0 modulo 3 associated with the normal frame 0 (maximal values of the function at i = 0, 3, 6, etc) . The specification of YRY(N)iYRY on the alphabet {A, C, G, T} leads to 64 i-motifs: CAC(N)iCAC, CAC(N)iCAT, ..., TGF(N)iTGT . The 64 autocorrelation functions associated with these 64 i-motifs in protein coding genes have all the periodicity modulo 3, but, surprisingly, not always the expected periodicity 0 modulo 3 . Two new types of periodicities are identified: a periodicity 1 modulo 3 associated with the shifted frame +1 (maximal values of the function at i = 1, 4, 7, etc) and a periodicity 2 modulo 3 associated with the shifted frame -1 (maximal values of the function at i = 2, 5, 8 etc) . Furthermore, the classification of i-motifs according to the type of periodicity demonstrates a strong coherence relation between the 64 i-motifs, which is, in addition, common to the three gene populations, as the same i-motifs in the three gene populations have the same periodicities . The three periodicities 0, 1 and 2 modulo 3 can be simulated by an evolutionary model at two successive processes . The simulated genes are generated by a process of gene construction, with a stochastic automaton followed by a process of gene evolution with random insertions and deletions of trinucleotides simulating RNA editing . For almost all i-motifs, the autocorrelation functions in these simulated genes are strongly correlated with those in protein coding genes, for both the type and the probability level of periodicities . This paper describes the process of ribosomal frameshifting leading to the shifted periodicities, which may reveal overlapping genes or concatenated genes from different frames . It also presents the evolutionary aspects of the shifted periodicities . The shifted periodicities cannot be associated with the RNY model (Eigen & Schuster, 1978, Naturwissenschaften 65, 341-369) or the RRY model (Crick et al., 1976, Origins of Life 7, 389-397), but are compatible with the oligonucleotide mixing model (Arques & Michel, 1990, Bull . math . Biol . 52, 741-772) . Finally, a variant of the primitive translation model of Crick et al . (1976) is proposed to explain the shifted periodicities.

Gene, 1995 Feb 3, 153(1), 123 - 7
Identification of four new prokaryotic bacterioferritins, from Helicobacter pylori, Anabaena variabilis, Bacillus subtilis and Treponema pallidum, by analysis of gene sequences; Evans DJ Jr et al.; The nucleotide (nt) sequence of the Helicobacter pylori (Hp) napA gene, encoding neutrophil-activating protein A (HPNAP) was determined . Alignment of this sequence with those of known bacterioferritins (Bfr) revealed sequence homology and conservation of a 7-amino-acid (aa) motif constituting the ferroxidase (Frx) center of Bfr in the HPNAP . The N-terminal aa sequence deduced from the iron-regulated mrgC gene of Bacillus subtilis {Chen et al., J . Bacteriol . 175 (1993) 5428-5437} is highly similar to that of HPNAP and contains five Frx center aa residues . The deduced aa sequences for proteins of unknown function in Treponema pallidum {Walfield et al., Infect . Immun . 57 (1989) 633-635} and in the cyanobacterium Anabaena variabilis {Sato, GenBank accession No . JU0384 (1991)} identify these two proteins as Bfr . Although the DNA-binding protein from starved cells of Escherichia coli {Almiron et al., Genes Dev . 6 (1992) 2646-2654} is clearly a HPNAP/Bfr homologue, a significant part of its Frx center is missing . It is unlikely that the intracellular function of HPNAP is related to its ability to activate neutrophils.

J Chromatogr B Biomed Appl, 1995 Feb 3, 664(1), 241 - 6
In vitro toxicity assays for dye ligands used in affinity chromatography; Santambien P et al.; Some reactive textile dyes have been used for years as biomimetic ligands in protein purification . There has been reluctance, however, to use these dyes on a large scale for therapeutically applicable proteins for fear of possible dye leakage and consequent contamination . Therefore, toxicological data are necessary to quantify the level of this hazard . This study deals with a series of in vitro toxicity investigations with eukaryotic cells (growth, polyploidy, etc.) and with prokaryotic cells (Escherichia coli) for genotoxic studies . Both approaches demonstrated a lack of or slight toxicity for Reactive Blue 2 and Reactive Red 120 and their derivatives over the range 10-62.5 micrograms/ml in several assays.

Eur J Biochem, 1995 Feb 1, 227(3), 734 - 44
Membrane lipid composition and cell size of Acholeplasma laidlawii strain A are strongly influenced by lipid acyl chain length; Wieslander A et al.; The small, cell-wall-less prokaryote Acholeplasma laidlawii strain A-EF22 could grow with membrane lipids having an average acyl chain length Cn varying over 14.5- almost 20 carbons by exogenous supplementation with selected fatty acids . For 16 < Cn < 18, the cells grew with lipids containing 100% (mol/100 mol) monounsaturated acyl chains, whereas for Cn < 16 and Cn > 18, cell growth only occurred with gradually lower fractions of unsaturated chains . Cn was actively increased and decreased by chain elongation or de novo fatty acid synthesis upon incorporation of short-chain and long-chain fatty acids, respectively . The membrane lipid composition was strongly affected by the acyl chain length and unsaturation, and the metabolic responses are readily explained as a regulation mechanism based on the established phase equilibria of the individual lipids in the A . laidlawii membrane . Monoglucosyldiacylglycerol (Glc-acyl2-Gro) was the dominating lipid with short chains but the fraction of this lipid decreased with increasing Cn, correlating with the decreasing lamellar to nonlamellar phase transition temperatures for this lipid . The fractions of diglucosyldiacylglycerol (Glc2-acyl2Gro) and phosphatidylglycerol (PtdGro), forming lamellar phases only, increased with increasing Cn over the entire chain-length interval . A weaker correlation was usually observed between the relative amount of a lipid and the extent of chain unsaturation; however, the fractions of Glc2-acyl2Gro and PtdGro increased clearly with an increasing degree of unsaturation . Moreover, the synthesis of the nonbilayer-forming lipids acyl2Gro and monoacyl-Glc-acyl2Gro was strongly stimulated by a high degree of chain saturation . Concomitantly, the phase equilibria of Glc-acyl2Gro are shifted towards lamellar phases at the growth temperature . The fraction of the three potentially nonbilayer-forming lipids varied over 10-80% (mol/100 mol) total lipids as a function of the acyl chain composition . The combined molar fractions of the three phospholipids increased strongly with chain unsaturation . However, the fraction of phosphate moieties in the different lipids was constant over the entire chain-length interval . It is concluded that the regulation of the membrane lipid composition aims at maintaining similar phase equilibria and surface charge densities of the lipid bilayer . The size of A . laidlawii cells was changed in a systematic manner and correlated qualitatively with the packing properties of the lipids . Cell diameters were increased by an increase in acyl chain length and saturation, and was affected by additives such an n-dodecane and acyl2Gro.

J Gen Virol, 1995 Feb, 76 ( Pt 2), 437 - 43
Identification of a novel viral protein in infectious bursal disease virus-infected cells; Mundt E et al.; Infectious bursal disease virus (IBDV), a member of the Birnaviridae, specifies two genomic double-stranded RNAs, segment A and segment B . Segment A encodes a 110 kDa polyprotein which is processed into virus proteins VP2, VP3 and VP4 . A second open reading frame (ORF), designated ORF A-2, immediately preceding and partially overlapping the 110 kDa protein gene has also been described . After prokaryotic expression of this ORF and immunization of rabbits with the expressed protein we obtained reagents that allowed the identification of the ORF A-2 gene product in IBDV-infected cells . The ORF A-2 protein exhibits an apparent molecular mass of 21 kDa which is larger than the size of 16.5 kDa calculated from the deduced amino acid sequence . Immunofluorescence studies demonstrated the presence of the ORF A-2 protein in bursa samples from IBDV-infected chicken . In summary, the IBDV ORF A-2 product represents the fifth IBDV protein described . Therefore, we propose to designate it as IBDV VP5.

J Bacteriol, 1995 Feb, 177(3), 792 - 8
Saccharomyces cerevisiae has a single glutamate synthase gene coding for a plant-like high-molecular-weight polypeptide; Cogoni C et al.; Purification of the glutamate synthase (GOGAT) enzyme from Saccharomyces cerevisiae showed that it is an oligomeric enzyme composed of three identical 199-kDa subunits . The GOGAT structural gene was isolated by screening a yeast genomic library with a yeast PCR probe . This probe was obtained by amplification with degenerate oligonucleotides designed from conserved regions of known GOGAT genes . The derived amino-terminal sequence of the GOGAT gene was confirmed by direct amino-terminal sequence analysis of the purified protein of 199 kDa . Northern (RNA) analysis allowed the identification of an mRNA of about 7 or 8 kb . An internal fragment of the GOGAT gene was used to obtain null GOGAT mutants completely devoid of GOGAT activity . The results show that S . cerevisiae has a single NADH-GOGAT enzyme, consisting of three 199-kDa monomers, that differs from the one found in prokaryotic microorganisms but is similar to those found in other eukaryotic organisms such as alfalfa.

FASEB J, 1995 Feb, 9(2), 167 - 74
The actin fold; Kabsch W et al.; X-ray structure analysis of actin and of the NH2-terminal domain of the heat-shock cognate protein Hsc70 has revealed an unexpected extensive structural similarity between these two molecules . Despite the absence of significant similarity of their amino acid sequences, both proteins share the same core architecture and a common nucleotide binding site resembling the structure of hexokinase . All three are ATPases or kinases and bind ATP in association with Mg2+ or Ca2+ . The common fold consists of two alpha/beta domains, which are connected by a putative hinge with an ATP-binding site situated between the domains . Each domain contains a five-stranded beta-sheet of identical topology, which suggests that the molecules may have evolved by gene duplication . From a comparison of the three aligned structures, a fingerprint sequence of the adenine nucleotide binding pocket was derived, which predicted that members of the glycerol kinase family should also have a similar fold of their nucleotide binding domain . This was later confirmed when the X-ray structure was published . Data base search with a refined consensus sequence has retrieved other sugar kinases, as well as the prokaryotic cell cycle proteins FtsA, MreB, and StbA, and two Escherichia coli phosphatases . These proteins are predicted to possess a structure similar to actin in the common core region . As exemplified for actin, Hsc70, and glycerol kinase, the diversity of biological function is provided by the polymorphism of the loops joining the beta-strands and helices in the core region and by inserted domains that show high variability.

Curr Opin Struct Biol, 1995 Feb, 5(1), 4 - 10
DNA modification by methyltransferases; Cheng X; Enzymatic methylation of DNA plays important roles in both prokaryotes and eukaryotes . Structural study of the HhaI DNA methyltransferase has provided considerable insight into the chemistry of C5-cytosine methylation . The DNA-protein complex reveals a substrate cytosine flipped out of the double helix during the reaction, and a novel two-loop DNA-binding motif used for both sequence recognition and flipping the base . Structural comparison of HhaI C5-cytosine methyltransferase, TaqI N6-adenine methyltransferase, and catechol O-methyltransferase reveals a common catalytic domain structure, which might be universal among S-adenosyl-L-methionine (SAM)-dependent methyltransferases.

Pharmacogenetics, 1995 Feb, 5(1), 1 - 17
Nomenclature for N-acetyltransferases; Vatsis KP et al.; A consolidated classification system is described for prokaryotic and eukaryotic N-acetyltransferases in accordance with the international rules for gene nomenclature . The root symbol (NAT) specifically identifies the genes that code for the N-acetyltransferases, and NAT* loci encoding proteins with similar function are distinguished by Arabic numerals . Allele characters, denoted by Arabic numbers or by a combination of Arabic numbers and uppercase Latin letters, are separated from gene loci by an asterisk, and the entire gene-allele symbols are italicized . Alleles at the different NAT* loci have been numbered chronologically irrespective of the species of origin . For designation of genotypes at a single NAT* locus, a slash serves to separate the alleles; in phenotype designations, which are not italicized, alleles are separated by a comma.

Immunol Cell Biol, 1995 Feb, 73(1), 73 - 80
Infection of human and murine macrophages with Leishmania major is associated with early parasite heat shock protein synthesis but fails to induce a host cell stress response; Kantengwa S et al.; Heat shock/stress proteins (HSP) represent the most conserved proteins expressed in prokaryotes and eukaryotes . These constitutive and inducible proteins function as molecular chaperones and are part of virulence factors . They participate in self/non-self discrimination and may protect phagocytes from the toxic effects of the reactive oxygen species generated by these cells during bacterial phagocytosis and infection . In this study, we investigated the early stress response of host cells {either human alveolar macrophages (AM) or murine peritoneal macrophages (PM)} during infection by an obligate intracellular parasite (Leishmania major), which lives within phagolysosomes . Immunoblotting with specific antibodies demonstrated that L . major had no effect on host stress protein synthesis, but synthesized high levels of its own stress proteins within AM and PM . The lack of induction of a host cell stress response may relate to the failure of L . major to activate the respiratory burst in these cells, whereas the upshift of L . major HSP within macrophages is part of an adaptive response of the parasite to the host.

J Ind Microbiol, 1995 Feb, 14(2), 119 - 25
Cyanobacterial metallothioneins: biochemistry and molecular genetics; Turner JS et al.; Metallothioneins have been extensively studied in many different eukaryotes where they sequester, and hence detoxify, excess amounts of certain metal ions . However, the precise functions of many of these molecules are not fully understood . This article reviews literature concerning their namesakes in prokaryotes.

Hum Mol Genet, 1995 Feb, 4(2), 243 - 9
The human SB1.8 gene (DXS423E) encodes a putative chromosome segregation protein conserved in lower eukaryotes and prokaryotes; Rocques PJ et al.; We report that the human gene SB1.8 (DXS423E) encodes a protein of 1233 amino acids that is highly homologous (30% identity) to the essential yeast protein SMC1 which is required for the segregation of chromosomes at mitosis . Both SB1.8 and SMC1 contain an N-terminal NTP binding site, a central coiled-coil region and a C-terminal helix-loop-helix domain, and have structural features in common with the force generating proteins myosin and kinesin . SB1.8 also exhibits regions of homology and overall structural similarity to the prokaryote (Mycoplasma hyorhinis) protein 115p . Thus SB1.8 and SMC1 are members of a highly conserved and ubiquitous family of proteins that appear to have a fundamental role in cell division . In addition we show that SB1.8 (DXS423E) maps to a cosmid contig that lies centromeric to the OATL2 locus at chromosome Xp11.2.

Plant Mol Biol, 1995 Feb, 27(4), 779 - 88
A protein is involved in accessibility of the inhibitor acetazolamide to the carbonic anhydrase(s) in the cyanobacterium Synechocystis PCC 6803; Beuf L et al.; A gene, zam (for resistance to acetazolamide), controlling resistance to the carbonic anhydrase inhibitor acetazolamide, is described . It has been cloned from a spontaneous mutant, AZAr-5b, isolated from the cyanobacterium Synechocystis PCC 6803, for its resistance to this drug (Bedu et al., Plant Physiol 93: 1312-1315, 1990) . This mutant, besides its resistance to acetazolamide, displayed an absence of catalysed oxygen exchange activity on whole cells, suggestive of a deficiency in carbonic anhydrase activity . The gene was isolated by screening a genomic library of AZAr-5b, and selecting for the capacity to transfer the AZAr phenotype to wild-type cells . A system leading to forced homologous recombination in the host chromosome, using a platform vector, was devised in order to bypass direct selection difficulties . The putative encoded protein, 782 amino acids long, showed some homology with four eukaryotic and prokaryotic proteins involved in different cellular processes, one of them suppressing a phosphatase deficiency . The mutated allele of AZAr-5b showed an in-frame 12 nucleotide duplication, which should not interfere with translation, and might result from transposition of a mobile element . Integration into a wild-type genome of either the spontaneous mutated allele or one inactivated by insertional mutagenesis conferred the character of resistance, but not the deficiency in oxygen exchange, indicating that the two phenotypic aspects of AZAr-5b corresponded to two independent mutations . A working hypothesis explaining the phenotypes of the mutants is that the presence of the Zam protein would be necessary for the inhibitor to reach (one of) the two carbonic anhydrases present in this strain . This, however, would be a secondary action, the physiological role of the protein still being cryptic.

Plant Mol Biol, 1995 Feb, 27(4), 753 - 67
Existence of two ferredoxin-glutamate synthases in the cyanobacterium Synechocystis sp . PCC 6803 . Isolation and insertional inactivation of gltB and gltS genes; Navarro F et al.; The first two genes of ferredoxin-dependent glutamate synthase (Fd-GOGAT) from a prokaryotic organism, the cyanobacterium Synechocystis sp . PCC 6803, were cloned in Escherichia coli . Partial sequencing of the cloned genomic DNA, of the 6.3 kb Hind III and 9.3 kb Cla I fragments, confirmed the existence of two different genes coding for glutamate synthases, named gltB and gltS . The gltB gene was completely sequenced and encodes for a polypeptide of 1550 amino acid residues (M(r) 168,964) . Comparative analysis of the gltB deduced amino acid sequence against other glutamate synthases shows a higher identity with the alfalfa NADH-GOGAT (55.2%) than with the corresponding Fd-GOGAT from the higher plants maize and spinach (about 43%), the red alga Antithamnion sp . (42%) or with the NADPH-GOGAT of bacterial source, such as Escherichia coli (41%) and Azospirillum brasilense (45%) . The detailed analysis of Synechocystis gltB deduced amino acid sequence shows strongly conserved regions that have been assigned to the 3Fe-4S cluster (CX5CHX3C), the FMN-binding domain and the glutamine-amide transferase domain . Insertional inactivation of gltB and gltS genes revealed that both genes code for ferredoxin-dependent glutamate synthases which were nonessential for Synechocystis growth, as shown by the ferredoxin-dependent glutamate synthase activity and western-blot analysis of the mutant strains.

Plant Physiol, 1995 Feb, 107(2), 393 - 400
Purification and cDNA isolation of chloroplastic phosphoglycerate kinase from Chlamydomonas reinhardtii; Kitayama M et al.; Chloroplastic phosphoglycerate kinase (PGK) was purified to homogeneity from a soluble fraction of chloroplasts of a cell-wall-deficient mutant strain of Chlamydomonas reinhardtii (cw-15) using ammonium sulfate fractionation, Reactive Blue-72 column chromatography, and native polyacrylamide gel electrophoresis . PGK activity was attributed to a single polypeptide with a molecular mass of 42 kD . Relative purity and identity of the isolated enzyme was confirmed by N-terminal amino acid sequence determination . Antiserum against this enzyme was raised and a western blot analysis of whole-cell lysate from cw-15 cells using this anti-chloroplastic PGK serum detected a single polypeptide with a molecular mass of 42 kD . The cDNA clone corresponding to the Chlamydomonas chloroplastic PGK was isolated from a Chlamydomonas cDNA expression library using the anti-PGK serum . The cDNA sequence was determined and apparently codes for the entire precursor peptide, which consists of 461 codons . The results from Southern and northern blot analyses suggest that the chloroplastic PGK gene exists as a single copy in the nuclear genome of C . reinhardtii and is expressed as a 1.8-kb transcript . The C . reinhardtii chloroplastic PGK cDNA has 71 and 66% homology with wheat chloroplastic PGK and spinach chloroplastic PGK, respectively . Based on the deduced amino acid sequence, the chloroplastic PGK of C . reinhardtii has more similarity to plant PGKs than to other PGKs, having both prokaryotic and eukaryotic features.

Proc Natl Acad Sci U S A, 1995 Jan 31, 92(3), 791 - 5
Programmed DNA rearrangement of a cyanobacterial hupL gene in heterocysts; Carrasco CD et al.; Programmed DNA rearrangements that occur during cellular differentiation are uncommon and have been described in only two prokaryotic organisms . Here, we identify the developmentally regulated rearrangement of a hydrogenase gene in heterocysts of the cyanobacterium Anabaena sp . strain PCC 7120 . Heterocysts are terminally differentiated cells specialized for nitrogen fixation . Late during heterocyst differentiation, a 10.5-kb DNA element is excised from within the hupL gene by site-specific recombination between 16-bp direct repeats that flank the element . The predicted HupL polypeptide is homologous to the large subunit of {NiFe} uptake hydrogenases . hupL is expressed similarly to the nitrogen-fixation genes; hupL message was detected only during the late stages of heterocyst development . An open reading frame, named xisC, identified near one end of the hupL DNA element is presumed to encode the element's site-specific recombinase . The predicted XisC polypeptide is homologous with the Anabaena sp . strain PCC 7120 site-specific recombinase XisA . Neither XisC nor XisA shows sequence similarity to other proteins, suggesting that they represent a different class of site-specific recombinase.

Philos Trans R Soc Lond B Biol Sci, 1995 Jan 30, 347(1319), 5 - 12
RecA protein mediates homologous recognition via non-Watson-Crick bonds in base triplets; Rao BJ et al.; E . coli RecA protein, the prototype of a class, forms a helical nucleoprotein filament on single-stranded DNA that recognizes homology in duplex DNA, and initiates the exchange of strands in homologous recombination . The discovery of this reaction some years ago posed a quandary on how a third strand recognizes homology in duplex DNA, whose Watson-Crick bonds face inward in a hydrophobic core of stacked bases . Recent studies have shown that RecA protein promotes homologous recognition via non-Watson-Crick bonds in base triplets . The intermediates in the RecA reaction differ distinctly from triplex DNA that forms non-enzymically . The biological significance of the novel set of DNA interactions by which RecA protein effects homologous recognition is indicated by the importance of this protein in recombination, and the widespread distribution of homologous proteins in prokaryotes and eukaryotes.

J Biol Chem, 1995 Jan 27, 270(4), 1859 - 65
Cloning and sequence analysis of the human mitochondrial translational initiation factor 2 cDNA; Ma L et al.; Complete cDNAs encoding human mitochondrial translational initiation factor 2 (IF-2mt) have been obtained from liver, heart, and fetal brain cDNA libraries . These cDNAs have a long open reading frame 2181 residues in length encoding a protein of 727 amino acids . Overall, human IF-2mt has 30-40% identity to the corresponding prokaryotic factors . Surprisingly, it is no more homologous to yeast IF-2mt than to the IF-2s from bacterial sources . The greatest region of conservation lies in the G-domain of this factor with less conservation in the COOH-terminal half of the protein and very little homology near the amino terminus . The 5'-untranslated leaders of the liver and heart cDNAs contain a number of short open reading frames . These sequences may play a role in the translational activity of the IF-2mt mRNA . Northern analysis indicates that the IF-2mt gene is expressed in all tissues but that the level of expression varies over a wide range.

Nucleic Acids Res, 1995 Jan 25, 23(2), 256 - 60
One short well conserved region of Alu-sequences is involved in human gene rearrangements and has homology with prokaryotic chi; Rudiger NS et al.; Alu elements have repeatedly been found involved in gene rearrangements in humans . Although these elements have been suggested to stimulate gene rearrangements, sparse information is available for the possible mechanism(s) of these events . Here we present a compilation of Alu elements that have been involved in recombinational events leading to gene rearrangements, indicating the presence of a common 26 bp core sequence at or close to the sites of recombination . Besides the obvious possibility of retrotransposition, gene rearrangements may be induced by sequences that stimulate genetic recombination . We suggest that the core sequence stimulates recombination and may thereby cause the frequent involvement of these elements in gene rearrangements . Curiously, the core sequence contains the pentanucleotide motif CCAGC, which is also part of chi, an 8 bp sequence known to stimulate recBC mediated recombination in Escherichia coli.

Nucleic Acids Res, 1995 Jan 25, 23(2), 203 - 10
The fission yeast gene pmt1+ encodes a DNA methyltransferase homologue; Wilkinson CR et al.; DNA methylation of cytosine residues is a widespread phenomenon and has been implicated in a number of biological processes in both prokaryotes and eukaryotes . This methylation occurs at the 5-position of cytosine and is catalyzed by a distinct family of conserved enzymes, the cytosine-5 methyltransferases (m5C-MTases) . We have cloned a fission yeast gene pmt1+ (pombe methyltransferase) which encodes a protein that shares significant homology with both prokaryotic and eukaryotic m5C-MTases . All 10 conserved domains found in these enzymes are present in the pmt1 protein . This is the first m5C-MTase homologue cloned from a fungal species . Its presence is surprising, given the inability to detect DNA methylation in yeasts . Haploid cells lacking the pmt1+ gene are viable, indicating that pmt1+ is not an essential gene . Purified, bacterially produced pmt1 protein does not possess obvious methyltransferase activity in vitro . Thus the biological significance of the m5C-MTase homologue in fission yeast is currently unclear.

Proc Natl Acad Sci U S A, 1995 Jan 17, 92(2), 636 - 40
Cyanobacterial protein with similarity to the chlorophyll a/b binding proteins of higher plants: evolution and regulation; Dolganov NA et al.; We have isolated, from the prokaryotic cyanobacterium Synechococcus sp . strain PCC 7942, a gene encoding a protein of 72 amino acids {designated high light inducible protein (HLIP)} with similarity to the extended family of eukaryotic chlorophyll a/b binding proteins (CABs) . HLIP has a single membrane-spanning alpha-helix, whereas both the CABs and the related early light inducible proteins have three membrane-spanning helices . Hence, HLIP may represent an evolutionary progenitor of the eukaryotic members of the CAB extended family . We also show that the gene encoding HLIP is induced by high light and blue/UV-A radiation . The evolution, regulation, and potential function of HLIP are discussed.

Proc Natl Acad Sci U S A, 1995 Jan 17, 92(2), 557 - 60
Periodic recurrence of methionines: fossil of gene fusion?
Kolker E, Trifonov EN.
As we have recently shown, approximately 20% of proteins are made of uniform size units of approximately 123 aa for eukaryotes and approximately 152 aa for prokaryotes . Such regularity may reflect certain past events in protein evolution by fusion (molecular recombination) of a spectrum of standard-size protein-coding DNA segments--the early genes . Consequently, methionines, as start residues, would mark those locations in proteins that correspond to the DNA recombination sites--the borders between the fused genes . This positional preference of the methionines may still survive as a fossil of the early protein sequence organization . In this study we address the question how methionines are distributed in modern protein sequences . This analysis of eukaryotic sequences shows that methionine residues do preferentially appear at the positions corresponding to the multiples of the unit size, as predicted.

Biochemistry, 1995 Jan 17, 34(2), 648 - 55
Identification of N2-(1-carboxyethyl)guanine (CEG) as a guanine advanced glycosylation end product; Papoulis A et al.; Reducing sugars such as glucose react nonenzymatically with protein amino groups to initiate a posttranslational modification process known as advanced glycosylation . Nucleotide bases also participate in advanced glycosylation reactions, producing DNA-linked advanced glycosylation endproducts (AGEs) that cause mutations and DNA transposition . Although several protein-derived AGEs have been isolated and structurally characterized, AGE-modified nucleotides have not yet been reported . We systematically examined the reactivities of the model nucleotide bases 9-methylguanine (9-mG), 9-methyladenine (9-mA), and 1-methylcytosine (1-mC) toward glucose and several glucose-derived reactants . In "fast" reactions performed at refluxing temperature and physiological pH, 1 equiv of nucleotide base was reacted with 10 equiv of D-glucose, D-glucose 6-phosphate (G-6-P), D-glucose 6-phosphate/lysine (G-6-P/Lys), the Schiff base 1-n-propylamino-N-D-glucoside (SB), or the Amadori product 1-n-propylamino-N-D-fructose (AP) . In every reaction involving 9-mG, N2-(1-carboxyethyl)-9-methylguanine (CEmG) was a major product which was produced . N2-(1-carboxyethyl)-9-methylguanine also formed from 9-mG and AP in long-term incubations performed at 37 degrees C . Direct treatment of 9-mG with methylglyoxal (MG), a Maillard reaction propagator that forms from the decomposition of AP, also produced CEmG in high yield . N2-(1-Carboxyethyl)-9-methylguanine appears to result from the nucleophilic addition of the primary amino group of guanine to the ketone group of MG followed by an intramolecular rearrangement . Methylglyoxal is a known prokaryotic mutagen and was shown additionally to be mutagenic in a eukaryotic shuttle vector assay system.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Jan 17, 34(2), 391 - 6
Isolation and in vitro phosphorylation of sensory transduction components controlling anaerobic induction of light harvesting and reaction center gene expression in Rhodobacter capsulatus; Inoue K et al.; Anaerobic induction of light harvesting and reaction center gene expression involves two transacting factors termed RegA and RegB . Sequence and mutational analysis has indicated that RegA and RegB constitute cognate components of a prokaryotic sensory transduction cascade with RegB comprising a membrane-spanning sensor kinase and RegA a cytosolic response regulator . In this study we have purified RegA, as well as a truncated portion of RegB (RegB') and undertaken an in vitro analysis of autophosphorylation and phosphotransfer activities . Incubation of RegB' with {gamma-32P}ATP and MgCl2 resulted in phosphorylation of RegB' (RegB' approximately P) over a 20-min incubation period . Incubation of RegB' approximately P with RegA resulted in rapid transfer of the phosphate from RegB' to RegA . In analogy to other characterized prokaryotic sensory transduction components, mutational and chemical stability studies also indicate that RegB' is autophosphorylated at a conserved histidine and that RegA accepts the phosphate from RegB at a conserved aspartate.

FEMS Microbiol Lett, 1995 Jan 15, 125(2-3), 149 - 58
rRNA operon restriction derived taxa for Streptomyces (RiDiTS); Fulton TR et al.; A method for grouping Streptomyces strains by fingerprints of their rRNA operons is described . In polyacrylamide gels, multicopy rRNA operon fragments in Streptomyces genomic MseI fingerprints produced intense bands which are well resolved from the less conspicuous low copy fragments interspersed between them . The high intensity multicopy rRNA bands are easily distinguished from the low intensity bands, eliminating the need for Southern blot hybridization to visualize the rRNA fragments . Direct evidence that the high-intensity bands in these polyacrylamide gels originated from rRNA operons was provided by a 'differential' Southern blot technique . We have used this method to assign 98 strains to 11 rRNA fingerprint type groups . This clustering method may be applicable to any prokaryote with a high G+C content genome.

Eur J Biochem, 1995 Jan 15, 227(1-2), 214 - 25
Molecular cloning and sequence of two novel P-type adenosinetriphosphatases from Plasmodium falciparum; Trottein F et al.; We have identified two novel P-type ATPase genes from Plasmodium falciparum and report the full-length nucleotide and derived amino acid sequence of the ATPase2 gene from P . falciparum (PfATPase2) . PfATPase2 is phylogenetically remote from the different members of prokaryotic and mammalian P-type ATPases but shares features with a putative membrane-spanning Ca2+ ATPase involved in ribosome function in yeast . PfATPase2 is expressed during the intraerythrocytic life cycle of the parasite and appears to be required in the late stages of its asexual development . We also present the partial sequence of another malarial gene displaying sequence similarity with the family of P-type transporting ATPases (PfATPase4) . We have analysed the organisation of the genes encoding the P-type ATPases of P . falciparum and show that they are a highly dispersed gene family.

Virology, 1995 Jan 10, 206(1), 707 - 12
Analysis of African cassava mosaic virus recombinants suggests strand nicking occurs within the conserved nonanucleotide motif during the initiation of rolling circle DNA replication; Stanley J; Intact clones containing partial repeats of the genomic components of African cassava mosaic (ACMV DNAs A and B) are infectious when mechanically coinoculated onto Nicotiana benthamiana . Monomeric genomic components may be generated either by homologous recombination or, when two copies of the origin of replication (ori) are present, by a modified rolling circle replication mechanism in which nascent single-stranded DNA is resolved by the introduction of nicks at both oris . DNA B partial repeats with duplicated common region sequences containing combinations of wild-type sequences and nonlethal mutations at nucleotides 151 and 155 within the putative stem-loop region have been constructed and introduced into plants in the presence of DNA A . Analysis of progeny indicates that monomers are generated by DNA strand nicking preferentially between nucleotides 151 and 155, suggesting a nonrandom replicative release mechanism involving the ubiquitous TAATATTAC motif (nucleotides 146-154) . Viable ACMV DNA A deletion mutants are known to revert to wild-type size during systemic infection by generating tandem repeats . The recombination point in one such revertant has been mapped between nucleotides 152 and 153 . Just as ori-nicking enzymes mediate recombinational events during prokaryotic rolling circle DNA replication, the result suggests that a nick has been introduced in the virion-sense strand within the nonanucleotide motif (TAATATT decreases AC) during the initiation of ACMV DNA replication.

J Biol Chem, 1995 Jan 6, 270(1), 437 - 44
Riboflavin biosynthesis in Saccharomyces cerevisiae . Cloning, characterization, and expression of the RIB5 gene encoding riboflavin synthase; Santos MA et al.; Saccharomyces cerevisiae has a monofunctional riboflavin synthase that catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine . We have isolated the gene encoding this enzyme from a yeast genomic library by functional complementation of a mutant, rib5-10, lacking riboflavin synthase activity . Deletion of the chromosomal copy of RIB5 led to riboflavin auxotrophy and loss of enzyme activity . Intragenic complementation between point and deletion mutant alleles suggested that the encoded protein (Rib5p) assembles into a multimeric complex and predicted the existence of a discrete functional domain located at the N terminus . Nucleotide sequencing revealed a 714-base pair open reading frame encoding a 25-kDa protein . Rib5p was purified to apparent homogeneity by a simple procedure . The specific activity of the enzyme was enriched 8500-fold . The N-terminal sequence of the purified enzyme was identical to the sequence predicted from the nucleotide sequence of the RIB5 gene . Initial structural characterization of riboflavin synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a trimer of identical 25-kDa subunits . The derived amino acid sequence of RIB5 shows extensive homology to the sequences of the alpha subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes . In addition, the sequence also shows internal homology between the N-terminal and the C-terminal halves of the protein . Taken together, these results suggest that the Rib5p subunit contains two structurally related (substrate-binding) but catalytically different (acceptor and donator) domains.

FEBS Lett, 1995 Jan 2, 357(1), 79 - 82
Purification and characterization of an iron superoxide dismutase from the nitrogen-fixing Azotobacter vinelandii; Pagani S et al.; Two electrophoretically distinct forms of superoxide dismutase (SOD; EC 1.15.1.1) which show different inhibition patterns to hydrogen peroxide have been identified in Azotobacter vinelandii . The SOD inhibited by hydrogen peroxide was purified to homogeneity, and turned out to be an iron superoxide dismutase . The enzyme is present in only one molecular form with an isoelectric point of 4.1, and it is composed of two identical subunits with an apparent molecular weight of 21,000 Da . Spectroscopic analyses indicated that this enzyme contains ferric iron (1.4-1.6 mol/mol protein) in the typical high-spin form present in other prokaryotic Fe-SODs . N-Terminal sequence alignments (up to the 49th residue) showed that A . vinelandii Fe-SOD has high similarity with other prokaryotic Fe-SODs.

Mol Biol Rep, 1995-96, 22(2-3), 177 - 80
Enzymatic RNA synthesis and RNase P . Evolutionary aspects; Krupp G; TransferRNA recognition was used as leit-motiv in the illustration of possible links between a hypothetical primordial RNA world and the contemporary DNA world . In an RNA world, 'proto-tRNA' could have functioned as replication origin and as primitive telomere . Possibly, this primitive structure is preserved in a 'universal substrate' for modern tRNA-specific enzymes . The combination of acceptor stem and T arm (plus a linker) was finally revealed as sufficient for the recognition by prokaryotic and eukaryotic RNase P, as well as other tRNA enzymes . In modern life forms, a tRNA-like element in viral RNAs still serves as replication origin, and furthermore, the recognition of similar structures as cryptic promoters is universally conserved for template-dependent RNA polymerases . Another common property of modern polymerases is their high, but clearly limited and condition-dependent substrate specificity . Very likely, also substrate recognition by primitive polymerases was not more stringent, and this lead to the occurrence of mixed nucleic acids as intermediates in the transition of genomic RNA to contemporary genomic DNA.

Mol Biol Rep, 1995-96, 22(2-3), 139 - 45
Ribonuclease P from plant nuclei and photosynthetic organelles; Schon A; RNase P consists of both protein and RNA subunits in all organisms and organelles investigated so far, with the exception of chloroplasts and plant nuclei where no enzyme-associated RNA has been detected to date . Studies on substrate specificity revealed that cleavage by plant nuclear RNase P is critically dependent on a complete and intact structure of the substrate . No clearcut answer is yet possible regarding the order of processing events at the 5' or 3' end of tRNAs in the case of nuclear or chloroplast processing enzymes . RNase P from a phylogenetically ancient photosynthetic organelle will be discussed in greater detail: The enzyme from the Cyanophora paradoxa cyanelle is the first RNase P from a photosynthetic organelle which has been shown to contain an essential RNA subunit . This RNA is strikingly similar to its counterpart from cyanobacteria, yet it lacks catalytic activity . Properties of the holoenzyme suggest an intermediate position in RNA enzyme evolution, with an Eukaryotic-type, inactive RNA and a prokaryotic-type small protein subunit . The possible presence of an RNA component in RNase P from plant nuclei and modern chloroplasts will be discussed, including a critical evaluation of some criteria that have been frequently applied to elucidate the subunit composition of RNase P from different organisms.

Tsitologiia, 1995, 37(11), 985 - 1009
Progress in understanding the phylogeny of flagellates; Sleigh MA; Heterotrophic free-living flagellates appear to provide the ancestry for all other eukaryote groups . Not only are the oldest surviving anaerobic eukaryotes flagellated protists, but also there appear to be survivors of a lineage of flagellate forms which could have been close to the sources of the main branches of eukaryote evolution . These 'stem' forms of flagellates developed more complex flagellation with anchoring fibres which became the main components of the cytoskeleton and supported the cytostome; by their phagotrophic activities these flagellates established symbiotic relationships, first with aerobic bacteria to form mitochondria, and later with various forms of prokaryotic and eukaryotic algal cells to form chloroplasts of a variety of types having different pigments, different structure and different food storage patterns . The specific patterns of flagellation, cytoskeleton, cytostome, secreted surface structures and cell aggregation into colonies enable groups of organisms to be recognised, whose most primitive survivors in almost all cases are heterotrophic flagellates . The current view of the phylogeny of eukaryotes suggests that heterotrophic flagellates have provided the origins of all major eukaryote groups, and that the phylogeny of these flagellates is the key to understanding the evolution of all eukaryotes . We anticipate that further rRNA analyses, supported by ultrastructural data, will confirm the central role played by these flagellates in eukaryote evolution.

Cell Mol Biol Res, 1995, 41(5), 461 - 5
Bacterial aspartate kinase-like activity in human platelet; Arenas-Diaz G et al.; One form of a group of enzymes known as aspartate kinases, primarily reported in prokaryotes and plants, might also exist in animal cells . Here we report the immunodetection of an aspartate kinase-like activity in human platelets using antibodies against the pure form of the enzyme purified from Escherichia coli . Moreover, the enrichment of platelet extracts with the bacterial kinase results in the phosphorylation of discrete forms mainly of membrane-bound endogenous polypeptides.

Cell Mol Biol Res, 1995, 41(5), 391 - 5
Melatonin immunoreactivity in the photosynthetic prokaryote Rhodospirillum rubrum: implications for an ancient antioxidant system; Manchester LC et al.; Rhodospirillum rubrum is a spiral anoxygenic photosynthetic bacterium that can exist under either aerobic or anaerobic conditions . The organism thrives in the presence of light or complete darkness and represents one of the oldest species of living organisms, possibly 2-3.5 billion years old . The success of this prokaryotic species may be attributed to the evolution of certain indole compounds that offer protection against life-threatening oxygen radicals produced by an evolutionary harsh environment . Melatonin, N-acetyl-5-methoxytryptamine, is an indolic highly conserved molecule that exists in protists, plants, and animals . This study was undertaken to determine the presence of an immunoreactive melatonin in the kingdom Monera and particularly in the photosynthetic bacterium, R . rubrum, under conditions of prolonged darkness or prolonged light . Immunoreactive melatonin was measured during both the extended day and extended night . Significantly more melatonin was observed during the scotophase than the photophase . This study marks the first demonstration of melatonin in a bacterium . The high level of melatonin observed in bacteria may provide on-site protection of bacterial DNA against free radical attack.

Acta Biochim Pol, 1995, 42(4), 381 - 93
Topoisomerase II as a target for anticancer chemotherapy; Kaufmann SH et al.; Type II DNA topoisomerases are required for the segregation of genomic DNA at cell division in prokaryotic and eukaryotic cells, and inhibitors of these enzymes are potential cytotoxic agents in both prokaryotes and eukaryotes . The bacterial member of the topoisomerase II family, DNA gyrase, and the chemotherapeutic agents which target it are the subject of a recent review (Maxwell, A . et al., 1993, in Molecular Biology of DNA Topoisomerases, Andoh, T . et al., eds., pp . 21-30, CRC Press, Boca Raton) . Here we present an overview of current knowledge of eukaryotic topoisomerase II and the anticancer agents which target this enzyme, focussing predominantly on new observations and recent reports and reviews.

J Basic Clin Physiol Pharmacol, 1995, 6(3-4), 251 - 63
Resistance mechanisms to arsenicals and antimonials; Rosen BP; Salts and organic derivatives of arsenic and antimony are quite toxic . Living organisms have adapted to this toxicity by the evolution of resistance mechanisms . Both prokaryotic and eukaryotic cells develop resistance when exposed to arsenicals or antimonials . In the case of bacteria resistance is conferred by plasmid-encoded arsenical resistance (ars) operons . The genes and gene products of the ars operon of the clinically-isolated conjugative R-factor R773 have been identified and their mechanism of action elucidated . The operon encodes an ATP-driven pump that extrudes arsenite and antimonite from the cells . The lowering of their intracellular concentration results in resistance . Arsenate resistance results from the action of the plasmid-encoded arsenate reductase that reduces arsenate to arsenite, which is then pumped out of the cell.

Adv Exp Med Biol, 1995, 380, 287 - 90
Identification of proteins specified by porcine epidemic diarrhoea virus; Utiger A et al.; Up to now, little was known about the proteins of porcine epidemic diarrhoea virus (PEDV) . Using (i) metabolic labelling, (ii) antisera directed against synthetic peptides and monoclonal antibodies, and (iii) eukaryotic and prokaryotic expression systems, we have started to identify and characterize these proteins . The nucleocapsid protein (N) of PEDV was identified as a phosphoprotein with a relative mobility (M(r)) of 57 k . No additional phosphoproteins were detected . At least three glycoproteins were virion associated . The 180/200 k band most probably represented the surface glycoprotein (S), whereas the 27 k protein was the membrane protein (M) . The 21 k species could not yet be identified . A rabbit anti-M protein antiserum was generated by using synthetic peptides corresponding to the C-terminus of M . The specificity of this serum was reconfirmed by transient expression of the M-gene in Vero cells followed by immunofluorescence . In order to generate antisera against the putative gene products of ORF3 and of the internal N reading frames (I-1, I-2, I-3), additional anti-peptide sera were raised . The putative ORF3 product which had been tagged by a 6 Histidine tail, was expressed in E . coli and purified by nickel chelate affinity chromatography before 2-dimensional polyacrylamide gel electrophoresis and immunostaining with a rabbit anti-peptide serum directed against the N-terminus of the ORF3 product . Transient expression of the I-1 and I-3 reading frames was used to confirm the specificity of the corresponding anti-peptide sera . The immunological tools presented in this paper are now being used to identify the putative corresponding gene products specified by PEDV.

Virus Genes, 1995, 11(2-3), 299 - 302
Molecular evolution of viruses: an interim summary; Becker Y; The origin and molecular evolution of viruses in this issue is dealt with at two levels: (1) tracing the past evolutionary pathways of viruses belonging to RNA virus families, retroviruses, and small and large DNA viruses; (2) tracing current changes in the RNA and DNA viral genomes that lead to the evolution of new virus mutants . In this interim summary, a time scale for the evolutionary processes is given, based on the accumulated published knowledge concerning the postulated origins of life on planet Earth, and the hypothesis that living cells with RNA genomes may have emerged (the "RNA world hypothesis") that then developed into cells with DNA genomes in eukaryotic and prokaryotic cells (1-3) . The ideas about the evolution of RNA and DNA viruses from ancient cellular RNA and DNA molecules over a period of 3.5 billion years are discussed . It may be possible that by studying virus genes and molecular processes in virus-infected cells, and their involvement in the shaping of the genomes of bacteria, yeast, plants, insects, mammals, and humans, it will be possible to understand the importance of viruses in past evolution and to predict their possible impact on current and future evolutionary trends in biology.

Virus Genes, 1995, 11(2-3), 81 - 94
Structure, function, and evolution of bacterial reverse transcriptase; Inouye S et al.; The discovery of retroelements in the prokaryotes raises intriguing questions concerning their roles in bacteria and the origin and evolution of reverse transcriptases . We first discuss a possible structure of bacterial reverse transcriptases on the basis of the known three-dimensional structure of HIV-1 reverse transcriptase, and how such a putative three-dimensional structure is able to recognize a single primer-template RNA molecule to initiate DNA chain elongation from the 2'-OH group of an internal G residue . This reaction leads to the production of a unique RNA-DNA complex called msDNA (multicopy single-stranded DNA) in which a single-stranded DNA branches out from an RNA molecule via a 2',5'-phosphodiester linkage . Second, the mobility of the bacterial retroelements called retrons, responsible for the production of msDNA, are discussed and compared with the mobility of group I and group II introns . Third, the original and evolution of bacterial reverse transcriptases are discussed in light of the question of whether the bacterial reverse transcriptases are older than eukaryotic reverse transcriptases.

Annu Rev Genet, 1995, 29, 509 - 52
Homologous recombination proteins in prokaryotes and eukaryotes; Camerini-Otero RD et al.; Genetic recombination is common to all forms of life and involves the exchange of DNA sequences between two chromosomes or DNA molecules . Such exchanges contribute to the generation of genetic diversity and the repair of damaged DNA . There are two major classes of recombination, site-specific recombination and general or homologous recombination . In homologous recombination the joining of the DNA duplexes exhibits a similar degree of precision or fidelity but, generally speaking, does not take place at specific sites . Since exchange can occur anywhere along the length of two homologous chromosomes, it follows that the proteins that catalyze homologous recombination are not sequence- or site-specific binding proteins . This review focuses on genetic and biochemical analyses of homologous recombination proteins that carry out conjugational recombination in E . coli and meiotic recombination in eukaryotes.

Viral Immunol, 1995, 8(2), 109 - 19
High efficiency prokaryotic expression and purification of a portion of the hepatitis C core protein and analysis of the immune response to recombinant protein in BALB/c mice; Hitomi Y et al.; Hepatitis C virus (HCV) produces chronic persistent liver infection in 1-2% of the U.S . population and is the leading cause of end stage liver disease in patients presenting for liver transplantation at our center . Efforts to cure persistent HCV infection are frequently unsuccessful, so the development of a HCV vaccine is a high priority . HCV envelope proteins are hypervariable so production of a recombinant surface antigen vaccine such as is available for hepatitis B is not likely to confer widespread, high level protective immunity . As the most highly conserved structural protein in the HCV genome, the core protein is one reasonable target for vaccine production . Presented here are data on the manufacture of recombinant core protein containing partial carboxy terminus deletions in an effort to increase the efficiency of core expression . The maltose binding protein (MBP) and glutathione S-transferase (GST) protein prokaryotic expression systems were used to study two different constructs, expressing the first 140 and 163 amino acids of the core region . Deletion of the 23 amino acids (aa) from aa141-163 led to a marked increase in the efficiency of protein production from < 1 to 3-4 mg/liter for both systems studied . Protein purification was accomplished using affinity chromatography (MBP) or inclusion body isolation (GST) as determined by SDS-PAGE gels and immunotransblot with HCV core protein-specific monoclonal antibody . Finally, the immune response to recombinant protein was assessed in BALB/c mice using a MBP HCV core fusion protein and an ELISA developed using GST HCV core protein as a target . In all mice of this strain, serum anti-HCV core antibody titer increased to 10(-4), two logs above background, following immunization in conjunction with Freund's complete adjuvant . These results represent an encouraging first step toward production of a core protein vaccine . Recombinant core protein is a useful tool to study the immune response to core protein and may be useful to further study the epidemiology and biology of the HCV virus.

Biochimie, 1995, 77(10), 803 - 7
Replication of DNA containing cisplatin lesions and its mutagenic consequences; Pillaire MJ et al.; Cisplatin {cis-diamminedichloroplatinum II} is widely used in the treatment of a broad range of tumors . A number of biological and biochemical results indicate that the reaction of cisplatin with DNA is responsible for the cytotoxic action of the drug . However, cisplatin can induce mutagenesis and may be carcinogenic in humans . Error prone replication of damaged DNA must be considered as a possible mechanism of mutagenesis . In this short review, we present data indicating that DNA containing cisplatin lesions can be replicated by prokaryotes and eukaryotes in a mutagenic fashion.

Virus Genes, 1995, 11(1), 31 - 5
Repetitive sequence in the Epstein-Barr virus EBNA-3C gene is related to a family of minisatellite arrays in the human genome; Fujiwara S et al.; A unique feature of the Epstein-Barr virus (EBV) genome is its high content of repetitive sequences . We identified a new human minisatellite element, tentatively designated MEB-1, that is similar to the 10 x "15bp" tandem repeat within the EBV nuclear antigen-3C (EBNA-3C) coding region . Southern blot analysis showed that the human genome has multiple copies of MEB-1-related repeats and that some of them are highly polymorphic . Both MEB-1 and the 10 x "15bp" repeat contain an octamer consensus GC{A/T}GG{A/T}GG, resembling the prokaryotic recombination signal chi . This octamer was also found in another EBV repeat sequence IR3 and the cellular GGA family of repeats that are related to IR3 . Since the octamer motif is generally considered to have a role in the generation of a group of minisatellite DNA, these findings suggest that the four viral and cellular repeats have been generated through similar mechanisms promoted by the motif.

Adv Exp Med Biol, 1995, 383, 255 - 69
Evidence that immunosuppression is an intrinsic property of the alpha-fetoprotein molecule; Semeniuk DJ et al.; Among the proteins that comprise the albumin family, alpha-fetoprotein (AFP) is the only member which exhibits immunoregulatory properties . However, some investigations have argued that AFP-mediated immunosuppression is not an inherent property of the molecule itself, but is instead, hypothesized to be either a function of a low molecular weight inhibitor bound to AFP or to a post-translational modification of the protein . AFP cannot be isolated from natural sources in quantities sufficient for the detailed biochemical and functional analyses required to resolve these issues . We have therefore produced recombinant forms of the protein (rAFP) by cloning the cDNA's for mouse and human AFP in both eukaryotic and prokaryotic expression systems . As described in this report, we were able to abundantly express rAFP's in bacterial, baculovirus and yeast expression systems . Recombinant proteins derived from each expression system were recognized by polyclonal and monoclonal anti-AFP antibodies as determined by immunoblot analysis . Pure recombinant protein samples, as characterized by polyacrylamide gel analyses, N-terminal sequencing and FPLC and HPLC chromatography, were evaluated for their immunoregulatory properties in murine and human in vitro immunological assays . The results of these studies establish that rAFP is functionally equivalent to natural fetal derived AFP molecules . Importantly, the data reported here demonstrate that AFP-mediated immunoregulation is an activity intrinsic to the molecule itself and cannot be attributed to either putative non-covalently bound moieties or to post-translational modifications such as glycosylation and sialylation . These studies provide a basis for initiating detailed investigations into the potential clinical usefulness of AFP as an immunotherapeutic agent.

Nucleic Acids Symp Ser, 1995, (33), 190 - 3
Peptidyl-tRNAs promote translational frameshifting; Vimaladithan A et al.; Programmed translational frameshifting is a ubiquitous, though rare, mechanism of gene expression in prokaryotes and eukaryotes . Research on many such sites has led to a general understanding that frameshifting depends on slippage of one or two ribosome-bound tRNAs on the mRNA . We recently found an example of an efficient frameshift in the Ty3 retrotransposon of the yeast Saccharomyces cerevisiae which occurs without tRNA slippage . Frameshifting appears to occur by misplacement of aminoacyl-tRNA in the ribosomal A site . Most of the eight tRNAs which induce measurable amounts of +1 frameshifting are predicted to slip only very poorly . In fact, frameshifting by tRNA slippage appears an unusual event in yeast, and where it occurs depends on peptidyl-tRNAs which employ two-out-of-three decoding . In addition, frameshifting either by slippage or by aminoacyl-tRNA misplacement depends on adequate availability of the first +1 frame tRNA . We present two models to explain how the tRNA which reads the shifted frame codon could promote +1 translational frameshifting.

Nucleic Acids Symp Ser, 1995, (33), 167 - 9
tRNA selection by a class II aminoacyl-tRNA synthetase: the role of accessory domains and inter-domain communication in RNA recognition; Yan W et al.; We have used a secondary site suppression approach to investigate the basis of tRNA selection by the E . coli histidyl-tRNA synthetase . This enzyme recognizes a unique G-1:C73 base pair located in the acceptor stems of prokaryotic histidyl-tRNAs . A genetic selection system was constructed in which growth on glycerol was dependent on histidine specific amber suppression of a triose phosphate isomerase (tpi) gene containing an amber codon at His 95 . Three independent revertants linked to hisS were isolated and sequenced, and the resulting mutant proteins were characterized biochemically . These studies are interpreted in light of the x-ray structure of the E . coli HisRS adenylate complex, and show that the C-terminal domain and its interactions with the catalytic domain play a biologically significant role in tRNA selection.

Micron, 1995, 26(5), 461 - 80
Nucleoid proteins; Hayat MA et al.; This article examines the published evidence in support of the classification of organisms into three groups (Bacteria, Archae, and Eukarya) instead of two groups (prokaryotes and eukaryotes) and summarizes the comparative biochemistry of each of the known histone-like, nucleoid DNA-binding proteins . The molecular structures and amino acid sequences of Archae are more similar to those of Eukarya than of Bacteria, with a few exceptions . Cytochemical methodology employed for localizing these proteins in archaeal and bacterial cells has also been reviewed . It is becoming increasingly apparent that these proteins participate both in the organization of DNA and in the control of gene expression . Evidence obtained from biochemical properties, structural and functional differences, and the ultrastructural location of these proteins, as well as from gene mutations clearly justifies the division of prokaryotes into bacterial and archaeal groups . Indeed, chromosomes, whether they be nuclear, prokaryotic, or organellar, are invariably complexed with abundant, small, basic proteins that bind to DNA with low sequence specificity . These proteins include the histones, histone-like proteins, and nonhistone high mobility group (HMG) proteins.

Biochimie, 1995, 77(7-8), 562 - 72
NADPH-P-450 reductase: structural and functional comparisons of the eukaryotic and prokaryotic isoforms; Sevrioukova IF et al.; The comparison of the properties of microsomal NADPH-P-450 reductase and the flavoprotein domain of P-450BM-3 (BMR) has revealed a significant difference in the mechanism of reduction of the hemoprotein P-450 by these flavoproteins . Microsomal NADPH-P-450 reductase transfers electrons to the hemoprotein by shuttling between hydroquinone and semiquinone forms of the FMN delivering one electron per cycle . Since the microsomal NADPH-P450 reductase has evolved as a component of multi-enzyme system, this type of mechanism may permit regulation of the steps of the P-450 reaction via variation in the affinity of the reductase for different P-450s, interaction with cytochrome b5, etc . In contrast, in the soluble, bacterial flavocytochrome P-450BM-3, the reductase domain has evolved together with a single unique heme domain . This enzyme was found to utilize the fastest and simplest way to reduce the heme iron, with the FMN moiety of BMR shuttling between the semiquinone and oxidized states . This mechanism of reduction provides the highest turnover number of any P-450 and tight coupling of the monooxygenation reaction . While there are clear differences in the intermediates involved in the reduction of P-450s by these two enzymes, the domain structure and presumably the mode of interaction between the reductase and P-450s has been maintained over evolutionary time.

Prog Brain Res, 1995, 106, 305 - 21
Some aspects of the pathophysiology of semicarbazide-sensitive amine oxidase enzymes; Callingham BA et al.; The widespread distribution of enzymes classed as semicarbazide-sensitive amine oxidases (SSAO enzymes) throughout a very wide range of eukaryotic as well as prokaryotic organisms encourages the aspirations of those who wish to demonstrate physiological, pathological or pharmacological importance . Such enzymes are found in several tissues of mammals, both freely soluble, as in blood plasma, and membrane-bound, for example, in smooth muscle and adipose tissue . While they are capable of deaminating many amines with the production of an aldehyde and hydrogen peroxide, doubt still surrounds the identity of the most important endogenous substrates for these enzymes . At present, methylamine and aminoacetone appear to head the list of candidates . The possibility that SSAO enzymes can convert amine substrates to highly toxic metabolites is illustrated by the production of acrolein from the xenobiotic amine, allylamine and formaldehyde and methylglyoxal from methylamine and aminoacetone, respectively . Activities of SSAO enzymes may be influenced by physiological changes, such as pregnancy or pathologically by disease states, including diabetes, tumours and burns . Increased deamination of aminoacetone by tissue and plasma SSAO enzymes as a result of its increased production from L-threonine in conditions such as exhaustion, starvation and diabetes mellitus may be harmful . Such dangers could be mitigated either physiologically by a compensatory reduction in SSAO activity or pharmacologically by treatment with inhibitors of SSAO.

Int Rev Cytol, 1995, 162B, 303 - 35
Targeting and association of proteins with functional domains in the nucleus: the insoluble solution; Leonhardt H et al.; The mammalian nucleus is highly organized into distinct functional domains separating different biochemical processes such as transcription, RNA processing, DNA synthesis, and ribosome assembly . A number of proteins known to participate in these processes were found to be specifically localized at their corresponding functional domains . A distinct targeting sequence, necessary and sufficient for the localization to DNA replication foci, was identified in the N-terminal, regulatory domain of DNA methyltransferase and DNA ligase I and might play a role in the coordination of DNA replication and DNA methylation . The fact that the targeting sequence is absent in lower eukaryotic and prokaryotic DNA ligase I homologs suggests that "targeting" is a rather recent development in evolution . Finally, targeting sequences have also been identified in some splicing factors and in viral proteins, which are responsible for their localization to the speckled compartment and to the nucleolus, respectively . These higher levels of organization are likely to contribute to the regulation and coordination of the complex and interdependent biochemical processes in the mammalian nucleus.

Int Rev Cytol, 1995, 163, 175 - 247
Biochemistry and molecular biology of chromoplast development; Camara B et al.; Plant cells contain a unique class of organelles, designated the plastids, which distinguish them from animal cells . According to the largely accepted endosymbiotic theory of evolution, plastids are descendants of prokaryotes . This process requires several adaptative changes which involve the maintenance and the expression of part of the plastid genome, as well as the integration of the plastid activity to the cellular metabolism . This is illustrated by the diversity of plastids encountered in plant cells . For instance, in tissues undergoing color changes, i.e., flowers and fruits, the chromoplasts produce and accumulate excess carotenoids . In this paper we attempt to review the basic aspects of chromoplast development.

J Bacteriol, 1995 Jan, 177(1), 191 - 9
In vitro studies of the domains of the nitrogen fixation regulatory protein NIFA; Berger DK et al.; The prokaryotic enhancer-binding protein NIFA is a multidomain transcriptional activator that catalyzes the formation of open complexes at nitrogen fixation (nif) promoters by a specialized form of RNA polymerase containing sigma 54 . The NIFA protein from Klebsiella pneumoniae consists of three domains: the N-terminal domain of unknown function; the central catalytic domain, which is sufficient for transcriptional activation; and the C-terminal DNA-binding domain . Purified fusion proteins between maltose-binding protein (MBP) and NIFA deleted of its N-terminal domain (MBP-delta N-NIFA) or its C-terminal domain (MBP-NIFA-delta C) activated transcription from the K . pneumoniae nifH promoter both in vitro and in vivo . We previously showed that the same was true for a fusion between MBP and the central domain of NIFA . These results indicate that NIFA is sufficiently modular for all fusions carrying its catalytic domain to be active . Unexpectedly, however, simple predictions regarding the location of determinants of the heat lability and insolubility of NIFA, which were based on previous studies of its isolated central and C-terminal domains, were not borne out . Contrary to a previous report from this laboratory, we found that the in vitro start site of transcription for the K . pneumoniae nifH operon could be either of two adjacent G residues, as others had reported in vivo . This was true independent of the activator, i.e., with MBP-NIFA and MBP-delta N-NIFA and with the homologous activator NTRC . When open complexes were formed with GTP as the activating nucleotide, the upstream G residue was probably as a consequence of initiation of transcription.

J Bacteriol, 1995 Jan, 177(1), 183 - 90
Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 {alpha beta} component in Escherichia coli; Skinner DD et al.; A cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH) of Streptomyces avermitilis has been cloned and sequenced . Open reading frame 1 (ORF1) (E1 alpha), 1,146 nucleotides long, would encode a polypeptide of 40,969 Da (381 amino acids) . ORF2 (E1 beta), 1,005 nucleotides long, would encode a polypeptide of 35,577 Da (334 amino acids) . The intergenic distance between ORF1 and ORF2 is 73 bp . The putative ATG start codon of the incomplete ORF3 (E2) overlaps the stop codon of ORF2 . Computer-aided searches showed that the deduced products of ORF1 and ORF2 resembled the corresponding E1 subunit (alpha or beta) of several prokaryotic and eukaryotic BCDH complexes . When these ORFs were overexpressed in Escherichia coli, proteins of about 41 and 34 kDa, which are the approximate masses of the predicted S . avermitilis ORF1 and ORF2 products, respectively, were detected . In addition, specific E1 {alpha beta} BCDH activity was detected in E . coli cells carrying the S . avermitilis ORF1 (E1 alpha) and ORF2 (E1 beta) coexpressed under the control of the T7 promoter.

J Virol, 1995 Jan, 69(1), 231 - 8
Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein-Barr virus DNA; Kempkes B et al.; We have assembled derivatives of Epstein-Barr Virus (EBV) that include 71 kbp of noncontiguous DNA sequences cloned into a prokaryotic F-factor plasmid . These mini-EBVs, when introduced into an EBV-containing lymphoblastoid cell, can be packaged by the endogenous helper virus . One such mini-EBV was found to have a single C residue deleted from its EBNA3a open reading frame . When packaged, this mini-EBV initiates proliferation of infected primary human B lymphocytes only in conjunction with a complementing helper virus . Proliferation of the infected cells, however, was maintained either alone by the mini-EBV containing the mutated EBNA3a open reading frame or alone by its derivative in which the EBNA3a open reading frame had been healed of its lesion by recombination with the helper virus . The mini-EBV with a wild-type EBNA3a open reading frame when packaged alone can both initiate and maintain proliferation upon infection of primary human B lymphocytes . These findings identify 41% of EBV DNA which is sufficient to immortalize primary human B lymphocytes and provide an assay to distinguish virus contributions to initiation or maintenance of cell proliferation or both . They also identify EBNA3a as a transforming gene, which contributes primarily to the initiation of cell proliferation.

Microbiology, 1995 Jan, 141 ( Pt 1), 85 - 93
Expression of the flagellin gene in Borrelia is controlled by an alternative sigma factor; Noppa L et al.; The flagellin genes from six Borrelia species were cloned, sequenced and characterized at the molecular level . The flagellin genes of two relapsing fever Borrelia species, B . hermsii and B . crocidurae, three Lyme disease genomic species, B . burgdorferi, B . afzelii and B . garinii, and the avian borreliosis agent, B . anserina, were compared and showed an 85-93% sequence identity to each other . Comparison of the fla genes from the different Lyme borreliosis spirochaetes revealed that they were 94-99% identical . Nucleotide sequencing of the fla gene and primer extension on isolated mRNA from both B . hermsii (as transcribed in Escherichia coli) and B . burgdorferi (as transcribed in the natural host) identified the putative transcriptional start points, the ribosomebinding sites and the promoter regions of these genes . The deduced promoter of the Borrelia flagellin gene resembled neither the sigma 70 promoter of prokaryotes, as seen for the genes for the outer-surface proteins A and B in Lyme disease Borrelia and the genes for the variable major proteins 7 and 21 of B . hermsii, nor the sigma 28 consensus promoter region of motility genes from other bacteria . Instead, the promoter of the fla gene in Borrelia has most similarity to the bacteriophage SP01 sigma gp33-34 promoter sequence of Bacillus subtilis.

Plant J, 1995 Jan, 7(1), 3 - 15
Starch branching enzymes belonging to distinct enzyme families are differentially expressed during pea embryo development; Burton RA et al.; cDNA clones for two isoforms of starch branching enzyme (SBEI and SBEII) have been isolated from pea embryos and sequenced . The deduced amino acid sequences of pea SBEI and SBEII are closely related to starch branching enzymes of maize, rice, potato and cassava and a number of glycogen branching enzymes from yeast, mammals and several prokaryotic species . In comparison with SBEI, the deduced amino acid sequence of SBEII lacks a flexible domain at the N-terminus of the mature protein . This domain is also present in maize SBEII and rice SBEIII and resembles one previously reported for pea granule-bound starch synthase II (GBSSII) . However, in each case it is missing from the other isoform of SBE from the same species . On the basis of this structural feature (which exists in some isoforms from both monocots and dicots) and other differences in sequence, SBEs from plants may be divided into two distinct enzyme families . There is strong evidence from our own and other work that the amylopectin products of the enzymes from these two families are qualitatively different . Pea SBEI and SBEII are differentially expressed during embryo development . SBEI is relatively highly expressed in young embryos whilst maximum expression of SBEII occurs in older embryos . The differential expression of isoforms which have distinct catalytic properties means that the contribution of each SBE isoform to starch biosynthesis changes during embryo development . Qualitative measurement of amylopectin from developing and maturing embryos confirms that the nature of amylopectin changes during pea embryo development and that this correlates with the differential expression of SBE isoforms.

FEMS Microbiol Rev, 1995 Jan, 16(1), 53 - 75
Amino acid transporters of lower eukaryotes: regulation, structure and topogenesis; Sophianopoulou V et al.; Lower eukaryotes such as the yeast Saccharomyces cerevisiae and the filamentous fungus Aspergillus nidulans possess a multiplicity of amino acid transporters or permeases which exhibit different properties with respect to substrate affinity, specificity, capacity and regulation . Regulation of amino acid uptake in response to physiological conditions of growth is achieved principally by a dual mechanism; control of gene expression, mediated by a complex interplay of pathway-specific and wide-domain transcription regulatory proteins, and control of transport activities, mediated by a series of protein factors, including a kinase, and possibly, by amino acids . All fungal and a number of bacterial amino acid permeases show significant sequence similarities (33-62% identity scores in binary comparisons), revealing a unique transporter family conserved across the prokaryotic-eukaryotic boundary . Prediction of the topology of this transporter family utilizing a multiple sequence alignment strongly suggests the presence of a common structural motif consisting of 12 alpha-helical putative transmembrane segments and cytoplasmically located N- and C-terminal hydrophilic regions . Interestingly, recent genetic and molecular results strongly suggest that yeast amino acid permeases are integrated into the plasma membrane through a specific intracellular translocation system . Finally, speculating on their predicted structure and on amino acid sequence similarities conserved within this family of permeases reveals regions of putative importance in amino acid transporter structure, function, post-translational regulation or biogenesis.

J Clin Endocrinol Metab, 1995 Jan, 80(1), 46 - 53
Human autoantibodies to the thyrotropin receptor: recognition of linear, folded, and glycosylated recombinant extracellular domain; Vlase H et al.; To examine the heterogeneity of autoantibodies to the human TSH receptor (hTSHR), we evaluated 20 sera from patients with Graves' disease for their recognition of prokaryotic (unglycosylated) and eukaryotic (insect cell glycosylated) recombinant hTSHR extracellular domain (ecd) in an unfolded (linear) and a folded (nonlinear) state . With the prokaryotic antigen, 12 (60%) bound folded hTSHR ecd monomer, 8 (40%) bound to the unfolded monomer, and 3 (15%) bound to a tetrameric species . Such binding to different hTSHR antigens was not mutually exclusive . In addition, 7 (35%) sera showed an apparently higher reactivity for the folded than the unfolded monomer . When reacted against the glycosylated insect cell hTSHR ecd, 9 (45%) sera recognized both the unfolded and folded monomer, and 5 (25%) recognized the tetrameric form . In all of our testing, 17 of the 20 sera (85%) bound to 1 or more of the recombinant hTSHR ecd antigens, and the recognition pattern appeared to be heterogeneous in at least 4 (20%) of the serum samples, with hTSHR antibodies recognizing linear, folded, and glycosylated hTSHR ecd monomers . We conclude, therefore, that patients with Graves' disease have autoantibodies that recognize multiple epitopes on the hTSHR ecd and that it is possible to classify them according to their recognition of linear, folded, and glycosylated products.

J Clin Invest, 1995 Jan, 95(1), 367 - 76
Inhibition of cytoplasmic and organellar protein synthesis in Toxoplasma gondii . Implications for the target of macrolide antibiotics; Beckers CJ et al.; We investigated potential targets for the activity of protein synthesis inhibitors against the protozoan parasite Toxoplasma gondii . Although nanomolar concentrations of azithromycin and clindamycin prevent replication of T . gondii in both cell culture and in vivo assays, no inhibition of protein labeling was observed in either extracellular or intracellular parasites treated with up to 100 microM drug for up to 24 h . Quantitative analysis of > 300 individual spots on two-dimensional gels revealed no proteins selectively depleted by 100 microM azithromycin . In contrast, cycloheximide inhibited protein synthesis in a dose-dependent manner . Nucleotide sequence analysis of the peptidyl transferase region from genes encoding the large subunit of the parasite's ribosomal RNA predict that the cytoplasmic ribosomes of T . gondii, like other eukaryotic ribosomes, should be resistant to macrolide antibiotics . Combining cycloheximide treatment with two-dimensional gel analysis revealed a small subset of parasite proteins likely to be synthesized on mitochondrial ribosomes . Synthesis of these proteins was inhibited by 100 microM tetracycline, but not by 100 microM azithromycin or clindamycin . Ribosomal DNA sequences believed to be derived from the T . gondii mitochondrial genome predict macrolide/lincosamide resistance . PCR amplification of total T . gondii DNA identified an additional class of prokaryotic-type ribosomal genes, similar to the plastid-like ribosomal genes of the Plasmodium falciparum . Ribosomes encoded by these genes are predicted to be sensitive to the lincosamide/macrolide class of antibiotics, and may serve as the functional target for azithromycin, clindamycin, and other protein synthesis inhibitors in Toxoplasma and related parasites.

Annu Rev Entomol, 1995, 40, 221 - 43
Cellular and molecular interrelationships between ticks and prokaryotic tick-borne pathogens; Munderloh UG et al.; Tick-borne prokaryotic pathogens share a very intimate relationship with the vectors . Ingestion during the bloodmeal places the microbe into the gut lumen whence it must travel to the salivary glands at the right time for transmission during a subsequent feeding . This crucial event requires coordination between pathogen development and arthropod host activities that may be mediated by the expression of genes specific for the vector phase of the pathogen . Invertebrate hormones or factors associated with tick tissues may provide the cues that signal changes in tick physiology that induce necessary steps in the pathogen, such as colonization of ovaries during egg development in preparation for transovarial transmission or dispersion to the salivary glands at the time of a bloodmeal . These hypotheses cannot easily be investigated within the complex environment of the tick, but tick cell culture offers a simplified system with which to examine many of these important interrelationships.

J Bacteriol, 1995 Jan, 177(1), 37 - 45
Phylogenetic comparison of retron elements among the myxobacteria: evidence for vertical inheritance; Rice SA et al.; Twenty-eight myxobacterial strains, representing members from all three subgroups, were screened for the presence of retron elements, which are novel prokaryotic retroelements encoding reverse transcriptase . The presence of retrons was determined by assaying strains for a small satellite DNA produced by reverse transcription called multicopy, single-stranded DNA (msDNA) . An msDNA-producing retron appeared to be absent from only one of the strains surveyed . DNA hybridization experiments revealed that retron elements similar to retron Mx162, first identified in Myxococcus xanthus, were found only among members of the Myxococcus subgroup; that is, each of the seven different genera which constitute this subgroup contained a Mx162 homolog . Another retron element also appeared to have a clustered distribution, being found exclusively within the Nannocystis subgroup of the myxobacteria . A retron element of the Mx162 type was cloned from Melittangium lichenicola, and its DNA sequence was compared with those of similar elements in M . xanthus and Stigmatella aurantiaca . Together, the degree of sequence diversity, the codon bias of the reverse transcriptase genes, and the clustered distribution of these retrons suggest a possible evolutionary scenario in which a common ancestor of the Myxococcus subgroup may have acquired this retroelement.

Virchows Arch, 1995, 426(3), 215 - 22
Microsatellite instability: new aspects in the carcinogenesis of colorectal carcinoma; Ruschoff J et al.; Very recently a new molecular mechanism in the tumorigenesis of colorectal carcinoma has been described which is closely linked to hereditary non-polyposis colonic cancer (HNPCC) . Ubiquitous changes in the length of simple repetitive DNA sequences between constitutional and tumour DNA occur in about 90% of cases of HNPCC and in about 15% of cases of non-familial, sporadic colorectal carcinoma . Such microsatellite instabilities have been shown to be the phenotypical marker of mutations in the human homologues of prokaryotic mismatch repair genes (MutS, MutL, MutH) . These data provide crucial new tools in the detection of patients at high risk of developing colon cancer and other HNPCC-related carcinomas . In addition, these developments provide new insights into a new, presumably primary event in oncogenesis, i.e . the occurrence of mutations in genomic stability genes leading to an increased cellular mutation rate ("mutator phenotype") and thus to cancer.

Curr Microbiol, 1995 Jan, 30(1), 27 - 32
ppGpp-mediated regulation of DNA replication and cell division in Escherichia coli; Schreiber G et al.; ppGpp serves as an alarmon in prokaryotes, distributing and coordinating different cellular processes according to the nutritional potential of the growth medium . This work is interpreted as favoring the view that, in addition to its previously documented role in regulating the rate of ribosome synthesis, ppGpp participates in coordinating DNA replication and cell division . We studied the effects of ppGpp on the cell division cycle, using cells containing plasmid pSM11 that codes for the 55-kDa truncated RelA protein under the inducible Ptac promoter . In this system it was found that the rate of initiation of new rounds of DNA replication is inversely correlated with the intracellular level of ppGpp . Furthermore, ppGpp levels similar to those found during the activation of stringent control inhibited replication initiation, in a manner comparable to that resulting from inhibition of protein synthesis by amino acid starvation or by chloramphenicol addition . However, in contrast to chloramphenicol treatment, elevated ppGpp levels did not block septum formation, and, in fact, there is some evidence for enhanced septation . As a result, the residual cell division following elevation in ppGpp levels was higher than after chloramphenicol treatment, resulting in cells with a size similar to that of stationary phase cells.

C R Acad Sci III, 1995 Jan, 318(1), 35 - 41
Presence of a protein specific of endocytobiosis (symbionin) in the weevil Sitophilus; Charles H et al.; Chaperonins are ubiquitous proteins found in all prokaryotic and eukaryotic cells . They are overproduced in several parasitic bacteria and are implicated in at least 2 types of endocytobiosis: in amoebae and in aphids . This work puts in evidence that a protein named symbionin, which shows an immunological homology with the E . coli protein GroEL, is present in the symbiotic relationship of 3 species of Sitophilus (S . oryzae, S . granarius, and S . zeamais) . This protein is neither found in the naturally asymbiotic specie S . linearis nor in the aposymbiotic strain of S . oryzae obtained in the laboratory . This symbionin is stored in a great quantity within endocytobiotes and its amino acid composition seems to corroborate its chaperonin resemblance rather than its possible function as one of the insect storage proteins already described in the literature.

Mol Microbiol, 1995 Jan, 15(1), 55 - 63
A small gene, designated comS, located within the coding region of the fourth amino acid-activation domain of srfA, is required for competence development in Bacillus subtilis; Hamoen LW et al.; The valine-activation domain-encoding portion of the srfA locus (srfA-d4) is not only involved in the non-ribosomal synthesis of surfactin, but is also required for the regulation of competence development . In this study we show that impairment of the adenylation activity of the valine-activating domain did not affect competence development . Deletion analysis and complementation studies delineated the competence-required portion of srfA-d4 to a 168 bp fragment, which contains a small open reading frame (ORF), designated comS, encoding a polypeptide of 46 amino acids, embedded within, but translated in, a frame different from that of srfA-d4 . Introduction of an amber mutation in the comS-coding frame prevented competence development, demonstrating the involvement of comS in this prokaryotic specialization process.

Mol Microbiol, 1995 Jan, 15(2), 355 - 66
Characterization of RNA polymerase and two sigma-factor genes from Mycobacterium smegmatis; Predich M et al.; A search for Mycobacterium smegmatis genes showing similarity to the conserved family encoding major sigma factors in diverse prokaryotes has identified two such determinants . Both genes are expressed in exponentially growing cells, as judged by Western immunoassays . A series of chromatographic steps was used to purify M . smegmatis RNA polymerase holoenzyme and it was shown that its ability to initiate in vitro transcription with a heterologous Bacillus subtilis promoter is dependent on the presence of these sigma factor(s) . Reconstitution of specific in vitro transcription activity was obtained upon mixing of M . smegmatis core RNA polymerase with the major sigma factor of Bacillus subtilis . We also demonstrated in vitro transcription of the M . smegmatis rrnB promoter by the M . smegmatis RNA polymerase . Significantly, highly active B . subtilis RNA polymerase holoenzyme was unable to transcribe this gene.

Parasite Immunol, 1995 Jan, 17(1), 11 - 9
Human immune recognition of recombinant proteins representing discrete domains of the Plasmodium falciparum gamete surface protein, Pfs230; Riley EM et al.; The 230 kD gametocyte/gamete-specific surface protein of Plasmodium falciparum, Pfs230, is a target of antibodies which inhibit the development of the parasite inside the mosquito vector . A transmission blocking vaccine based on Pfs230 may be a powerful tool for malaria control . As a first step, Pfs230 has been expressed in E . coli as a series of recombinant proteins, fused to maltose binding protein . We have used the fusion proteins to assess cellular and humoral immune responses to Pfs230 in malaria-immune adult Gambian blood donors; responses to the fusion proteins have been compared with responses to native Pfs230 . The tetrapeptide repeat region of the molecule appears to be immunodominant for both antibody-producing cells and peripheral blood T cells . We postulate that this may represent a mechanism for immune evasion since the N-terminal repeat region of the molecule is cleaved from the mature protein and shed into the plasma . Responses to fusion proteins representing the seven-cysteine motifs were correlated within individual donors, suggesting that cross-reactive epitopes occur within the motifs . Antibody responses to recombinant proteins were poorly correlated with responses to native Pfs230 suggesting that dominant epitopes of the native protein are not adequately represented in the recombinant proteins . Although prokaryotic expression products may be suitable for induction of cellular immune responses to Pfs230, alternative expression systems may be needed for creation of appropriate B cell epitopes.

J Eukaryot Microbiol, 1995 Jan-Feb, 42(1), 83 - 91
Induction of antibiotic resistance in Paramecium tetraurelia by the bacterial gene APH-3'-II; Haynes WJ et al.; We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neor) from the transposon Tn5 . The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium . After the neor ORF was inserted, the plasmid was referred to as pPXV-NEO . Delivery of approximately 10-20 picoliters of linearized PXV-NEO at > or = 2000 copies/pl into the macronucleus effected 100% transformation . Southern and Northern blot hybridization showed the presence of neor-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones . The degree of resistance to G-418, and the concentrations of neor-specific DNA and neor-specific RNA in the clones were proportional to the concentration of the vector injected . We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.

Brain Res Mol Brain Res, 1995 Jan, 28(1), 94 - 100
Antibodies to the rat beta 3 subunit of the gamma-aminobutyric acid A receptors; Fernando LP et al.; Polymerase chain reaction was used to amplify the cDNA region that codes for the large intracellular loop of the beta 3 subunit of the gamma-aminobutyric acidA/benzodiazepine receptors (GABAAR/BZDR) from rat brain . The amplified cDNA was inserted into the prokaryotic expression vector pGEX-3X and a fusion protein containing glutathione-S-transferase and beta 3 intracellular loop moieties was expressed in bacteria . The fusion protein was affinity-purified and it was used to raise a rabbit anti-beta 3 antiserum . The anti-beta 3 antiserum immunoprecipitated the gamma-aminobutyric acidA receptor from rat and bovine brain . Immunoblots of the affinity-purified GABAAR/BZDR from bovine brain revealed that the anti-beta 3 antiserum reacted with a 57 kDa peptide, whereas the monoclonal antibody 62-3G1 that recognized both beta 2 and beta 3 reacted with 55 and 57 kDa peptides . The anti-beta 3 antiserum showed specificity for the beta 3 subunit vs beta 2 and beta 1.

Bioessays, 1995 Jan, 17(1), 53 - 62
Nm23/nucleoside diphosphate kinase: toward a structural and biochemical understanding of its biological functions; de la Rosa A et al.; The nm23 gene, a putative metastasis suppressor gene, was originally identified by its reduced expression in highly metastatic K-1735 murine melanoma cell lines, as compared to related, low metastatic melanoma cell lines . Transfection of nm23 cDNA has been reported to suppress malignant progression in Drosophila and mammalian cells . Highly conserved homologues of nm23 have been found in organisms ranging from the prokaryote Myxococcus xanthus to Drosophila, where the gene is involved in normal development and differentiation . The product of the nm23 gene exhibits a nucleoside diphosphate kinase activity, yet the nucleoside diphosphate kinase activity of Nm23 does not correlate with its apparent biological functions . We review recent cellular, genetic, biochemical and X-ray crystallographic data to formulate and evaluate hypotheses concerning the molecular mechanism of nm23 action.

Eur J Pediatr, 1995, 154(7 Suppl 2), S28 - 32
Regulation of galactose-1-phosphate uridyltransferase gene expression; Heidenreich RA; Analysis of both the human and rat galactose-1-phosphate uridyltransferase (GALT) genes reveal 5' regulatory consensus sequences suggestive of a housekeeping gene . This is in accord with the finding of GALT activity in all tissues . However, the complications seen in galactosemia, in particular ovarian dysfunction and verbal dyspraxia, suggest organ-specific sensitivity to lack of GALT activity . Analysis of steady-state GALT mRNA and specific activity levels in adult rat organs reveals a marked difference between organs that correlates with the degree of organ dysfunction in humans with galactosemia . The organ variation in GALT mRNA and activity thus appears to be due to genetic regulation . Discernment of the pathophysiologic basis for the organ-specific complications requires an understanding of the basis for the differences in organ regulation . The present state of knowledge of the regulation of the Leloir enzymes in general, GALT in particular, from prokaryotes to mammals is discussed.

Annu Rev Biophys Biomol Struct, 1995, 24, 293 - 318
Structure and function of DNA methyltransferases; Cheng X; In prokaryotes, the major role of DNA methylation is to protect host DNA against degradation by restriction enzymes . In eukaryotes, DNA methylation has been implicated in the control of several cellular processes, including differentiation, gene regulation, and embryonic development . Structural work on HhaI DNA methyltransferase demonstrates that the substrate nucleotide is completely flipped out of the helix during the modification reaction and has provided much insight into the enzymatic properties of S-adenosyl-L-methionine (SAM)-dependent DNA-modifying enzymes . Structural comparison of three enzymes, HhaI C5-cytosine methyltransferase, TaqI N6-adenine methyltransferase, and catechol O-methyltransferase, reveals a striking similarity in protein folding and indicates that many SAM-dependent methyltransferases have a common catalytic-domain structure . This feature permits the prediction of tertiary structure for other DNA, RNA, protein, and small-molecule methyltransferases from their amino acid sequences, including the eukaryotic CpG methyltransferases.

Biochimie, 1995, 77(3), 162 - 6
Production and purification of active FGF2 via recombinant fusion protein; Lemaitre G et al.; Basic fibroblast growth factor (FGF2) is involved in both cell proliferation and differentiation processes . Heparin may interfere in the stability and biological activities of FGFs . However, it is difficult to obtain FGF preparation without traces of heparin since heparin affinity chromatographies are routinely used to prepare this growth factor . We have therefore devised a means of production of active recombinant FGF2 devoid of heparin traces . The bovine FGF2 gene was inserted into the pMAL-c prokaryotic expression vector and the recombinant protein was synthesised as a fusion product between the maltose binding protein (MBP) and FGF2 . Purification of the FGF2 fusion protein was performed by an amylose affinity chromatography . Yields were similar to those obtained by using a traditional heparin affinity column purification procedure . The fusion protein (MBP-FGF2) and the cleaved-off FGF2 were tested for some of their biological properties and compared to recombinant FGF2 purified by heparin affinity chromatography . Mitogenic activity on Chinese hamster lung fibroblasts (CCL39) and neurite outgrowth on pheochromocytoma culture cells (PC12) were used as biological assays . The cleaved-off FGF2 was as active as commercially available recombinant FGF2 (ED50 at 0.16 and 0.04 nM respectively) . However MBP-FGF2 was less active (ED50 at 0.9 nM) in both tests.

Adv Exp Med Biol, 1995, 369, 99 - 110
Impact of biotechnology in the diagnostic and therapeutic management of cardiovascular disorders; Fareed J et al.; Although biotechnology has been useful in the development of new diagnostic methods and drugs for the management of cardiovascular disorders, there are several issues which raise certain questions on the global use of biotechnology based drugs and diagnostic methods (Piascik, 1991; Fareed, 1993a; Fareed, 1994a) . The cost is rather prohibitive in the development of this type of technology . Most diagnostic methods and drugs developed utilizing biotechnology based methods are relatively expensive . The second important consideration is the equivalence of the newer biotechnology derived drugs to the natural products . Many of the biotechnology derived drugs are obtained in prokaryotic systems (E . coli) . Post-transcriptional modifications such as glycosylation are often important in determining the function of various proteins . On the other hand, biotechnology based diagnostic methods exhibit somewhat different specificity in comparison to conventional methods . Thus, it is rather important to assess the developments in this area in a careful manner . Furthermore, validation of the clinical, diagnostic and therapeutic efficacy of biotechnology derived diagnostic devices and drugs is a prerequisite for their use in cardiovascular medicine.

Proc Int Conf Intell Syst Mol Biol, 1995, 3, 127 - 35
Reconstruction of metabolic networks using incomplete information; Gaasterland T et al.; This paper describes an approach that uses methods for automated sequence analysis (Gaasterland et al . August 1994) and multiple databases accessed through an object+attribute view of the data (Baehr et al . 1992), together with metabolic pathways, reaction equations, and compounds parsed into a logical representation from the Enzyme and Metabolic Pathway Database (Selkov, Yunus, & et.al . 1994), as the sources of data for automatically reconstructing a weighted partial metabolic network for a prokaryotic organism . Additional information can be provided interactively by the expert user to guide reconstruction.

Adv Enzyme Regul, 1995, 35, 147 - 62
A new family of protein kinases--the mitochondrial protein kinases; Harris RA et al.; Molecular cloning has provided evidence for a new family of protein kinases in eukaryotic cells . These kinases show no sequence similarity with other eukaryotic protein kinases, but are related by sequence to the histidine protein kinases found in prokaryotes . These protein kinases, responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase complexes, are located exclusively in mitochondrial matrix space and have most likely evolved from genes originally present in respiration-dependent bacteria endocytosed by primitive eukaryotic cells . Long-term regulatory mechanisms involved in the control of the activities of these two kinases are of considerable interest . Dietary protein deficiency increases the activity of branched-chain alpha-ketoacid dehydrogenase kinase associated with the branched-chain alpha-ketoacid dehydrogenase complex . The amount of branched-chain alpha-ketoacid dehydrogenase kinase protein associated with the branched-chain alpha-ketoacid dehydrogenase complex and the message level for branched-chain alpha-ketoacid dehydrogenase kinase are both greatly increased in the liver of rats starved for protein, suggesting increased expression of the gene encoding branched-chain alpha-ketoacid dehydrogenase kinase . The increase in branched-chain alpha-ketoacid dehydrogenase kinase activity results in greater phosphorylation and lower activity of the branched-chain alpha-ketoacid dehydrogenase complex . The metabolic consequence is conservation of branched chain amino acids for protein synthesis during periods of dietary protein deficiency . Two isoforms of pyruvate dehydrogenase kinase have been identified and cloned . Pyruvate dehydrogenase kinase 1, the first isoform cloned, corresponds to the 48 kDa subunit of the pyruvate dehydrogenase kinase isolated from rat heart tissue . Pyruvate dehydrogenase kinase 2, the second isoform cloned, corresponds to the 45 kDa subunit of this enzyme . In addition, it also appears to correspond to a possibly free or soluble form of pyruvate dehydrogenase kinase that was originally named kinase activator protein . Assuming that differences in kinetic and/or regulatory properties of these isoforms exist, tissue specific expression of these enzymes and/or control of their association with the complex will probably prove to be important for the long term regulation of the activity of the pyruvate dehydrogenase complex . Starvation and the diabetic state are known to greatly increase activity of the pyruvate dehydrogenase kinase in the liver, heart and muscle of the rat . This contributes in these states to the phosphorylation and inactivation of the pyruvate dehydrogenase complex and conservation of pyruvate and lactate for gluconeogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Parasitol Res, 1995, 81(6), 459 - 64
Carbohydrate involvement in the association of a prokaryotic cell with Trichomonas vaginalis and Tritrichomonas foetus; Benchimol M et al.; Previous studies have shown that Trichomonas vaginalis are capable of ingesting bacteria . This observation was confirmed in the present study and extended to Tritrichomonas foetus . Using a special strain of Escherichia coli grown under conditions that produced fimbriae presenting a lectin-like molecule recognizing mannose, we showed that the bacteria attached to and were ingested by trichomonads through a mechanism involving cell-to-cell recognition . Absence of the fimbriae or addition of alpha-methyl-D-mannoside to the interaction medium blocked attachment of the bacteria to the protozoa cell surface . Ingested bacteria were later digested within the cytoplasmic vacuole.

Mol Biol Rep, 1995, 21(1), 43 - 7
Relationships between proteasomes and RNA; Schmid HP et al.; The 20S proteasome (prosome) is a highly organized multi-protein complex with approximate molecular weight of about 700 kDa . Whilst the role of the proteasome in the processing and turnover of cellular proteins is becoming clearer, its relationship with RNA remains obscure . Over the last decade the possibility of association of proteasomes with specific RNAs or mRNPs have been particularly controversial . Proteasomes were reported to inhibit translation of viral mRNAs and to be tightly associated with RNase activity . It is possible that proteasomes are also involved in cellular RNA breakdown and RNA processing like prokaryotic RNase E.

Mutat Res, 1995 Jan, 336(1), 91 - 100
Host-cell reactivation of reporter genes introduced into cells by adenovirus as a convenient way to measure cellular DNA repair; Valerie K et al.; In order to conveniently measure cellular DNA repair in immortalized and primary human cells we have combined the features of high cellular infectivity of adenovirus (Ad) with that of host-cell reactivation (HCR) of ultraviolet light (UV)-damaged reporter genes . We show that Ads having either the cat (chloramphenicol acetyltransferase) or seap (secreted alkaline phosphatase) reporter gene under control of a strong constitutive promoter can be used to measure relative levels of DNA repair by HCR . Most importantly, the SEAP assay allows for a convenient, inexpensive, and sensitive colorimetric microtiter assay . Only a few steps are involved and it is possible to process many samples simultaneously in a relatively short time, which is not as easily done with other reporter gene assays . Furthermore, we show that co-infection of UV-damaged SEAP Ad with an Ad carrying a prokaryotic repair gene significantly increased the HCR levels in xeroderma pigmentosum cells . The Ad gene delivery system, and the SEAP assay in particular, should simplify existing HCR assays considerably . By using non-lytic Ad as a vehicle it should be possible to quantitatively introduce normal or dominant negative mutant DNA repair genes into bulk cell populations for DNA repair studies.

Endeavour, 1995, 19(3), 112 - 7
A powerful bacterial world; Mathieu LG et al.; Bacteria (prokaryotes) were the sole form of life on earth for some two billion years--roughly half its history . During this time they evolved into a giant, global superorganism and developed a remarkable mechanism for the creation and exchange of genetic material . Apart from its intrinsic interest, this has practical significance, for example in the development of multiple resistance to antibiotics of pathogenic bacteria such as those of tuberculosis . Eukaryotes, with nucleated cells, may have developed from a permanent symbiosis of three or more prokaryotes.

Methods Enzymol, 1995, 252, 186 - 95
Dihydrolipoamide dehydrogenase: activity assays; Patel MS et al.; We have described the most commonly used assay procedures determination of the DHLipDH activities from prokaryotic and eukaryotic cells . We have also described the procedures for the preparation of tissue extracts to determine the enzymatic activity . We have briefly alluded to the problems inherent in these assay procedures and suggested some precautions for using the assays to determine DHLipDH activity . We have also included a statement about the purification procedures and described some of the properties of this enzyme, which might help the reader in planning studies . We have, by no means, attempted to be comprehensive in this regard; therefore, the reader should consult the appropriate references (and references therein).

Cell, 1994 Dec 30, 79(7), 1233 - 43
Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA; Krishna TS et al.; The crystal structure of the processivity factor required by eukaryotic DNA polymerase delta, proliferating cell nuclear antigen (PCNA) from S . cerevisiae, has been determined at 2.3 A resolution . Three PCNA molecules, each containing two topologically identical domains, are tightly associated to form a closed ring . The dimensions and electrostatic properties of the ring suggest that PCNA encircles duplex DNA, providing a DNA-bound platform for the attachment of the polymerase . The trimeric PCNA ring is strikingly similar to the dimeric ring formed by the beta subunit (processivity factor) of E . coli DNA polymerase III holoenzyme, with which it shares no significant sequence identity . This structural correspondence further substantiates the mechanistic connection between eukaryotic and prokaryotic DNA replication that has been suggested on biochemical grounds.

Biochem Biophys Res Commun, 1994 Dec 30, 205(3), 1497 - 502
The extrinsic proteins of photosystem II in photosynthetic organisms: distribution, properties and evolutionary implications; Fairweather MS et al.; Photosystem II from higher plant chloroplasts contains extrinsic proteins of molecular weights 33, 23 and 16 kDa . Cyanobacteria, which are thought to be closely related to the evolutionary progenitor of chloroplasts, have a protein homologous to the largest of these, but lack homologues of the smaller ones . Here we report immunological evidence for the occurrence of the 33 kDa extrinsic protein in red and chromophyte algae and its absence from anoxygenic photosynthetic bacteria . The 23 kDa protein was absent from all three groups . The red algal protein was found to be hydrophilic and rather more loosely associated with the thylakoid membrane than its higher plant equivalent . These findings support an origin for all plastids among the oxygenic prokaryotes, in contrast to the phylogeny inferred from ribulose bisphosphate carboxylase-oxygenase sequence data.

J Biol Chem, 1994 Dec 30, 269(52), 32871 - 8
The thylakoid translocation of subunit 3 of photosystem I, the psaF gene product, depends on a bipartite transit peptide and proceeds along an azide-sensitive pathway; Karnauchov I et al.; Subunit 3 of photosystem I (PSI-3), the product of the nuclear psaF gene, is the docking protein for plastocyanin during photosynthetic electron transport in thylakoid membranes and is synthesized in the cytosol with a transit peptide that resembles structurally the bipartite targeting signals of hydrophilic, lumenal components such as plastocyanin . In organello import experiments performed with the authentic PSI-3 precursor and chimeric polypeptides consisting of residue-correct fusions of transit peptides and mature proteins derived from different plastid proteins demonstrate that the PSI-3 transit peptide is indeed capable of translocating proteins into the thylakoid lumen and that, conversely, mature PSI-3 depends on a bipartite transit peptide for its thylakoid transfer . Of the three recently described translocation/integration pathways for nucleus-encoded proteins carrying bipartite transit peptides that are distinct in their physiological requirements and strictly protein-specific, PSI-3, like plastocyanin and the 33-kDa protein of the oxygen-evolving complex, is translocated by a pathway that involves stromal factors but no proton gradient across the membrane . It is not affected by saturating amounts of the precursor for the 23-kDa protein of the oxygen-evolving complex that follows the latter route . Thylakoid translocation of PSI-3 is, however, impaired in the presence of sodium azide, which indicates that a homolog to the bacterial SecA protein might be involved in this process suggesting, thus, a prokaryote-like translocation pathway . The azide-sensitive factor appears to interact predominantly with the transit peptide of a precursor protein, since chimeras consisting of a presequence from an azide-resistant precursor and a mature part of an azide-sensitive polypeptide are still translocated in the presence of the inhibitor.

Biochim Biophys Acta, 1994 Dec 30, 1188(3), 447 - 9
Nucleotide sequence of the petE gene encoding plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica; Arudchandran A et al.; We have determined the nucleotide sequence of the petE gene encoding plastocyanin from the chlorophyll a/b-containing photosynthetic prokaryote, Prochlorothrix hollandica . Comparison of the deduced amino acid sequence encoded by the gene with the N-terminal sequence of the purified protein revealed that plastocyanin is synthesized as a precursor bearing an N-terminal domain of 34 amino acids having some structural similarity to thylakoid lumenal transit peptides identified in other organisms . The mature protein has an apparent isoelectric point of 8.37 and a molecular mass of 10,236 Da.

FEBS Lett, 1994 Dec 19, 356(2-3), 261 - 6
Poly (ADP-ribose) polymerase inhibits DNA replication by human replicative DNA polymerase alpha, delta and epsilon in vitro; Eki T; The influence of poly (ADP-ribose) polymerase (PARP) and poly ADP-ribosylation on DNA synthesis supported by human replicative DNA polymerase (DNA pol) alpha, delta, and epsilon has been examined using the replication system containing poly(dA)4500-oligo(dT)12-18 as the template primer . PARP alone inhibited the pol activities in a dose-dependent manner even in the presence of the accessory factors for DNA pol delta, proliferating cell nuclear antigen (PCNA) and activator 1 (Al; RF-C) . Both DNA pol alpha and epsilon activities were decreased approximately 10-fold under the poly ADP-ribosylating condition . In contrast, DNA synthesis by DNA pol delta holoenzyme was not affected by poly ADP-ribosylation like prokaryotic DNA pol's . The analysis of poly(dT) formed by DNA pol alpha and epsilon indicated that poly ADP-ribosylation mainly reduced the frequency of replication . These observations suggest a possibility that PARP acts as a negative regulator for the initiation of DNA replication upon cellular DNA damage.

FEBS Lett, 1994 Dec 19, 356(2-3), 165 - 8
Purification and crystallization of the ternary complex of elongation factor Tu:GTP and Phe-tRNA(Phe); Nissen P et al.; Elongation factor Tu (EF-Tu) is the most abundant protein in prokaryotic cells . Its general function in protein biosynthesis is well established . It is a member of the large family of G-proteins, all of which bind guanosine phosphates (GDP or GTP) as cofactors . In its active GTP bound state EF-Tu binds aminoacylated tRNA (aa-tRNA) forming the ternary complex EF-Tu:GTP:aa-tRNA . The ternary complex interacts with the ribosome where the anticodon on tRNA recognises a codon on mRNA, GTPase activity is induced and inactive EF-Tu:GDP is released . Here we report the successful crystallization of a ternary complex of Thermus aquaticus EF-Tu:GDPNP and yeast Phe-tRNA(Phe) after its purification by HPLC.

J Biol Chem, 1994 Dec 16, 269(50), 31410 - 7
The subunit structure of elongation factor 1 from Artemia . Why two alpha-chains in this complex?
Janssen GM, van Damme HT, Kriek J, Amons R, Moller W.
Elongation factor 1 (EF-1) regulates the specific interaction of aminoacyl-tRNA with the ribosome during the elongation phase of protein biosynthesis . Although individual functions of its separate chains have been well defined, to date there is hardly information about the structure and function of the whole complex . We describe here the complete subunit structure of elongation factor 1, and discuss its change during development of Artemia . Elongation factor 1 consists of a pentameric complex, composed of four different subunits alpha, beta, gamma, and delta in a molar ratio of 2:1:1:1 . Although one molecule of EF-1 alpha dissociates easily from the complex EF-1 alpha 2 beta gamma delta under the influence of aminoacyl-tRNA and GTP, the second molecule of EF-1 alpha was found to remain firmly attached . Thus, in eukaryotic protein synthesis, movement of transfer RNAs to the ribosome seems under the influence of two distinct molecules of EF-1 alpha, a result possibly related to the presumed consumption of two molecules of GTP by EF-Tu during the elongation step of prokaryotic protein synthesis.

Nature, 1994 Dec 15, 372(6507), 701 - 3
A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor; Frolova L et al.; The termination of protein synthesis in ribosomes is governed by termination (stop) codons in messenger RNAs and by polypeptide chain release factors (RFs) . Although the primary structure of prokaryotic RFs and yeast mitochrondrial RF is established, that of the only known eukaryotic RF (eRF) remains obscure . Here we report the assignment of a family of tightly related proteins (designated eRF1) from lower and higher eukaryotes which are structurally and functionally similar to rabbit eRF . Two of these proteins, one from human and the other from Xenopus laevis, have been expressed in yeast and Escherichia coli, respectively, purified and shown to be active in the in vitro RF assay . The other protein of this family, sup45 (sup1) of Saccharomyces cerevisiae, is involved in omnipotent suppression during translation . The amino-acid sequence of the eRF1 family is highly conserved . We conclude that the eRF1 proteins are directly implicated in the termination of translation in eukaryotes.

Cancer Res, 1994 Dec 15, 54(24), 6464 - 8
Cross-resistance between cisplatin and antimony in a human ovarian carcinoma cell line; Naredi P et al.; The metal compound cisplatin (DDP) is a widely used anticancer agent but naturally occurring and acquired resistance to DDP limits its effectiveness . Resistance is associated with a decreased accumulation of DDP and increased levels of glutathione and metallothioneins . Such changes also serve as protective and detoxification mechanisms for other metal salts in prokaryotes and lower eukaryotes . The aim of this study was to find metal salts for which the cross-resistance profile was the same as for DDP in sublines of the parental 2008 human ovarian carcinoma cells selected with either DDP (2008/C13*5.25) or CdCl2 and ZnCl2 (2008/MT) . Among the metal salts tested the resistance profile of trivalent antimony most closely resembled that of DDP . DDP-selected cells were 15-fold resistant to DDP and 4.4-fold cross-resistant to antimony potassium tartrate, whereas of the cations tested (Cd2+, Zn2+, Ni2+ and Co2+) cross-resistance was observed only for Cd2+ (2.4-fold) . When 2008 cells were selected for resistance to antimony (6.6-fold) they were found to be 16-fold cross-resistant to DDP . Accumulation of the DDP analogue cis-{3H}dichloro(ethylenediamine)platinum(II) was 59% lower in the DDP-selected subline and 48% lower in the antimony-selected variant than in the parental cell line . We conclude from the mutual cross-resistance to DDP and antimony potassium tartrate and from the impaired uptake of {3H}DEP in both the DDP and antimony-selected variants that DDP and antimony share a common mechanism of resistance . The significance of this observation lies in the fact that several evolutionarily conserved mechanisms for antimony detoxification are already known in lower organisms which may point the way to identification of additional DDP resistance mechanisms in mammalian cells.

Nucleic Acids Res, 1994 Dec 11, 22(24), 5354 - 9
The CpG-specific methylase SssI has topoisomerase activity in the presence of Mg2+; Matsuo K et al.; A prokaryotic CpG-specific methylase from Spiroplasma, SssI methylase, is now widely used to study the effect of CpG methylation in mammalian cells, and can processively modify cytosines in CpG dinucleotides in the absence of Mg2+ . In the presence of Mg2+, we found (i) that the methylation reaction is distributive rather than processive as a result of the decreased affinity of SssI methylase for DNA, and (ii) that a type I-like topoisomerase activity is present in SssI methylase preparations . This topoisomerase activity was still present in SssI methylase further purified by either SDS-polyacrylamide or isoelectric focusing gel electrophoresis . We show that methylase and topoisomerase activities are not functionally interdependent, since conditions exist where only one or the other enzymatic activity is detectable . The catalytic domains of SssI methylase and prokaryotic topoisomerases show similarity at the amino acid level, further supporting the idea that the topoisomerase activity is a genuine activity of SssI methylase . Mycoplasmas, including Spiroplasma, have the smallest genomes of all living organisms; thus, this condensation of two enzymatic activities into the same protein may be a result of genome economy, and may also have functional implications for the mechanism of methylation.

J Bacteriol, 1994 Dec, 176(24), 7566 - 73
Identification and molecular genetic characterization of a sensor kinase responsible for coordinately regulating light harvesting and reaction center gene expression in response to anaerobiosis; Mosley CS et al.; Our laboratory recently demonstrated that anaerobic induction of light harvesting and reaction center structural gene expression involved a trans-acting factor, RegA, which exhibits sequence similarity to the class of prokaryotic sensory transduction proteins known as response regulators (M . W . Sganga and C . E . Bauer, Cell 68:945-954, 1992) . In this study, we performed a screen for additional genes involved in inducing anaerobic expression of light harvesting and reaction center structural genes . This search resulted in the isolation of four strains that were shown by complementation and marker rescue analysis to harbor mutations allelic to the originally described regA mutation and one strain with a mutation found to be linked but nonallelic to regA . Sequence analysis indicated that this additional gene, regB, codes for a polypeptide that exhibits sequence similarity to the prokaryotic family of histidine sensor kinases . Analysis of photosynthesis gene expression in regB mutants indicates that the disruption of regB results in a phenotype that is very similar to that described for regA mutants, namely, a failure to trans activate anaerobic expression of the puf, puh, and puc operons . In analogy to other prokaryotic sensory transduction systems, we propose that RegB functions as a membrane-spanning sensor kinase that controls the anaerobic phosphorylation state of RegA, which in turn controls the induction of light harvesting and reaction center structural genes.

Virology, 1994 Dec, 205(2), 417 - 29
Cloning, characterization, and expression of the murine cytomegalovirus homologue of the human cytomegalovirus 28-kDa matrix phosphoprotein (UL99); Cranmer LD et al.; We have identified, characterized, and expressed in bacteria and recombinant vaccinia viruses a protein which likely represents the murine cytomegalovirus (MCMV) homologue of the human cytomegalovirus (HCMV) 28-kDa matrix phosphoprotein, the product of the HCMV UL99 open reading frame (ORF) . This protein, referred to as the MCMV UL99, is encoded by a 336-nucleotide ORF within the HindIII G fragment of MCMV strain Smith (K181) . Using a DNA probe that corresponded to the amino terminus of the ORF, we detected a transcript of 4.8 kb at 8 hr and additional transcripts of 0.88, 2.4, and 5.7 kb at 24-48 hr postinfection (p.i.) of NIH 3T3 cells with MCMV . The smallest transcript is unspliced, initiates 235 nucleotides upstream from the start of the ORF, and utilizes a polyadenylation site located 62 nucleotides downstream from the end of the ORF . The ORF encodes a protein of 112 amino acids, with a predicted mass of 11.8 kDa . Comparison of the derived amino acid sequence with that of the HCMV UL99 gene product reveals 34.8% identity in an overlap of 66 amino acids . Within the amino acid sequence are at least two potential protein kinase C and one potential casein kinase II target motifs for phosphorylation . The ORF was cloned into the pGEX-KG prokaryotic expression vector and bacterially expressed protein was used to generate a specific rabbit antiserum against the protein . Western blotting of MCMV-infected NIH 3T3 cells showed that the ORF was expressed as a doublet of 16.3 and 15.2 kDa at 48 hr p.i . only in the absence of phosphonoacetic acid, thus demonstrating that this protein is a member of the true late gene class . The immunoreactive protein in MCMV-infected cells comigrated with that produced in cells infected with recombinant vaccinia virus containing the ORF . The protein appears to be part of the MCMV virion, is phosphorylated in vivo, and generates a strong humoral immune response following MCMV infection of BALB/c mice.

Mol Cell Biol, 1994 Dec, 14(12), 8391 - 8
The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase; Gangloff S et al.; We have previously shown that cells mutant for TOP3, a gene encoding a prokaryotic-like type I topoisomerase in Saccharomyces cerevisiae, display a pleiotropic phenotype including slow growth and genome instability . We identified a mutation, sgs1 (slow growth suppressor), that suppresses both the growth defect and the increased genomic instability of top3 mutants . Here we report the independent isolation of the SGS1 gene in a screen for proteins that interact with Top3 . DNA sequence analysis reveals that the putative Sgs1 protein is highly homologous to the helicase encoded by the Escherichia coli recQ gene . These results imply that Sgs1 creates a deleterious topological substrate that Top3 preferentially resolves . The interaction of the Sgs1 helicase homolog and the Top3 topoisomerase is reminiscent of the recently described structure of reverse gyrase from Sulfolobus acidocaldarius, in which a type I DNA topoisomerase and a helicase-like domain are fused in a single polypeptide.

Infect Immun, 1994 Dec, 62(12), 5538 - 44
Mechanisms involved in uptake of Bordetella bronchiseptica by mouse dendritic cells; Guzman CA et al.; The invasion and intracellular survival of Bordetella bronchiseptica in mouse dendritic cells were investigated . The results obtained suggest that B . bronchiseptica binds specifically to glycosylated receptors present on the plasma membrane of dendritic cells, thereby inducing a signal that triggers an actin polymerization-dependent phagocytic process, probably via a protein kinase-dependent transducing phosphorylation signal . The energy required for the uptake process by host cells is provided mainly by the glycolytic pathway . An intact microtubule system and de novo protein synthesis in eukaryotic and prokaryotic cells are essential for efficient uptake and intracellular survival . The interaction of B . bronchiseptica with dendritic cells may be pertinent to natural infections that follow a chronic clinical course and predispose to secondary infections, and to the T-cell response involved in protective immunity following infections caused by Bordetella spp.

Blood, 1994 Dec 1, 84(11), 3915 - 24
X-linked sideroblastic anemia: identification of the mutation in the erythroid-specific delta-aminolevulinate synthase gene (ALAS2) in the original family described by Cooley; Cotter PD et al.; In 1945, Thomas Cooley described the first cases of X-linked sideroblastic anemia (XLSA) in two brothers from a large family in which the inheritance of the disease was documented through six generations . Almost 40 years later the enzymatic defect in XLSA was identified as the deficient activity of the erythroid-specific form of delta-aminolevulinate synthase (ALAS2), the first enzyme in the heme biosynthetic pathway . To determine the nature of the mutation in the ALAS2 gene causing XLSA in Cooley's original family, genomic DNAs were isolated from two affected hemizygotes, and each ALAS2 exon was PCR amplified and sequenced . A single transversion (A to C) was identified in exon 5 . The mutation predicted the substitution of leucine for phenylalanine at residue 165 (F165L) in the first highly conserved domain of the ALAS2 catalytic core shared by all species . No other nucleotide changes were found by sequencing each of the 11 exons, including intron/exon boundaries, 1 kb of 5'-flanking and 350 nucleotides of 3'-flanking sequence . The mutation introduced an Mse I site and restriction analysis of PCR-amplified genomic DNA confirmed the presence of the lesion in the two affected brothers and in three obligate heterozygotes from three generations of this family . Carrier diagnosis of additional family members identified the mutation in one of the proband's sisters . After prokaryotic expression and affinity purification of both mutant and normal ALAS2 fusion proteins, the specific activity of the F165L mutant enzyme was about 26% of normal . The cofactor, pyridoxal 5'-phosphate, activated and/or stabilized the purified mutant recombinant enzyme in vitro, consistent with the pyridoxine-responsive anemia in affected hemizygotes from this family.

Curr Opin Genet Dev, 1994 Dec, 4(6), 823 - 31
Introns: evolution and function; Mattick JS; The debate continues on the issue of whether nuclear introns were present in eukaryotic protein-coding genes from the beginning (introns-early) or invaded them later in evolution (introns-late) . Recent studies concerning the location of introns with respect to gene and protein structure have been interpreted as providing strong support for both positions, but the weight of argument is clearly moving in favour of the latter . Consistent with this, there is now good evidence that introns can function as transposable elements, and that nuclear introns derived from self-splicing group II introns, which then evolved in partnership with the spliceosome . This was only made possible by the separation of transcription and translation . If introns did colonize eukaryotic genes after their divergence from prokaryotes, the original question as to the evolutionary forces that have seen these sequences flourish in the higher organisms, and their significance in eukaryotic biology, is again thrown open . I suggest that introns, once established in eukaryotic genomes, might have explored new genetic space and acquired functions which provided a positive pressure for their expansion . I further suggest that there are now two types of information produced by eukaryotic genes--mRNA and iRNA--and that this was a critical step in the development of multicellular organisms.

Comp Biochem Physiol B Biochem Mol Biol, 1994 Dec, 109(4), 557 - 66
The large subunit of the pig heart mitochondrial membrane-bound beta-oxidation complex is a long-chain enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme; Yang SY; The subunit locations of the component enzymes of the pig heart trifunctional mitochondrial beta-oxidation complex are suggested by analyzing the primary structure of the large subunit of this membrane-bound multienzyme complex {Yang S.-Y . et al . (1994) Biochem . biophys . Res . Commun . 198, 431-437} with those of the subunits of the E . coli fatty acid oxidation complex and the corresponding mitochondrial matrix beta-oxidation enzymes . Long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase are located in the amino-terminal and the central regions of the 79 kDa polypeptide, respectively, whereas the long-chain 3-ketoacyl-CoA thiolase is associated with the 46 kDa subunit of this complex . The pig heart mitochondrial bifunctional beta-oxidation enzyme is more homologous to the large subunit of the prokaryotic fatty acid oxidation complex than to the peroxisomal trifunctional beta-oxidation enzyme . The evolutionary trees of 3-hydroxyacyl-CoA dehydrogenases and enoyl-CoA hydratases suggest that the mitochondrial inner membrane-bound bifunctional beta-oxidation enzyme and the corresponding matrix monofunctional beta-oxidation enzymes are more remotely related to each other than to their corresponding prokaryotic enzymes, and that the genes of E . coli multifunctional fatty acid oxidation protein and pig heart mitochondrial bifunctional beta-oxidation enzyme diverged after the appearance of eukaryotic cells.

Vaccine, 1994 Dec, 12(16), 1520 - 5
Nucleic acid immunization: a prophylactic gene therapy?
Cichutek K.
Nucleic acid (NA) vaccines may offer the safety of subunit or inactivated vaccines and, at the same time, provide the advantages of live recombinant vaccines, such as induction of a protective cellular immune response . In Germany, the so-called 'Gene Law' regulates the genetic modification of organisms such as prokaryotic or eukaryotic cells for the construction of recombinant NAs intended for use as NA vaccines . Neither NAs nor human beings treated with NAs are subject to Gene Law regulations but preclinical laboratory experiments are regulated by the Gene Law . Gene therapy, as defined in a recent draft of a European guideline for the production of gene therapeutics, includes the genetic modification of human somatic cells via transfer of NAs and thus includes NA vaccines . The guideline provides recommendations for the production of NA vaccines for human use and for preclinical safety testing . NA vaccines are products derived by biotechnological processes, as defined in part A of the annex of Council Regulation (EEC) No . 2309/93 of 22 July 1993 . Applications for marketing authorization in Member States of the European Union will thus be reviewed by the European Agency for the Evaluation of Medicinal Products starting from 1 January 1995 . Inoculation of NAs encompassing a full-length but int/nef-defective simian immunodeficiency provirus allowing limited replication of viruses released is being investigated at the Paul-Ehrlich-Institute as a model for a NA vaccine against AIDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Plant Cell, 1994 Dec, 6(12), 1889 - 97
A soybean 101-kD heat shock protein complements a yeast HSP104 deletion mutant in acquiring thermotolerance; Lee YR et al.; A cDNA clone encoding a 101-kD heat shock protein (HSP101) of soybean was isolated and sequenced . Genomic DNA gel blot analysis indicated that the corresponding gene is a member of a multigene family . The mRNA for HSP101 was not detected in 2-day-old etiolated soybean seedlings grown at 28 degrees C but was induced by elevated temperatures . DNA sequence comparison has shown that the corresponding gene belongs to the Clp (caseinolytic protease) (or Hsp100) gene family, which is evolutionarily conserved and found in both prokaryotes and eukaryotes . On the basis of the spacer length between the two conserved ATP binding regions, this gene has been identified as a member of the ClpB subfamily . Unlike other Clp genes previously isolated from higher plants, the expression of this soybean Hsp101 gene is heat inducible, and it does not have an N-terminal signal peptide for targeting to chloroplasts . Transformation of the soybean Hsp101 gene into a yeast HSP104 deletion mutant complemented restoration of acquired thermotolerance, a process in which cells survive an otherwise lethal heat stress after they are given a permissive heat treatment.

Microbiol Rev, 1994 Dec, 58(4), 755 - 805
Desiccation tolerance of prokaryotes; Potts M; The removal of cell-bound water through air drying and the addition of water to air-dried cells are forces that have played a pivotal role in the evolution of the prokaryotes . In bacterial cells that have been subjected to air drying, the evaporation of free cytoplasmic water (Vf) can be instantaneous, and an equilibrium between cell-bound water (Vb) and the environmental water (vapor) potential (psi wv) may be achieved rapidly . In the air-dried state some bacteria survive only for seconds whereas others can tolerate desiccation for thousands, perhaps millions, of years . The desiccated (anhydrobiotic) cell is characterized by its singular lack of water--with contents as low as 0.02 g of H2O g (dry weight)-1 . At these levels the monolayer coverage by water of macromolecules, including DNA and proteins, is disturbed . As a consequence the mechanisms that confer desiccation tolerance upon air-dried bacteria are markedly different from those, such as the mechanism of preferential exclusion of compatible solutes, that preserve the integrity of salt-, osmotically, and freeze-thaw-stressed cells . Desiccation tolerance reflects a complex array of interactions at the structural, physiological, and molecular levels . Many of the mechanisms remain cryptic, but it is clear that they involve interactions, such as those between proteins and co-solvents, that derive from the unique properties of the water molecule . A water replacement hypothesis accounts for how the nonreducing disaccharides trehalose and sucrose preserve the integrity of membranes and proteins . Nevertheless, we have virtually no insight into the state of the cytoplasm of an air-dried cell . There is no evidence for any obvious adaptations of proteins that can counter the effects of air drying or for the occurrence of any proteins that provide a direct and a tangible contribution to cell stability . Among the prokaryotes that can exist as anhydrobiotic cells, the cyanobacteria have a marked capacity to do so . One form, Nostoc commune, encompasses a number of the features that appear to be critical to the withstanding of a long-term water deficit, including the elaboration of a conspicuous extracellular glycan, synthesis of abundant UV-absorbing pigments, and maintenance of protein stability and structural integrity . There are indications of a growing technology for air-dried cells and enzymes . Paradoxically, desiccation tolerance of bacteria has virtually been ignored for the past quarter century . The present review considers what is known, and what is not known, about desiccation, a phenomenon that impinges upon every facet of the distributions and activities of prokaryotic cells.

Microbiol Rev, 1994 Dec, 58(4), 631 - 40
The arginine repressor of Escherichia coli; Maas WK; This review tells the story of the arginine repressor of Escherichia coli from the time of its discovery in the 1950s until the present . It describes how the research progressed through physiological, genetic, and biochemical phases and how the nature of the repressor and its interaction with its target sites were unraveled . The studies of the repression of arginine biosynthesis revealed unique features at every level of the investigations . In the early phase of the work they showed that the genes controlled by the arginine repressor were scattered over the linkage map and were not united, as in other cases, in a single operon . This led to the concept of the regulon as a physiological unit of regulation . It was also shown that different alleles of the arginine repressor could result in either inhibition of enzyme formation, as in E . coli K-12, or in stimulation of enzyme formation, as in E . coli B . Later it was shown that the arginine repressor is a hexamer, whereas other repressors of biosynthetic pathways are dimers . As a consequence the arginine repressor binds to two palindromic sites rather than to one . It was found that the arginine repressor not only acts in the repression of enzyme synthesis but also is required for the resolution of plasmid multimers to monomers, a completely unrelated function . Finally, the arginine repressor does not possess characteristic structural features seen in other prokaryotic repressors, such as a helix-turn-helix motif or an antiparallel beta-sheet motif . The unique features have sustained continuous interest in the arginine repressor and have made it a challenging subject of investigation.

Plant Physiol, 1994 Dec, 106(4), 1303 - 12
Molecular genetics of the maize (Zea mays L.) aspartate kinase-homoserine dehydrogenase gene family; Muehlbauer GJ et al.; Aspartate kinase (AK) and homoserine dehydrogenase (HSDH) are enzymes in the aspartate-derived amino acid biosynthetic pathway . Recent biochemical evidence indicates that an AK-HSDH bifunctional enzyme exists in maize (Zea mays L.) . In this report, we characterize three genes that encode subunits of AK-HSDH . Two cDNAs, pAKHSDH1 and pAKHSDH2, containing the full-coding sequence, and one partial cDNA, pAKHSDH3, encode amino acid sequences similar to the reported monofunctional AK and HSDH enzymes from prokaryotes and yeast (Saccharomyces cerevisiae) and to AK-HSDH bifunctional enzymes of prokaryotes, yeast, carrot (Daucus carota), and Arabidopsis thaliana . Immunological and biochemical analyses verify that the cDNAs encode AK-HSDH and indicate that both the AK and HSDH activities are feedback inhibited by threonine . RNA blots identify a 3.2-kb transcript in all maize tissues examined . pAKHSDH1 and pAKHSDH2 map to chromosomes 4L and 2S, respectively . This study shows that maize contains AK-HSDH bifunctional enzyme(s) encoded by a small gene family of at least three genes . Maize AK-HSDH has conserved sequences found in communication modules of prokaryotic two-component regulatory systems, which has led us to propose that maize AK-HSDH may be involved in a similar regulatory mechanism.

J Mol Evol, 1994 Dec, 39(6), 620 - 4
Evolutionary role of abortive transcript as a primer for DNA replication; Matsumoto J; Abortive cycling features transcription initiation by RNA polymerase in both prokaryote and eukaryote . It is known that T7 RNA polymerase produces abortive transcripts up to eight ribonucleotides in length depending on the initial sequence of the DNA message . On the other hand, T7 RNA polymerase initiates DNA replication from the T7 primary origin by synthesizing primers . And the shortest primer from the phi l.lB promoter in the primary origin also seems to be eight ribonucleotides in length . Therefore, it is likely that the longest abortive transcript serves as the shortest primer for T7 DNA replication from the primary origin . Considering that promoters often exist in DNA replication origins for example, E . coli oriC and many eukaryotic origins, the early DNA replication system appears to have taken advantage of the abortive cycling of RNA-dependent RNA polymerase that already existed before the emergence of DNA world . The evolutionary primitive RNA polymerase could do both transcription and priming of DNA replication . Accordingly, abortive cycling would play an important role in evolution at the emergence of DNA world . The priming activity of the primitive RNA polymerase would be taken over by primase later, which seems to be a specialized RNA polymerase for abortive cycling.

J Cell Biol, 1994 Dec, 127(6 Pt 1), 1505 - 14
NAP57, a mammalian nucleolar protein with a putative homolog in yeast and bacteria; Meier UT et al.; We report the identification and molecular characterization of a novel nucleolar protein of rat liver . As shown by coimmunoprecipitation this protein is associated with a previously identified nucleolar protein, Nopp140, in an apparently stoichiometric complex and has therefore been termed NAP57 (Nopp140-associated protein of 57 kD) . Immunofluorescence and immunogold electron microscopy with NAP57 specific antibodies show colocalization with Nopp140 to the dense fibrillar component of the nucleolus, to coiled bodies, and to the nucleoplasm . Immunogold staining in the nucleoplasm is occasionally seen in the form of curvilinear tracks between the nucleolus and the nuclear envelope, similar to those previously reported for Nopp140 . These data suggest that Nopp140 and NAP57 are indeed associated with each other in these nuclear structures . The cDNA deduced primary structure of NAP57 shows a protein of a calculated molecular mass of 52,070 that contains a putative nuclear localization signal near its amino and carboxy terminus and a hydrophobic amino acid repeat motif extending across 84 residues . Like Nopp140, NAP57 lacks any of the known consensus sequences for RNA binding which are characteristic for many nucleolar proteins . Data bank searches revealed that NAP57 is a highly conserved protein . A putative yeast (S . cerevisiae) homolog is 71% identical . Most strikingly, there also appears to be a smaller prokaryotic (E . coli and B . subtilis) homolog that is nearly 50% identical to NAP57 . This indicates that NAP57 and its putative homologs might serve a highly conserved function in both pro- and eukaryotes such as chaperoning of ribosomal proteins and/or of preribosome assembly.

Immunol Lett, 1994 Dec, 43(1-2), 99 - 107
Genetic regulation of leishmanial and mycobacterial infections: the Lsh/Ity/Bcg gene story continues; Blackwell JM et al.; A common basis to genetic regulation of leishmanial and mycobacterial infections is provided by the action of the murine Lsh/Ity/Bcg gene in controlling the priming/activation of macrophages for antimicrobial activity . This relies on the TNF-alpha-dependent sustained expression of the inducible nitric oxide synthase (iNOS) gene responsible for the generation of large amounts of toxic nitric oxide (NO) . The Lsh/Ity/Bcg gene has many pleiotropic effects, including differential expression of the early response gene KC following stimulation of macrophages with bacterial lipopolysaccharide (LPS) and mycobacterial lipoarabinomannan (LAM) . The major signal transduction pathway involved in KC induction requires the generation of low levels of NO via constitutive nitric oxide synthase (cNOS) activity, leading to activation of guanylate cyclase and the cGMP-dependent kinase pathway . NO therefore appears to provide a common link between the early influence of Lsh in regulating the expression of genes which mediate many pleiotropic effects, and the later production of NO as the final effector mechanism for kill . The recently cloned candidate for Lsh/Ity/Bcg, designated Nramp for Natural resistance associated macrophage protein, encodes a polytopic integral membrane protein that has structural features common to prokaryotic and eukaryotic transporters and includes a conserved binding-protein-dependent transport motif which may be involved in interaction with peripheral ATP-binding subunits . The N-terminal sequence also carries a proline/serine rich putative SH3 binding domain, consistent with a role for tyrosine kinases in regulating Nramp function . (ABSTRACT TRUNCATED AT 250 WORDS)

Protein Eng, 1994 Dec, 7(12), 1449 - 53
LexA repressor and iron uptake regulator from Escherichia coli: new members of the CAP-like DNA binding domain superfamily; Holm L et al.; Comparison of structures can reveal surprising connections between protein families and provide new insights into the relationship between sequence, structure and function . The solution structure of LexA repressor from Escherichia coli reveals an unexpected structural similarity to a widespread class of prokaryotic and eukaryotic regulatory proteins, which is typified by catabolite gene activator protein (CAP) . The use of combined sequence profiles allows the identification of two new prokaryotic members of the superfamily: listeriolysin regulatory protein (PrfA) and ferric uptake regulatory protein (Fur) . LexA, PrfA and Fur are the first examples of prokaryotic regulatory proteins in which DNA recognition is mediated by a variant of the classical helix-turn-helix motif, with an insertion in the turn region.

J Virol Methods, 1994 Dec, 50(1-3), 219 - 25
Detection of human T-cell leukaemia virus 1 permissive cells using cell lines producing selectable recombinant virions; Copeland KF et al.; A selectable retrovirus vector based on a full length HTLV-1 provirus clone, pCS-HTLV-1, was constructed by replacing the coding regions for tax, rex and the 3' region of env with the prokaryotic neomycin resistance gene under the control of the CMV promoter . This vector, pHTLV-1-CMVneo, was transfected into HTLV-1 infected human lymphocytes and fibroblasts . The production of recombinant virus by these cells was measured by the transfer of G418 resistance to target cells . Infection of target cells showed a preference for human lymphocytes in addition to two human fibroblast cell lines, Hos7 and RD4, and the African green monkey kidney cell line, Cos7 . This system provides a method to study the cellular tropism of HTLV-1 and additionally provides a model to facilitate molecular studies of the natural events of HTLV-1 infection and integration.

Curr Biol, 1994 Dec 1, 4(12), 1115 - 7
Molecular evolution . Who are the parents of eukaryotes?
Irwin DM.
Phylogenetic analysis of heat shock protein 70 sequences suggests that the eukaryotic nuclear genome may be a hybrid, originating from the fusion of distinct prokaryotic cells.

Glycoconj J, 1994 Dec, 11(6), 501 - 6
Lectin domains in the toxin of Bordetella pertussis: selectin mimicry linked to microbial pathogenesis; Sandros J et al.; The pathogenesis of many infectious diseases is critically determined by prokaryotic lectins which enable differential recognition and activation of targeted eukaryotic cells . Some bacterial adhesins mimic and co-opt eukaryotic cell-cell adhesion motifs . This is illustrated by the toxin of Bordetella pertussis . Pertussis toxin mediates intoxication of eukaryotic cells by elevation of cAMP and it serves as an adhesin binding the bacteria to ciliated cells and respiratory macrophages . These activities are mediated by the lectin-like properties of the binding oligomer of the toxin . A comparison of pertussis toxin and the selectins involved in leukocyte trafficking indicates that these prokaryotic and eukaryotic C-type lectins share some element of primary sequence similarity, three dimensional structure, and biological activities . Such mimicry suggests a link between eukaryotic cell-cell adhesion motifs and microbial pathogenesis.

Experientia, 1994 Nov 30, 50(11-12), 1061 - 6
Hsp70: a carrier molecule with built-in adjuvanticity; Del Giudice G; One problem associated with the development of subunit vaccines is their limited immunogenicity, due to their physico-chemical structure, their inability to encounter the correct MHC restriction element, or the need for strong adjuvants to be delivered along with them . These problems are usually solved by conjugating target epitopes (peptides or oligosaccharides) with carrier proteins which provide a source of T-cell epitopes recognised by a large proportion of the vaccinated individuals . We have shown that mycobacterial hsp65 and hsp70 exert a strong helper effect in vivo when conjugated to synthetic peptides or oligosaccharides . Interestingly, this helper effect did not require the need for any adjuvant, either in mice or in monkeys . The helper effect mediated by the hsp65 required that animals were previously primed with either live BCG or the hsp65 alone; on the other hand, such a priming was not required when the hsp70 was used in the conjugates . Similar results were obtained with HSP molecules from Escherichia coli . This may suggest that the adjuvant-free helper effect observed applies not only to mycobacterial HSP, but also to HSP from other prokaryotes . These findings suggest that microbial hsp70 could be considered for the design of conjugated vaccine constructs for eventual human use.

J Biol Chem, 1994 Nov 25, 269(47), 29903 - 7
Dexamethasone enhancement of gene expression after direct hepatic DNA injection; Malone RW et al.; The critical physiological functions of the liver make hepatocytes important targets for therapeutic gene delivery . This study reports significant gene expression following direct injection of plasmid DNA into the livers of rats and cats . Transfection was characterized using luciferase and Lac Z expression from plasmids with the cytomegalovirus immediate early promoter/enhancer (CMV IE) or the Rous sarcoma virus long terminal repeat (RSV LTR) . Dexamethasone treatment enhanced and prolonged transfected gene expression, possibly by activating gene expression . Southern analysis of total DNA extracted from liver at various times following injection detected persistent unintegrated plasmid DNA which maintained a prokaryotic methylation pattern . This study demonstrates the feasibility of direct DNA injection in the experimental analysis of hepatic gene expression in vivo.

Nucleic Acids Res, 1994 Nov 25, 22(23), 5031 - 7
Histidylation by yeast HisRS of tRNA or tRNA-like structure relies on residues -1 and 73 but is dependent on the RNA context; Rudinger J et al.; Residue G-1 and discriminator base C73 are the major histidine identity elements in prokaryotes . Here we evaluate the importance of these two nucleotides in yeast histidine aminoacylation identity . Deletion of G-1 in yeast tRNA(His) transcript leads to a drastic loss of histidylation specificity (about 500-fold) . Mutation of discriminator base A73, common to all yeast tRNA(His) species, into G73 has a more moderate but still significant effect with a 22-fold decrease in histidylation specificity . Changes at position 36 in the anticodon loop has negligible effect on histidylation . The role of residues -1 and 73 for specific aminoacylation by yeast HisRS was further investigated by studying the histidylation capacities of seven minihelices derived from the Turnip Yellow Mosaic Virus tRNA-like structure . Changes in the nature of nucleotides -1 and 73 modulate this activity but do not suppress it . The optimal mini-substrate for HisRS presents a G.A mismatch at the position equivalent to residues G-1.A73 in yeast tRNA(His), confirms the importance of this structural feature in yeast histidine identity . The fact that the minisubstrates contain a pseudoknot in which position -1 is mimicked by an internal nucleotide from the pseudoknot highlights further the necessity of a stacking interaction of this position over the amino acid accepting branch of the tRNA during the aminoacylation process . Individual transplantation of G-1 or A73 into yeast tRNA(Asp) transcript improves the histidylation efficiency of the engineered tRNA(Asp) . However, a tRNA(Asp) transcript presenting simultaneously both residues G-1 and A73 becomes a less good substrate for HisRS, suggesting the importance of the structural context and/or the presence of antideterminants for an optimal expression of these two identity elements.

Nucleic Acids Res, 1994 Nov 25, 22(23), 4983 - 8
The pseudodisaccharides: a novel class of group I intron splicing inhibitors; Rogers J et al.; Lysinomicin, a naturally-occurring pseudodisaccharide, inhibits translation in prokaryotes . We report that lysinomicin (and three related compounds) are able to inhibit the self-splicing of group I introns, thus identifying pseudodisaccharides as a novel class of group I intron splicing inhibitors . Lysinomicin inhibited the self-splicing of the sunY intron of phage T4 with a Ki of 8.5 microM (+/- 5 microM) and was active against other group I introns . Inhibition was found to be competitive with the substrate guanosine, unlike aminoglycoside antibiotics, which act non-competitively to inhibit the splicing of group I introns . Competitive inhibitors of group I intron splicing known to date all contain a guanidino group that was thought to be required for inhibition; lysinomicin lacks a guanidino group.

Nucleic Acids Res, 1994 Nov 25, 22(23), 4869 - 71
Human and E.coli excinucleases are affected differently by the sequence context of acetylaminofluorene-guanine adduct; Mu D et al.; Synthetic DNA substrates containing an acetylaminofluorene (AAF) adduct at each of the three guanine in the G1G2CG3CC sequence were constructed and tested as substrates for reconstituted E.coli (A)BC excinuclease and human excinuclease in HeLa cell-free extract (CFE) . The (A)BC excinulcease repaired the three substrates with relative efficiencies of G1:G2:G3 of 100:18:66 in agreement with an earlier report {Seeberg, E., and Fuchs, R.P.P . (1990) Proc . Natl Acad . Sci . USA 87, 191-194} . The same lesions were repaired by the human excinuclease with the strikingly different efficiencies of G1:G2:G3 as 38:100:68 . These results reveal that the human excinuclease is affected by the sequence context of the lesion in a different manner than its prokaryotic counterpart.

Nucleic Acids Res, 1994 Nov 25, 22(23), 4953 - 7
Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs; Chen H et al.; The prokaryotic mRNA ribosome binding site (RBS) usually contains part or all of a polypurine domain UAAGGAGGU known as the Shine-Dalgarno (SD) sequence found just 5' to the translation initiation codon . It is now clear that the SD sequence is important for identification of the translation initiation site on the mRNA by the ribosome, and that as a result, the spacing between the SD and the initiation codon strongly affects translational efficiency (1) . It is not as clear, however, whether there is a unique optimal spacing . Complications involving the definition of the spacing as well as secondary structures have obscured matters . We thus undertook a systematic study by inserting two series of synthetic RBSs of varying spacing and SD sequence into a plasmid vector containing the chloramphenicol acetyltransferase gene . Care was taken not to introduce any secondary structure . Measurements of protein expression demonstrated an optimal aligned spacing of 5 nt for both series . Since aligned spacing corresponds naturally to the spacing between the 3'-end of the 16S rRNA and the P-site, we conclude that there is a unique optimal aligned SD-AUG spacing in the absence of other complicating issues.

J Theor Biol, 1994 Nov 21, 171(2), 215 - 23
Third codon G + C periodicity as a possible signal for an "internal" selective constraint; Lio P et al.; Quasi-local analysis methods, such as window Fast Fourier Transform and an information theoretical quantity known as mutual information, have allowed us to gain some further insights on the importance and the contextual dependence of a pattern found in DNA sequences showing a periodicity of three with a G or C base in the third position . We have screened for such a periodicity, in terms of the alternative "strong" (S = C or G) versus "weak" (W = A or T) base, a large sample of DNA coding and non-coding sequences from both prokaryotes and eukaryotes, with the aim of testing whether this pattern could be considered as a significant signal for past or present constraints regarding DNA organization and/or function . This periodicity was indeed found in a number of sequences always associated with open reading frames, generally confined in prokaryotes living in extreme environments or in highly conserved eukaryotic genes . Moreover, codon usage was found to be very similar even in genes coding for very different functions . The data are discussed in view of their possible implications for an adaptive value of such a periodicity, in terms of more accurate translation processing and better overall stability.

Biochem Biophys Res Commun, 1994 Nov 15, 204(3), 1074 - 80
Isolation and characterization of human Nramp cDNA; Kishi F; The mouse gene locus Lsh/Ity/Bcg regulates macrophage activation for antimicrobial activity against intracellular pathogens . A candidate gene, designated natural resistance-associated macrophage protein gene (Nramp), recently isolated from a mouse pre-B cell cDNA library, encodes an integral membrane protein that has structural homology with known prokaryotic and eukaryotic transport systems . In the present study, the cDNA for human Nramp was isolated by screening a human monocyte cDNA library . The cDNA was 2245 bp in length and coded for a protein of 483 amino acid residues with a molecular weight mass of 52.8 kDa . The deduced amino acid sequence was 89% homologous with that of mouse . Southern blot analysis indicated a single gene for Nramp counterpart in the human genome . Northern blot analysis revealed a single species of mRNA of approximately 2.5 kb.

Eur J Biochem, 1994 Nov 15, 226(1), 15 - 22
A super-family of medium-chain dehydrogenases/reductases (MDR) . Sub-lines including zeta-crystallin, alcohol and polyol dehydrogenases, quinone oxidoreductase enoyl reductases, VAT-1 and other proteins; Persson B et al.; The protein super-family of medium-chain alcohol dehydrogenases (and glutathione-dependent formaldehyde dehydrogenase), polyol dehydrogenases, threonine dehydrogenase, archaeon glucose dehydrogenase, and eye lens reductase-active zeta-crystallins also includes Escherichia coli quinone oxidoreductase, Torpedo VAT-1 protein, and enoyl reductases of mammalian fatty acid and yeast erythronolide synthases . In addition, two proteins with hitherto unknown function are shown to belong to this super-family of medium-chain dehydrogenases and reductases (MDR) . Alignment of zeta-crystallins/quinone oxidoreductases/VAT-1 reveals 38 strictly conserved residues, of which approximately half are glycine residues, including those at several space-restricted turn positions and critical coenzyme-binding positions in the alcohol dehydrogenases . This indicates a conserved three-dimensional structure at the corresponding parts of these distantly related proteins and a conserved binding of a coenzyme in the two proteins with hitherto unknown function, thus ascribing a likely oxidoreductase function to these proteins . When all forms are aligned, including enoyl reductases, a zeta-crystallin homologue from Leishmania and the two proteins with hitherto unknown function, only three residues are strictly conserved among the 106 proteins characterised within the superfamily, and significantly these residues are all glycines, corresponding to Gly66, Gly86 and Gly201 of mammalian class I alcohol dehydrogenase . Notably, these residues are located in different domains . Hence, a distant origin and divergent functions, but related forms and interactions, appear to apply to the entire chains of the many prokaryotic and eukaryotic members . Additionally, in the zeta-crystallins/quinone oxidoreductases, a highly conserved tyrosine residue is found . This residue, in the three-dimensional structure of the homologous alcohol dehydrogenase, is positioned at the subunit cleft that contains the active site and could therefore be involved in catalysis . If so, this residue and its role may resemble the pattern of a conserved tyrosine residue in the different family of short-chain dehydrogenases/reductases (SDR).

EMBO J, 1994 Nov 15, 13(22), 5355 - 60
Interference of DNA sequence divergence with precise recombinational DNA repair in mammalian cells; Belmaaza A et al.; Studies done in prokaryotes and eukaryotes have indicated that DNA sequence divergence decreases the frequency of homologous recombination . To determine which step(s) of homologous recombination is sensitive to DNA sequence divergence in mammalian cells we have used an assay that does not rely on the recovery of functional products . The assay is based on the acquisition by homologous recombination of endogenous LINE-1 sequences by exogenous LINE-1 sequences . In parallel experiments, we introduced into mouse cells two gapped exogenous LINE-1 sequences, one from the mouse, L1Md-A2, and the other from the rat, L1Rn-3 . Although L1Rn-3 is on average less than 85% homologous to the LINE-1 elements of the mouse, the frequency of homologous recombination with endogenous LINE-1 elements obtained with L1Rn-3 was the same as the one obtained with L1Md-A2 which is on average 95% homologous to the LINE-1 elements of the mouse . The endogenous LINE-1 sequences rescued by L1Rn-3 were 8-18% divergent from L1Rn-3 sequences, whereas those rescued by L1Md-A2 were 2-5% divergent from L1Md-A2 sequences . The gap which had been introduced into the exogenous LINE-1 sequences had been precisely repaired in 50% of the recombinants obtained with L1Md-A2 . None of the L1Rn-3 recombinants showed precise gap repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Genomics, 1994 Nov 15, 24(2), 280 - 7
Isolation and expression of a cDNA encoding the precursor for a novel member (ACADSB) of the acyl-CoA dehydrogenase gene family; Rozen R et al.; The acyl-CoA dehydrogenases (ACDs) are a family of mitochondrial enzymes that oxidize straight chain or branched chain acyl-CoAs in the metabolism of fatty acids or branched chain amino acids . Deficiencies in members of this gene family are important causes of human disease . A cDNA encoding the human precursor for a novel member (gene symbol ACADSB) of the ACD gene family has been isolated and characterized . The open reading frame of 1.3 kb encodes a precursor protein of 431 amino acids, which is processed in vitro to yield a mature protein of 399 amino acids . The cDNA has significant sequence similarity to other members of the acyl-CoA dehydrogenase family, with the greatest homology (38%) to the short chain acyl-CoA dehydrogenase . The cDNA was expressed in eukaryotic (COS) and prokaryotic (Escherichia coli) cells, producing a protein of the expected size, with activity toward the short branched chain acyl-CoA derivatives ((S)-2-methylbutyryl-CoA, isobutyryl-CoA, and 2-methylhexanoyl-CoA), as well as toward the short straight chain acyl-CoAs (butyryl-CoA and hexanoyl-CoA).

J Mol Biol, 1994 Nov 11, 243(5), 950 - 6
Sequence analysis identifies the proline dehydrogenase and delta 1-pyrroline-5-carboxylate dehydrogenase domains of the multifunctional Escherichia coli PutA protein; Ling M et al.; The PutA protein of Escherichia coli has two enzymatic activities: proline dehydrogenase (PDH) and delta 1-pyrroline-5-carboxylate dehydrogenase (P5CDH) . It associates with the cytoplasmic membrane as PDH and P5CDH and with put control region DNA as put repressor . Reduction of the PutA flavin by proline, a PutA conformational change and association of PutA with membranes are coincident . The nucleotide base sequence of E . coli putA was determined, that of S . typhimurium putA was updated and the deduced PutA protein sequences were surveyed for catalytic domains and ligand binding sites . The two sequences were very similar (80.5% and 95% on the nucleic acid and protein levels, respectively) . Residues 650 through 1130 of PutA were very similar to the sequences of P5C dehydrogenases and aldehyde dehydrogenases from both prokaryotes and eukaryotes . Glutamate 883 and cysteine 917 of PutA were conserved with the corresponding residues in P5C dehydrogenases and with those proposed to be active site residues in the aldehyde dehydrogenases . Those relationships suggest that gamma-glutamic semialdehyde, believed to equilibrate spontaneously with P5C, is the substrate for P5C dehydrogenases . Residues 340 through 590 of PutA were similar in sequence to proline dehydrogenases from Saccharomyces cerevisiae and Drosophila melanogaster . Limited similarities were also found between residues 315 through 357 of PutA and a consensus sequence near a putative active site and FAD-binding region shared by succinate dehydrogenase sequences from several organisms . Since residues 228 through 358 of PutA were similar in sequence to several serine-pyruvate aminotransferases, PutA is proposed to catalyze the hydrolysis of P5C (a Schiff's base intermediate) to gamma-glutamic semialdehyde . A carboxyl-terminal sequence that resembles a leucine zipper motif may be involved in association of PutA with put control region DNA.

J Biol Chem, 1994 Nov 11, 269(45), 28487 - 93
Effect of redox environment on the in vitro and in vivo folding of RTEM-1 beta-lactamase and Escherichia coli alkaline phosphatase; Walker KW et al.; The oxidative folding mechanisms of two Escherichia coli periplasmic proteins, alkaline phosphatase and RTEM-1 beta-lactamase, have been examined in vitro and in vivo . In contrast to eukaryotic proteins, which require a relatively reducing environment for optimal folding rates, both alkaline phosphatase and beta-lactamase fold fastest under very oxidizing conditions . For example, bovine pancreatic ribonuclease exhibits an optimal folding rate in a redox buffer consisting of 1 mM GSH and 0.2 mM GSSG (Lyles, M . M., and Gilbert, H . F . (1991) Biochemistry 30, 613-619); however, both E . coli alkaline phosphatase and beta-lactamase exhibit optimal in vitro folding rates at low concentrations of GSH (< 0.4 mM) and very high concentrations of GSSG (4-8 mM) . For both bacterial proteins, GSH inhibits oxidative folding . Under optimal redox conditions, the rate-limiting step for the in vitro oxidative folding of alkaline phosphatase depends on the concentration of the protein, consistent with a mechanism involving rapid oxidation followed by slow dimerization . With beta-lactamase, the oxidative folding mechanism involves a competition between disulfide bond formation and folding of the molecule into a catalytically active conformation that buries the 2 reduced cysteines in the core of the enzyme . The effects of including a thiol reductant in the growth medium on the in vivo folding of alkaline phosphatase and beta-lactamase are similar to the effects observed during in vitro folding of these enzymes . The levels of both oxidized proteins are decreased by GSH in the growth medium . However, addition of a disulfide oxidant to the growth medium does not positively affect the production of either enzyme . These observations are consistent with the idea that the oxidative folding mechanisms of E . coli periplasmic proteins and, by inference, proteins of the eukaryotic endoplasmic reticulum have evolved to accommodate constraints placed on the folding reaction by the folding environment . The consequences of differences between the folding mechanisms in eukaryotic and prokaryotic disulfide-containing proteins on the expression of eukaryotic proteins in the bacterial periplasm are discussed.

Biochim Biophys Acta, 1994 Nov 11, 1201(2), 217 - 22
Heparin and heparan sulfate enhancement of the inhibitory activity of plasminogen activator inhibitor type 1 toward urokinase type plasminogen activator; Urano T et al.; To study effects of glycosaminoglycan on the interaction between two chain urokinase type plasminogen activator (tcu-PA) (EC 3.4.21.31) and plasminogen activator inhibitor type 1 (PAI-1) the second order rate constant (k1) between high molecular weight tcu-PA and active recombinant prokaryotic PAI-1 (rpPAI-1) was determined employing a continuous method using chromogenic substrate S-2444 either in the presence or absence of various kinds of glycosaminoglycans . k1 was (5.9 +/- 1.6).10(6)/mol per s in the absence of effector molecule, and following addition of heparin (1.0 U/ml) k1 was enhanced to (3.22 +/- 0.73).10(7) . A significant enhancement of k1 was also obtained by heparan sulfate (1.87 +/- 0.25).10(7) . Dermatan sulfate or chondroitin sulfate did not show a significant effect on k1 although a slight decrease was obtained by mono-dextran sulfate (4.2 +/- 1.2).10(6) . The intrinsic fluorescence of rpPAI-1 was shown to be slightly increased following addition of heparin (1.49 +/- 0.22%, n = 6), suggesting that heparin may enhance the inhibitory activity of PAI-1 toward tcu-PA both by a template mechanism and by a modification of PAI-1 structure.

Biochemistry, 1994 Nov 8, 33(44), 13013 - 21
Site-directed mutagenesis of residues within helix VI in subunit I of the cytochrome bo3 ubiquinol oxidase from Escherichia coli suggests that tyrosine 288 may be a CuB ligand; Thomas JW et al.; The heme-copper oxidase superfamily contains all of the mammalian mitochondrial cytochrome c oxidases, as well as most prokaryotic respiratory oxidases . All members of the superfamily have a subunit homologous to subunit I of the mammalian cytochrome c oxidases . This subunit provides the amino acid ligands to a low-spin heme component as well as to a heme-copper binuclear center, which is the site where dioxygen is reduced to water . The amino acid sequence of transmembrane helix VI of subunit I is the most highly conserved within the superfamily . Previous efforts have demonstrated that one of the residues in this region, H284, is critical for oxidase activity and for the assembly of CuB . This paper presents the analysis of additional site-directed mutants in which other highly conserved residues in helix VI (P285, E286, Y288, and P293) have been substituted . Most of the mutants are enzymatically inactive . Structural perturbations reported by Fourier transform infrared absorption difference spectroscopy of CO adducts of the mutant oxidases confirm the previous suggestion that this region is adjactent to CuB . Furthermore, the analysis of five different substitutions for Y288 indicates that all lack CuB . On the basis of these data, it is proposed that Y288 may be a CuB ligand along with H333, H334, and H284, and a plausible molecular model of the CuB site is presented.

J Biol Chem, 1994 Nov 4, 269(44), 27663 - 9
Purification of a DNA topoisomerase II from the hyperthermophilic archaeon Sulfolobus shibatae . A thermostable enzyme with both bacterial and eucaryal features; Bergerat A et al.; A type II DNA topoisomerase has been purified to homogeneity from the hyperthermophilic archaeon Sulfolobus shibatae . The enzyme is composed of two subunits of 60 and 47 kDa . It has a Stokes radius of 69 A and has a sedimentation coefficient of 7.8 S which gives a calculated native molecular mass of approximately 230 kDa, indicating a heterotetrameric structure . This enzyme is ATP and Mg2+ dependent and can relax both negatively and positively supercoiled DNA, but presents no supercoiling activity . The S . shibatae DNA topoisomerase II is more efficient in decatenation than in relaxation . The optimal temperature for the enzymatic activity is approximately 80 degrees C . This archaeal enzyme is not inhibited by the gyrase inhibitor novobiocin but is sensitive to several inhibitors of eucaryotic DNA topoisomerases of type II such as amsacrines, ellipticine, and the quinolone CP-115,953 . Like all prokaryotic DNA topoisomerase II, the S . shibatae DNA topoisomerase II is a heterotetramer but the absence of supercoiling activity, the strong decatenase activity, and the pattern of antibiotic sensitivity of the S . shibatae DNA topoisomerase II is reminiscent of eucaryotic enzymes.

J Biol Chem, 1994 Nov 4, 269(44), 27297 - 302
Isolation, purification, and characterization of an Amadori product binding protein from a Pseudomonas sp . soil strain; Gerhardinger C et al.; Sugars react nonenzymatically with protein amino groups to form a ketoamine adduct (Amadori product), which leads to the formation of advanced glycation end-products . These compounds are involved in the development of tissue modifications such as cross-linking and fluorescence in diabetes and aging . Searching for an enzyme to reverse protein glycation, we isolated a Pseudomonas sp . soil strain growing selectively on the Amadori product epsilon-fructosyl-aminocaproate . An Amadori product binding protein (ABP) was purified from the bacterial extract by single-step affinity chromatography on glycated lysine-Sepharose . The protein, a monomer of 45 kDa, did not bind to unglycated or NaBH4-reduced glycated lysine-Sepharose suggesting specificity for the Amadori compound . The concentration-dependent binding of glycated aminocaproate showed saturation with Kd = 1.49 microM and Bmax = 17.63 nmol/mg of protein corresponding to 0.8 mol/mol of protein . The binding of epsilon-1-{14C}fructosyl-aminocaproate to the protein was inhibited by other glucose-derived Amadori products, but not by free sugars, unglycated amines, or ribated lysine . The sequence of the first 16 NH2-terminal amino acids and a GenBank search revealed that ABP is a novel protein . Its synthesis was inducible by growth of the organism in Amadori product . Immunoblotting studies showed that ABP is not found in cell extracts from other prokaryotes, yeast, or liver homogenate and does not bind Amadori products in glycated proteins . ABP has no enzymatic activity toward glycated substrates and may thus have transport or permease function for glycated amino acids.

J Biol Chem, 1994 Nov 4, 269(44), 27183 - 5
In vivo conversion of L-serine to D-alanine in a ribosomally synthesized polypeptide; Skaugen M et al.; In the course of characterizing the bacteriocin lactocin S and its encoding gene, we discovered three alanine-for-serine substitutions which, apparently, is a violation of the genetic code . Subsequent chiral analysis of lactocin S hydrolysates revealed a correlation between D-alanine content and the three substitutions, implying a conversion of L-serine to D-alanine in lactocin S maturation . In order to explain this observation, we suggest a sequence of events initiated by the dehydration of serine, which is common in the biosynthesis of the lanthionine-containing polycyclic lantibiotics (Schnell, N., Entian, K.-D., Schneider, U., Gotz, F., Zahner, H., Kellner, R . & Jung, G . (1988) Nature 333, 276-278; Jung, G . (1991) Angew, Chem . Int . Ed . Engl . 30, 1051-1068; Bierbaum, G . & Sahl, H.-G . (1993) Zentralbl . Bakteriol . 278, 1-22) and completed by the stereospecific reduction of dehydroalanine residues . The occurrence of non-lanthionine alpha-carbon stereoinversion in lactocin S maturation substantiates the hypothetical alpha-epimerization scheme originally put forward by Bycroft (Bycroft, B . W . (1969) Nature 224, 595-597), and we propose a revision of this model to accommodate the lactocin S-type stereoinversion . Lactocin S is the first prokaryotic exception to the rule that only L-amino acids are included in ribosomally synthesized peptides.

Gene, 1994 Nov 4, 149(1), 173 - 8
Inhibition of prokaryotic and eukaryotic transcription by the 2:1 2,9-dimethyl-1,10-phenanthroline-cuprous complex, a ligand specific for open complexes; Perrin DM et al.; The redox-stable, tetrahedral cuprous chelate of neocuproine (2,9-dimethyl-1,10-phenanthroline) binds to the single-stranded DNA formed in open complexes and is an effective inhibitor of eukaryotic and prokaryotic transcription . Despite the many kinetic and structural differences between prokaryotic and eukaryotic transcription systems, they are all similarly inhibited by neocuproine copper, suggesting that all open complexes may share a homologous structure.

Cell, 1994 Nov 4, 79(3), 449 - 58
A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro; Hirano T et al.; We report here a chromosomal protein that plays an essential role in mitotic chromosome condensation in Xenopus egg extracts . Two polypeptides, designated XCAP-C and XCAP-E, were found to associate with each other in the extracts, presumably forming a heterodimer . During chromosome assembly in mitotic extracts, XCAP-C/E was recruited to the chromatin and formed a discrete internal structure within assembled chromosomes . Antibody blocking experiments showed that XCAP-C function is required for both assembly and structural maintenance of mitotic chromosomes in vitro . Deduced amino acid sequences revealed that the two polypeptides share common structural motifs, consisting of an N-terminal NTP-binding domain, two central coiled-coil regions, and a C-terminal conserved domain . These motifs are highly conserved in a protein family, members of which have been identified recently in both prokaryotes and eukaryotes.

J Biol Chem, 1994 Nov 4, 269(44), 27747 - 55
Identification of the regulatory domain of the mammalian multifunctional protein CAD by the construction of an Escherichia coli hamster hybrid carbamyl-phosphate synthetase; Liu X et al.; Carbamyl-phosphate synthetases from different organisms have similar catalytic mechanisms and amino acid sequences, but their structural organization, sub-unit structure, and mode of regulation can be very different . Escherichia coli carbamyl-phosphate synthetase (CPSase), a monofunctional protein consisting of amido-transferase and synthetase subunits, is allosterically inhibited by UMP and activated by NH3, IMP, and ornithine . In contrast, mammalian CPSase II, part of the large multifunctional polypeptide, CAD, is inhibited by UTP and activated by 5-phosphoribosyl-1-pyrophosphate (PRPP) . Previous photoaffinity labeling studies of E . coli CPSase showed that allosteric effectors bind near the carboxyl-terminal end of the synthetase subunit . This region of the molecule may be a regulatory subdomain common to all CPSases . An E . coli mammalian hybrid CPSase gene has been constructed and expressed in E . coli . The hybrid consists of the E . coli CPSase synthetase catalytic subdomains, residues 1-900 of the 1073 residue polypeptide, fused to the amino-terminal end of the putative 190-residue regulatory subdomain of the mammalian protein . The hybrid CPSase had normal activity, but was no longer regulated by the prokaryotic allosteric effectors . Instead, the glutamine- and ammonia-dependent CPSase activities and both ATP-dependent partial reactions were activated by PRPP and inhibited by UTP, indicating that the binding sites of both of these ligands are located in a regulatory region at the carboxyl-terminal end of the CPSase domain of CAD . The apparent ligand dissociation constants and extent of inhibition by UTP are similar in the hybrid and the wild type mammalian protein, but PRPP binds 4-fold more weakly to the hybrid . The allosteric ligands affected the steady state kinetic parameters of the hybrid differently, suggesting that while the linkage between the catalytic and regulatory subdomains has been preserved, there may be qualitative differences in interdomain signal transmission . Nevertheless, switching prokaryotic and eukaryotic allosteric controls argues for remarkable conservation of structure and regulatory mechanisms in this family of proteins.

J Bacteriol, 1994 Nov, 176(22), 6842 - 51
rRNA-mRNA base pairing stimulates a programmed -1 ribosomal frameshift; Larsen B et al.; Base pairing between the 3' end of 16S rRNA and mRNA is shown to be important for the programmed -1 frameshifting utilized in decoding the Escherichia coli dnaX gene . This pairing is the same as the Shine-Dalgarno pairing used by prokaryotic ribosomes in selection of translation initiators, but for frameshifting the interaction occurs within elongating ribosomes . For dnaX -1 frameshifting, the 3' base of the Shine-Dalgarno sequence is 10 nucleotides 5' of the shift site . Previously, Shine-Dalgarno rRNA-mRNA pairing was shown to stimulate the +1 frameshifting necessary for decoding the release factor 2 gene . However, in the release factor 2 gene, the Shine-Dalgarno sequence is located 3 nucleotides 5' of the shift site . When the Shine-Dalgarno sequence is moved to the same position relative to the dnaX shift site, it is inhibitory rather than stimulatory . Shine-Dalgarno interactions by elongating ribosomes are likely to be used in stimulating -1 frameshifting in the decoding of a variety of genes.

Clin Exp Immunol, 1994 Nov, 98(2), 224 - 8
Specificity of antibodies induced after immunization of mice with the mycobacterial heat shock protein of 65 kD; Barrios C et al.; We have previously shown in mice and monkeys that mycobacterial heat shock proteins (hsp) of 65 and 70 kD exert a strong in vivo helper effect when conjugated to synthetic peptides or bacterial oligosaccharides and given in the absence of any adjuvants . Considering the degree of homology existing in the phylogeny among hsp belonging to the same family, we studied whether antibodies induced in mice with this protocol of immunization with the mycobacterial 65-kD hsp (hsp65) would cross-react, and to what extent, with hsp homologues from other origins, notably with the Escherichia coli GroEL protein and with the human homologue (hsp60) . The results obtained show that antibodies to the mycobacterial hsp65 cross-reacted with the E . coli GroEL protein, both in ELISA and Western blot experiments, but not with the human hsp60 . In competitive ELISA experiments, the binding of these antibodies to solid-phase hsp65 was very effectively inhibited by low concentrations of the mycobacterial hsp65; however, for human hsp60, 100 times higher concentrations were required in order to obtain similar patterns of inhibition . Finally, murine antibodies to the mycobacterial hsp65 always failed to give positive results in Western blot experiments using extracts of murine cells . Taken together, these data suggest that, after immunization of mice with the mycobacterial hsp65 conjugated to peptides or oligosaccharides in the absence of adjuvants, anti-hsp65 antibodies are induced which cross-react well with hsp homologues from other prokaryotes (e.g . E . coli GroEL), but which weakly bind the human hsp homologue . These results may have implications for the potential use of microbial hsp molecules in the design of conjugated vaccine constructs.

Infect Immun, 1994 Nov, 62(11), 5201 - 4
Subcloning and expression of the Brucella abortus L7/L12 ribosomal gene and T-lymphocyte recognition of the recombinant protein; Oliveira SC et al.; The Brucella abortus L7/L12 ribosomal gene was amplified by PCR and subcloned into the prokaryotic expression vector pMAL-c2 . Escherichia coli DH5 alpha was transformed with the pMAL-L7/L12 construct, and gene expression was induced by IPTG (isopropyl-beta-D-thiogalactopyranoside) . The resulting fusion protein was purified by affinity chromatography and confirmed by Western blot (immunoblot) analysis using an anti-maltose-binding protein antibody . Additionally, purified recombinant L7/L12 protein induced T-lymphocyte proliferation of B . abortus-primed bovine peripheral blood mononuclear cells . Phenotypic analysis of the proliferating cell population demonstrated an increase in the percentage of CD4+ T lymphocytes when peripheral blood mononuclear cells were cultured with recombinant L7/L12 compared with cells cultured in medium alone . Subcloning and expression of a B . abortus gene encoding a previously demonstrated immunodominant protein for bovine lymphocytes are important steps in selecting Brucella proteins that have potential as a component of a genetically engineered candidate vaccine.

Curr Genet, 1994 Nov-Dec, 26(5-6), 557 - 63
Structure and expression characteristics of the chloroplast DNA region containing the split gene for tRNA(Gly) (UCC) from mustard (Sinapis alba L.); Liere K et al.; The mustard chloroplast gene trnG-UCC is split by a 717-bp group-II intron . Northern hybridization and RNase protection experiments suggest cotranscription with the upstream psbK-psbI operon, but not with the downstream trnR-UCU gene . The ends of most RNase-protected fragments between psbI and trnG correlate with the position of two potential stem-loop structures in this region, which could act as RNA processing elements . However, one RNA 5' end, approximately 75 bp upstream of the trnG 5' exon, does not so correlate and is preceded by prokaryotic-type '-10' and '-35' sequence elements . This suggests the possibility that a fraction of the trnG transcripts is initiated here . All precursor transcripts spanning the trnG region seem to have a common 3' end, which was located 117 bp downstream from the 3' exon, immediately after a stem-loop region . During seedling development, the major 0.8-0.9-kb trnG precursor transcripts show a transient maximum level at around 48 h after sowing, at a time when the mature tRNA begins to accumulate to constant levels . No significant differences in transcript patterns were observed either in the light or in darkness.

Anal Biochem, 1994 Nov 1, 222(2), 305 - 9
Prokaryotic test system for evaluation of oligonucleotide-affected antisense inhibition; Puskas LG et al.; A new test system is introduced for the estimation of the antisense effect of various types of oligodeoxy-nucleotides . This system is based upon the inhibition of translation of P2 phage repressor protein . Evidence for a direct correlation between phage yield and antisense inhibition was obtained by comparing the concentration-dependent effectiveness of an antisense phosphorothioate 18-mer, its mutated version, and the unmodified oligonucleotide . Oligonucleotides of complementary sequences were synthesized against the ribosome binding site and different sites of the coding region . In our test system only small differences were found based on the location of the target sequence . Rather, the degree of inhibition was related to the calculated ability to hybridize based on Tm values . In this way the sensitivity of the test system could also be demonstrated.

J Exp Biol, 1994 Nov, 196, 197 - 212
'Active' sugar transport in eukaryotes; Wright EM et al.; Sugar transporters in prokaryotes and eukaryotes belong to a large family of membrane proteins containing 12 transmembrane alpha-helices . They are divided into two classes: one facilitative (uniporters) and the other concentrative (cotransporters or symporters) . The concentrative transporters are energised by either H+ or Na+ gradients, which are generated and maintained by ion pumps . The facilitative and H(+)-driven sugar transporters belong to a gene family with a distinctive secondary structure profile . The Na(+)-driven transporters belong to a separate, small gene family with no homology at either the primary or secondary structural levels . It is likely that the Na(+)- and H(+)-driven sugar cotransporters share common transport mechanisms . To explore these mechanisms, we have expressed cloned eukaryote Na+/sugar cotransporters (SGLT) in Xenopus laevis oocytes and measured the kinetics of sugar transport using two-electrode voltage-clamp techniques . For SGLT1, we have developed a six-state ordered model that accounts for the experimental data . To test the model we have carried out the following experiments . (i) We measured pre-steady-state kinetics of SGLT1 using voltage-jump techniques . In the absence of sugar, SGLT1 exhibits transient carrier currents that reflect voltage-dependent conformational changes of the protein . Time constants for the carrier currents give estimates of rate constants for the conformational changes, and the charge movements, integrals of the transient currents, give estimates of the number and valence of SGLT1 proteins in the plasma membrane . Ultrastructural studies have confirmed these estimates of SGLT1 density . (ii) We have perturbed the kinetics of the cotransporter by site-directed mutagenesis of selected residues.(ABSTRACT TRUNCATED AT 250 WORDS)

J Exp Biol, 1994 Nov, 196, 15 - 49
Structure, function and evolution of solute transporters in prokaryotes and eukaryotes; Hediger MA; In both prokaryotes and eukaryotes, transport systems of organic solutes can be classified as passive transporters, such as channels and facilitated transporters, and active transporters, which utilize diverse energy-coupling mechanisms . In the past decade, our understanding of the biochemistry and molecular biology of transporters from Escherichia coli has progressed significantly, whereas the analysis of mammalian transporters has initially been limited by the ability to purify membrane proteins . The recent development of methods to detect the activity of recombinant proteins in individual cells, however, has led to the cloning of several novel mammalian transporter cDNAs . One of the most useful expression cloning systems is Xenopus oocytes in conjunction with uptake studies and electrophysiological experiments . Overall, the sequence information and the functional data derived from many transporters has revealed unifying designs, similar energy-coupling mechanisms and common evolutionary origins . Here, I will provide a general survey of the known transport systems in bacteria, yeast, plants, insects and vertebrates and illustrate the different types of transport systems in mammals by discussing transporters recently studied in our laboratory.

Microbiology, 1994 Nov, 140 ( Pt 11), 3167 - 76
Endogenous ADP-ribosylation during development of the prokaryote Myxococcus xanthus; Eastman D et al.; We examined endogenous ADP-ribosylation of proteins during the development of the prokaryote Myxococcus xanthus . In vivo and in vitro endogenous ADP-ribosylation of M . xanthus proteins was detected and the profile of modified proteins changed during development . Adenosine and nicotinamide inhibited ADP-ribosylation . Nicotinamide stimulated cells at low density to develop, in a manner similar to that previously observed with adenosine . Higher concentrations of nicotinamide inhibited aggregation . The in vivo effects of nicotinamide on developing M . xanthus cells correlate with its in vitro effects on ADP-ribosylation and the developmental profile of putative ADP-ribosylation substrates . These results suggest that ADP-ribosylation may regulate developmental proteins in M . xanthus.

Trends Genet, 1994 Nov, 10(11), 402 - 7
Prokaryotic translation: the interactive pathway leading to initiation; McCarthy JE et al.; Finding answers to the many open questions concerning the mechanism and control of prokaryotic translation remains one of the central challenges of molecular biology . In fact, recent experimental data even force us to reconsider aspects that were previously thought to be established fact . Here, we attempt a synthesis of new and not-so-new information, which leads to a revised and testable working hypothesis for translational initiation.

Protein Sci, 1994 Nov, 3(11), 2023 - 32
Engineering the quaternary structure of an enzyme: construction and analysis of a monomeric form of malate dehydrogenase from Escherichia coli; Breiter DR et al.; The citric acid cycle enzyme, malate dehydrogenase (MDH), is a dimer of identical subunits . In the crystal structures of 2 prokaryotic and 2 eukaryotic forms, the subunit interface is conformationally homologous . To determine whether or not the quaternary structure of MDH is linked to the catalytic activity, mutant forms of the enzyme from Escherichia coli have been constructed . Utilizing the high-resolution structure of E . coli MDH, the dimer interface was analyzed critically for side chains that were spatially constricted and needed for electrostatic interactions . Two such residues were found, D45 and S226 . At their nearest point in the homodimer, they are in different subunits, hydrogen bond across the interface, and do not interact with any catalytic residues . Each residue was mutated to a tyrosine, which should disrupt the interface because of its large size . All mutants were cloned and purified to homogeneity from an mdh- E . coli strain (BHB111) . Gel filtration of the mutants show that D45Y and D45Y/S226Y are both monomers, whereas the S226Y mutant remains a dimer . The monomeric D45Y and D45Y/S226Y mutants have 14,000- and 17,500-fold less specific activity, respectively, than the native enzyme . The dimeric S226Y has only 1.4-fold less specific activity . All forms crystallized, indicating they were not random coils . Data have been collected to 2.8 A resolution for the D45Y mutant . The mutant is not isomorphous with the native protein and work is underway to solve the structure by molecular replacement.

Insect Mol Biol, 1994 Nov, 3(4), 213 - 7
Detection of Buchnera, the primary prokaryotic endosymbiont of aphids, using the polymerase chain reaction; Rouhbakhsh D et al.; Members of the genus Buchnera constitute a distinct prokaryotic lineage containing the primary endosymbionts of aphids (Homoptera: Aphidoidea) . Using synthetic oligonucleotides in conjunction with the polymerase chain reaction, we propose three approaches for the identification of members of this genus . The first is based on unique sequences within rrs (gene coding for 16S ribosomal RNA) . The second is based on a different and unique organization of the ribosomal RNA operons of Buchnera and the close proximity of aroE upstream of rrl (gene coding for 23S rRNA) . The third is based on the linkage relationship of argS which is upstream of rrs . Validation of these three approaches requires their more extensive application.

Mol Cell Endocrinol, 1994 Nov, 105(2), R1 - 9
Monoclonal antibodies that recognize the native human thyrotropin receptor; Johnstone AP et al.; Monoclonal antibodies have been produced that recognize the native human thyrotropin receptor by using a sensitive screening protocol based on flow cytofluorimetry combined with recombinant eukaryotic cells expressing high levels of the full-length functional receptor . The more standard screening method of ELISA preferentially selected antibodies that only reacted with the denatured receptor . Mice were immunized with recombinant receptor produced in either eukaryotic or prokaryotic systems; after screening and cloning, three stable hybridoma lines were established . An IgM antibody (7B5) produced in response to the eukaryotic material recognized only the native receptor (by flow cytofluorimetry) and did not react with denatured material on ELISA or immunoblotting, suggesting that its epitope is conformational . In contrast, two IgG1 antibodies (2C11 and 3B12) produced in response to the prokaryotic material recognized both native and denatured receptor (by flow cytofluorimetry, immunoprecipitation and immunoblotting) . The use of different recombinant constructs in the immunoblotting procedure allowed the epitopes for both the IgG1 antibodies to be assigned to the region 125-369 . None of the antibodies stimulated production of cAMP by recombinant cells expressing the full-length functional receptor, but one of the IgG1 antibodies (2C11) did inhibit binding of radiolabelled thyrotropin to these same cells . These antibodies, and others that can now be produced with this screening protocol, will help define the relationship between structure and function of this important receptor.

J Mol Evol, 1994 Nov, 39(5), 506 - 18
Evolution of secondary structure in the family of 7SL-like RNAs; Labuda D et al.; Primate and rodent genomes are populated with hundreds of thousands copies of Alu and B1 elements dispersed by retroposition, i.e., by genomic reintegration of their reverse transcribed RNAs . These, as well as primate BC200 and rodent 4.5S RNAs, are ancestrally related to the terminal portions of 7SL RNA sequence . The secondary structure of 7SL RNA (an integral component of the signal recognition particle) is conserved from prokaryotes to distant eukaryotic species . Yet only in primates and rodents did this molecule give rise to retroposing Alu and B1 RNAs and to apparently functional BC200 and 4.5S RNAs . To understand this transition and the underlying molecular events, we examined, by comparative analysis, the evolution of RNA structure in this family of molecules derived from 7SL RNA . RNA sequences of different simian (mostly human) and prosimian Alu subfamilies as well as rodent B1 repeats were derived from their genomic consensus sequences taken from the literature and our unpublished results (prosimian and New World Monkey) . RNA secondary structures were determined by enzymatic studies (new data on 4.5S RNA are presented) and/or energy minimization analyses followed by phylogenetic comparison . Although, with the exception of 4.5S RNA, all 7SL-derived RNA species maintain the cruciform structure of their progenitor, the details of 7SL RNA folding domains are modified to a different extent in various RNA groups . Novel motifs found in retropositionally active RNAs are conserved among Alu and B1 subfamilies in different genomes . In RNAs that do not proliferate by retroposition these motifs are modified further . This indicates structural adaptation of 7SL-like RNA molecules to novel functions, presumably mediated by specific interactions with proteins; these functions were either useful for the host or served the selfish propagation of RNA templates within the host genome.

Mutat Res, 1994 Nov 1, 311(1), 31 - 8
Deletions induced in the white and vermilion genes of Drosophila melanogaster by the antitumor drug cis-dichlorodiammineplatinum(II); Cizeau J et al.; This paper describes the analysis of cisplatin induced mutations at the white (w) and vermilion (v) loci located on the X chromosome of Drosophila melanogaster . Twenty-eight w and eight v mutants have been found in a male genetic context and 42 w mutants in a female genetic context . In these latter experiments, genetic analysis showed the presence of multi-locus deficiencies in 18 out of 42 w mutants . Eighteen w and three v intragenic mutations were analyzed at the molecular level . Seventeen w and three v mutants carry deletions within the gene, ranging in size from 4 to 109 base pairs . Sequence analysis of the mutants indicates that most of them were produced by non-homologous recombinational events occurring between short (2-5 bp) sequence repeats on both sides of the deletion, one repeat being retained at the new junction . These results differ largely from those obtained in prokaryotic and other eukaryotic cells.

Gene, 1994 Oct 21, 148(2), 351 - 6
A recombinant DNA bio-assay for selenium in blood; Zhao C et al.; The trace element selenium (Se) is included in the form of selenocysteine (Sec) at the active site of several prokaryotic and eukaryotic proteins known as selenoproteins (SePro) . The growing implications of SePro in cell physiology and human health point to the need for an adequate means of assessing Se status in biological fluids . Here, we describe a new approach based on a recombinant DNA construct, in which the expression of the 'lacZ gene in Escherichia coli is proportionally and specifically driven by UGA-directed Sec incorporation . Se status is determined in samples of rat blood first treated by acid hydrolysis for protein degradation . As compared to other methods, this simple, sensitive bioassay (BIO) for determining Se status seems to be unique in its ability to measure all functional Sec residues in SePro in blood serum.

Gene, 1994 Oct 21, 148(2), 187 - 93
A frequently amplified region in Leishmania contains a gene conserved in prokaryotes and eukaryotes; Myler PJ et al.; A 27.5-kb sequence that is present in an approx . 2-Mb chromosome in Leishmania also occurs as an inverted dimer in a multicopy, 55-kb circular molecule (LD1) in Leishmania infantum ITMAP263 . Sequence analysis of a 7100-bp cloned segment from the circular molecule revealed three open reading frames (ORFs) . The ORFs are likely to have protein coding function by a number of criteria, including Northern blot analyses . The amino acid (aa) sequences deduced from two ORFs showed no similarity to other sequences in the databases . The C-terminal aa sequence from the third ORF is related (22-29% identity, 57-71% similarity) to a family of genes conserved in bacteria and humans . One member (sfhB) of the gene family in Escherichia coli appears to have a role in regulation of cell growth.

J Biol Chem, 1994 Oct 21, 269(42), 26518 - 24
Cloning, nucleotide sequence, and expression of an isovaleryl pepstatin-insensitive carboxyl proteinase gene from Pseudomonas sp . 101; Oda K et al.; A unique carboxyl proteinase (EC 3.4.23.33), insensitive to the classical inhibitor isovaleryl pepstatin and isolated from Pseudomonas sp . 101 (PCP), is the first example of a prokaryotic enzyme of this class . The gene coding for PCP was cloned, sequenced, and expressed in Escherichia coli . The gene consists of 1,761 base pairs encoding a protein of 587 amino acid residues . The NH2-terminal 215-amino acid preprosequence flanks the 372-amino acid mature protein, which is identical with the primary structure of an authentic PCP determined by chemical methods . E . coli carrying a plasmid containing the cloned wild-type PCP gene produced a 62-kDa protein . This molecule was processed and secreted into the periplasm as a 43-kDa protein, which converted to mature PCP under acidic conditions . This autocatalytic conversion was completely blocked by tyrostatin, a PCP-specific peptidic inhibitor from Kitasatosporia sp . 55 . The purified recombinant PCP has the same characteristics as authentic PCP . When several preprosequence deletion mutants were expressed in E . coli, mutant proteins were accumulated as insoluble forms with no proteinase activities . These results suggest that the prepropeptide of PCP plays an essential role in the formation of functional PCP.

Nature, 1994 Oct 20, 371(6499), 695 - 7
High abundance of Archaea in Antarctic marine picoplankton; DeLong EF et al.; Archaea (archaebacteria) constitute one of the three major evolutionary lineages of life on Earth . Previously these prokaryotes were thought to predominate in only a few unusual and disparate niches, characterized by hypersaline, extremely hot, or strictly anoxic conditions . Recently, novel (uncultivated) phylotypes of Archaea have been detected in coastal and subsurface marine waters, but their abundance, distribution, physiology and ecology remain largely undescribed . Here we report exceptionally high archaeal abundance in frigid marine surface waters of Antarctica . Pelagic Archaea constituted up to 34% of the prokaryotic biomass in coastal Antarctic surface waters, and they were also abundant in a variety of other cold, pelagic marine environments . Because they can make up a significant fraction of picoplankton biomass in the vast habitats encompassed by cold and deep marine waters, these pelagic Archaea represent an unexpectedly abundant component of the Earth's biota.

Mol Gen Genet, 1994 Oct 17, 245(1), 61 - 8
Transcriptional analysis of groEL genes in Streptomyces coelicolor A3(2); Duchene AM et al.; In Streptomyces coelicolor A3(2), synthesis of the groES, groES-groEL1 and groEL2 transcripts is induced either by heat shock or by undefined physiological stress signals present at a certain stage of growth . Under all conditions tested, transcription of groES and groES-groEL1 originated from a unique start site upstream of groES, whereas transcription of groEL2 originated from a unique site upstream of groEL2 . RNA polymerase isolated either from heat-shocked or control mycelia allowed in vitro transcription from the P1 promoter of groES/EL1 and the P2 promoter of groEL2 . The fact that these two RNA polymerase preparations both initiated transcription with equal efficiency from the same sites suggested that a heat shock-specific sigma factor is not responsible for the temperature-induced transcription of groE genes . Instead, regulation of these genes from vegetative-type promoters may be effected by a DNA-binding protein observed in gel retardation assays, which recognizes a motif found in the groE and dnaK promoter regions of many prokaryotic genes.

J Biol Chem, 1994 Oct 14, 269(41), 25381 - 6
Protein expression using cotranslational fusion and cleavage of ubiquitin . Mutagenesis of the glutathione-binding site of human Pi class glutathione S-transferase; Baker RT et al.; Expression of cloned genes in prokaryotes such as Escherichia coli is a widely used technique in both basic research and biotechnology . Despite the availability of several E . coli expression vector systems, adequate levels of expression may not be achieved . Expressing proteins as fusions to the highly conserved eukaryotic protein ubiquitin has been reported by several investigators to enhance protein yield in both bacterial and eukaryotic systems . We have modified this technique by the co-expression in E . coli of a ubiquitin-fusion protein and the Saccharomyces cerevisiae ubiquitin-specific protease Ubp2 . This allows the co-translational cleavage of engineered ubiquitin-fusion proteins expressed in E . coli . This system was used to express a human Pi class glutathione S-transferase (GST) GSTP1 as well as two mutant GSTP1 derivatives, Trp39-->Cys and Gln52-->Glu . The yield of these enzymes was improved 40-fold by using the ubiquitin-fusion/co-translational cleavage technique, and no uncleaved product was detected . The Trp39-->Cys mutant was totally devoid of GST activity, while the activity of the Gln52-->Glu mutant was reduced to 6% of wild-type GSTP1-1 . As both of the mutated residues map within the glutathione-binding site, the reduced GST activity is consistent with a marked reduction in glutathione binding ability.

Virology, 1994 Oct, 204(1), 436 - 41
Exchange of functional domains between Rev proteins of HIV-1 and SIVmac239 results in a dominant negative phenotype; Berchtold S et al.; The Rev proteins of primate immunodeficiency viruses are essential transactivators to switch from early to late phase in the viral replication cycle . Surprisingly, the Rev protein of HIV-1 is able to substitute those of HIV-2 and, as shown in here, of SIVmac239, but not vice versa . To understand the underlying mechanism of this incomplete functional reciprocity, we constructed a series of chimeric HIV-1/SIVmac239 Rev proteins and tested for transcomplementation efficacy on Rev-dependent indicator plasmids . In addition, we analyzed the prokaryotically expressed wild type and chimeric proteins for RNA-binding properties in a gel-shift assay in vitro . The functional defect of SIVmac239 on the HIV-1 Rev response element (RRE) is not due to a lack of binding or multimerization . In cotransfection experiments, SIVmac239 Rev and the chimeric proteins were tested for potential inhibitory effects on HIV-1 Rev function using the HIV-1 based indicator plasmid . Some of these proteins turned out to be transdominant inhibitors almost as potent as the HIV-1 Rev mutant M10 which is localized in the activation domain and is one of the strongest transdominant inhibitors . Surprisingly, M10 was not able to inhibit the function of either Rev protein on SIVmac239 RRE, whereas a corresponding SIVmac239 Rev mutant (SIV M10) was a transdominant inhibitor of SIVmac239 Rev function on its homologous RRE.

Virology, 1994 Oct, 204(1), 266 - 78
Control of expression, glycosylation, and secretion of HIV-1 gp120 by homologous and heterologous signal sequences; Li Y et al.; The HIV-1 gp120 signal sequence of 30 amino acids is longer than most glycoprotein signal sequences and contains an average of 5 positively charged amino acids . The HIV-1 gp120 gene with its natural signal sequence expressed in any prokaryotic or eukaryotic expression systems showed extremely low levels of synthesis and secretion . However, deletion of the HIV-1 gp120 signal sequence results in production of large quantities of a nonglycosylated form of gp120 in Spodoptera frugiperda cells . Substitution of the gp120 natural signal sequences with the signal sequences from honeybee mellitin or murine interleukin 3 promotes a high level of expression of a glycosylated form of gp120 and efficient secretion . These heterologous signal sequences contain one (mellitin) or no (IL-3) positively charged amino acids and led us to investigate the role of the positively charged amino acids in the signal sequence of HIV-1 gp120 . Four charge-altered forms of the gp120 signal sequence of HIV-1 were constructed by site-directed mutagenesis in which the positively charged amino acids were sequentially substituted with neutral amino acids . The results of these experiments showed that the expression and secretion of gp120 was progressively increased by eliminating the positively charged amino acids in a stepwise fashion . However, the substitution of all positively charged amino acids resulted in the accumulation of nonglycosylated gp120 within the cells with decreased amounts of the glycosylated form of gp120 . These results demonstrate that the positively charged amino acids in the signal sequence of HIV-1 gp120 are key factors in determining its poor expression and secretion . Analyses of intracellular transport and folding of gp120 further indicate that the presence of a highly charged, uncleaved signal sequence is an important factor limiting transport of gp120 from the rough ER to the Golgi apparatus.

Jpn J Genet, 1994 Oct, 69(5), 489 - 502
Evolution of glutamine synthetase genes is in accordance with the neutral theory of molecular evolution; Tateno Y; Evolution of glutamine synthetase gene is discussed on the results of DNA sequence analysis of the gene . Thirty DNA sequences of various organisms spanning from prokaryotes to eukaryotes were collected from the DNA data banks and translated first, they were aligned next, then evolutionary distances were computed, and molecular phylogeny was finally estimated . The results of the alignment reveal that functionally important regions of glutamine synthetase have been evolutionarily more conserved than the remaining regions . The evolutionary distances computed also show that the rate of synonymous substitution is higher than that of nonsynonymous substitution . These are well in accordance with the neutral theory of molecular evolution . Besides, the molecular phylogeny obtained shows that the origin of glutamine synthetase gene is much earlier than the divergence between eukaryotes and prokaryotes, suggesting that the gene is one of the oldest genes functioning now.

J Mol Evol, 1994 Oct, 39(4), 387 - 99
Mitochondrial DNA of the sea anemone, Metridium senile (Cnidaria): prokaryote-like genes for tRNA(f-Met) and small-subunit ribosomal RNA, and standard genetic code specificities for AGR and ATA codons; Pont-Kingdon GA et al.; The nucleotide sequence of a segment of the mitochondrial DNA (mtDNA) molecule of the sea anemone Metridium senile (phylum Cnidaria, class Anthozoa, order Actiniaria) has been determined, within which have been identified the genes for respiratory chain NADH dehydrogenase subunit 2 (ND2), the small-subunit rRNA (s-rRNA), cytochrome c oxidase subunit II (COII), ND4, ND6, cytochrome b (Cyt b), tRNA(f-Met), and the large-subunit rRNA (1-rRNA) . The eight genes are arranged in the order given and are all transcribed from the same strand of the molecule . The overall order of the M . senile mt-genes differs from that of other metazoan mtDNAs . In M . senile mt-protein genes, AGA and AGG codons appear to have the standard genetic code specification of arginine, rather than serine as found for other invertebrate mt-genetic codes . Also, ATA has the standard genetic code specification of isoleucine . TGA occurs in three M . senile mt-protein genes and may specify tryptophan as in other metazoan, protozoan, and some fungal mt-genetic codes . The M . senile mt-rRNA(f-Met) gene has primary and secondary structure features closely resembling those of the Escherichia coli initiator tRNA, including standard dihydrouridine and T psi C loop sequences and a mismatch pair at the top of the aminoacyl stem . Determinations of the 5' and 3' end nucleotides of the M . senile mt-s-rRNAs indicated that these molecules have a homogenous size of 1,081 ntp, larger than any other known metazoan mt-s-rRNAs . Consistent with its larger size, the M . senile mt-s-rRNA can be folded into a secondary structure that more closely resembles that of the E . coli 16S rRNA than can any other metazoan mt-s-rRNA . These findings concerning M . senile mtDNA indicate that most of the unusual features regarding metazoan mt-genetic codes, rRNAs, and probably tRNAs developed after divergence of the Cnidarian line from the ancestral line common to other metazoa.

Plant Mol Biol, 1994 Oct, 26(2), 553 - 6
Experimental and theoretical definition of geminivirus origin of replication; Arguello-Astorga G et al.; Geminiviruses are plant pathogens that replicate by a rolling-circle mechanism, analogous to that used by several prokaryotic ssDNA replicons . Recent reports provide important progress in understanding the structure and functioning of replication origin from these viruses . We have used these data to propose models for the initiation of replication in dicot- and monocot-infecting geminiviruses.

Mol Cell Biol, 1994 Oct, 14(10), 6627 - 34
Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes; Voos W et al.; We characterized a 24-kDa protein associated with matrix hsp70 (mt-hsp70) of Neurospora crassa and Saccharomyces cerevisiae mitochondria . By using specific antibodies, the protein was identified as MGE, a mitochondrial homolog of the prokaryotic heat shock protein GrpE . MGE extracted from mitochondria was quantitatively bound to hsp70 . It was efficiently released from hsp70 by the addition of Mg-ATP but not by nonhydrolyzable ATP analogs or high salt . A mutant mt-hsp70, which was impaired in release of bound precursor proteins, released MGE in an ATP-dependent manner, indicating that precursor proteins and MGE bind to different sites of hsp70 . A preprotein accumulated in transit across the mitochondrial membranes was specifically coprecipitated by either antibodies directed against MGE or antibodies directed against mt-hsp70 . The preprotein accumulated at the outer membrane was not coprecipitated by either antibody preparation . After being imported into the matrix, the preprotein could be coprecipitated only by antibodies against mt-hsp70 . We propose that mt-hsp70 and MGE cooperate in membrane translocation of preproteins.

J Gen Virol, 1994 Oct, 75 ( Pt 10), 2635 - 44
Reactivity of primate sera to foamy virus Gag and Bet proteins; Hahn H et al.; In order to establish criteria for the serodiagnosis of foamy virus infections we investigated the extent to which sera from infected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays . Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein . Both the Gag precursor molecules of 70 and 74K apparent M(r) and the cytoplasmic 60K M(r) Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells . Rabbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey and African green monkey origin . This was reflected by a broad cross-reactivity of the respective monkey sera to the Gag proteins of the various foamy virus isolates . Cross-reactivity of antisera against the Bet protein was restricted to viruses from man and the great apes . Recombinant Gag and Bet proteins expressed in prokaryotes or in insect cells were readily recognized by foamy virus-positive primate sera . Screening serum samples from chimpanzees with HFV Gag and Bet proteins expressed by recombinant baculoviruses revealed that 18 out of 35 (52%) were positive for Gag antibodies . Of these, 13 (72%) showed antibodies against the Bet protein, indicating that Bet antigen is of value in serological screening for foamy virus infections.

J Gen Virol, 1994 Oct, 75 ( Pt 10), 2559 - 65
Endosymbiotic bacteria associated with circulative transmission of potato leafroll virus by Myzus persicae; van den Heuvel JF et al.; In order to understand the molecular mechanisms underlying circulative transmission of potato leafroll virus (PLRV) by aphids, we screened Myzus persicae proteins as putative PLRV binding molecules using a virus overlay assay of protein blots . In this way, we found that purified PLRV particles exhibited affinity for five aphid proteins . The one most readily detected has an M(r) of 63K, and was identified as symbionin . This is the predominant protein synthesized by the bacterial endosymbiont of the aphid and is released into the haemolymph . Since further studies clearly showed that PLRV particles also bind to native symbionin, it was envisaged that virus particles when acquired into the haemocoel of an aphid interact with symbionin . Inhibition of prokaryotic protein synthesis by feeding M . persicae nymphs on an antibiotic-containing artificial diet prior to PLRV acquisition reduced virus transmission by more than 70% . The major coat protein of the virus was found to be degraded in the antibiotic-treated aphids; this would obviously have resulted in an increased exposure of viral RNA to enzymic breakdown and concomitant loss of infectivity . For these reasons we conclude that endosymbiotic bacteria play a crucial role in determining the persistent nature of PLRV in the aphid haemolymph and that symbionin is probably the key protein in this interaction.

J Bacteriol, 1994 Oct, 176(20), 6304 - 11
Integral proteins of the extracellular matrix fibrils of Myxococcus xanthus; Behmlander RM et al.; The extracellular matrix fibrils of Myxococcus xanthus are mediators of cell-cell cohesion and as such are required for the maintenance of the social lifestyle characteristic of these prokaryotes . The fibrils have also been implicated as factors involved in contact-mediated cell interactions and in signal exchange . The fibrils are extracellular carbohydrate structures with associated proteins . All of the major proteins associated with the fibrils react with monoclonal antibody 2105 and can be removed from the fibrils only by boiling with sodium dodecyl sulfate (SDS) and beta-mercaptoethanol . For consistency with their integral association with the fibrils, we have designated this class of proteins as integral fibrillar proteins class 1 (IFP-1) . IFP-1 comprises five major proteins whose molecular sizes range from 66 to 14 kDa . All of the proteins in IFP-1 have been purified from isolated fibrils by electroelution after size separation on SDS-PAGE gels . Analysis of the purified proteins suggested that the forms with different molecular sizes result from the aggregation of a single small-molecular-size subunit . Fingerprint analysis and amino acid composition profiles confirmed the identity among the different members of IFP-1 . The sequence of the 31 amino-terminal amino acids of the 31-kDa form of IFP-1 (IFP-1:31) was determined . There was no significant homology to other known protein sequences . During development there is a dramatic shift in the banding pattern of IFP-1 proteins without any apparent overall loss of total protein.

J Bacteriol, 1994 Oct, 176(20), 6175 - 87
A new type of NtrC transcriptional activator; Foster-Hartnett D et al.; The enteric NtrC (NRI) protein has been the paradigm for a class of bacterial enhancer-binding proteins (EBPs) that activate transcription of RNA polymerase containing the sigma 54 factor . Activators in the NtrC class are characterized by essentially three properties: (i) they bind to sites distant from the promoters that they activate (> 100 bp upstream of the transcriptional start site), (ii) they contain a conserved nucleotide-binding fold and exhibit ATPase activity that is required for activation, and (iii) they activate the sigma 54 RNA polymerase . We have characterized the NtrC protein from a photosynthetic bacterium, Rhodobacter capsulatus, which represents a metabolically versatile group of bacteria found in aquatic environments . We have shown that the R . capsulatus NtrC protein (RcNtrC) binds to two tandem sites that are distant from promoters that it activates, nifA1 and nifA2 . These tandem binding sites are shown to be important for RcNtrC-dependent nitrogen regulation in vivo . Moreover, the conserved nucleotide-binding fold of RcNtrC is required to activate nifA1 and nifA2 but is not required for DNA binding of RcNtrC to upstream activation sequences . However, nifA1 and nifA2 genes do not require the sigma 54 for activation and do not contain the highly conserved nucleotides that are present in all sigma 54-type, EBP-activated promoters . Thus, the NtrC from this photosynthetic bacterium represents a novel member of the class of bacterial EBPs . It is probable that this class of EBPs is more versatile in prokaryotes than previously envisioned.

Cancer Res, 1994 Oct 1, 54(19), 5081 - 5
Potent carcinogenicity of uncovered halogen lamps in hairless mice; D'Agostini F et al.; Uncovered halogen quartz lamps, which are potently genotoxic both in prokaryotic and human cells due to the emission of far-UV radiation (UVB and UVC), were assayed for carcinogenicity in three strains of hairless mice (SKH-1, MF-1, and C3H) of both sexes . As assessed in 5 independent experiments, no spontaneous skin tumor was observed, even after more than 2 years, in 49 animals used as unexposed controls or in 29 animals exposed to halogen lamps equipped with a common glass cover . In contrast, almost all of the 185 mice exposed to the light emitted by low-voltage (12 V) and low-power (50 W) tungsten bulbs, equipped with dichroic diffusers, contracted multiple skin tumors of various histological type, both benign and malignant . Tumors were induced over a range of illuminance levels (1,000, 3,333, 5,000, and 10,000 lux) and daily exposure times (1.5, 3, 6, and 12 h) . The tumor latency times were quite short and significantly correlated with the daily exposure times, as well as with the square of the distance (46-194 cm) from the illumination source . A carcinogenic effect was even observed when exposure was discontinued well before the appearance of skin lesions . Both in vitro genotoxicity data and animal carcinogenicity data support the view that the light emitted by uncovered halogen lamps, to which an enormous number of individuals are exposed, may pose carcinogenic risks to humans . Without renouncing the benefits of this modern illumination system, UV-blocking devices should be compulsory.

Blood, 1994 Oct 1, 84(7), 2171 - 4
Cloning and expression of a new mammalian chaperonin gene from a multipotent hematopoietic progenitor clone; Xie X et al.; Molecular chaperones assist in the folding and assembly of proteins in cells . Although chaperonins have been shown in prokaryotes, mitochondria, and chloroplasts long-ago, a cytoplasmic heteromeric chaperonin complex was isolated only recently and found to contain at least five to six polypeptides, one of which was identified as the product of the T complex polypeptide-1 (TCP-1) gene . We have isolated and cloned a novel gene called A45 from a cDNA library constructed from poly (A)+ RNA of a multipotent hematopoietic progenitor clone . The A45 cDNA encodes a predicted polypeptide of M(r) 58,118 that exhibits 32% overall amino acid sequence identity to TCP-1 and contains the putative adenosine triphosphate-binding domain and two characteristic consensus regions that are conserved in all chaperonins . The A45 gene is expressed in hematopoietic precursors cells at a much higher level than in nonhematopoietic cells and tissues . We conclude that A45 represents a new member of the mammalian chaperonins that is involved in the folding and assembly of polypeptides.

J Vet Med Sci, 1994 Oct, 56(5), 961 - 4
Comparative analysis of the 5' non-coding region of pestivirus RNA detected from live virus vaccines; Harasawa R; Comparative analysis of nucleotide sequences in the 5' non-coding region (NCR) of pestivirus RNA detected from live porcine and human virus vaccines indicated that the contaminants are of bovine viral diarrhea virus (BVDV), and that there are at least three genotypes, which are distinct from hog cholera virus, among the BVDV strains . Most of the nucleotide changes in variable regions of the 5' NCR were covariant, with complementary substitutions at other positions for secondary structures . The proposed secondary structure in the 5' NCR was similar to the prokaryotic rho independent terminator . Short open reading frames in the 5' NCR were well conserved among pestiviruses.

Biotechniques, 1994 Oct, 17(4), 686, 688, 690 - 1
A versatile and general prokaryotic expression vector, pLACT7; Chong S et al.; We have previously reported the constitutive over-expression of the tRNA-guanine transglycosylase (TGT) from plasmid pTGT1 in Escherichia coli . To obtain a controllable expression system for TGT, we have subsequently cloned the tgt gene into pET21b . Though the overexpression of TGT is inducible in pET21b, the plasmid has a low copy number, a poor yield of single-stranded DNA and relies on an E . coli strain that produces T7 RNA polymerase for protein expression . We have combined the features of pTZ18U and pET21b and have constructed a versatile plasmid pLACT7 that has a high copy number, a high yield of single-stranded DNA and both the T7 and lac promoters for protein expression in a wide variety of E . coli strains.

Mol Microbiol, 1994 Oct, 14(2), 199 - 205
Building bridges: disulphide bond formation in the cell; Bardwell JC; Disulphides are often vital for the folding and stability of proteins . Dedicated enzymatic systems have been discovered that catalyse the formation of disulphides in the periplasm of prokaryotes . These discoveries provide compelling evidence for the actual catalysis of protein folding in vivo . Disulphide bond formation in Escherichia coli is catalysed by at least three 'Dsb' proteins; DsbA, -B and -C . The DsbA protein has an extremely reactive, oxidizing disulphide which it simply donates directly to other proteins . DsbB is required for the reoxidation of DsbA . DsbC is active in disulphide rearrangements and appears to work synergistically with DsbA . The relative rarity of disulphides in cytoplasmic proteins appears to be dependent upon a disulphide-destruction machine . One pivotal cog in this machine is thioredoxin reductase.

Mol Microbiol, 1994 Oct, 14(1), 61 - 71
New clusters of genes required for gliding motility in Myxococcus xanthus; MacNeil SD et al.; Gliding is the directed movement of cells across surfaces which occurs in the absence of external organelles such as flagella . Gliding of the complex prokaryote, Myxococcus xanthus, results from the action of two independent sets of genes known as the A (adventurous motility) and S (social motility) genes . Strains with mutations in both systems (A-S-) do not spread on agar surfaces because both individual and group movement is abolished . To generate regulated, transcriptional fusions with operons including A and S genes, we introduced TN5-lac into A- and S- strains to obtain non-motile A-S::Tn5-lac and A::Tn5-lac S- double mutants . These insertions identify five separate clusters of A genes and three separate clusters of S genes on the M . xanthus genome . Some Tn5-lac insertions map near two of the five previously identified motility gene clusters, but at least five new clusters were identified in this search . Single mutations at only one locus, mglA, block motility; the mglA locus is epistatic to A and S motility genes . A- and S- Tn5-lac insertions were transduced into mgl+ and delta mgl strains . The levels of beta-galactosidase activity produced from each A- or S- Tn5-lac insertion are similar in otherwise isogenic mgl+ and delta mgl strains, showing that MglA does not regulate the transcription of many A and S genes.

Mol Microbiol, 1994 Oct, 14(1), 1 - 5
Prokaryotic HU and eukaryotic HMG1: a kinked relationship; Bianchi ME; HU and IHF proteins have long been considered the prokaryotic analogues of eukaryotic histones . Their ability to bend DNA, however, is distinctly similar to that of eukaryotic HMG-box proteins, a recently identified family of chromatin components and transcription factors . In some conditions, HU and HMG1-like proteins can even be swapped, both in vitro and in vivo . In spite of this, HU/IHF and HMG-box proteins are not evolutionarily related, and represent two independent solutions for the same biochemical problem.

J Cell Biochem, 1994 Oct, 56(2), 171 - 6
Two-dimensional protein crystals (S-layers): fundamentals and applications; Sleytr UB et al.; Two-dimensional crystalline surface layers (S-layers) composed of protein or glycoprotein subunits are one of the most commonly observed prokaryotic cell envelope structures . Isolated S-layer subunits are endowed with the ability to assemble into monomolecular arrays in suspension, on surfaces or interfaces by an entropy-driven process . S-layer lattices are isoporous structures with functional groups located on the surface in an identical position and orientation . These characteristic features have already led to applications of S-layers as (1) ultrafiltration membranes with well-defined molecular weight cut-offs and excellent antifouling characteristics, (2) immobilization matrices for functional molecules as required for affinity and enzyme membranes, affinity microcarriers and biosensors, (3) conjugate vaccines, (4) carriers for Langmuir-Blodgett films and reconstituted biological membranes, and (5) patterning elements in molecular nanotechnologyPublication Types:

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