J Cell Biol, 1992 Jul, 118(2), 481 - 90 Topology and phosphorylation of soybean nodulin-26, an intrinsic protein of the peribacteroid membrane; Miao GH et al.; Soybean nodulin-26, a homologue of bovine eye lens major intrinsic protein (MIP-26), is an integral protein of the peribacteroid membrane in symbiotic root nodules . It comprises 271 amino acids with six potential transmembrane domains and lacks an amino-terminal signal sequence . A full-length nodulin-26 cDNA and its various deletion derivatives were transcribed in vitro after linking them to bacteriophage T3 promoter . In vitro translation of these transcripts in a rabbit reticulocyte lysate, in the presence or absence of canine pancreatic microsomal membranes, suggested that nodulin-26 is cotranslationally inserted into the microsomes without a cleavable signal peptide . The first two transmembrane domains (103 amino acids) of the protein are sufficient for microsomal membrane insertion . Membrane-translocated nodulin-26 binds to Con-A and is sensitive to endoglycosidase-H treatment, suggesting that it is glycosylated . Native nodulin-26 from root nodules retains its sugar moiety as it, too, binds to Con-A . Chemical cleavage mapping at cysteine residues, a trypsin protection assay, and the Con-A binding affinity of nodulin-26 suggested that both the NH2 and COOH termini of this protein are on the cytoplasmic surface of the peribacteroid membrane, while the glycosidic residue is on the surface of the membrane facing the bacteroids . In vitro phosphorylation experiments showed that nodulin-26 is a major phosphorylated protein in the peribacteroid membrane . This phosphorylation is mediated by a Ca(2+)-dependent, calmodulin-independent protein kinase located in the peribacteriod membrane . Externally supplied acid phosphatase dephosphorylates this protein, but alkaline phosphatase does not . Based on its homology with several eukaryotic and prokaryotic channel-type membrane proteins, nodulin-26 may form a channel translocating specific molecules to the bacteroids during endosymbiosis in legume plants. Eur J Biochem, 1992 Jul 1, 207(1), 1 - 11 The polyanion-binding domain of cytoplasmic Lys-tRNA synthetase from Saccharomyces cerevisiae is not essential for cell viability; Martinez R et al.; Cytoplasmic Lys-tRNA synthetase (LysRS) from Saccharomyces cerevisiae is a dimeric enzyme made up of identical subunits of 68 kDa . By limited proteolysis, this enzyme can be converted to a truncated dimer without loss of activity . Whereas the native enzyme strongly interacts with polyanionic carriers, the modified form displays reduced binding properties . KRS1 is the structural gene for yeast cytoplasmic LysRS . It encodes a polypeptide with an amino-terminal extension composed of about 60-70 amino acid residues, compared to its prokaryotic counterpart . This segment, containing 13 lysine residues, is removed upon proteolytic treatment of the native enzyme . The aim of the present study was to probe in vivo the significance of this amino-terminal extension . We have constructed derivatives of the KRS1 gene, encoding enzymes lacking 58 or 69 amino-terminal residues and, by site-directed mutagenesis, we have changed four or eight lysine residues from the amino-terminal segment of LysRS into glutamic acids . Engineered proteins were expressed in vivo after replacement of the wild-type KRS1 allele . The mutant enzymes displayed reduced specific activities (2-100-fold) . A series of carboxy-terminal deletions, encompassing 3, 10 or 15 amino acids, were introduced into the LysRS mutants with modified amino-terminal extensions . The removal of three residues led to a 2-7-fold increase in the specific activity of the mutant enzymes . This partial compensatory effect suggests that interactions between the two extreme regions of yeast LysRS are required for a proper conformation of the native enzyme . All KRS1 derivatives were able to sustain growth of yeast cells, although the mutant cell lines displaying a low LysRS activity grew more slowly . The expression, as single-copy genes, of mutant enzymes with a complete deletion of the amino-terminal extension or with four Lys----Glu mutations, that displayed specific activities close to that of the wild-type LysRS, had no discernable effect on cell growth . We conclude that the polycationic extensions of eukaryotic aminoacyl-tRNA synthetases are dispensable, in vivo, for aminoacylation activities . The results are discussed in relation to the triggering role in in situ compartmentalization of protein synthesis that has been ascribed to the polypeptide-chain extensions that characterize most, if not all, eukaryotic aminoacyl-tRNA synthetases. EMBO J, 1992 Jul, 11(7), 2611 - 7 Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain; Bestor TH; Mammalian DNA (cytosine-5) methyltransferase contains a C-terminal domain that is closely related to bacterial cytosine-5 restriction methyltransferase . This methyltransferase domain is linked to a large N-terminal domain . It is shown here that the N-terminal domain contains a Zn binding site and that the N- and C-terminal domains can be separated by cleavage with trypsin or Staphylococcus aureus protease V8; the protease V8 cleavage site was determined by Edman degradation to lie 10 residues C-terminal of the run of alternating lysyl and glycyl residues which joins the two domains and six residues N-terminal of the first sequence motif conserved between the mammalian and bacterial cytosine methyltransferases . While the intact enzyme had little activity on unmethylated DNA substrates, cleavage between the domains caused a large stimulation of the initial velocity of methylation of unmethylated DNA without substantial change in the rate of methylation of hemimethylated DNA . These findings indicate that the N-terminal domain of DNA methyltransferase ensures the clonal propagation of methylation patterns through inhibition of the de novo activity of the C-terminal domain . Mammalian DNA methyltransferase is likely to have arisen via fusion of a prokaryotic-like restriction methyltransferase and an unrelated DNA binding protein . Stimulation of the de novo activity of DNA methyltransferase by proteolytic cleavage in vivo may contribute to the process of ectopic methylation observed in the DNA of aging animals, tumors and in lines of cultured cells. J Bacteriol, 1992 Jul, 174(13), 4356 - 60 Tyrosine phosphorylation of a membrane protein from Pseudomonas solanacearum; Atkinson M et al.; We have investigated a tyrosine kinase activity from Pseudomonas solanacearum, an economically important plant pathogen . In vitro incubation of membrane fractions with {gamma-32P}ATP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an 85-kDa phosphoprotein . Phosphorylation of this protein on tyrosine residues was demonstrated by phosphoamino acid analysis of base hydrolysis products and by immunoanalysis of Western blots (immunoblots) with antiphosphotyrosine monoclonal antibody . In vitro incubation of membranes with ATP was not required for recognition by the antibody, indicating that the 85-kDa protein is phosphorylated in vivo . These results demonstrate that membranes from P . solanacearum exhibit a tyrosine kinase activity toward an endogenous membrane protein . This bacterium provides an opportunity to study the structure and function of a prokaryotic tyrosine kinase. Arch Biochem Biophys, 1992 Jul, 296(1), 321 - 7 Purification and characterization of a catalase-peroxidase from the fungus Septoria tritici; Levy E et al.; Three classes of heme proteins, commonly designated hydroperoxidases, are involved in the metabolism of hydrogen peroxide: catalases, peroxidases, and catalase-peroxidases . While catalases and peroxidases are widely spread in animals, plants, and microorganisms, catalase-peroxidases were characterized only in prokaryotes . We report here, for the first time, on a catalase-peroxidase in a eukaryotic organism . The enzyme was purified from the fungal wheat pathogen Septoria tritici, and is one of three different hydroperoxidases synthesized by this organism . The S . tritici catalase-peroxidase, designated StCP, is similar to the enzymes previously isolated from the bacteria Rhodobacter capsulatus, Escherichia coli, and Klebsiella pneumoniae, although it is significantly more sensitive to denaturing conditions . In addition to its catalatic activity StCP catalyzes peroxidatic activity with o-dianisidine, diaminobenzidine, pyrogallol, NADH, and NADPH as electron donors . The enzyme is a tetramer with identical subunits of 61,000 Da molecular weight . StCP shows a typical high-spin ferric heme spectrum with a Soret band at 405 nm and a peak at 632 nm, and binding of cyanide causes a shift of the Soret band to 421 nm, the appearance of a peak at 537 nm, and abolition of the peak at 632 nm . Reduction with dithionite results in a decrease in the intensity of the Soret band and its shift to 436 nm, and in the appearance of a peak at 552 nm . The pH optimum is 6-6.5 and 5.4 for the catalatic and peroxidatic activities, respectively . Fifty percent of the apparent maximal activity is reached at 3.4 mM and 0.26 mM for the catalatic and peroxidatic activities, respectively . The enzyme is inactivated by ethanol/chloroform, and is inhibited by KCN and NaN3, but not by the typical catalase inhibitor 3-amino-1,2,4-triazole. Comp Biochem Physiol B, 1992 Jul, 102(3), 437 - 44 Structure and possible function of heat-shock proteins in Falciparum malaria; Sharma YD; Like many prokaryotes and eukaryotes, the malaria parasite also synthesizes several stress proteins . Most widely studied stress proteins of this parasite are the heat-shock proteins (hsps) . Their discovery in malaria is a gift of recombinant DNA technology . Five hsp genes from Plasmodium falciparum have been identified which are located on different chromosomes . Thus the inheritance and expression of hsp genes are independent of each other . They share a large amount of sequence homology at N-terminus with the hsps of other organisms . Their gene regulatory sequences and other elements, important for gene expression, are yet to be determined . The biological role of these proteins in malaria is not fully understood but it is possible that they provide protection to the parasite from various stresses encountered in the host . In this process hsps probably bind to the toxic molecules as well as damaged proteins to flush them out of the parasite . Their involvement in the stage-specific parasite transformation to increase the infectivity and virulence, as observed in other parasites, remains to be determined . Malarial hsps are antigenic in humans . This antigenicity could be attributed to the non-homologous sequences in the C-terminus region . The potential of one of them (pfhsp 70I) for a future malaria vaccine and immunodiagnostics requires re-evaluation of the data. Mol Gen Genet, 1992 Jul, 234(1), 147 - 54 New tools for mitochondrial genetics of Chlamydomonas reinhardtii: manganese mutagenesis and cytoduction; Bennoun P et al.; A novel and efficient genetic procedure is described for generating mitochondrial mutants of the green alga Chlamydomonas reinhardtii . The development of a mutagenesis procedure using manganese cations and the application of cytoduction techniques resulted in a combined approach for the generation and analysis of mitochondrial mutants . Although mitochondrial mutations are inherited in sexual crosses from the minus mating type parent, the cytoduction technique can be used to transfer mitochondrial mutations into recipient strains with different genetic backgrounds, irrespective of their mating type . Cytoduction allows the transfer of mitochondrial markers from diploid to haploid cells also, which is of great benefit since diploid cells do not germinate in C . reinhardtii . We report here the isolation and characterisation of eight mutants, which are resistant to the antibiotics myxothiazol and mucidin . The mutants all have point mutations in the mitochondrial gene for apocytochrome b . Using in vitro-amplified cytb gene fragments as probes for direct DNA sequencing, three different types of single base pair substitutions were revealed in all mutants tested . In particular, amino acid substitutions in the mutant apocytochrome b polypeptide have been identified at residues 129, 132 and 137, which have been implicated in forming part of an antibiotic-binding niche . The amino acid substitution at position 132 has not been so far described for mutant apocytochrome b in any other organism, prokaryotic or eukaryotic . The genetic approach presented here confirms C . reinhardtii as a model system that is unique among plant cells. Int J Biochem, 1992 Jul, 24(7), 1125 - 33 Ribosomes from trichomonad protozoa have prokaryotic characteristics; Champney WS et al.; 1 . Ribosomes from cells of the genera Trichomonas and Tritrichomonas have been isolated and characterized . The ribosomes from each organism had a sedimentation coefficient of 70S in calibrated sucrose gradients and the subunits sedimented as 50S and 30S particles under the same conditions . 2 . The major ribosomal RNAs from each species were identical in size to prokaryotic ribosomal RNAs when examined by denaturing gel electrophoresis . The ribosomes contained both 5.8S and 5S RNAs . 3 . The ribosomal proteins were compared by the methods of two-dimensional gel electrophoresis and reversed phase HPLC . Electrophoresis of the ribosomal proteins in two different gel systems indicated the presence of 56 proteins in T . gallinae, 40 in T . bactrachorum and 45 in the Tritrichomonas sp . The protein molecular mass range was 8.5-40 kDa . 4 . The HPLC analysis confirmed the protein number established by the gel methods . 5 . Both methods of analysis revealed greater similarities between the ribosomal proteins of the 2 Tritrichomonas sp . than between those of the more distantly related T . gallinae and T . bactrachorum. Plant Cell, 1992 Jul, 4(7), 785 - 98 A novel light-regulated promoter is conserved in cereal and dicot chloroplasts; Christopher DA et al.; The chloroplast psbD-psbC genes encode D2 and cp43, a reaction center protein and chlorophyll-binding antenna protein of photosystem II, respectively . We have previously shown that differential accumulation of light-induced psbD-psbC mRNAs in barley chloroplasts is due to transcription from a blue light-responsive promoter (LRP) . It is hypothesized that the light-induced mRNAs help to maintain levels of the D2 polypeptide, which is photodamaged and degraded in illuminated plants . To determine if light-induced accumulation of psbD-psbC mRNAs was a conserved phenomenon in chloroplasts, the expression of psbD-psbC operons from five cereals (barley, wheat, rice, maize, and sorghum) and three dicot (tobacco, spinach, and pea) species was examined . Cereal and dicot psbD-psbC operons differ due to several DNA rearrangements that moved psbK-psbI proximal to psbD-psbC, allowing cotranscription of these genes and production of several unique transcripts in cereals . Despite differences in the structure and expression of the cereal and dicot psbD-psbC operons, the accumulation of light-induced psbD-psbC mRNAs was conserved in all species studied . An unusual feature of the light-induced mRNAs was the occurrence of 5' end microheterogeneity . The multiple 5' termini were mapped to several consecutive nucleotides (8 to 25 bp) within a highly conserved (61%) DNA region that represents the transcription initiation site for the mRNAs in barley and tobacco . The novel LRP differs in sequence from typical plastid promoters that have prokaryotic "-10" and "-35" elements and is centered 570 bp (cereals), 900 bp (tobacco, spinach), or 1100 bp (pea) upstream from the psbD translational start codon . We propose that physiological and gene regulatory demands of the chloroplast act as constraints that preserved the linkage of the LRP with psbD despite DNA inversions involving the psbD upstream region. Protein Sci, 1992 Jul, 1(7), 925 - 34 Theory of chaperonin action: inertial model for enhancement of prokaryotic Rubisco assembly; Roy H et al.; We have performed a computational simulation of the aggregation and chaperonin-dependent reconstitution of dimeric prokaryotic ribulose bisphosphate carboxylase/oxygenase (Rubisco), based on the data of P . Goloubinoff et al . (1989, Nature 342, 884-889) and P . V . Viitanen et al . (1990, Biochemistry 29, 5665-5671) . The aggregation is simulated by a set of 12 differential equations representing the aggregation of the Rubisco folding intermediate, Rubisco-I, with itself and with aggregates of Rubisco-I, leading up to dodecamers . Four rate constants, applying to forward or reverse steps in the aggregation process, were included . Optimal values for these constants were determined using the ellipsoid algorithm as implemented by one of us (Ecker, J.G . & Kupferschmid, M., 1988, Introduction to Operations Research, Wiley, New York, pp . 315-322) . Intensive exploration of simpler aggregation models did not identify an alternative that could simulate the data as well as this one . The activity of the chaperonin in this system was simulated by using this aggregation model, combined with a model similar to that proposed by Goloubinoff et al . (1989) . The model assumes that the chaperonin can bind the folding intermediate rapidly, and that the chaperonin complex releases the Rubisco molecule slowly, permitting time for its spontaneous folding while interacting with the chaperonin . This is followed by self-association of the folded Rubisco monomer to yield the active dimeric Rubisco . A modification of the model that simulates temperature effects was also constructed . The most important results we obtained indicate that the chaperonin-dependent reconstitution of Rubisco can be simulated adequately without invoking any catalysis of folding by the chaperonin . In addition, the simulations predict values for the association rate constant of Rubisco-I with the chaperonin, and other variables, that are subject to experimental verification. Trends Biochem Sci, 1992 Jul, 17(7), 262 - 6 Evolutionary conservation of the active site of soluble inorganic pyrophosphatase; Cooperman BS et al.; Soluble inorganic pyrophosphatases (PPases) are essential enzymes that are important for controlling the cellular levels of inorganic pyrophosphate (PPi) . Although prokaryotic and eukaryotic PPases differ substantially in amino acid sequence, recent evidence now demonstrates clearly that PPases throughout evolution show a remarkable level of conservation of both an extended active site structure, which has the character of a mini-mineral, and a catalytic mechanism . PPases require several (three or four) Mg2+ ions at the active site for activity and many of the 15-17 fully conserved active site residues are directly involved in the binding of metal ions . Each of the eight microscopic rate constants that has been evaluated for the PPases from both Escherichia coli and Saccharomyces cerevisiae is quite similar in magnitude for the two enzymes, supporting the notion of a conserved mechanism. Genes Dev, 1992 Jul, 6(7), 1269 - 79 Trans and cis requirements for intron mobility in a prokaryotic system; Clyman J et al.; Intron mobility requires cleavage of an intronless allele by an intron-encoded endonuclease, followed by transfer of the intron into the cleaved recipient . The mobile phage introns provide an opportunity to identify accessory functions involved in the intron inheritance process . To test for trans and cis requirements of mobility in Escherichia coli, we have exploited the td intron of phage T4 in both phage T4 and lambda genetic backgrounds . Mobility depends on host or phage recombinase functions, RecA or UvsX, respectively . The process also requires a phage-encoded 5'----3' exonuclease activity and associated annealing function that can be provided by phage lambda . Finally, host-encoded 3'----5' exonuclease activities are also implicated in intron inheritance . We demonstrated further that restriction enzymes could substitute for the intron-encoded endonuclease, indicating that the endonuclease does not have an essential role in recombination . Neither the precise position nor the nature of the double-strand break was critical to intron transfer . These features provide insight into the recombination pathway and are factors impacting on the spread of introns throughout natural populations. FEBS Lett, 1992 Jun 29, 305(2), 91 - 6 Fibronectin type III-like sequences and a new domain type in prokaryotic depolymerases with insoluble substrates; Hansen CK; Fibronectin type III-like sequences are present in many proteins from higher eukaryotes and are involved in protein-protein interactions, heparin binding and cell adhesion . A nine-member family of bacterial sequences is shown to be significantly homologous to the type III-like sequences . All the sequences are contained in secreted depolymerases acting on complex, energy-rich insoluble substrates, in which they apparently do not participate in catalysis or substrate-binding, their exact function remaining unclear . Furthermore, a new family of sequences, present in some cellulases, is presented. Proc R Soc Lond B Biol Sci, 1992 Jun 22, 248(1323), 273 - 81 Amplification and rearrangement of a prokaryotic metallothionein locus smt in Synechococcus PCC 6301 selected for tolerance to cadmium; Gupta A et al.; Metal-tolerant cyanobacteria have been isolated from metal-polluted aquatic environments and also selected in culture, but no genes which confer metal tolerance have been described . To investigate the possibility that amplification of a prokaryotic metallothionein gene (smtA), or rearrangement of the smt locus, could be involved in the development of Cd tolerance in Synechococcus PCC 6301, Cd-tolerant lines were selected by stepwise adaptation of a Synechococcus culture . An increase in smtA gene copy number and the appearance of unique additional smtA restriction fragments (both larger and smaller) were detected in these tolerant lines (tolerant to 0.8 microM Cd, 1.3 microM Cd and 1.7 microM Cd) . Stepwise adaptation was repeated by using a culture of Synechococcus PCC 6301 inoculated from a single plated colony to obtain four new lines (tolerant to 1.4 microM Cd, 1.8 microM Cd, 2.6 microM Cd and 3.2 microM Cd) . Amplification of the smtA gene and development of unique smtA restriction fragments (larger and smaller) were once again detected in these tolerant lines . Amplification and rearrangement of the smt locus were only detected in the seven Cd-tolerant lines, with no evidence of amplification or rearrangement in the non-tolerant lines from which they were derived . As a control, another gene, psaE, was also monitored in these cell lines . There was no evidence of amplification or rearrangement of psaE in the non-tolerant or any of the Cd-tolerant lines. J Mol Biol, 1992 Jun 20, 225(4), 933 - 8 From adenylate cyclase to guanylate cyclase . Mutational analysis of a change in substrate specificity; Beuve A et al.; Adenylate and guanylate cyclases, having different but related substrates, are a paradigm for the study of substrate discrimination . A prokaryotic adenylate cyclase gene, phylogenetically related to eukaryotic counterparts, was screened for mutants remodelling the enzyme's specificity . In a first step, a mutant was selected displaying a significant level of guanylate cyclase activity . This was due to a point mutation destroying most of the adenylate cyclase activity . A second selection step restored most of the original activity . This resulted from an additional mutation in the same region, thus permitting the first identification of a functional domain in adenylate and guanylate cyclases. Biochem J, 1992 Jun 15, 284 ( Pt 3), 855 - 60 Expression of rat liver NAD(P)H:quinone-acceptor oxidoreductase in Escherichia coli and mutagenesis in vitro at Arg-177; Chen HH et al.; A prokaryotic expression plasmid, pKK-DT2, containing the cDNA of rat liver NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.99.2; DT-diaphorase) was constructed and used to transform Escherichia coli strain JM109 . The rat liver quinone reductase was expressed in strain in JM109 and was inducible with isopropyl beta-D-thiogalactopyranoside (IPTG) . The expressed rat protein was purified by affinity chromatography and had kinetic and physical properties identical with the protein purified from rat liver in that it could utilize either NADH or NADPH as the electron donor and its activity was inhibited by dicoumarol . In addition, we have generated four mutants, Arg-177----His (R177H), Arg-177----Ala (R177A), Arg-177----Cys (R177C) and Arg-177----Leu (R177L), using this expression system . Several of the mutants behaved anomalously on SDS/PAGE, but all of the mutant proteins had the expected M(r) as determined by electrospray m.s . These results and those obtained from enzyme kinetic analysis, u.v./visible absorption spectral analysis, and flavin and tryptophan fluorescence analysis of the wild-type enzyme and four mutants indicated that mutations at Arg-177 changed the conformation of the enzyme, resulting in a decrease in enzyme activity . Replacing Arg-177 with leucine altered the protein conformation and decreased FAD incorporation. FEBS Lett, 1992 Jun 15, 304(2-3), 119 - 23 Characterisation of a chloroplast-encoded secY homologue and atpH from a chromophytic alga . Evidence for a novel chloroplast genome organisation; Scaramuzzi CD et al.; secY is a prokaryotic gene that encodes the SecY protein, an integral membrane component of the prokaryotic protein translocation apparatus . A chloroplast-encoded secY homologue has been identified in the unicellular, chromophytic alga, Pavlova lutherii . The gene predicts a protein composed of ten membrane-spanning regions, that is approximately 25% homologous and 50% similar to bacterial and plastid SecY proteins . The secY gene from P . lutherii is independent of the ribosomal protein (rp) gene cluster to which it is closely linked in other organisms . In P . lutherii secY is located 5' to atpI and atpH . Since, in higher plants the atpIHFA gene cluster and the rp gene cluster are separated by approximately 50 kb, we conclude, this indicates a novel chloroplast gene arrangement in P . lutherii. Gene, 1992 Jun 15, 115(1-2), 167 - 72 Regulation of secondary metabolism and cell differentiation in Streptomyces: A-factor as a microbial hormone and the AfsR protein as a component of a two-component regulatory system; Horinouchi S et al.; A-factor is a microbial hormone that functions as a key switch for secondary metabolite formation and morphogenesis in Streptomyces griseus . Genetic and biochemical studies on the A-factor-binding protein have implied that the binding protein present in the cytoplasm plays a role in repressing streptomycin (Sm) production and sporulation while the binding of A-factor to the binding protein releases this repression . The A-factor signal is transferred, probably via some additional regulatory proteins in the A-factor-regulatory cascade, to the strR gene, a regulator for Sm biosynthesis . A positive regulatory protein binds about 430-330 bp upstream from the transcription start point of the strR promoter and activates its transcription . The StrR product, in turn, activates the other Sm-biosynthesis genes . A global regulatory gene, afsR, of Streptomyces coelicolor A3(2) encodes a 993-amino acid protein that is phosphorylated by a specific phosphokinase, AfsK, encoded by the region just upstream from the afsR gene . Site-directed mutagenesis of afsR has revealed that phosphorylated AfsR globally stimulates transcription of antibiotic-production genes . It is most likely that AfsR and AfsK compose a two-component regulatory system . Although AfsR shows no significant homology with typical regulators of the two-component systems in other prokaryotes, such as OmpR and PhoB of Escherichia coli, it shows considerable homology with regulatory proteins in antibiotic biosynthetic gene clusters of Streptomyces spp., such as actII ORF4, dnrR1 ORF1 and redD ORF1.(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Biochem, 1992 Jun 15, 206(3), 927 - 34 Structural and functional characterization of Escherichia coli peptidyl-prolyl cis-trans isomerases; Compton LA et al.; Peptidyl-prolyl cis-trans isomerases (PPIases), enzymes that catalyze the cis-trans isomerization of peptide bonds to which proline contributes the nitrogen, were purified from Escherichia coli . In this organism, at least two PPIases are present . Both the cationic (periplasmic) and anionic (cytoplasmic) PPIases are inhibited by cyclosporin A with a Ki of 25-50 microM, a concentration 1000-fold higher than that required for eukaryotic PPIases . Although isoelectric focusing indicates that the two enzymes differ in isoelectric point by at least 4.0 pH units, the specific activities of the enzymes toward the tetrapeptide substrate succinyl-Ala-Ala-Pro-Phe-methyl-coumarylamide are equivalent . The activity of both enzymes for a series of substituted succinyl-Ala-Xaa-Pro-Phe-para-nitroanilide tetrapeptides suggests that the structure and function of the active site of the prokaryotic proteins is similar to that of eukaryotic cyclophilins . Both enzymes are capable of catalyzing the refolding of thermally denatured type III collagen . Antibodies against the periplasmic PPIase do not recognize the cytoplasmic enzyme, indicating significant differences in epitopes between the two forms . Circular dichroism spectroscopy indicates that the secondary structure of the cationic protein consists of 17% alpha-helix, 34% beta-sheet, 17% turns, 33% random coil and is very similar to human cytosolic PPIase. FEMS Microbiol Lett, 1992 Jun 15, 72(3), 209 - 12 The effects of growth temperature on the methyl sterol and phospholipid fatty acid composition of Methylococcus capsulatus (Bath); Jahnke LL; Growth of Methylococcus capsulatus (Bath) at temperatures ranging from 30 to 50 degrees C resulted in changes to the whole cell lipid constituents . As temperature was lowered, the overall proportion of hexadecenoic acid (C16:1) increased, and the relative proportions of the delta 9, delta 10 and delta 11 C16:1 double bond positional isomers changed . Methyl sterol content also increased as the growth temperature was lowered . The highest amounts of methyl sterol were found in 30 degrees C cells and the lowest in 50 degrees C cells (sterol-phospholipid ratios of 0.077 and 0.013, respectively) . The data are consistent with a membrane modulating role for the sterol produced by this prokaryotic organism. FEMS Microbiol Lett, 1992 Jun 15, 72(3), 243 - 7 'uidA-antibiotic-resistance cassettes for insertion mutagenesis, gene fusions and genetic constructions; Bardonnet N et al.; We have constructed a series of promoterprobe cassettes that provides powerful tools for insertion mutagenesis, transcription fusions and genetic constructions . These cassettes contain the Tn9 chloramphenicol (CmR) and the Tn903 kanamycin (KmR) resistance genes which are expressed in a large variety of microorganisms; these antibiotic-resistance markers were associated with the uidA promoterless gene . This beta-glucuronidase-encoding gene of Escherichia coli K-12 has been successfully used as reporter gene for various organisms including prokaryotes and eukaryotes . The resulting 'uidA-KmR and 'uidA-CmR cassettes (truncated at the ') can be excized with most of the commonly used restriction enzymes . Furthermore, they are borne by ApR or CmR plasmids which facilitate their utilization . These promoter-probe cassettes allow transcriptional signal localization and regulation studies. Biochem J, 1992 Jun 15, 284 ( Pt 3), 861 - 7 Overproduction in Escherichia coli of the dehydroquinate synthase domain of the Aspergillus nidulans pentafunctional AROM protein; van den Hombergh JP et al.; The pentafunctional AROM protein of Aspergillus nidulans is encoded by the complex aromA locus and catalyses steps 2-6 in the synthesis of chorismate, the common precursor for the aromatic amino acids and p-aminobenzoic acid . DNA sequences encoding the 3-dehydroquinate synthase (DHQ synthase) and 3-dehydroquinase domains of the AROM protein have been amplified with the inclusion of a translational stop codon at the C-terminus by PCR technology . These amplified fragments of DNA have been subcloned into the prokaryotic expression vector pKK233-2 and expressed in Escherichia coli . As a result, the DHQ synthase domain is overproduced in E . coli, forming 30% of total cell protein, and can be purified to greater than 80% homogeneity by a simple two-step protocol . The 3-dehydroquinase domain is produced at a specific activity 8-fold greater than the corresponding activity encoded by the aromA gene in A . nidulans . The qutB gene of A . nidulans encoding quinate dehydrogenase has similarly been subjected to PCR amplification and expression in E . coli . The quinate dehydrogenase is not overproduced, but is active in E . coli as a shikimate dehydrogenase, as the presence of the qutB gene allows the growth of an E . coli mutant strain lacking shikimate dehydrogenase on minimal medium lacking aromatic-amino-acid supplementation. Cell, 1992 Jun 12, 69(6), 1043 - 50 A cytoplasmic chaperonin that catalyzes beta-actin folding; Gao Y et al.; We have isolated a cytoplasmic chaperonin based on its ability to catalyze the folding of denatured beta-actin . The cytoplasmic chaperonin is organized as a multisubunit toroid and requires Mg2+ and ATP for activity . The folding reaction proceeds via the rapid ATP-independent formation of a binary complex, followed by a slower ATP-dependent release of the native product . Electron microscopic observations reveal a striking structural change that occurs upon addition of Mg2+ and ATP . The eukaryotic cytoplasm thus contains a chaperonin that is functionally analagous to its prokaryotic, mitochondrial, and chloroplastic counterparts. Biochim Biophys Acta, 1992 Jun 11, 1107(1), 39 - 43 Isoprenoid modification of proteins distinct from membrane acyl proteins in the prokaryote Acholeplasma laidlawii; Nystrom S et al.; Isoprenylation is an important posttranslational modification that affects the activity, subunit interactions and membrane anchoring of different eukaryotic proteins . The small, cell-wall-less prokaryote Acholeplasma laidlawii has more than 20 membrane acyl-proteins enriched in myristoyl and palmitoyl chains . Radioactive mevalonate, a precursor to isoprenoids, was incorporated into several specific membrane proteins of 20 to 45 kDa and two soluble proteins of 23-25 kDa, respectively . No acyl proteins and none of the polar acyl lipids became labelled but these are all labelled by radioactive fatty acids . Mevalonate was incorporated mainly into a minor neutral, non-saponifiable lipid which migrated just above a C30-isoprenoid (squalene) on TLC-plates . The isoprenoid chains could not be released by mild alkaline hydrolysis from most of the isoprenylated proteins, although this procedure releases acyl chains from lipids and all acylated proteins . Isoprenylated proteins were enriched in the detergent phase upon partition with the non-ionic detergent Triton X-114 . This behaviour is similar to the acyl proteins of this organism and indicates that the isoprenoid chains give the proteins a hydrophobic character. Nucleic Acids Res, 1992 Jun 11, 20(11), 2667 - 71 An African swine fever virus gene with homology to DNA ligases; Hammond JM et al.; Sequence analysis of the SalI g region of the genome of a virulent isolate of ASFV (Malawi Lil 20/1) has revealed an open reading frame with the potential to encode a 48 kilodalton (kD) polypeptide which has significant homology with eukaryotic and prokaryotic DNA ligases . This ASFV encoded gene also contains the putative active site region of DNA ligases including the lysine residue which is necessary for enzyme-adenylate adduct formation, but lacks the C-terminal basic region conserved in other eukaryotic DNA ligases . A novel {32P}-labelled potential DNA ligase-adenylate adduct of M(r) 45 kD was observed upon incubation of ASFV infected cell cytoplasmic extracts with alpha-{32P}-ATP and subsequent analysis of products by SDS/PAGE . These data together suggest that ASFV encodes its own DNA ligase. J Biol Chem, 1992 Jun 5, 267(16), 11553 - 8 The sequence of the flavoprotein subunit of bovine heart succinate dehydrogenase; Birch-Machin MA et al.; The cDNA sequence of the flavoprotein subunit of bovine heart succinate dehydrogenase is reported . This is the first complete eukaryotic sequence of the flavoprotein subunit to be characterized, and it encodes a 665-amino acid protein that consists of a presequence and a 621-residue mature protein . The deduced bovine sequence shows homology to the corresponding peptides of prokaryotic succinate dehydrogenase and the related fumarate reductases; in particular, there is good overall homology (48%) to the flavoprotein subunit of Escherichia coli succinate dehydrogenase . The conserved sequences comprising the active site and those involved in FAD binding are also found in the bovine protein . The active site of the bovine polypeptide contains a cysteine that confers sensitivity of the enzyme to sulfhydryl reagents; this cysteine is only present in some sequences and thus provides a discriminatory biochemical marker . A putative flavoprotein subunit of human placental succinate dehydrogenase (partial sequence) that lacks this critical cysteine (Malcovati, M., Marchetti, T., Zanelli, T., and Tenchini, M . L . (1991) in Flavins and Flavoproteins 1990 (Curti, B., Ronchi, S., and Zanetti, G., eds) pp . 727-730, Walter de Gruyter & Co., Berlin) has only 16% homology to the bovine heart flavoprotein subunit . However, we show that the enzyme from human placenta is as sensitive to N-ethylmaleimide as that from bovine tissues . In addition, a transcript in human placenta and muscle hybridizes to the bovine heart flavoprotein cDNA and is the same size as that in bovine tissues. Plant Mol Biol, 1992 Jun, 19(3), 355 - 65 Conserved relationship between psbH and petBD genes: presence of a shared upstream element in Prochlorothrix hollandica; Greer KL et al.; Prochlorophytes are an unusual group of prokaryotic oxygenic photoautotrophs that morphologically appear to bridge the gap between cyanobacteria and the chloroplasts of eukaryotic plants . Molecular data place this group evolutionarily within the cyanobacteria, but they have a photosynthetic apparatus that is very similar to that found in chloroplasts . We have sequenced from the prochlorophyte Prochlorothrix hollandica a set of genes (psbB, psbH, petB and petD) that has a conserved organization in chloroplast genomes that is different from the organization in cyanobacterial genomes . The four genes are linked as an operon in chloroplasts, but only petB and petD are closely linked and cotranscribed in cyanobacteria . Although the prochlorophyte gene arrangement resembles that of cyanobacteria, one feature suggests the coordinated regulation of the unlinked genes . A 93 bp region of absolute conservation occurs upstream of the psbH gene and the petBD operon, near the site of transcription initiation in each gene set . This conserved element may indicate an alternative to cotranscription for achieving co-regulation of the psbH and petBD genes in the prochlorophyte. FEBS Lett, 1992 Jun 1, 303(2-3), 159 - 63 Cyanobacterial metallothionein gene expressed in Escherichia coli . Metal-binding properties of the expressed protein; Shi J et al.; The recently isolated Synechococcus gene smtA encodes the only characterised prokaryotic protein designated to be a metallothionein (MT) . To examine the metal-binding properties of its product the smtA gene was expressed in Escherichia coli as a carboxyterminal extension of glutathione-S-transferase . The pH of half dissociation of Zn, Cd and Cu ions from the expressed protein was determined to be 4.10, 3.50, 2.35, respectively, indicating a high affinity for these ions (in particular for Zn in comparison to mammalian MT) . E . coli expressing this gene showed enhanced (ca . 3-fold) accumulation of Zn. Biochem J, 1992 Jun 1, 284 ( Pt 2), 469 - 76 Human homologues of the bacterial heat-shock protein DnaJ are preferentially expressed in neurons; Cheetham ME et al.; The bacterial heat-shock protein DnaJ has been implicated in protein folding and protein complex dissociation . The DnaJ protein interacts with the prokaryotic analogue of Hsp70, DnaK, and accelerates the rate of ATP hydrolysis by DnaK . Several yeast homologues of DnaJ, with different proposed subcellular localizations and functions, have recently been isolated and are the only eukaryotic forms of DnaJ so far described . We have isolated cDNAs corresponding to two alternatively spliced transcripts of a novel human gene, HSJ1, which show sequence similarity to the bacterial DnaJ protein and the yeast homologues . The cDNA clones were isolated from a human brain-frontal-cortex expression library screened with a polyclonal antiserum raised to paired-helical-filament (PHF) proteins isolated from extracts of the brains of patients suffering from Alzheimer's disease . The similarity between the predicted human protein sequences and the bacterial and yeast proteins is highest at the N-termini, this region also shows a limited similarity to viral T-antigens and is a possible common motif involved in the interaction with DnaK/Hsp70 . Northern-blot analysis has shown that human brain contains higher levels of mRNA for the DnaJ homologue than other tissues examined, and hybridization studies with riboprobes in situ show a restricted pattern of expression of the mRNA within the brain, with neuronal layers giving the strongest signal . These findings suggest that the DnaJ-DnaK (Hsp70) interaction is general to eukaryotes and, indeed, to higher organisms. Eur J Biochem, 1992 Jun 1, 206(2), 503 - 10 Cloning and nucleotide sequence of the psrA gene of Wolinella succinogenes polysulphide reductase; Krafft T et al.; The polysulphide reductase (formerly sulphur reductase) of Wolinella succinogenes is a component of the phosphorylative electron transport system with polysulphide as the terminal acceptor . Using an antiserum raised against the major subunit (PsrA, 85 kDa) of the enzyme, the corresponding gene (psrA) was cloned from a lambda-gene bank . The N-terminal amino acid sequence of PsrA mapped within the psrA gene product, which also contained an apparent signal peptide . Downstream of the psrA gene two more open reading frames (psrB and psrC) were found . The three genes may form a transcriptional unit with the transcription start site in front of psrA . The three genes were present only once on the genome . PsrA is a hydrophilic protein homologous to the largest subunits of six prokaryotic molybdoenzymes . PsrB is predicted to be hydrophilic, to contain ferredoxin-like cysteine clusters and to be homologous to the smaller hydrophilic subunits of four molybdoenzymes . PsrC is predicted to be a hydrophobic protein that could possibly serve as the membrane anchor of the enzyme. Proc Natl Acad Sci U S A, 1992 Jun 1, 89(11), 5188 - 91 Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages; Cianciotto NP et al.; Legionella pneumophila is an intracellular parasite of freshwater protozoa and human macrophages . Recent studies determined that the macrophage infectivity potentiator (Mip) surface protein, a prokaryotic homolog of the FK506-binding proteins, is required for optimal infection of macrophages . To determine whether Mip is also involved in L . pneumophila infection of protozoa, we examined the ability of a strain lacking Mip to parasitize Hartmannella amoebae and Tetrahymena ciliates . After 3 days of incubation, approximately 1000-fold fewer bacteria were recovered from protozoan cocultures infected with the Mip- strain than from those cocultures infected with an isogenic Mip+ strain . The mip mutant was, however, not impaired in its ability to bind to amoebae cell surfaces, indicating that Mip is involved in bacterial resistance to intracellular killing and/or intracellular multiplication . These data suggest that L . pneumophila employs similar genes and mechanisms to infect human cells and protozoa . Furthermore, they support the hypothesis that the ability of L . pneumophila to parasitize macrophages and hence to cause human disease is a consequence of its prior adaptation to intracellular growth within protozoa. EMBO J, 1992 Jun, 11(6), 2219 - 28 The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent; Austin S et al.; The prokaryotic activator protein NTRC binds to enhancer-like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme . Formation of open complexes requires the phosphorylated form of NTRC and the reaction is ATP dependent . We find that NTRC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NTRC binding sites. Res Microbiol, 1992 Jun, 143(5), 467 - 70 Two unrelated alkaliphilic Bacillus species possess identical deviations in sequence from those of other prokaryotes in regions of F0 proposed to be involved in proton translocation through the ATP synthase; Ivey DM et al.; The a and c subunits of two unrelated alkaliphilic Bacillus species contain unusual motifs in regions previously implicated by others in H(+)-coupled oxidative phosphorylation . The facultative alkaliphile B . firmus OF4 apparently does not contain genes encoding an alternative F0, supporting other evidence that a single species of proton-translocating F1F0-ATPase catalyses oxidative phosphorylation both at low and high pH . The unusual F0 sequence motifs may be part of the adaptation of the extreme alkaliphiles to growth at very high pH. Immunol Lett, 1992 Jun, 33(1), 53 - 9 Rabbit single domain antibodies specific to protein C expressed in prokaryotes; Suter M et al.; VDJ genes were amplified by the polymerase chain reaction from mRNA isolated from peripheral blood B cells of rabbits immunized with protein C . The amplified genes were cloned into a lambda phage expression vector and packaged . A library of 6 x 10(5) recombinant phages was screened with labelled protein C and 30 positive clones were found . Three of them were plaque purified and the affinity of the single domain antibodies to the antigen determined to be 10(6)-10(7) l M-1 . The data indicate the feasibility of generating single domain antibody, specific to protein antigen, from rabbit. FASEB J, 1992 Jun, 6(9), 2660 - 6 ATP-dependent bacterial transporters and cystic fibrosis: analogy between channels and transporters; Ames GF et al.; The traffic ATPases superfamily includes known transporters, both prokaryotic and eukaryotic, including the medically important proteins, P-glycoprotein, and the cystic fibrosis gene product (CFTR), which is known to be a Cl- channel . The structure and mechanism of action of the best-studied members of the superfamily, the periplasmic permeases, are described and related to that of CFTR and eukaryotic traffic ATPases in general . The contention is put forward that the distinction between the architecture and mechanisms of action of channels and transporters is blurred. Immunol Invest, 1992 Jun, 21(3), 241 - 57 Immunoreactivity of recombinant carcinoembryonic antigen proteins expressed in Escherichia coli; Kuroki M et al.; Immunoreactivities of recombinant carcinoembryonic antigen (CEA) proteins expressed in Escherichia coli (E . coli) were analyzed in relation to the CEA domain structure {domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M} . We reconstructed in a prokaryotic expression vector, pUCPL-cI, the cDNAs fro CEA-N, CEA-I, CEA-II, and CEA-III-M . The latter three were expressed as fusion products with bacterial beta-galactosidase . The recombinant proteins were solubilized by sonication in 1% sodium dodecyl sulfate (SDS) and purified by preparative SDS-polyacrylamide gel electrophoresis followed by electroelution . Their molecular weights judged from Western blotting coincided with those calculated from their cDNA sequences, respectively . By solid-phase enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 21 distinct anti-CEA monoclonal antibodies (MAbs) which had been found to recognize the peptide epitopes of the CEA molecule and to be reactive with the recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells . Fourteen of the 21 MAbs reacted with the recombinant CEA proteins expressed in E . coli and confirmed the localization of the epitopes identified by using the recombinant CEA proteins expressed in CHO cells . The reactivities of 5 MAbs with the recombinant proteins expressed in E . coli were remarkably low when compared with those of the proteins expressed in CHO cells but also confirmed the localization of the epitopes identified with the recombinant CEA proteins expressed in CHO cells . The remaining 2 MAbs did not react with any recombinant protein expressed in E . coli . These results indicate that the fusion CEA-proteins expressed in E . coli are useful in the localization of the epitopes on the polypeptide chains when they reacted with the MAbs tested . However, one third of the epitopes of CEA peptides may be profoundly affected by the presence of disulfide bonds and/or sugar chains which do not seem to be formed well in E . coli. J Virol, 1992 Jun, 66(6), 3918 - 24 Gene for the major antigenic structural protein (p100) of human herpesvirus 6; Neipel F et al.; A human herpesvirus 6 (HHV-6) structural protein of 100 kDa (p100) is the polypeptide most frequently and intensively reactive in immunoblotting analyses with human sera on HHV-6-infected cells or partially purified virions . The gene for p100 was identified by screening a bacteriophage lambda library with monospecific rabbit antisera . The gene codes for a polypeptide of 870 amino acids with a calculated molecular size of 97 kDa . Its amino-terminal third is weakly homologous to the immunogenic basic matrix phosphoprotein pp150 of human cytomegalovirus . Five fragments representing more than 93% of HHV-6 p100 were prokaryotically expressed . The antigenic epitopes of p100 were preliminary mapped by immunoblotting with human sera . They are located within the carboxy-terminal part which is neither homologous nor cross-reactive to pp150 of human cytomegalovirus . Availability of the gene for the immunodominant structural protein should provide tools for studies of pathogenesis by HHV-6. Virology, 1992 Jun, 188(2), 938 - 47 A gene homologous to topoisomerase II in African swine fever virus; Garcia-Beato R et al.; A putative topoisomerase II gene of African swine fever virus was mapped using a degenerate oligonucleotide probe derived from a region highly conserved in type II topoisomerases . The gene is located within EcoRI fragments P and H of the African swine fever virus genome . Sequencing of this region has revealed a long open reading frame, designated P1192R, encoding a protein of 1192 amino acids, with a predicted molecular weight of 135,543 . Open reading frame P1192R is transcribed late after infection into a 4.6-kb RNA . The deduced amino acid sequence of this open reading frame shares significant similarity with topoisomerase II sequences from different sources, with percentages of identity between 23 and 29% . The evolutionary relationships among the topoisomerase II sequences of ASF virus, eukaryotes and prokaryotes were analyzed and a phylogenetic tree was established . The tree indicates that the ASF virus topoisomerase II gene was present in the virus genome before protozoa, yeasts, and metazoa diverged. J Biol Chem, 1992 May 15, 267(14), 9654 - 62 Guanine nucleotide-binding proteins in the intestinal parasite Giardia lamblia . Isolation of a gene encoding an approximately 20-kDa ADP-ribosylation factor; Murtagh JJ Jr et al.; Giardia lamblia is a protozoan intestinal parasite that has characteristics of both eukaryotes and prokaryotes . To determine whether genes for guanine nucleotide-binding proteins are present in Giardia, genomic DNA and cDNA libraries were screened by polymerase chain reaction and by hybridization with mixed oligonucleotide probes complementary to sequences encoding conserved GTP-binding domains . A gene with a high degree of sequence identity with mammalian ADP-ribosylation factors (ARFs), believed to be important in vesicular transport, was identified . The Giardia ARF gene had a 573-base open reading frame encoding 191 amino acids which are 63-70% identical with known mammalian and yeast ARFs . Sequence conservation among ARFs was greatest in putative GTP-binding domains . A single ARF mRNA species of approximately 750 bases was found in two different Giardia isolates . Primer extension and RNA sequencing of the Giardia ARF transcript revealed a short (6-base) 5'-untranslated region similar in size to those found in other Giardia transcripts . Giardia extracts contained ARF activity, as shown by stimulation of cholera toxin-catalyzed ADP-ribosylation and a Giardia ARF expressed in Escherichia coli as a fusion protein likewise exhibited biochemical activity . Its presence in Giardia is consistent with the view that ARF emerged before the divergence of this protozoan from other eukaryotes (approximately 1.5 billion years ago), and that an ARF-like protein may have been the ancestor of several other classes of signal-transducing guanine nucleotide-binding proteins, including the alpha subunits of the heterotrimeric G proteins. Gene, 1992 May 15, 114(2), 239 - 43 A selectable bifunctional beta-galactosidase::phleomycin-resistance fusion protein as a potential marker for eukaryotic cells; Baron M et al.; The Sh ble gene, conferring phleomycin resistance (PhR), was fused in frame to both the 3' and 5' ends of the Escherichia coli lacZ gene . The bifunctionality of the resulting 130-kDa hybrid proteins was demonstrated in E . coli and in the fungus, Tolypocladium geodes . PhR transformants of both organisms could be selected for . All transformants from E . coli and most from T . geodes displayed beta Gal activity . In the fungal host, higher transformation frequencies and greater levels of beta Gal activity were observed in clones harboring the lacZ::Sh ble fusion, as compared to the Sh ble::lacZ configuration . This system appears to be a potentially useful tool for the direct selection of transformants, and the evaluation of gene expression and regulation in a wide variety of prokaryotic and eukaryotic hosts. Eur J Biochem, 1992 May 15, 206(1), 69 - 77 Purification and characterization of the ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp . PCC 6301; Marques S et al.; Ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp . PCC 6301 has been purified using, as main steps, ethanol fractionation in the presence of high ionic strength, ion-exchange chromatography and ferredoxin-Sepharose affinity chromatography . The overall process yielded an homogeneous enzyme with a specific activity of 30 U/mg protein, after a purification of 2800-fold with a recovery of 43% . The molecular mass of the native protein was 156 kDa, as calculated from its Stokes radius (rS, 4.32 nm) and sedimentation coefficient (S20,w, 8.46 S) . The size was also estimated by SDS/PAGE as 160 kDa, indicating that the native protein was a monomer . The enzyme exhibited absorption maxima at 279, 370 and 438 nm and a A279/A438 absorbance ratio of 11 . One molecule of FMN, but not FAD, was found/molecule native protein . The addition of dithionite resulted in the loss of the absorption peak at 438 nm, which was restored by the addition of 2-oxoglutarate, thus indicating that the prosthetic group is functional in catalysis . Classical hyperbolic kinetics with substrate inhibition was seen for 2-oxoglutarate . The Km values determined for glutamine and ferredoxin were 0.7 mM and 7 microM, respectively, and the apparent Km for 2-oxoglutarate was estimated to be 1.7 mM . Azaserine and 6-diazo-5-oxo-L-norleucine were potent inhibitors of the activity, while pyridoxal 5-phosphate, known to react with Lys residues, partially inactivated the enzyme . This ferredoxin-dependent glutamate synthase is, as far as we know, the first purified from prokaryotic organisms and resembles its counterpart from chloroplasts, suggesting that cyanobacterial glutamate synthase may have been the ancestor of ferredoxin-glutamate synthase in plants. Proc Natl Acad Sci U S A, 1992 May 15, 89(10), 4437 - 41 A gene homologous to chloroplast carbonic anhydrase (icfA) is essential to photosynthetic carbon dioxide fixation by Synechococcus PCC7942; Fukuzawa H et al.; To understand the CO2-concentrating mechanism in cyanobacteria, a genomic DNA fragment that complements a temperature-sensitive high-CO2 (5%)-requiring mutant of Synechococcus PCC7942 has been isolated . An open reading frame (ORF272) encoding a polypeptide of 272 amino acids (Mr, 30,184) was found within the genomic region located 20 kilobases downstream from the genes for ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcLS) . Insertion of a kanamycin-resistance gene cartridge within the ORF272 in wild-type cells led to a high-CO2-requiring phenotype . Strains carrying a gene disabled by insertional mutagenesis accumulated inorganic carbon in the cells, but they could not fix it efficiently, even though ribulose-1,5-bisphosphate carboxylase activity was comparable to that of the wild-type strain . Therefore, the ORF272 was designated as a gene icfA, which is essential to inorganic carbon fixation . Furthermore, the predicted icfA gene product shared significant sequence similarities with plant chloroplast carbonic anhydrases (CAs) from pea (22%) and spinach (22%) and also with the Escherichia coli cynT gene product (31%), which was recently identified to be E . coli CA . These results indicate that the putative CA encoded by icfA is essential to photosynthetic carbon dioxide fixation in cyanobacteria and that plant chloroplast CAs may have evolved from a common ancestor of the prokaryotic CAs, which are distinct from mammalian CAs and Chlamydomonas periplasmic CAs. Cell, 1992 May 15, 69(4), 677 - 84 Signal peptides open protein-conducting channels in E . coli; Simon SM et al.; Plasma membrane vesicles and protoplasts of Escherichia coli were fused to planar lipid bilayers and studied with electrophysiological techniques . Large transmembrane aqueous channels were opened when 0.2 nM LamB signal peptide was added to the cytoplasmic side of the membrane . These aqueous pores are similar in conductance to those previously observed in mammalian endoplasmic reticulum when puromycin is used to release and thus unplug nascent translocating chains . Signal sequences have been previously shown to be necessary and sufficient for targeting proteins to cellular membranes . These results demonstrate that signal peptides are sufficient for opening the protein-conducting channels . We suggest that they are the physiological ligands that open protein-conducting channels at the initiation of protein translocation across prokaryotic plasma membrane and mammalian endoplasmic reticulum. Nucleic Acids Res, 1992 May 11, 20(9), 2287 - 91 Isolation and characterization of the cDNA encoding human DNA methyltransferase; Yen RW et al.; We have cloned a series of overlapping cDNA clones encoding a 5194 bp transcript for human DNA methyltransferase (DNA MTase) . This sequence potentially codes for a protein of 1495 amino acids with a predicted molecular weight of 169 kDa . The human DNA MTase cDNA has eighty percent homology at the nucleotide level, and the predicted protein has seventy-four percent identity at the amino acid level, to the DNA MTase cDNA cloned from mouse cells . Like the murine DNA MTase, the amino terminal two-thirds of the human protein contains a cysteine-rich region suggestive of a metal-binding domain . The carboxy terminal one-third of the protein shows considerable similarity to prokaryotic (cytosine-5)-methyltransferases . The arrangement of multiple motifs conserved in the prokaryotic genes is preserved in the human DNA MTase, including the relative position of a proline-cysteine dipeptide thought to be an essential catalytic site in all (cytosine-5)-methyltransferases . A single 5.2 kb transcript was detected in all human tissues tested, with the highest levels of expression observed in RNA from placenta, brain, heart and lung . DNA MTase cDNA clones were used to screen a chromosome 19 genomic cosmid library . The DNA MTase-positive cosmids which are estimated to span a genomic distance of 93 kb have been localized to 19p13.2-p13.3 by fluorescence in situ hybridization . Isolation of the cDNA for human DNA MTase will allow further study of the regulation of DNA MTase expression, and of the role of this enzyme in establishing DNA methylation patterns in both normal and neoplastic cells. FEBS Lett, 1992 May 4, 302(1), 1 - 4 Pseudouridine in the large-subunit (23 S-like) ribosomal RNA . The site of peptidyl transfer in the ribosome? Lane BG, Ofengand J, Gray MW. On evolutionary grounds, it has been advocated for more than 40 years that RNA generally, and more recently rRNA in particular, may participate, catalytically, in protein biosynthesis . A specific molecular mechanism has never been proposed . We suggest here that the N-1 position(s) in one or more of the approximately 4 pseudouridine (omega) residues in E . coli 23 S rRNA catalyzes transfer of the aminoacyl moiety from teh 3'-terminus of peptidyl tRNA in the P site to aminoacyl tRNA in the A site of the ribosome . Evidence that supports the proposal in the case of E . coli ribosomes, and relevant information pertaining to eukaryotic ribosomes, is summarized . Essential features of the evidence are that (i) the N-1 position in 1-acetylthymine (a direct analogue of 1-acetylpseudouridine) has an especially high potential for acyl-group transfer, comparable to that found for N-acetylimidazole (Spector, L.B . and Keller, E.B . (1958) J . Biol . Chem . 232, 185-192), (ii) most of the omega residues in prokaryotic 23 S rRNA are confined to the peptidyl transferase center in E . coli ribosomes, and (iii) Um-Gm-omega, the most densely modified sequence in eukaryotic 26 S rRNA, is universally conserved at a fixed site in the putative peptidyl transferase center of all eukaryotic ribosomes. Mol Microbiol, 1992 May, 6(10), 1253 - 7 Membrane-associated GTPases in bacteria; March PE; Members of the GTPase superfamily are extremely important in regulating membrane signalling pathways in all cells . This review focuses on membrane-associated GTPases that have been described in prokaryotes . In bacteria, LepA and NodQ are very similar to protein synthesis elongation factors but apparently have membrane-related functions . The amino acid sequences of FtsY and Ffh are clearly related to eukaryotic factors involved in protein secretion . Obg and Era are not closely related to any GTPase subgroup according to amino acid sequence comparisons, but they are essential for viability . In spite of similarities to well-studied eukaryotic proteins the signalling pathways of these cellular regulators, with the exception of NodQ, have not yet been elucidated. Bioessays, 1992 May, 14(5), 295 - 301 DNA precursor asymmetries, replication fidelity, and variable genome evolution; Mathews CK et al.; Balanced pools of deoxyribonucleoside triphosphates (dNTPs) are essential for DNA replication to occur with maximum fidelity . Conditions that create biased dNTP pools stimulate mutagenesis, as well as other phenomena, such as recombination or cell death . In this essay we consider the effective dNTP concentrations at replication sites under normal conditions, and we ask how maintenance of these levels contributes toward the natural fidelity of DNA replication . We focus upon two questions . (1) In prokaryotic systems, evidence suggests that replication is driven by small, localized, rapidly replenished dNTP pools that do not equilibrate with the bulk dNTP pools in the cell . Since these pools cannot be analyzed directly, what indirect approaches can illuminate the nature of these replication-active pools? (2) In eukaryotic cells, the normal dNTP pools are highly asymmetric, with dGTP being the least abundant nucleotide . Moreover, the composition of the dNTP pools changes as cells progress through the cell cycle . To what extent might these natural asymmetries contribute toward a recently described phenomenon, the differential rate of evolution of different genes in the same genome? Cell, 1992 May 1, 69(3), 439 - 56 DMC1: a meiosis-specific yeast homolog of E . coli recA required for recombination, synaptonemal complex formation, and cell cycle progression; Bishop DK et al.; DMC1 is a new meiosis-specific yeast gene . Dmc1 protein is structurally similar to bacterial RecA proteins . dmc1 mutants are defective in reciprocal recombination, accumulate double-strand break (DSB) recombination intermediates, fail to form normal synaptonemal complex (SC), and arrest late in meiotic prophase . dmc1 phenotypes are consistent with a functional relationship between Dmc1 and RecA, and thus eukaryotic and prokaryotic mechanisms for homology recognition and strand exchange may be related . dmc1 phenotypes provide further evidence that recombination and SC formation are interrelated processes and are consistent with a requirement for DNA-DNA interactions during SC formation . dmc1 mutations confer prophase arrest . Additional evidence suggests that arrest occurs at a meiosis-specific cell cycle "checkpoint" in response to a primary defect in prophase chromosome metabolism . DMC1 is homologous to yeast's RAD51 gene, supporting the view that mitotic DSB repair has been recruited for use in meiotic chromosome metabolism. Genes Dev, 1992 May, 6(5), 825 - 36 Polar localization of a bacterial chemoreceptor; Alley MR et al.; The bacterial chemotaxis signal transducer MCP is an integral membrane receptor protein . The chemoreceptor is localized at the flagellum-bearing pole of Caulobacter crescentus swarmer cells . Amino-terminal sequences of the MCP target the protein to the membrane while the carboxy-terminal portion of the protein is responsible for polar localization . The C . crescentus and Escherichia coli MCPs have highly conserved carboxy-terminal domains, and when an E . coli MCP is expressed in C . crescentus, it is targeted to the swarmer cell progeny . These results suggest that subcellular localization of a prokaryotic protein involves interaction of specific regions of the protein with unique cell sites that contain either localized binding proteins or a specific secretory apparatus. Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 4028 - 32 Enzymatic defect in "X-linked" sideroblastic anemia: molecular evidence for erythroid delta-aminolevulinate synthase deficiency; Cotter PD et al.; Recently, the human gene encoding erythroid-specific delta-aminolevulinate synthase was localized to the chromosomal region Xp21-Xq21, identifying this gene as the logical candidate for the enzymatic defect causing "X-linked" sideroblastic anemia . To investigate this hypothesis, the 11 exonic coding regions of the delta-aminolevulinate synthase gene were amplified and sequenced from a 30-year-old Chinese male with a pyridoxine-responsive form of X-linked sideroblastic anemia . A single T----A transition was found in codon 471 in a highly conserved region of exon 9, resulting in an Ile----Asn substitution . This mutation interrupted contiguous hydrophobic residues and was predicted to transform a region of beta-sheet structure to a random-coil structure . Prokaryotic expression of the normal and mutant cDNAs revealed that the mutant construct expressed low levels of enzymatic activity that required higher concentrations of pyridoxal 5'-phosphate to achieve maximal activation than did the normal enzyme . The amino acid substitution occurred in the exon containing the putative pyridoxal 5'-phosphate binding site and may account for the reduced ability of the cofactor to catalyze the formation of delta-aminolevulinic acid. J Bacteriol, 1992 May, 174(9), 2929 - 34 Comparative physiology of two protozoan parasites, Leishmania donovani and Trypanosoma brucei, grown in chemostats; ter Kuile BH et al.; Cultures of the insect stage of the protozoan parasites Leishmania donovani and Trypanosoma brucei were grown in chemostats with glucose as the growth rate-limiting substrate . L . donovani has a maximum specific growth rate (mu max) of 1.96 day-1 and a Ks for glucose of 0.1 mM; the mu max of T . brucei is 1.06 day-1 and the Ks is 0.06 mM . At each steady state (specific growth rate, mu, equals D, the dilution rate), the following parameters were measured: external glucose concentration (Glcout), cell density, dry weight, protein, internal glucose concentration (Glcin), cellular ATP level, and hexokinase activity . L . donovani shows a relationship between mu and yield that allows an estimation of the maintenance requirement (ms) and the yield per mole of ATP (YATP) . Both the ms and the YATP are on the higher margin of the range found for prokaryotes grown on glucose in a complex medium . L . donovani maintains the Glcin at a constant level of about 50 mM as long as it is not energy depleted . T . brucei has a decreasing yield with increasing mu, suggesting that it oxidizes its substrate to a lesser extent at higher growth rates . Glucose is not concentrated internally but is taken up by facilitated diffusion, while phosphorylation by hexokinase is probably the rate-limiting step for glucose metabolism . The Ks is constant as long as glucose is the rate-limiting substrate . The results of this study demonstrate that L . donovani and T . brucei have widely different metabolic strategies for dealing with varying external conditions, which reflect the conditions they are likely to encounter in their respective insect hosts. Infect Immun, 1992 May, 60(5), 1729 - 33 Consequences of microbial attachment: directing host cell functions with adhesins; Hoepelman AI et al.; We take the view that adherence is not just a static process of holding hands but rather elicits a response in the targeted cell . From this point of view, adherence is an active process with an outcome . This outcome or fate is predictable only when several parameters of the host cell-adhesin interaction are known: is the adhesin acting alone or in series with other products, is the receptor up- or down-regulated at the time of ligation, which domain of the receptor is bound, and finally, which intracellular response circuits are connected to the receptor in the cell type targeted? Variations in these parameters are the basis for the ability of the adhesins of pathogens to orchestrate outcomes as disparate as simple address recognition versus actin nucleation, cytokine induction, activation of plasmin, derangement of leukocyte migration, or deposition of antibody on host cell membranes . The recognition of the relatedness of some eukaryotic and prokaryotic adhesive domains and the shared use of existing eukaryotic cell-cell interaction systems between host and pathogen suggest that the cellular interactions of interest in eukaryotic cell biology can be revealed by taking clues from the pathogens, which have studied and adapted to them the longest. J Virol, 1992 May, 66(5), 2934 - 42 Regulated expression of foreign genes in vaccinia virus under the control of bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor; Alexander WA et al.; The gene encoding bacteriophage T7 RNA polymerase (T7gene1) was placed under the control of regulatory elements from the Escherichia coli lac operon to construct an inducible vaccinia virus expression system consisting entirely of prokaryotic transcriptional machinery . Regulated expression of T7 RNA polymerase was necessary to construct a stable recombinant vaccinia virus harboring a T7 promoter; otherwise, uncontrolled expression led to interference with endogenous virus replication . To this end, the gene encoding the repressor protein of the lac operon was fused to a viral early/late promoter so that it was expressed constitutively, and the lac operator was interposed between a viral major late promoter and T7gene1 . Greater than 99% repression of T7 RNA polymerase, which was relieved approximately 80-fold in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), was obtained . An expression cassette containing a T7 promoter-controlled beta-galactosidase reporter gene was recombined into a different region of the viral genome containing T7gene1 . A stable, double recombinant virus was isolated and grown to a high titer . In the absence of inducer, beta-galactosidase expression was substantially repressed . Addition of increasing amounts of IPTG induced expression of beta-galactosidase to the point of suppression of viral replication . This hybrid vaccinia virus system (Vac/Op/T7) has potential applications for the efficient bioproduction of a wide variety of gene products. J Neurochem, 1992 May, 58(5), 1782 - 9 Expression of functional membrane-bound and soluble catechol-O-methyltransferase in Escherichia coli and a mammalian cell line; Malherbe P et al.; Human catechol-O-methyltransferase (hCOMT) cDNA was used to express the recombinant hCOMT enzyme in sufficient quantities in prokaryotic as well as in eukaryotic cells to allow kinetic studies . When human membrane-bound catechol-O-methyltransferase (MB-COMT; amino acids 1-271) and the soluble catechol-O-methyltransferase COMT (S-COMT; delta membrane anchor hCOMT; amino acids 27-271), with the latter lacking the first 26 hydrophobic amino acids, were expressed in Escherichia coli, a relatively high-level synthesis of catalytically active enzymes was obtained . Insertion of the human MB-COMT-coding sequence into an eukaryotic expression vector under transcriptional control of the cytomegalovirus (CMV) promoter and enhancer yielded large quantities of hCOMT in human kidney 293 cells . Subcellular fractionation of 293 cells transfected with pBC12/CMV-hCOMT showed hCOMT to be located predominantly in the membrane fraction . The catechol-O-methyltransferase (COMT) activity was measured in cytosolic and membrane fractions at 37 degrees C, giving values of 33 and 114 units/mg of protein, respectively (1 unit produces 1 nmol of guaiacol/h) . Km values were 10 microM for MB-COMT and 108 microM for S-COMT, indicating that recombinant MB-COMT exhibits a higher affinity for catechol as the substrate than the soluble form . RNA blot analysis of human hepatome cells (Hep G2), kidney, liver, and fetal brain revealed only one species of hCOMT mRNA of approximately 1.4 kb . Its level in these various tissues was similar to those of COMT protein in each tissue.(ABSTRACT TRUNCATED AT 250 WORDS) Antimicrob Agents Chemother, 1992 May, 36(5), 1119 - 24 The unstable tetracycline resistance gene of Streptomyces lividans 1326 encodes a putative protein with similarities to translational elongation factors and Tet(M) and Tet(O) proteins; Dittrich W et al.; Streptomyces lividans contains a genetically unstable tetracycline resistance determinant . Nucleotide sequencing revealed an open reading frame of 1,917 nucleotides . The transcriptional start site was mapped at about 110 bp upstream of the ATG codon . The proposed promoter contains an 8-bp perfect inverted repeat between the -10 and -35 regions . The deduced amino acid sequence showed several motifs which are commonly found in many GTP-binding proteins . On the basis of its amino acid sequence, the presumptive S . lividans 1326 protein belongs to the Tet(M)-Tet(O) group of tetracycline resistance proteins and shows significant similarity to translational elongation factors of prokaryotes and eukaryotes. Drug Saf, 1992 May-Jun, 7(3), 214 - 22 Mutagenicity of quinolone antibacterials; Fort FL; The literature is summarised on the activity of quinolone antibacterial compounds in assays which are commonly used for risk assessment of new pharmaceuticals . These include assays for DNA damage, sister chromatid exchanges, chromosome aberrations and mutation induction . The general pattern of activity exhibited by these compounds is induction of DNA damage in both prokaryotic and eukaryotic cells, and induction of mutations in DNA repair-proficient bacteria and at the thymidine kinase locus in mammalian cells . They do not appear as a class to induce mutations at the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) or Na+,K(+)-ATPase loci or to cause chromosome aberrations . It is suggested that these actions may be the result of interference with eukaryotic topoisomerase and that this interference differs in some respects from the topoisomerase interference caused by certain antitumour compounds . The postulated mechanism of action has important implications for assessment of risk from consumption of quinolone antibacterials . The risk of adverse genotoxic events should vary directly with the concentration of drug reaching the intracellular enzyme target and the affinity of the drug for the target . Results of carcinogenicity studies conducted to date with the quinolone antibacterials suggest minimal risk from long term consumption of the newer, second-generation compounds. Mol Microbiol, 1992 May, 6(10), 1375 - 83 Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPlase) activity; Fischer G et al.; Legionella pneumophila is an intracellular parasite which is able to survive and multiply in human monocytes and alveolar macrophages . The Mip (macrophage infectivity potentiator) protein has been shown to be an essential virulence factor . A search of translated nucleic acid data bases has shown that the Mip protein from strain Wadsworth possesses regions homologous to those found in the FK506-binding proteins (FKBPs) of several different eukaryotic organisms . FKBPs are able to bind to the immunosuppressant macrolide FK506 and possess peptidyl-prolyl cis/trans isomerase (PPIase) activity . The gene coding for the Mip protein was cloned from the chromosome of L . pneumophila strain Philadelphia I and sequenced . It was synthesized in Escherichia coli K-12 and after purification it exhibited PPIase activity catalysing the slow cis/trans isomerization of prolyl peptide bonds in oligopeptides . Mip is inhibited by FK506 and fully resistant to cyclosporin A, as was also found for the recently characterized FKBP-type PPIases of eukaryotes . However, the N-terminal extension of Mip and/or the substitutions of the variable amino acids in the C-terminal FKBP core leads to variations, when compared with eukaryotic FKBPs, in substrate specificity with the oligopeptide substrates of type Suc-Ala-Xaa-Pro-Phe-4-nitroanilide . Nevertheless, the Legionella Mip factor represents a bacterial gene product which shares some characteristics normally found in eukaryotic proteins . In view of the activity of PPIases in protein-folding reactions, such prokaryotic FKBP analogues may represent a new class of bacterial pathogenicity factors. Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 3756 - 60 Phage display of ricin B chain and its single binding domains: system for screening galactose-binding mutants; Swimmer C et al.; We demonstrate that the B chain of ricin toxin preserves its lectin activity when expressed as a fusion protein on the surface of fd phage . Moreover, B chain, which folds into two topologically similar globular domains, can be dissected into amino-terminal and carboxyl-terminal domains to form single binding domains (SBDs) of B chain, each of which displays specificity for complex galactosides . The specific binding exhibited by the fusion protein of these SBDs was eliminated when amino acid substitutions Gly-46 in SBD1 or Gly-255 in SBD2 for native asparagine were introduced to alter key residues implicated in hydrogen bonding with substrate . These data demonstrate that it is possible to use a prokaryotic expression system to stably express and screen ricin B chain and its SBDs for sugar-binding mutants . Expression of ricin B chain on the surface of fd phage provides a method that can be used to efficiently select mutants with altered binding activities from a randomly generated library. EMBO J, 1992 May, 11(5), 1785 - 96 Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo; Cardenas ME et al.; The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis . Phosphorylation has been shown to stimulate topoisomerase II activity in vitro . Here we show that topoisomerase II is a phosphoprotein in yeast and that the level of incorporated phosphate is significantly higher at mitosis than in G1 . Comparison of tryptic phosphopeptide maps reveals that the major phosphorylation sites in vivo are targets for casein kinase II . Incorporation of phosphate into topoisomerase II is nearly undetectable at the non-permissive temperature in a conditional casein kinase II mutant . The sites modified by casein kinase II are located in the extreme C-terminal domain of topoisomerase II . This domain is absent in prokaryotic and highly divergent among eukaryotic type II topoisomerases, and may serve to regulate functions of topoisomerase II that are unique to eukaryotic cells. J Bacteriol, 1992 May, 174(10), 3242 - 9 The lysP gene encodes the lysine-specific permease; Steffes C et al.; Escherichia coli transports lysine by two distinct systems, one of which is specific for lysine (LysP) and the other of which is inhibited by arginine ornithine . The activity of the lysine-specific system increases with growth in acidic medium, anaerobiosis, and high concentrations of lysine . It is inhibited by the lysine analog S-(beta-aminoethyl)-L-cysteine (thiosine) . Thiosine-resistant (Tsr) mutants were isolated by using transpositional mutagenesis with TnphoA . A Tsr mutant expressing alkaline phosphatase activity in intact cells was found to lack lysine-specific transport . This lysP mutation was mapped to about 46.5 min on the E . coli chromosome . The lysP-phoA fusion was cloned and used as a probe to clone the wild-type lysP gene . The nucleotide sequence of the 2.7-kb BamHI fragment was determined . An open reading frame from nucleotides 522 to 1989 was observed . The translation product of this open reading frame is predicted to be a hydrophobic protein of 489 residues . The lysP gene product exhibits sequence similarity to a family of amino acid transport proteins found in both prokaryotes and eukaryotes, including the aromatic amino acid permease of E . coli (aroP) and the arginine permease of Saccharomyces cerevisiae (CAN1) . Cells carrying a plasmid with the lysP gene exhibited a 10- to 20-fold increase in the rate of lysine uptake above wild-type levels . These results demonstrate that the lysP gene encodes the lysine-specific permease. J Bacteriol, 1992 May, 174(9), 3011 - 20 The lemA gene required for pathogenicity of Pseudomonas syringae pv . syringae on bean is a member of a family of two-component regulators; Hrabak EM et al.; The lemA gene of the plant pathogen Pseudomonas syringae pv . syringae is required for disease lesion formation on bean plants . Cosmid clones that complemented a lemA mutant in trans were isolated previously . The lemA gene was localized by subcloning and transposon mutagenesis . The lemA region and flanking DNA were sequenced, and an open reading frame of 2.7 kb was identified . The nucleotide and predicted amino acid sequences of the lemA gene showed sequence similarity to a family of prokaryotic two-component regulatory proteins . Unlike most of the previously described two-component systems, the lemA gene product contained homology to both components in one protein . Mutations introduced upstream and downstream of the lemA gene failed to locate a gene for a second protein component but identified the putative cysM gene of P . syringae pv . syringae . The cysM gene was located upstream of the lemA gene and was divergently transcribed . The lemA gene product was expressed at low levels in P . syringae pv . syringae and appeared to be positively auto-regulated. J Biol Chem, 1992 Apr 25, 267(12), 8007 - 11 Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae; Chang YH et al.; A yeast gene for a methionine aminopeptidase, one of the central enzymes in protein synthesis, was cloned and sequenced . The DNA sequence encodes a precursor protein containing 387 amino acid residues . The mature protein, whose NH2-terminal sequence was confirmed by Edman degradation, consists of 377 amino acids . The function of the 10-residue sequence at the NH2 terminus, containing 1 serine and 6 threonine residues, remains to be established . In contrast to the structure of the prokaryotic enzyme, the yeast methionine aminopeptidase consists of two functional domains: a unique NH2-terminal domain containing two motifs resembling zinc fingers, which may allow the protein to interact with ribosomes, and a catalytic COOH-terminal domain resembling other prokaryotic methionine aminopeptidases . Furthermore, unlike the case for the prokaryotic gene, the deletion of the yeast MAP1 gene is not lethal, suggesting for the first time that alternative NH2-terminal processing pathway(s) exist for cleaving methionine from nascent polypeptide chains in eukaryotic cells. Nature, 1992 Apr 23, 356(6371), 725 - 8 A eukaryotic DNA glycosylase/lyase recognizing ultraviolet light-induced pyrimidine dimers; Hamilton KK et al.; Cyclobutane pyrimidine dimers (CPDs) are the predominant product of photodamage in DNA after exposure of cells to ultraviolet light and are cytotoxic, mutagenic and carcinogenic in a variety of cellular and animal systems . In prokaryotes, enzymes and protein complexes have been characterized that remove or reverse CPDs in DNA . Micrococcus luteus and T4 phage-infected Escherichia coli contain a specific N-glycosylase/apurinic-apyrimidinic lyase that catalyses a two-step DNA incision process at sites of CPDs, thus initiating base excision repair of these lesions . It is well established that CPDs are recognized and removed from eukaryotic DNA by excision repair processes but very little information exists concerning the nature of the proteins involved in CPD recognition and DNA incision events . We report here that an enzyme functionally similar to the prokaryotic N-glycosylase/apurinic-apyrimidinic lyases exists in Saccharomyces cerevisiae . To our knowledge, this is the first time such an activity has been found in a eukaryote and is also the first example of an organism having both direct reversal and base excision repair pathways for the removal of CPDs from DNA. J Mol Biol, 1992 Apr 20, 224(4), 1039 - 54 Mutational analysis of an inherently defective translation initiation site; Ivey-Hoyle M et al.; In a reverse of many studies of translational initiation sites, we have explored the basis for the inactivity of an apparently defective initiation site . Gene VII of the filamentous phage f1 has a translational start site with highly unusual functional properties and a sequence dissimilar to a prokaryotic ribosome binding site . The VII site shows no activity in assays of independent initiation, even in a deletion series designed to remove potentially interfering RNA secondary structure . Activity from the VII site is only observed if the site is coupled to a source of translation immediately upstream, but its efficiency is low at a one-nucleotide spacing from the stop codon of the upstream cistron and extremely sensitive to the distance between the stop codon and the gene VII AUG . These and other atypical characteristics of coupling distinguish the VII site from most coupled initiation sites . To identify the pattern of nucleotide substitutions that give the VII site the capacity for independent initiation, a series of designed and random point mutations were introduced in the sequence . Improving the Shine-Dalgarno complementarity from GG to GGAG or GGAGG made activity detectable, but at only low levels . Random substitutions, each increasing activity above background by a small increment, were found at 16 positions throughout the region of ribosome contact . These substitutions lengthened the Shine-Dalgarno complementarity or changed the G and C residues present in the wild-type site to A or T . Significant activity was not observed unless a strong Shine-Dalgarno sequence and a number of the up-mutations were present together . The nature and distribution of the substitutions and their agreement with the known preferences for nucleotides in initiation sites provide evidence that the VII site's major defect is its primary sequence overall . It appears to lack the specialized sequence required to bind free 30 S ribosomes, and thus depends on the translational coupling process to give it limited activity. FEMS Microbiol Lett, 1992 Apr 15, 71(2), 175 - 80 Identification of putative multifunctional peptide synthetase genes using highly conserved oligonucleotide sequences derived from known synthetases; Borchert S et al.; Many peptide antibiotics in prokaryotes and lower eukaryotes are produced non-ribosomally by multi-enzyme complexes . Analysis of gene-derived amino acid sequences of some peptide synthetases of bacterial and fungal origins revealed a high degree of conservation (35-50% identity) . The genes encoding those peptide synthetases are clustered into large operons with repetitive domains (about 600 amino acids), in the case of synthetases activating more than one amino acid . We used two 35-mer oligonucleotides derived from two highly conserved regions of known peptide synthetases to identify the surfactin synthetase operon in Bacillus subtilis ATCC 21332, a strain not accessible to genetic manipulation . We show that the derived oligonucleotides can be used not only for the identification of unknown peptide synthetase genes by hybridization experiments but also in sequencing reactions as primers to identify internal domain sequences . Using this method, a 25.8-kb chromosomal DNA fragment bearing a part of the surfactin biosynthesis operon was cloned and partial sequences of two internal domains were obtained. Proc Natl Acad Sci U S A, 1992 Apr 15, 89(8), 3536 - 40 RNA polymerase-associated transcription specificity factor encoded by vaccinia virus; Ahn BY et al.; Vaccinia virus encodes a multisubunit DNA-dependent RNA polymerase (EC 2.7.7.6) that is packaged in the infectious virus particle . This polymerase was found to contain a submolar polypeptide of approximately 85 kDa in addition to the core subunits, which consist of two larger and several smaller polypeptides . The polymerase containing the 85-kDa polypeptide was separated from the core polymerase by column chromatography . Although the core polymerase actively transcribed heterologous single-stranded DNA, only the form with the associated 85-kDa polypeptide could act in conjunction with an early stage-specific factor to transcribe double-stranded DNA containing a vaccinia virus early promoter . Peptide sequencing established that the RNA polymerase-associated 85-kDa protein was derived from the vaccinia virus H4L open reading frame, which encodes a 94-kDa polypeptide that we named RAP94 . RAP94 is not closely related to prokaryotic sigma 70 or eukaryotic RAP30 RNA polymerase-binding proteins, although there are short regions of sequence similarity . The specific association of RAP94 with viral RNA polymerase was corroborated with antibody raised to a recombinant fusion protein . Unlike the previously defined subunits of vaccinia virus RNA polymerase, RAP94 is synthesized exclusively late in infection, and synthesis could be prevented by a DNA replication inhibitor . The role of RAP94 in mediating specific transcription was demonstrated by using an extract from cells in which the H4L open reading frame had been transiently expressed. Proc Natl Acad Sci U S A, 1992 Apr 15, 89(8), 3516 - 20 The DnaK chaperone modulates the heat shock response of Escherichia coli by binding to the sigma 32 transcription factor; Liberek K et al.; The heat shock response and the heat shock proteins have been conserved across evolution . In Escherichia coli, the heat shock response is positively regulated by the sigma 32 transcriptional factor and negatively regulated by a subset of the heat shock proteins themselves . In an effort to understand the regulation of the heat shock response, we have purified the sigma 32 polypeptide to homogeneity . During the purification procedure, we found that a large fraction of the overexpressed sigma 32 polypeptide copurified with the universally conserved DnaK heat shock protein (the prokaryotic equivalent of the 70-kDa heat shock protein, HSP70) . Further experiments established that purified sigma 32 bound to DnaK and that this complex was disrupted in the presence of ATP . Consistent with the fact that dnaK756 mutant bacteria overexpress heat shock proteins at all temperatures, purified DnaK756 mutant protein did not appreciably bind to sigma 32. J Biol Chem, 1992 Apr 15, 267(11), 7758 - 60 Specificities of RNA N-glycosidase activity of Mirabilis antiviral protein variants; Habuka N et al.; Mirabilis antiviral protein (MAP), a ribosome-inactivating protein, inactivates both eukaryotic and prokaryotic ribosomes by means of site-specific RNA N-glycosidase activity . In order to identify the site of this activity, some amino acid residues of MAP, conserved in homologous ribosome-inactivating proteins, were altered to other amino acids by replacing DNA fragments of the total synthetic gene of MAP . When the in vitro proteins synthesis of rabbit reticulocyte was treated with MAP variants secreted into culture media of Escherichia coli transformants, the inhibitory effect of R26L and R48L (R26L designates MAP variant with Arg-26 changed to Leu) was found to be similar to that of native MAP . Both purified Y72F and Y118F had the same effect as native MAP, and E168D had a slightly weaker effect . In contrast, on the protein synthesis of E . coli, Y118F had one-tenth the effect of native MAP, and Y72F and E168D approximately one-hundredth the effect . These three variant proteins also exhibited reduced RNA N-glycosidase activity on substrate E . coli ribosomes . These results suggest that Tyr-72 and Glu-168 are involved in RNA N-glycosidase activity . When the R171K gene was expressed in E . coli, an N-glycosidic bond of the 23 S rRNA of the host ribosome was found to be cleaved, although no product of the gene could be detected . This suggests that MAP variants can maintain their N-glycosidase activity when the conserved Glu-168 and Arg-171 are changed to similarly charged residues. Proc Natl Acad Sci U S A, 1992 Apr 15, 89(8), 3394 - 8 Identification of a mammalian 10-kDa heat shock protein, a mitochondrial chaperonin 10 homologue essential for assisted folding of trimeric ornithine transcarbamoylase in vitro; Hartman DJ et al.; We have identified a 10-kDa stress-inducible mitochondrial protein . The protein is synthesized at elevated rates in cultured rat hepatoma cells challenged with heat shock or amino acid analogues and, therefore, designated heat shock protein 10 (Hsp10) . Hsp10 was purified to homogeneity from rat liver and found to exhibit a native molecular mass of 65 kDa, as opposed to a monomeric molecular mass of 10,813.4 +/- 0.41 Da . The amino acid sequence of rat Hsp10 disclosed extensive sequence similarity with bacterial chaperonin (Cpn) 10 . Rat Hsp10 and Escherichia coli Cpn60 were used to reconstitute functional trimeric rat ornithine transcarbamoylase from a chemically denatured state with high efficiency . This process depended completely upon rat Hsp10 and was abolished in the presence of a nonhydrolyzable ATP analogue . We conclude that Hsp10 is a eukaryotic Cpn10 homologue and, therefore, together with Cpn60 essential for mitochondrial protein biogenesis . The Cpn-mediated protein-folding apparatus, thus, exhibits a high degree of conservation between prokaryotes and mitochondria of higher eukaryotes. Nucleic Acids Res, 1992 Apr 11, 20(7), 1785 - 91 Modifications of the E.coli Lac repressor for expression in eukaryotic cells: effects of nuclear signal sequences on protein activity and nuclear accumulation; Fieck A et al.; Eukaryotic expression vectors designed to produce E . coli Lac repressor protein targeted to the nucleus of mammalian cells were constructed . These constructions carry the lac repressor gene (lacI) fused at different positions to a nuclear localization sequence (NLS) from either the SV40 large T antigen or the adenovirus E1a . When the NLS's were fused to the lacI gene at the 5' end, the protein produced exhibited tighter repression of beta-galactosidase expression than the unmodified LacI protein . Localization sequences at the extreme 3' end of the gene generally diminished induction by IPTG, while introduction of the SV40 NLS nine base pairs upstream of the 3' end eliminated repressor activity . When either NLS was placed at the 3' end behind a random nine base pair linker, the activity of the LacI protein depended on the sequence of the linker, and in 9 of 10 linkers tested, activity of the protein was adversely affected . The one exception was the fusion protein from p3'ss, which had the NLS at the 3' end of lacI behind the nine base pair linker, AGC AGC CTG (ser-ser-leu) . This protein exhibited efficient nuclear accumulation, strong repressor activity and greater sensitivity to IPTG induction . The functional linker from the p3'ss fusion protein extends the leucine zipper heptad repeat located at the C-terminus of the protein . These data support the role of the leucine zipper in tetramer formation and predict that extension of this zipper will further stabilize the protein . This modified lacI gene should be valuable for improved adaptation of the prokaryotic regulatory system to eukaryotic cells. Biochemistry, 1992 Apr 7, 31(13), 3507 - 13 Purification and characterization of delta helicase from fetal calf thymus; Li X et al.; A DNA helicase (delta helicase) which partially copurifies with DNA polymerase delta has been highly purified from fetal calf thymus . delta helicase differs in physical and enzymatic properties from other eukaryotic DNA helicases described thus far . The enzyme has an apparent mass of 57 kDa by gel filtration and is associated with polypeptides of 56 and 52 kDa by SDS-polyacrylamide gel electrophoresis . Photo-cross-linking of the purified enzyme with {alpha-32P}ATP resulted in labeling of a polypeptide of approximately 58 kDa, suggesting that the active site is present on the larger polypeptide . Unwinding of a partial duplex requires a nucleoside triphosphate which can be either ATP or dATP but not a nonhydrolyzable analogue of ATP . Other ribo- and deoxyribonucleoside triphosphates have little or no activity as cofactors . delta helicase also has DNA-dependent ATPase activity which has a relatively low Km for ATP (40 microM) . delta helicase binds to single-stranded DNA but has little or no affinity for double-stranded DNA or single-stranded RNA . Similar to replicative DNA helicases from prokaryotes and the herpes simplex virus type 1 helicase-primase, delta helicase translocates in the 5'-3' direction along the strand to which it is bound and preferentially unwinds DNA substrates with a forklike structure. J Biol Chem, 1992 Apr 5, 267(10), 6801 - 6 Site-directed mutagenesis of a conserved domain in vaccinia virus thymidine kinase . Evidence for a potential role in magnesium binding; Black ME et al.; Alignment of prokaryotic and vertebrate type II thymidine kinases (TK) (EC 2.7.1.21), such as that encoded by vaccinia virus (VVTK), reveals three conserved regions: designated domains I, III, and VII . Domains I and III of VVTK contain residues which closely resemble segments A (ATP) and B (Mg2+), respectively, of a Mg.ATP binding descriptor proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J . (1982) EMBO J . 1, 945-951) . In support of this hypothesis, domain I of the VVTK enzyme has previously been identified as the ATP binding site (Black and Hruby, 1990b) . With regard to Mg2+ binding, several features of the VVTK domain III suggest that it may be responsible for this activity: 1) sequence similarity to a magnesium binding motif proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J . (1982) EMBO J . 1, 945-951); 2) alignment of the predicted secondary structure of type II TK enzymes with other magnesium-binding enzymes such as adenylate kinase, EF-TU, and p21 reveals a conserved aspartic acid residue preceded by several hydrophobic residues with domain III; and 3) the conserved VVTK domain III aspartic acid residue (D82) aligns with D93 residue of adenylate kinase which is has been shown by NMR to participate in Mg2+ binding (Yan, H., and Tsai, M.-D., Biochemistry, in press) . To directly examine the potential contribution of the conserved domain III D82 residue of VVTK in magnesium binding, site-directed mutagenesis was performed on positions D82 and G84 to generate four mutants, N82, L82, I82, and V84 . Each mutant was analyzed for enzyme activity, divalent cation requirements, tetramer formation, and ATP binding ability . The results obtained were consistent with D82 playing a direct role in Mg2+ binding and suggest that while the aspartic acid does not appear to participate directly with ATP binding it may instead act to facilitate ATP hydrolysis by binding Mg2+ which aids to correctly position ATP for nucleophilic attack. J Biol Chem, 1992 Apr 5, 267(10), 6455 - 8 Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product; Hartman J et al.; The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product . We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508) . Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP . Irreversible NBD-1 labeling by an ATP analog was demonstrated using {32P}8-azido-ATP . This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain . These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide . Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding. New Biol, 1992 Apr, 4(4), 389 - 95 The product of unr, the highly conserved gene upstream of N-ras, contains multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif; Doniger J et al.; We show that the open reading frame transcribed from the unr gene (immediately upstream of N-ras) in mammals consists of multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif found in prokaryotic cold-shock proteins, and eukaryotic DNA-binding proteins . Alignment of the CSD sequences of unr with those from other proteins reveals a core of similarity for which a consistent secondary structure prediction can be derived . This prediction suggests that the CSD consists primarily of beta-sheet, in contrast to most known eukaryotic DNA-binding proteins . Sequence analysis of the 3' end of the guinea pig unr gene shows that the core of one CSD repeat is encoded in a single exon, consistent with the modular assembly of the gene from ancestral CSD-coding units. New Biol, 1992 Apr, 4(4), 290 - 8 The Y-box factors: a family of nucleic acid binding proteins conserved from Escherichia coli to man; Wolffe AP et al.; The Y-box factors interact specifically with both DNA and RNA . Biologically they have roles in both transcriptional and translational regulation . Conserved through evolution from prokaryotic to eukaryotic organisms they represent a new family of nucleic acid binding proteins. Brain Res Mol Brain Res, 1992 Apr, 13(3), 179 - 87 Cu/Zn superoxide dismutase mRNA and enzyme activity, and susceptibility to lipid peroxidation, increases with aging in murine brains; de Haan JB et al.; To protect against reactive oxygen species, prokaryotic and eukaryotic cells have developed an antioxidant defence mechanism where O2- is converted to H2O2 by superoxide dismutase (Sod), and in a second step, H2O2 is converted to H2O by catalase (Cat) and/or glutathione peroxidase (Gpx) . If Sod levels are increased without a concomitant Gpx increase, then the intermediate H2O2 accumulates . This intermediate could undergo the Fenton's reaction, generating hydroxyl radicals which may lead to lipid peroxidation in cells . In this study, we investigate the expression of Sod1, Gpx1 and susceptibility to lipid peroxidation during the aging process in mouse brains . We demonstrate that the mRNA levels and enzyme activity of Sod1 are higher in brains from adult mice compared to neonatal mice . Furthermore, we show that a linear increase in Sod1 mRNA and enzyme activity occurs with aging (1-100 weeks) . On the contrary, we find that the mRNA and enzyme activity for Gpx1 does not increase with aging in mouse brains . In addition, our results demonstrate that the susceptibility of murine brains to lipid peroxidation increases with aging . The data in this study are consistent with the notion that reactive oxygen species may contribute to the aging process in mammalian brains . These results are discussed in relation to the normal aging process in mammals, and to the premature aging and mental retardation in Down syndrome. Gene, 1992 Apr 1, 113(1), 55 - 65 Codon usage in the G+C-rich Streptomyces genome; Wright F et al.; The codon usage (CU) patterns of 64 genes from the Gram+ prokaryotic genus Streptomyces were analysed . Despite the extremely high overall G+C content of the Streptomyces genome (estimated at 0.74), individual genes varied in G+C content from 0.610 to 0.797, and had third codon position G+C contents (GC3s) that varied from 0.764 to 0.983 . The variation in GC3s explains a significant proportion of the variation in CU patterns . This is consistent with an evolutionary model of the Streptomyces genome where biased mutation pressure has led to a high average G+C content with random variation about the mean, although the variation observed is greater than that expected from a simple binomial model . The only gene in the sample that can be confidently predicted to be highly expressed, EF-Tu of Streptomyces coelicolor A3(2) (GC3s = 0.927), shows a preference for a third position C in several of the four codon families, and for CGY and GGY for Arg and Gly codons, respectively (Y = pyrimidine); similar CU patterns are found in highly expressed genes of the G+C-rich Micrococcus luteus genome . It thus appears that codon usage in Streptomyces is determined predominantly by mutation bias, with weak translational selection operating only in highly expressed genes . We discuss the possible consequences of the extreme codon bias of Streptomyces and consider how it may have evolved . A set of CU tables is provided for use with computer programs that locate protein-coding regions. EMBO J, 1992 Apr, 11(4), 1487 - 92 RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system; Dingermann T et al.; We have investigated whether the RNA polymerase III-driven transcription of eukaryotic tRNA genes can be regulated by the prokaryotic tetracycline operator-repressor system . The bacterial tet operator (tetO) was inserted at two different positions (-7 and -46) upstream of a tRNA(Glu) (amber) suppressor gene . Both constructs are transcribed in Saccharomyces cerevisiae and yield functional tRNAs as scored by suppression of an amber nonsense mutation in the met8-1 allele . Controlled expression of Tet repressor was achieved by fusing the bacterial tetR gene to the yeast gal1 promoter . This leads to expression of Tet repressor in yeast on galactose--but not on glucose--containing media . Regulation of the su-tRNA gene with the tetO fragment inserted at position -7 has been demonstrated . Under conditions which allow tetR expression, cells exhibit a met- phenotype . This methionine auxotrophy can be conditionally reverted to prototrophy by adding tetracycline . However, a su-tRNA gene with the tetO fragment inserted at position -46 cannot be repressed . Our results demonstrate clearly that the bacterial repressor protein binds to its operator in the yeast genome . Formation of this complex in the vicinity of the pol III transcription initiation site reduces the level of su-tRNA at least 50-fold as concluded from quantitative primer extension analyses . This indicates for the first time that class III gene expression can be regulated by a DNA binding protein with its target site in the 5'-flanking region and that a prokaryotic repressor can confer regulation of a suitably engineered tRNA gene. Mol Cell Biol, 1992 Apr, 12(4), 1680 - 6 Fine-structure map of the human ribosomal protein gene RPS14; Diaz JJ et al.; We have used polymerase chain reaction-mediated chemical mutagenesis (J.-J . Diaz, D . D . Rhoads, and D . J . Roufa, BioTechniques 11:204-211, 1991) to analyze the genetic fine structure of a human ribosomal protein gene, RPS14 . Eighty-three DNA clones containing 158 random single-base substitution mutations were isolated . Mutant RPS14 alleles were tested for biological activity by transfection into cultured Chinese hamster cells . The resulting data permitted us to construct a map of the S14-coding sequence that is comparable to available fine-structure genetic maps of many prokaryotic and lower eukaryotic gene loci . As predicted from the multiplicity of protein-protein and protein-RNA interactions required for ribosomal protein transport and assembly into functional ribosomal subunits, the distribution of null mutations indicated that S14 is composed of multiple, functionally distinct polypeptide domains . Two of the protein's internal domains, designated domains B and D, were essential for S14 biological activity . In contrast, mutations which altered or deleted S14's amino-terminal 20 amino acid residues (domain A) had no observable effect on the protein's assembly and function in mammalian ribosomes . Interestingly, S14 structural domains deduced by in vitro mutagenesis correlate well with the RPS14 gene's exon boundaries. J Virol, 1992 Apr, 66(4), 2051 - 6 Activation of the human immunodeficiency virus type 1 long terminal repeat by vaccinia virus; Stellrecht KA et al.; A variety of DNA viruses are known to activate gene expression directed by the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) . In light of the proposed use of recombinant vaccinia virus for HIV-1 vaccines, evaluation of the role of vaccinia virus in HIV-1 activation is warranted . To investigate whether vaccinia virus induces HIV LTR-directed gene expression, transient expression assays in Jurkat cells persistently infected with vaccinia virus (Jvac) using plasmid DNA containing the LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were performed . CAT activity in Jvac cells was always recorded, although the level appears to fluctuate independently of virus titers . Dual intracytoplasmic staining and fluorescence-activated cell sorter analysis showed that CAT activity was expressed in the infected cells . CAT expression was not due to plasmid replication, since plasmid DNA extracted from Jvac cells 48 h after transfection was restricted only by enzymes which recognize methylated sequences, indicating a prokaryotic source for the DNA . These findings suggest that a factor(s) present in vaccinia virus-infected cells is capable of activating the LTR of HIV-1. Proc Natl Acad Sci U S A, 1992 Apr 1, 89(7), 2742 - 6 Sequence of Prochloron didemni atpBE and the inference of chloroplast origins; Lockhart PJ et al.; The prochlorophytes, oxygenic photosynthetic prokaryotes containing chlorophylls a and b, have been put forward as descended from the organisms that gave rise to chloroplasts of green plants and algae by endosymbiosis, although this has always been controversial . To assess the phylogenetic position of the prochlorophyte Prochloron didemni, we have cloned and sequenced its atpBE genes . Phylogenetic inference under a range of models gives moderate to strong support for a cyanobacterial grouping rather than a chloroplast one . Possible systematic errors in this and previous analyses of prochlorophyte sequences are discussed. Curr Genet, 1992 Apr, 21(4-5), 409 - 16 The nucleotide sequence of the small subunit ribosomal RNA gene from Symbiodinium pilosum, a symbiotic dinoflagellate; Sadler LA et al.; The complete sequence of the small subunit ribosomal RNA (SSU rRNA) gene was determined for the symbiotic dinoflagellate Symbiodinium pilosum . This sequence was compared with sequences from two other dinoflagellates (Prorocentrum micans and Crypthecodinium cohnii), five Apicomplexa, five Ciliata, five other eukaryotes and one archaebacterium . The corresponding structurally conserved regions of the molecule were used to determine which portions of the sequences could be unambiguously aligned . Phylogenetic relationships were inferred from an analysis of distance matrices, where pair-wise distances were determined using a maximum likelihood model for transition and transversion ratios, and from maximum parsimony analysis, with bootstrap resampling . By either analytical approach, the dinoflagellates appear distantly related to prokaryotes, and are most closely related to two of the Apicomplexa, Sarcocystis muris and Theileria annulata . Among the dinoflagellates, C . cohnii was found to be more closely affiliated with the Apicomplexa than either P . micans or S . pilosum. Hum Antibodies Hybridomas, 1992 Apr, 3(2), 81 - 5 Bacteriophage