J Mol Biol, 1991 Sep 5, 221(1), 81 - 95 mRNA degradation by processive 3'-5' exoribonucleases in vitro and the implications for prokaryotic mRNA decay in vivo; McLaren RS et al.; Two 3'-5' exoribonucleases, polynucleotide phosphorylase and ribonuclease II play a central role in the degradation of bacterial mRNA to ribonucleotides . Sequences with the potential to form stem-loop structures can stabilize upstream mRNA against 3'-5' exoribonucleolytic attack in vivo by blocking the processive activities of these enzymes . For many mRNA species stem-loop structures appear to provide a very efficient block to decay from the 3' end, such that the rate-determining step for mRNA decay occurs elsewhere in the transcript . We have examined the stalling of 3'-5' exoribonucleases at stem-loop structures in vitro . Although stem-loop structures alone can impede the progress of both enzymes, the duration of stalling at these structures in vitro is insufficient to account for the increased half-lives that they confer on mRNA in vivo . These data suggest that an additional factor, such as a stem-loop binding protein, is required for stabilization of mRNA by stem-loop structures in vivo . The implications for the regulation of mRNA stability are discussed. J Biol Chem, 1991 Sep 5, 266(25), 16324 - 30 Interaction of animal mitochondrial EF-Tu.EF-Ts with aminoacyl-tRNA, guanine nucleotides, and ribosomes; Schwartzbach CJ et al.; The mammalian mitochondrial complex consisting of elongation factors EF-Tu and EF-Ts (EF-Tu.Tsmt) is capable of efficiently binding aminoacyl-tRNA to the ribosome in the presence and absence of guanine nucleotides . In the presence of GTP the binding reaction is catalytic . In the absence of guanine nucleotides, or in the presence of a non-hydrolyzable GTP analog, only one round of ribosome binding occurs . EF-Tu.Tsmt is capable of forming a ternary complex with GTP and Escherichia coli Phe-tRNA as demonstrated by gel filtration chromatography, nitrocellulose filter binding, and by protection of the aminoacyl-tRNA bond from hydrolysis . GDP and the non-hydrolyzable GTP analog guanyl-5'-yl imidodiphosphate are also capable of facilitating ternary complex formation with EF-Tu.Tsmt, but are less effective . No kinetic advantage results from the formation of this ternary complex prior to ribosome binding, and EF-Tu.Tsmt may actually bind aminoacyl-tRNA directly to the ribosome prior to binding GTP . These results suggest that a variation of the prokaryotic elongation cycle is occurring in animal mitochondria . N-Ethylmaleimide inhibits the activity of EF-Tu.Tsmt in polymerization and in ribosome binding . However, the activity of the EF-Tsmt which can be measured independently, is not altered. Biochemistry, 1991 Sep 3, 30(35), 8690 - 7 Quantitating and engineering the ion specificity of an EF-hand-like Ca2+ binding; Falke JJ et al.; The Escherichia coli D-galactose and D-glucose receptor, an aqueous periplasmic receptor that triggers sugar sensing and transport, possesses a single Ca2+ binding site similar in structure and specificity to the EF-hand class of sites found in eukaryotic Ca2+ signaling proteins including calmodulin and its homologues . A universal feature of these sites is the use of a pentagonal bipyramidal array of seven oxygens to coordinate bound Ca2+ . Here we investigate the mechanisms used by this coordinating array to control ion specificity . To vary the cavity size and charge of the array, we have replaced axial glutamine 142 in the prokaryotic site with asparagine, glutamate, and aspartate . The ion selectivities of the resulting engineered sites have been quantitated by measuring dissociation constants for a series of spherical metal ions, differing in increments of radius and charge, from groups Ia, IIa, and IIIa and the lanthanides . Dramatic specificity changes are observed: sites containing an engineered smaller side chain (Asn or Asp) bind the largest cations up to 50-fold more tightly than the native site; and sites containing an engineered negative side chain (Glu or Asp) exhibit preferences for trivalent over divalent cations up to 1900-fold higher than the native site . The results indicate that the cavity size and negative charge of the coordination array play key roles in selective Ca2+ binding and that the array can be engineered to preferentially bind other cations. Mol Cell Endocrinol, 1991 Sep, 80(1-3), 183 - 92 Hydroxyphenyl acetate derivatives inhibit protein tyrosine kinase activity and proliferation in Nb2 rat lymphoma cells and insulin-induced lipogenesis in rat adipocytes; Shechter Y et al.; The ortho, meta, and para forms of hydroxyphenyl acetate were found to be inhibitory in the order of ortho greater than para greater than meta in three distinct biological assays: (a) insulin-dependent assimilation of glucose into lipids in intact adipocytes, (b) growth and proliferation of Nb2 rat lymphoma cells, and (c) tyrosine phosphorylation of copolymer (Glu4Tyr) under cell-free conditions . Although relatively high concentrations of o-hydroxyphenyl acetate (OHPA) were required to inhibit these processes, the inhibitor exhibited a low index of cytotoxicity and high specificity toward inhibiting tyrosine- (but not serine-) specific kinases . Cell cycle analysis of the DNA histograms in Nb2 cells revealed that exposure to OHPA did not change the initiation of the G0/G1----S transition but drastically reduced its rate and a subsequent cell proliferation . Kinetic experiments in which the inhibitor was added or withdrawn through different phases of cell cycle confirmed this conclusion . OHPA inhibition of cell growth appears to be limited to eukaryotic cells as the growth of either gram-positive or gram-negative bacteria was unaffected by the presence of the inhibitor . The study supports the following conclusions: (a) Events that are dependent on tyrosine phosphorylation are indeed essential for mammalian cell growth and proliferation . (b) Neither the initial nor intermediate events of the proliferative cascade that occur in the Nb2 cells prior to DNA synthesis are dependent on the activity of protein tyrosine kinase(s) that are inhibited by OHPA . (c) Cell growth of prokaryotic cells and yeast may lack protein tyrosine kinase activity or be less dependent on events requiring tyrosine phosphorylation . (d) Inhibition of the insulin-dependent lipogenesis is subsequent to the inhibition of insulin receptor tyrosine kinase activity. Microbiol Rev, 1991 Sep, 55(3), 349 - 70 Aromatic amino acid biosynthesis in the yeast Saccharomyces cerevisiae: a model system for the regulation of a eukaryotic biosynthetic pathway; Braus GH; This review focuses on the gene-enzyme relationships and the regulation of different levels of the aromatic amino acid biosynthetic pathway in a simple eukaryotic system, the unicellular yeast Saccharomyces cerevisiae . Most reactions of this branched pathway are common to all organisms which are able to synthesize tryptophan, phenylalanine, and tyrosine . The current knowledge about the two main control mechanisms of the yeast aromatic amino acid biosynthesis is reviewed . (i) At the transcriptional level, most structural genes are regulated by the transcriptional activator GCN4, the regulator of the general amino acid control network, which couples transcriptional derepression to amino acid starvation of numerous structural genes in multiple amino acid biosynthetic pathways . (ii) At the enzyme level, the carbon flow is controlled mainly by modulating the enzyme activities at the first step of the pathway and at the branch points by feedback action of the three aromatic amino acid end products . Implications of these findings for the relationship of S . cerevisiae to prokaryotic as well as to higher eukaryotic organisms and for general regulatory mechanisms occurring in a living cell such as initiation of transcription, enzyme regulation, and the regulation of a metabolic branch point are discussed. New Biol, 1991 Sep, 3(9), 886 - 95 rhlB, a new Escherichia coli K-12 gene with an RNA helicase-like protein sequence motif, one of at least five such possible genes in a prokaryote; Kalman M et al.; A newly recognized gene we name rhlB, after RNA helicase-like genes, has been found in the 85-minute region of the Escherichia coli chromosome . This gene encodes protein sequence motifs similar to those known for "D-E-A-D box" gene products . Proteins in this gene family occur in eukaryotes as well as prokaryotes, and, as far as tested, have been found to participate in ATP-dependent RNA helicase or RNA-dependent ATPase activities . The functions of these enzymes are poorly understood . In yeast, mutant phenotypes of various D-E-A-D genes suggest that they function in RNA splicing, processing, or translation . We find that rhlB is necessary for viability only in some genetic backgrounds . Conditional rhlB lethality is not complemented by another E . coli RNA helicase-like gene (srmB) . Using primers with homology to consensus sequences in D-E-A-D box proteins, we have recovered DNA fragments amplified from E . coli genomic DNA by polymerase chain reactions . Sequence analysis of these fragments suggests that E . coli possesses at least five RNA helicase-like (rhl) D-E-A-D box genes at widely separated chromosomal locations . The multiplicity of such genes in a prokaryote raises the possibility of important roles for the corresponding class of biologically widespread proteins. Mol Gen Genet, 1991 Sep, 228(3), 385 - 92 The Mycobacterium tuberculosis shikimate pathway genes: evolutionary relationship between biosynthetic and catabolic 3-dehydroquinases; Garbe T et al.; The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined . The deduced dehydroquinate synthase amino acid sequence from M . tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi . Surprisingly, the deduced M . tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases) . A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases . This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes . Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M . tuberculosis for use as live vaccines. Arch Biochem Biophys, 1991 Sep, 289(2), 208 - 13 Correlation of secondary structure with biological activity for a leader peptide: circular dichroism-derived structure and in vitro biological activities of preproparathyroid hormone peptide and its analogs; Caulfield MP et al.; Leader or signal sequences are specialized domains within precursor proteins which serve an essential role in interacting with the cellular secretory apparatus to enable intracellular transport and secretion of proteins . Despite many differences in primary amino acid sequences, signal domains interact with a common set of intracellular components, presumably because the signal sequences share an overall conformational similarity . In a few instances, mutant signal peptides from prokaryotes have been studied and their structures correlated with function (export) in vivo . A series of analogs of the precursor-specific region of preproparathyroid hormone have been prepared which contain substitutions of either proline or a charged amino acid within the hydrophobic core . These synthetic "mutants" have previously been evaluated in several in vitro assays to determine their functionality with regard to protein secretion and suitability as substrates for signal peptidase . The secondary structural content of each peptide, as well as the native sequence and sulfur-free analog, was determined in aqueous and nonaqueous conditions by circular dichroism (CD) as a function of time . The structures obtained were correlated with in vitro bioactivities . Unlike the findings or previous CD studies, all the peptides examined here had low to undetectable alpha-helical content in both aqueous and nonaqueous buffers . The unsubstituted and sulfur-free analogs had high (80-85%) beta-structure in aqueous conditions which was reduced to approximately 30% in nonaqueous solvent . The proline- and charged-substituted peptides contained about half the beta-structure content (35-55%) in aqueous buffer; in nonaqueous solvent their structure was similar to the unsubstituted peptides . The structure-activity correlates found were as follows: a high degree of structure (aqueous conditions) correlated with interaction with signal recognition particle and substrate suitability for signal peptidase; a low degree of structure (nonaqueous environment) correlated with activity in the translocation assay. Carcinogenesis, 1991 Sep, 12(9), 1693 - 9 Rotation about the C6-O6 bond in O6-methylguanine: the syn and anti conformers can be of similar energies in duplex DNA as estimated by molecular modeling techniques; Loechler EL; O6-Methylguanine (O6MeGua) is generally regarded as the most important premutagenic lesion formed from carcinogenic methylating agents, so its structure and mechanism of action have received considerable attention . Two conformations for the methyl group in O6MeGua are possible: in one the methyl group is syn with respect to the N(1)-position of the purine, points into the helix, and disrupts hydrogen bonding potential; in the second the methyl group is anti with respect to the N1-position of the purine, and points into the major groove . Syn-O6MeGua has been observed when paired with thymine in duplex DNA as determined by NMR, while anti-O6MeGua has been observed when paired with thymine in X-ray diffraction studies . Herein, molecular modeling/computational chemistry is used to evaluate this apparent discrepancy . {N6-Methyladenine (N6MeAde) was also studied, because it is isoelectronic with O6MeGua, and because more information is available about its energetics . Syn-N6MeAde is computed to be favored in small molecules; however, the fraction of the anti-conformer is computed to be approximately 7%, which agrees well with experimentally determined values (4-12%) . In contrast, the anti conformation for N6MeAde is calculated to be favored in duplex DNA, which is consistent with what has been observed experimentally using both NMR and X-ray diffraction techniques . The agreement between the calculated and experimentally determined results with N6MeAde suggest that the methods are reasonable.} For O6MeGua, a syn/anti ratio of approximately 10(3) is computed for small molecules . In duplex DNA, syn-O6MeGua is computed to be favored, but the anti-conformer is less than approximately 1 kcal/mol higher in energy . Whether syn- or anti-O6MeGua predominates may depend upon sequence context, as well as environmental factors . The comparison between O6MeGua and N6MeAde suggests a rationale for the puzzling observation that O6MeGua appears to be a cytotoxic lesion in eukaryotic, but not prokaryotic, cells. Eur J Biochem, 1991 Sep 1, 200(2), 537 - 43 Characteristics of short-chain alcohol dehydrogenases and related enzymes; Persson B et al.; Different short-chain dehydrogenases are distantly related, constituting a protein family now known from at least 20 separate enzymes characterized, but with extensive differences, especially in the C-terminal third of their sequences . Many of the first known members were prokaryotic, but recent additions include mammalian enzymes from placenta, liver and other tissues, including 15-hydroxyprostaglandin, 17 beta-hydroxysteroid and 11 beta-hydroxysteroid dehydrogenases . In addition, species variants, isozyme-like multiplicities and mutants have been reported for several of the structures . Alignments of the different enzymes reveal large homologous parts, with clustered similarities indicating regions of special functional/structural importance . Several of these derive from relationships within a common type of coenzyme-binding domain, but central-chain patterns of similarity go beyond this domain . Total residue identities between enzyme pairs are typically around 25%, but single forms deviate more or less (14-58%) . Only six of the 250-odd residues are strictly conserved and seven more are conserved in all but single cases . Over one third of the conserved residues are glycine, showing the importance of conformational and spatial restrictions . Secondary structure predictions, residue distributions and hydrophilicity profiles outline a common, N-terminal coenzyme-binding domain similar to that of other dehydrogenases, and a C-terminal domain with unique segments and presumably individual functions in each case . Strictly conserved residues of possible functional interest are limited, essentially only three polar residues . Asp64, Tyr152 and Lys156 (in the numbering of Drosophila alcohol dehydrogenase), but no histidine or cysteine residue like in the completely different, classical medium-chain alcohol dehydrogenase family . Asp64 is in the suggested coenzyme-binding domain, whereas Tyr152 and Lys156 are close to the center of the protein chain, at a putative inter-domain, active-site segment . Consequently, the overall comparisons suggest the possibility of related mechanisms and domain properties for different members of the short-chain family. J Bacteriol, 1991 Sep, 173(17), 5359 - 62 An unusual symbiont from the gut of surgeonfishes may be the largest known prokaryote; Clements KD et al.; Symbionts first reported from the gut of a Red Sea surgeonfish, Acanthurus nigrofuscus (family Acanthuridae), were subsequently described as Epulopiscium fishelsoni . The taxonomic position of this very large (up to 576 microns in length) microorganism has previously been designated in the literature as either uncertain or eukaryotic . We suggest that similar symbionts from Great Barrier Reef surgeonfish may be prokaryotes, which together with E . fishelsoni from the Red Sea may represent the largest known forms of this cell type . Features identifying the symbionts as prokaryotes include the presence of bacterial-type flagella and a bacterial nucleoid and the absence of a nucleus or any other membrane-bound organelle. Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7509 - 13 RNA polymerase II-associated proteins are required for a DNA conformation change in the transcription initiation complex; Buratowski S et al.; Proteins purified on the basis of their affinity for RNA polymerase II effectively substitute for previously defined transcription initiation factors . In two assays, formation of initiation complexes and transcription in vitro, the RNA polymerase II-associated proteins behaved identically to a fraction containing transcription factors IIE and IIF . Both fractions greatly stabilized the association of polymerase with the promoter and were required for the formation of complete initiation complexes . By using the DNA-cleaving reagent phenanthroline.copper in footprinting reactions, the RNA polymerase II-associated proteins were shown to be required for a DNA conformation change near the initiation site of the promoter . Based on similarity to the prokaryotic transcription complex, this conformation change is likely to represent a transition from a closed to an open complex. Virology, 1991 Sep, 184(1), 235 - 41 Mutagenesis of conserved region I in the DNA polymerase from human adenovirus serotype 2; Joung I et al.; The functional importance of the conserved region I (YGDTDSLF) found in several prokaryotic, eukaryotic, and viral DNA polymerases has been probed by site-directed mutagenesis of the adenovirus DNA polymerase (Ad Pol) . Three different adenovirus-specific assays have been used to measure the in vitro activity of region I mutants of Ad Pol expressed in transiently transfected CMT-4 cells . In general, both conservative and nonconservative changes generally showed a greater than 5- to 10-fold reduction in activity in three different assays for activity . However, several replacements at the glycine residue showed activities closer to wild-type levels . For example, replacements of this glycine with cysteine (found in bacteriophage phi 29, another protein primed replication system), with serine, or with methionine had little effect on the activity observed in adenovirus-specific assays, such as initiation and elongation . These studies confirm the importance of this region of Ad Pol in specific initiation and elongation reactions on Ad DNA templates. Chromosoma, 1991 Sep, 100(8), 510 - 8 Molecular cloning and immunolocalization of two variants of the major basic nuclear protein (HCc) from the histone-less eukaryote Crypthecodinium cohnii (Pyrrhophyta); Sala-Rovira M et al.; Two clones that encode variants (HCc1 and HCc2) of the major basic nuclear protein of the dinoflagellate Crypthecodinium cohnii, were identified by immunoscreening of a cDNA expression library . The first clone carries a full-length cDNA with an open reading frame (HCc1) encoding 113 amino acids . The cDNA from the second clone lacks some of the 5' end, and the coding sequence is only 102 residues . The two proteins display 77% sequence similarity and their NH2-ends are homologous to the NH2-peptide of the HCc protein determined by P . Rizzo . The amino acid composition, which confirms the basic nature of lysine-rich HCc proteins, differs markedly from other known DNA-binding proteins such as histones, HMGs or prokaryotic histone-like proteins . No convincing homology was found with other proteins . HCc antigens were localized on C . cohnii by immunofluorescence, and by electron microscopy (EM) with immunogold labelling . HCc proteins are mainly detected at the periphery of the permanently condensed chromosomes, where active chromatin is located, as well as in the nucleolar organizing region (NOR) . This suggests that these basic, non-histone proteins, with a moderate affinity for DNA, are involved at some level in the regulation of gene expression. J Mol Evol, 1991 Sep, 33(3), 259 - 66 Distinct patterns in the dinucleotide nearest neighbors to G/C and A/T oligomers in eukaryotic sequences; Nussinov R; The eukaryotic and prokaryotic databases are scanned for potential nearest-neighbor doublet preferences at the 5' and 3' flanks of some oligomers . Here we focus on oligomers containing alternating nucleotides, i.e., UV, UVUV, and UUVV where U not equal to V . Strong, consistent trends are observed in eukaryotic sequences . A/T alternation oligomers are preferentially flanked by A/T . G/C flanks are disfavored . G/C alternation oligomers are preferentially flanked by G/C . A/T flanks are disfavored . These trends are consistent with those observed previously for homooligomer tracts (Nussinov et al . 1989a,b) . G/C tracts are preferentially flanked by G/C . A/T nearest neighbors are disfavored . The reverse holds for A/T tracts . Additional patterns are described here as well . The possible origin of these DNA composition and sequence trends is discussed . These trends are suggested to stem from protein-DNA interaction constraints. Mol Endocrinol, 1991 Sep, 5(9), 1203 - 14 Proposed role of drug-metabolizing enzymes: regulation of steady state levels of the ligands that effect growth, homeostasis, differentiation, and neuroendocrine functions; Nebert DW; Every ligand known to bind to a receptor in the nuclear hormone receptor superfamily is involved in a variety of signal transduction pathways effecting growth, morphogenesis, homeostasis, proliferation, and neuroendocrine functions . Often these ligands are associated with increases in particular subsets of cytochromes P450 and other drug-metabolizing enzymes . Interestingly, certain of these enzymes participate in the metabolism (synthesis as well as degradation) of these ligands . It appears that genes coding for certain drug-metabolizing enzymes might have existed on this planet at least 1 billion years before the presence of plants, animals, and drugs . An early role for oxidative enzymes in prokaryotes most likely involved energy substrate utilization: insertion of oxygen into various inaccessible carbon and other food sources, thereby rendering them accessible to further metabolism . It is proposed that a later development of these "drug-metabolizing enzymes" in prokaryotes and early eukaryotes might be related to their metabolic ability to control the steady state levels of the ligands that modulate cell division, growth, morphogenesis, and mating, and that this role has diversified in numerous additional signal transduction pathways and exists today in all eukaryotes. Eur J Biochem, 1991 Sep 1, 200(2), 271 - 84 Quinoproteins: enzymes containing the quinonoid cofactor pyrroloquinoline quinone, topaquinone or tryptophan-tryptophan quinone; Duine JA; The presently best known and largest group of quinoproteins consists of enzymes using the cofactor 2,7,9-tricarboxy-1H-pyrrolo{2,3-f}quinoline- 4,5-dione (PQQ), a compound having a pyrrole ring fused to a quinoline ring with an o-quinone group in it . Representatives of this group are found among the bacterial, NAD(P)-independent, periplasmic dehydrogenases . Despite their high midpoint redox potential, the overall behaviour of quinoprotein dehydrogenases is similar to that of their counterparts, those using a flavin cofactor or a nicotinamide coenzyme . Apart from an exceptional Gram-positive one, the sole organisms where the presence of PQQ has really been established are Gram-negative bacteria . Evidence for the occurrence of covalently bound PQQ is lacking since it has now been shown that several enzymes previously considered to contain this prosthetic group do not in fact do so . Another group of quinoproteins, consisting of amine oxidoreductases, has a protein chain containing one of the following quinonoid aromatic amino acids: 6-hydroxy-phenylalanine-3,4-dione (TPQ) or 4-(2'-tryptophyl)-tryptophan-6,7-dione (TTQ) . There is no doubt that these o-quinones play a role as cofactor, in the case of TPQ in prokaryotic as well as eukaryotic amine oxidases . It appears, therefore, that a novel class of amino-acid-derived cofactors is emerging, ranging from the free radical form of tyrosine and tryptophan to those containing a dicarbonyl group (like the already known pyryvoyl group and the o-quinones here described. Biochem Biophys Res Commun, 1991 Aug 30, 179(1), 319 - 26 Prokaryotic expression of the thyrotropin receptor and identification of an immunogenic region of the protein using synthetic peptides; Takai O et al.; Graves' disease is characterized by hypersecretion of thyroid hormones due to binding of autoantibodies to the thyrotropin receptor (TSHR) . In order to study immunological aspects of the TSHR we expressed the extracellular domain of the rat TSHR (ETSHR) as a fusion protein with beta-galactosidase in a prokaryotic system . The identity of this ETSHR-fusion protein was confirmed by Western blot, using antibodies to synthetic peptides derived from TSHR . Patients' sera reacted to a significantly greater extent with the affinity purified ETSHR relative to control sera . Similarly, sera from patients with Graves' disease displayed significant reactivity with only one of five peptides, RH2 (residues 352-366), when compared with normal sera . These data, together with the predicted hydrophilicity of the peptide RH2, suggest that amino acids 352-366 which lie within one of the unique regions of the extracellular domain of the TSHR may be important for antibody binding. Gene, 1991 Aug 30, 105(1), 9 - 15 pEXPRESS: a family of expression vectors containing a single transcription unit active in prokaryotes, eukaryotes and in vitro; Forman BM et al.; We have constructed a family of expression vectors containing a single transcription unit that is active in Escherichia coli, eukaryotic cells, and in coupled in vitro transcription-translation systems . These vectors use the Rous sarcoma virus-long terminal repeat (RSV-LTR) as the promoter/enhancer for eukaryotic cells . In vitro transcription is made possible by inclusion of a bacteriophage T7 promoter . This same promoter is actively transcribed in E . coli that produce T7 RNA polymerase . Other features of this transcription unit include a high-efficiency eukaryotic translation start codon, a phage f1 origin of DNA replication for site-directed mutagenesis and a three-frame stop codon that facilitates C-terminal deletion mutagenesis . We term this vector family, pEXPRESS. Nature, 1991 Aug 22, 352(6337), 689 - 95 Cloning of a human gene encoding the general transcription initiation factor IIB; Ha I et al.; Transcription factor IIB (TFIIB) has a central role in transcription of class II genes . The purification of the human TFIIB protein and isolation of a complementary DNA encoding TFIIB activity is reported here . The sequence of TFIIB, which seems to be encoded by a single gene, contains a repeated motif, in addition to a motif with similarity to the prokaryotic sigma-factors . The recombinant protein expressed in bacteria substituted for all the functions attributed to the human TFIIB protein. Biochem Biophys Res Commun, 1991 Aug 15, 178(3), 1153 - 9 The primary structure of rat ribosomal protein L23; Chan YL et al.; The amino acid sequence of the rat 60S ribosomal subunit protein L23 was deduced from the sequence of nucleotides in two recombinant cDNAs . Ribosomal protein L23 has 140 amino acids and a molecular weight of 14,856 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7-9 copies of the L23 gene . The mRNA for the protein is about 600 nucleotides in length . Rat L23 is homologous to Saccharomyces cerevisiae L17a and related to Escherichia coli L14 and other members of the prokaryotic L14 family. Nucleic Acids Res, 1991 Aug 11, 19(15), 4127 - 32 A single-strand specific endonuclease activity copurifies with overexpressed T5 D15 exonuclease; Sayers JR et al.; The T5 D15 exonuclease purified from an overproducing strain of E . coli was shown to possess a low level of endonucleolytic activity specific for single-stranded DNA when assayed with 1-10 mM Mg2+ as co-factor . Endonuclease activity on double-stranded circular DNA could not be detected under these conditions . Nicked circular DNA was first gapped by the enzyme's exonucleolytic activity, creating a single-stranded region . This gapped substrate was then endonucleolytically cleaved and rapidly degraded . We show that a gapped and not a nicked substrate is required for this activity as previously suggested (Moyer, R . W . and Roth, C . T . 1977, J . Virol . 24, 177-193) . The single-strand endonuclease activity could be selectively suppressed by using low concentrations of Mg2+ as co-factor (less than 1 mM), thus allowing nicked double-stranded circular DNA to be gapped to a single-stranded circular species . We also report on sequence similarities between the T5 exonuclease and several prokaryotic DNA polymerases. Biochemistry, 1991 Aug 6, 30(31), 7809 - 17 A mammalian tryptophanyl-tRNA synthetase shows little homology to prokaryotic synthetases but near identity with mammalian peptide chain release factor; Garret M et al.; Determination of the amino acid sequence of beef pancreas tryptophanyl-tRNA synthetase was undertaken through both cDNA and direct peptide sequencing . A full-length cDNA clone containing a 475 amino acid open reading frame was obtained . The molecular mass of the corresponding peptide chain, 53,728 Da, was in agreement with that of beef tryptophanyl-tRNA synthetase, as determined by physicochemical methods (54 kDa) . Expression of this clone in Escherichia coli led to tryptophanyl-tRNA synthetase activity in cell extracts . The open reading frame included two sequences analogous to the consensus sequences, HIGH and KMSKS, found in class I aminoacyl-tRNA synthetases . The homology with prokaryotic and yeast mitochondrial tryptophanyl-tRNA synthetases was low and was limited to the regions of the consensus sequences . However, a 90% homology was observed with the recently described rabbit peptide chain release factor (eRF) {Lee et al . (1990) Proc . Natl . Acad . Sci . 87, 3508-3512} . Such a strong homology may reveal a new group of genes deriving from a common ancestor, the products of which could be involved in tRNA aminoacylation (tryptophanyl-tRNA synthetase) or translation termination (eRF). Mol Cell Biol, 1991 Aug, 11(8), 3972 - 7 V(D)J recombination: evidence that a replicative mechanism is not required; Hsieh CL et al.; We examined a series of extrachromosomal DNA substrates for V(D)J recombination under replicating and nonreplicating conditions . Complete and partial replications were examined by monitoring the loss of prokaryote-specific adenine methylation at 14 to 22 MboI-DpnI restriction sites (GATC) on the substrates . Some of these sites are within 2 bases of the signal sequence ends . We found that neither coding joint nor signal joint formation requires substrate replication . After ruling out replication as a substrate requirement, we determined whether replication had any effect on the efficiency of V(D)J recombination . Quantitation of V(D)J recombination efficiency on nonreplicating substrates requires some method of monitoring the entry of substrate molecules into the cells . We devised such a method by monitoring DNA repair of substrates into which we had substituted deoxyuridine for 10 to 20% of the thymidine nucleotides in the DNA . The substrates which enter the lymphoid cells were repaired efficiently in vivo by the eukaryotic uracil DNA repair system . Upon plasmid harvest, we distinguished repaired (entered) from unrepaired (not entered) plasmids by cleaving unrepaired molecules with uracil DNA glycoylase and Escherichia coli endonuclease IV in vitro . This method of monitoring DNA entry does not appear to underestimate or overestimate the amount of DNA entry . By using this method, we found no significant quantitative effect of DNA replication on V(D)J recombination efficiency. Radiat Res, 1991 Aug, 127(2), 220 - 5 X-ray induction of O6-alkylguanine-DNA alkyltransferase protects against some of the biological effects of N-methyl-N'-nitro-N-nitrosoguanidine in C3H 10T1/2 cells; von Hofe E et al.; We have shown previously that the repair of O6-methylguanine can be induced in murine fibroblasts (C3H 10T1/2 cells) by exposure to X rays . The magnitude of the response is less, however, than is observed in the well-characterized adaptive response of various prokaryotes to methylating agents . To determine whether the induction of O6-alkylguanine-DNA alkyltransferase in C3H 10T1/2 cells is sufficient for protection against the genotoxic effects of the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cells were challenged with MNNG after alkyltransferase induction by 1.5 Gy X rays and assayed for cytotoxicity, mutagenicity, and neoplastic transformation . Preirradiated cells were significantly more resistant to the mutagenic effects of MNNG as scored by formation of ouabain-resistant colonies . The protective effect was greatest in cells challenged with a low dose (0.2 or 0.4 micrograms/ml) of MNNG . Protection against neoplastic transformation by MNNG was also observed, although the protective effect in this case was significant only in cells treated with a high dose (1.0 micrograms/ml) of MNNG . In cells that were preirradiated, there was no reduction in the cytotoxicity caused by MNNG or the chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) . These data indicate that alkyltransferase induction in C3H 10T1/2 cells is sufficient to protect cells against some of the genotoxic effects of the alkylating agent MNNG . The data also suggest that formation of O6-alkylguanine may not be the only means by which alkylating agents can transform C3H 10T1/2 cells. New Biol, 1991 Aug, 3(8), 759 - 68 Trans-dominant Tat mutants with alterations in the basic domain inhibit HIV-1 gene expression; Modesti N et al.; The Tat protein of the human immunodeficiency virus type 1 (HIV-1) is required for efficient viral gene expression . By means of mutational analyses, several domains of the Tat protein that are required for complete activation of HIV-1 gene expression have been defined . These include an amino-terminal activating domain, a cysteine-rich dimerization domain, and a basic domain important in the binding of Tat to the trans-activation response element (TAR) and in Tat nuclear localization . Recently, we described a mutation, known as delta tat, which resulted in a protein with a truncated basic domain . This protein had a "trans-dominant" phenotype in that it inhibited wild-type Tat activation of the HIV-1 LTR . To further characterize the requirements for generating a Tat trans-dominant phenotype, we constructed a variety of Tat proteins with truncations or substitutions in the basic domain . A number of these proteins showed a trans-dominant phenotype . These Tat mutants also inhibited activation of the HIV-1 LTR by a protein composed of Tat fused to the prokaryotic R17 (phage MS2) RNA-binding protein in which the R17 recognition element was inserted in the HIV-1 LTR in place of TAR . Thus, an intact TAR element was not required for this inhibition . We also studied the cellular localization of Tat and a trans-dominant Tat mutant by means of immunofluorescence staining with the use of antibodies reactive to different domains of the Tat protein . The results indicated that Tat becomes localized predominantly in the nucleus both in the presence and absence of the trans-dominant Tat construct, suggesting that the trans-dominant mutant does not inhibit Tat nuclear localization . These studies further define the requirements for the creation of trans-dominant Tat mutants, and suggest that the mechanism of trans-dominant Tat inhibition may be either the formation of an inactive complex between wild-type and mutant Tat or sequestration of cellular factors involved in regulating HIV-1 gene expression. Klin Wochenschr, 1991 Aug 1, 69(11), 463 - 73 Chlamydiae as pathogens--an overview of diagnostic techniques, clinical features, and therapy of human infections; Oehme A et al.; Chlamydiae are Gram-negative bacteria with obligate intracellular reproduction and disability to synthesize high-energy compounds such as ATP . Their cycle of development is unique among the prokaryotes: the host cells, mainly epithelial cells, are infected by so-called elementary bodies (EB) which undergo reorganization to form metabolically active reticulate bodies (RB) . These RB multiply by binary fission, and after transition into infectious EB they are released within 48-72 hours . Chlamydiae cause prolonged subclinical infections of the conjunctiva, lung, cervix, and urethra . Complications in newborns are inclusion conjunctivitis, nasopharyngitis and pneumonia; in females, salpingitis, infertility, and perihepatitis; in male patients, epididymitis and prostatitis; and in both sexes, Chlamydiae-induced arthritis . Identification of the pathogenic agent confirms clinical diagnosis; tissue culture identification remains the diagnostic method of choice . Therapeutical drugs are tetracycline, erythromycin, josamycin, and in certain cases quinolone derivatives. Neuron, 1991 Aug, 7(2), 177 - 81 Mechanisms for diversity in gene expression patterns; Struhl K; Despite the relatively low number of transcriptional regulatory proteins, the number of possible combinations that act in particular cell types at specific times and in response to appropriate extracellular stimuli is enormous . In considering the regulatory patterns of a particular gene, the critical determinants of diversity are the specific promoter sequences that govern the potential DNA-binding proteins which function either directly or indirectly in association with other proteins; constellations of proteins in the nucleus and their transcriptional activities; and synergistic or antagonistic protein-protein interactions . Although some of these regulatory principles operate in prokaryotes, the combinatorial nature of the transcriptional activation process, the existence of multiprotein families, and the prevalence of heteromeric protein complexes are characteristic of eukaryotic cells and are essential for the extraordinary complexity of gene expression patterns in multicellular organisms. FEBS Lett, 1991 Jul 29, 286(1-2), 176 - 80 Cysteinyl-tRNA synthetase is a direct descendant of the first aminoacyl-tRNA synthetase; Avalos J et al.; The gene encoding the cysteinyl-tRNA synthetase of E . coli was cloned from an E . coli genomic library made in lambda 2761, a lambda vector which can integrate and which carries a chloramphenicol resistance gene . A thermosensitive cysS mutant of E . coli was lysogenised and chloramphenicol-resistant colonies able to grow at 42 degrees C were selected to isolate phages containing the wild-type cysS gene . The sequence of the gene was determined . It codes for a 461 amino-acid protein and includes the sequences HIGH and KMSK known to be involved in the ATP and tRNA binding respectively of class I synthetases . The cysteinyl enzyme has segments in common with the cytoplasmic leucyl-tRNA synthetase of Neurospora crassa, the tryptophanyl-tRNA synthetase of Bacillus stearothermophilus, and the phenylalanyl-tRNA synthetase of Saccharomyces cerevisiae . Sequence comparisons show that the amino end of the cysteinyl-tRNA synthetase has similarities with prokaryotic elongation factors Tu; this region is close to the equivalent acceptor binding domain of the glutaminyl-tRNA synthetase of E . coli . There is a further similarity with the seryl enzyme (a class II enzyme) which has led us to propose that both classes had a common origin and that this was the ancestor of the cysteinyl-tRNA synthetase. J Biol Chem, 1991 Jul 25, 266(21), 13712 - 8 Identification, sequence, and expression of the gene encoding a Mr 35,000 subunit of the vaccinia virus DNA-dependent RNA polymerase; Amegadzie BY et al.; The gene rpo35, encoding a subunit of the vaccinia virus DNA-dependent RNA polymerase, was identified, and its RNA and protein products were characterized . An Mr 35,000 polypeptide, which bound antibody to the purified RNA polymerase, was synthesized in reticulocyte lysates programmed with viral mRNA that hybridized to a 2,300-base pair segment of the viral genome . Determination of the sequence of the DNA segment revealed four potential protein coding regions, none of which had evident similarity to any described RNA polymerase subunit of prokaryotes or eukaryotes . One open reading frame that could encode a 35,400-Da protein was identified as rpo35 on the basis of mRNA hybridization, cell-free translation, and immunoprecipitation . The identification was confirmed by sequencing tryptic peptides of the authentic Mr 35,000 RNA polymerase subunit . Antiserum to the purified recombinant protein, expressed in bacteria, reacted specifically with a Mr 35,000 polypeptide that was detected starting 2 h after virus infection and that co-sedimented with RNA polymerase purified from virions . RNA analyses indicated that the 5'-end of an early transcript started 25 nucleotides upstream of rpo35, which is consistent with the location of an early promoter consensus sequence. Biochemistry, 1991 Jul 23, 30(29), 7277 - 82 Interaction between virginiamycin S and ribosomes is partly provided by a salt bridge with a Mg2+ ion; Di Giambattista M et al.; Type B streptogramins, such as virginiamycin S (VS), are cyclic hexadepsipeptides, inhibiting protein synthesis in prokaryotes . L-Thr connects a 3-hydroxypicolinyl residue (3-OH-Pic) to the peptide lactone ring . The fluorescence intensity of 3-OH-Pic is strongly increased by chelation to alkaline earth cations or binding to ribosomes . Similar behavior of the ribosome-VS complex and the VS-Mg chelate provides strong evidence for the presence of a VS-Mg chelate within the ribosomal binding site . Different models involving the ribosome binding of either members of the VS-Mg2+ chelate or both have been tested by fluorescence lifetime measurements, equilibrium titrations, and stopped-flow spectrofluorometry . Our data strongly suggest that (a) the interaction between VS and the ribosome is partly provided by a salt bridge between suitable acceptor atoms of the ribosome and the 3-OH-Pic residue, (b) Mg2+ can be exchanged by Mn2+ without dissociation of the ribosome-VS complex, (c) Mg2+ coordinates to the negative form of the 3-OH-Pic residue, probably via an interaction with the phenolate oxygen and the amide carboxyl group, and (d) the picolinyl residue is essential for the biological activity, as indicated by the lack of activity when the latter is replaced by a serine derivative. Gene, 1991 Jul 22, 103(2), 171 - 7 Modulation of firefly luciferase stability and impact on studies of gene regulation; Thompson JF et al.; Two of the reporter enzymes most commonly used in studies of eukaryotic gene expression are chloramphenicol acetyl-transferase (CAT) and firefly luciferase (Luc) . CAT has a half-life of about 50 h in mammalian cells, making it useful for transient transfection assays but less suitable for assays with stable cell lines . Luc has a half-life of only 3 h in mammalian cells, making it much more responsive in stable cell lines . Luc instability arises from its sensitivity to proteolysis both in vivo and in vitro . Compounds that resemble its natural substrate, luciferin, act as effective competitive inhibitors in vitro . When these compounds (e.g., phenylbenzothiazole) are added to either prokaryotic or eukaryotic cells, more than tenfold increases in Luc activity can be observed . This increased activity results from a lower rate of degradation of the enzyme in vivo and can be mimicked in vitro as phenylbenzothiazole protects Luc from trypsin digestion while it has no effect on the rate of digestion of alkaline phosphatase. FEBS Lett, 1991 Jul 22, 285(2), 182 - 8 Protein export in prokaryotes and eukaryotes . Theme with variations; Wiech H et al.; Protein export in prokaryotes as well as in eukaryotes can be defined as protein transport across the plasma membrane . In both types of organisms there are various apparently ATP-dependent transport mechanisms which can be distinguished from one another and which show similarities when the prokaryotic mechanism is compared with the respective eukaryotic mechanism . First, one can distinguish between transport mechanisms which involve so-called signal or leader peptides and those which do not . The latter mechanisms seem to employ ATP-dependent transport systems which belong to the family of oligopeptide permeases and multiple drug resistance proteins . Second, in signal or leader peptide-dependent transport one can distinguish between transport mechanisms which involve ribonucleoparticles and those which employ molecular chaperones . Both mechanisms appear to converge at the level of ATP-dependent translocases. Proc R Soc Lond B Biol Sci, 1991 Jul 22, 245(1312), 43 - 51 Electron transport in cytochromes P-450 by covalent switching; Baldwin JE et al.; The mechanism of electron transfer in cytochrome P-450cam is presented in terms of a covalent switching mechanism . We present a model of putidaredoxin built by homology, which helps explain protein-protein interactions . The mechanism is general enough to account for the genetic variations found in the superfamily of cytochromes P-450 . The detail should assist in the design of novel P-450 inhibitors and may have wider implications . The sequence analysis supports our protein model, and highlights the role of cystein and aromatic residues in electron-transport mechanisms . Eukaryotic cytochromes P-450 appear to have evolved their own intramolecular tryptophan electron-transfer mediator, unlike prokaryotic P . putida P-450cam, which still relies upon the C-terminal tryptophan of its attendant electron-transport protein, putidaredoxin . On this basis our protein model is capable of rationalizing the transfer of electrons from NADH to the active site of P-450 . At the electronic level the covalent switching that transfers pairs of electrons not only provides a plausible mechanism, but may also have ramifications in a wider context. Biochem Biophys Res Commun, 1991 Jul 15, 178(1), 322 - 8 The primary structure of rat ribosomal protein L17; Suzuki K et al.; The amino acid sequence of the rat 60S ribosomal subunit protein L17 was deduced from the sequence of nucleotides in two recombinant cDNAs . Ribosomal protein L17 has 184 amino acids and has a molecular weight of 21,383 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 17-19 copies of the L17 gene . The mRNA for the protein is about 720 nucleotides in length . Rat L17 is homologous to human L17 and related to Saccharomyces cerevisiae YL17, Halobacterium marismortui L23, Halobacterium halobium L22e, Escherichia coli L22 and other members of the prokaryotic L22 family. Gene, 1991 Jul 15, 103(1), 45 - 52 Characterization of an Escherichia coli gene encoding betaine aldehyde dehydrogenase (BADH): structural similarity to mammalian ALDHs and a plant BADH; Boyd LA et al.; An open reading frame of 1476 nucleotides, cloned from a region of the Escherichia coli genome encoding betaine biosynthesis functions, was shown to encode a betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) . Either of two adjacent codons (5'-GTGATG) could function as a start codon, producing a presumptive polypeptide of 491 or 490 amino acids . The deduced primary structure of the E . coli BADH showed 39-43% positional identity, over its entire length, to aldehyde dehydrogenases (ALDH: EC 1.2.1.3) of mammalian origin . This similarity increased to 75-77% when conservative aa substitutions were also taken into consideration . Spinach BADH was also similar to the bacterial BADH, showing 38% identity and 80% overall similarity . Other homologs included a fungal and a putative bacterial ALDH . Although E . coli BADH was specific for the substrate, betaine aldehyde, it showed the highest levels of similarity to the prototype human ALDH-2 . Only one gap in each sequence had to be introduced for optimal alignment . The conservation between E . coli BADH and the ALDHs was also evident in the predicted secondary structures and hydrophilicity profiles of the polypeptides, suggesting a similarity in the overall folding patterns of ALDH and BADH . These observations suggest a common ancestry for BADH and ALDH, preceding prokaryote-eukaryote divergence. FEMS Microbiol Lett, 1991 Jul 15, 66(1), 15 - 8 A new cyclitol derivative influences inositol metabolism in Neurospora crassa; Szabo G et al.; Cyclitol derivatives have been synthesized and screened for growth inhibitory effect upon prokaryotic and eukaryotic organisms . One derivative, (2S,3R,5R)-3-azido-2-benzoyloxy-5-hydroxycyclohexanone, was studied in detail: it has no effect upon bacteria, but it is inhibitory to Neurospora crassa . In Neurospora crassa it increased the amount of myo-inositol-1-phosphate synthase and inhibited the activity of myo-inositol-monophosphatase . The enhanced synthesis of myo-inositol-1-phosphate synthase was the consequence of lowering the intracellular inositol concentration . Li+ treatment of Neurospora crassa has effects similar to those of P.I.-658. Nucleic Acids Res, 1991 Jul 11, 19(13), 3489 - 98 Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases; Cusack S et al.; Class 2 aminoacyl-tRNA synthetases, which include the enzymes for alanine, aspartic acid, asparagine, glycine, histidine, lysine, phenylalanine, proline, serine and threonine, are characterised by three distinct sequence motifs 1,2 and 3 (reference 1) . The structural and evolutionary relatedness of these ten enzymes are examined using alignments of primary sequences from prokaryotic and eukaryotic sources and the known three dimensional structure of seryl-tRNA synthetase from E . coli . It is shown that motif 1 forms part of the dimer interface of seryl-tRNA synthetase and motifs 2 and 3 part of the putative active site . It is further shown that the seven alpha 2 dimeric synthetases can be subdivided into class 2a (proline, threonine, histidine and serine) and class 2b (aspartic acid, asparagine and lysine), each subclass sharing several important characteristic sequence motifs in addition to those characteristic of class 2 enzymes in general . The alpha 2 beta 2 tetrameric enzymes (for glycine and phenylalanine) show certain special features in common as well as some of the class 2b motifs . In the alanyl-tRNA synthetase only motif 3 and possibly motif 2 can be identified . The sequence alignments suggest that the catalytic domain of other class 2 synthetases should resemble the antiparallel domain found in seryl-tRNA synthetase . Predictions are made about the sequence location of certain important helices and beta-strands in this domain as well as suggestions concerning which residues are important in ATP and amino acid binding . Strong homologies are found in the N-terminal extensions of class 2b synthetases and in the C-terminal extensions of class 2a synthetases suggesting that these putative tRNA binding domains have been added at a later stage in evolution to the catalytic domain. FEBS Lett, 1991 Jul 8, 285(1), 1 - 5 Pyrophosphate-dependent phosphofructokinase, an anaerobic glycolytic enzyme? Mertens E. Recent evidence indicates that in as diverse organisms as unicellular eukaryotes, higher plants and prokaryotes, anaerobic glycolysis relies on a pyrophosphate-dependent phosphofructokinase instead of the classical ATP-dependent enzyme . This difference in phosphoryl donor specificity does not necessarily reflect a primitive metabolism, as thought earlier, but could rather be the result of convergent evolution, fostered by the energetic advantage conferred to the cell when glycolysis is the sole source of ATP. J Theor Biol, 1991 Jul 7, 151(1), 89 - 110 A new method for the analysis of the dynamics of the molecular genetic control systems . II . Application of the method of generalized threshold models in the investigation of concrete genetic systems; Prokudina EI et al.; Mathematical models of the prokaryotic control systems of tryptophan biosynthesis (both normal and with cloned blocks) and arabinose catabolism have been built using the method of generalized threshold models . Kinetic curves for molecular components (mRNAs, proteins, metabolites) of the systems considered are obtained . It has been shown that the method of generalized threshold models gives a more detailed qualitative picture of the dynamics of the molecular genetic control systems in comparison with the heuristic method of threshold models . The qualitative analysis of the functioning of the following mechanisms of control of the tryptophan biosynthesis: (1) inhibition of the activity of anthranilate synthetase by tryptophan, (2) repression and (3) attenuation of transcription of the tryptophan operon on the basis of the mathematical model of the control system of the tryptophan biosynthesis demonstrates that feedback inhibition is the most operative of the considered mechanisms while repression allows the bacterium to economize intracellular resources . As regards the control system of the arabinose catabolism the results of modelling enable us to state the following . The induction by arabinose within a wide range of parameter values causes two subsystems (araBAD and transport operons) of the arabinose regulon with a low rate of arabinose utilization to pass into a stationary regime and one subsystem (araC operon) to pass into a stable periodical regime . A study of the system characterized by the effective utilization of arabinose has shown that under induction by arabinose stable oscillations with small amplitudes of the concentration of regulatory protein and oscillations with large amplitudes of the concentrations of arabinose-isomerase and transport protein may occur . The period of the oscillation depends on the mean lifetime of the "activator-DNA" complex and on the rate constant of arabinoseisomerase degradation. Biochemistry, 1991 Jul 2, 30(26), 6465 - 75 Conformational transition required for efficient splicing of transcripts from hybrid lambda promoter yeast tRNA gene fusion; Shapero MH et al.; Fusion of a prokaryotic promoter to a yeast tRNA gene provides a means for uncoupling analyses of mutations affecting splicing from requirements for transcription and other processing steps . For this purpose, a phage lambda promoter was fused to the Saccharomyces cerevisiae tRNATyr(SUP3a) coding sequence . This fusion allows the synthesis of an end-mature precursor by in vitro transcription with Escherichia coli RNA polymerase . This precursor was accurately spliced by purified yeast endonuclease and ligase fractions . However, both the initial rate and the extent of the endonuclease cleavage reaction were reduced in comparison to those for substrates produced by yeast RNA polymerase III . Efficient splicing could be restored in a magnesium- and temperature-dependent renaturation step, suggesting a conformational transition was required . Enzymatic solution structure probing of transcripts from wild-type and intron-variant templates revealed that the essential conformational transition involved a segment of the tRNA-like portion of the precursor . These results (1) suggest that the primary sequence of the precursor alone may not be sufficient to ensure formation of the active conformer during synthesis, (2) provide direct evidence that endonuclease recognizes mature tRNA-like structure in the precursor, and (3) suggest a general caution for the use of semisynthetic transcripts in RNA processing reactions . Potentially, transcription and processing of tRNATyr in yeast may provide a useful paradigm for examining active control of conformation in RNA biosynthesis. J Bacteriol, 1991 Jul, 173(14), 4379 - 85 Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis; Revill WP et al.; The erythromycin A-producing polyketide synthase from the gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has evident structural similarity to fatty acid synthases, particularly to the multifunctional fatty acid synthases found in eukaryotic cells . Fatty acid synthesis in S . erythraea has previously been proposed to involve a discrete acyl carrier protein (ACP), as in most prokaryotic fatty acid synthases . We have cloned and sequenced the structural gene for this ACP and find that it does encode a discrete small protein . The gene lies immediately adjacent to an open reading frame whose gene product shows sequence homology to known beta-ketoacyl-ACP synthases . A convenient expression system for the S . erythraea ACP was obtained by placing the gene in the expression vector pT7-7 in Escherichia coli . In this system the ACP was efficiently expressed at levels 10 to 20% of total cell protein . The recombinant ACP was active in promoting the synthesis of branched-chain acyl-ACP species by extracts of S . erythraea . Electrospray mass spectrometry is shown to be an excellent method for monitoring the efficiency of in vivo posttranslational modification of ACPs. Mol Microbiol, 1991 Jul, 5(7), 1593 - 7 Regulation of methionine synthesis in Escherichia coli; Weissbach H et al.; The biosynthesis of methionine in Escherichia coli is under complex regulation . The repression of the biosynthetic pathway by methionine is mediated by a repressor protein (MetJ protein) and S-adenosyl-methionine which functions as a corepressor for the MetJ protein . Recently, a new regulatory locus, metR, has been identified . The MetR protein is required for both metE and metH gene expression, and functions as a transactivator of transcription of these genes . MetR is a unique prokaryotic transcription activator in that it possesses a leucine zipper motif, first described for eukaryotic DNA-binding proteins . The transcriptional activity of MetR is modulated by homocysteine, the metabolic precursor of methionine . Finally, it is known that vitamin B12 can repress expression of the metE gene . This effect is mediated by the MetH holoenzyme, which contains a cobamide prosthetic group. Comput Appl Biosci, 1991 Jul, 7(3), 287 - 93 Compositional variations in DNA sequences; Nussinov R; Biologically occurring nucleotide sequences differ from randomly generated ones . Here we describe general patterns found in prokaryotic and in eukaryotic DNA . In the accompanying paper (Nussinov, 1991) we also describe DNA signals recognized by their corresponding protein factors . In particular, we focus on modes of searches for such patterns and signals and on the potential properties such sequences may possess. J Mol Evol, 1991 Jul, 33(1), 4 - 12 Elements in microbial evolution; Arber W; Spontaneous mutation, selection, and isolation are key elements in biological evolution . Molecular genetic approaches reveal a multitude of different mechanisms by which spontaneous mutants arise . Many of these mechanisms depend on enzymes, which often do not act fully at random on the DNA, although a large number of sites of action can be observed . Of particular interest in this respect are DNA rearrangement processes, e.g., by transposition and by site-specific recombination systems . The development of gene functions has thus to be seen as the result of both DNA rearrangement processes and sequence alterations brought about by nucleotide substitutions and small local deletions, insertions, and duplications . Prokaryotic microorganisms are particularly appropriate for studying the effects of spontaneous mutation and thus microbial evolution, as they have haploid genomes, so that genetic alterations become rapidly apparent phenotypically . In addition, bacteria and their viruses and plasmids have relatively small genomes and short generation times, which also facilitate research on evolutionary processes . Besides the strategy of development of gene functions in the vertical transmission of genomes from generation to generation, the acquisition of short DNA segments from other organisms appears to be an important strategy in microbial evolution . In this process of horizontal evolution natural vector DNA molecules are often involved . Because of acquisition barriers, the acquisition strategy works best for relatively small DNA segments, hence at the level of domains, single genes, or at most operons . Among the many enzymes and functional systems involved in vertical and horizontal microbial evolution, some may serve primarily for essential life functions in each individual and only secondarily contribute to evolution.(ABSTRACT TRUNCATED AT 250 WORDS) J Biochem (Tokyo), 1991 Jul, 110(1), 159 - 62 Effects of prokaryotic termination signals on RNA polymerase II transcription in HeLa cells; Nakagoshi H et al.; Three prokaryotic termination signals, the Escherichia coli trp attenuator, lambda 4S RNA terminator, and lambda tR1 terminator, were examined as to their effects on transcription in vivo in HeLa cells . The trp attenuator inhibited the expression of downstream genes in an orientation-dependent manner, but both the lambda 4S RNA and lambda tR1 terminators did not . Furthermore, a point mutation of the attenuator that disrupted the secondary structure of mRNA abolished this inhibitory effect . These results suggest that an attenuator-like secondary structure is effective in inhibiting transcriptional elongation by RNA polymerase II. Bioessays, 1991 Jul, 13(7), 317 - 22 Mapping replication origins in yeast chromosomes; Brewer BJ et al.; The replicon hypothesis, first proposed in 1963 by Jacob and Brenner, states that DNA replication is controlled at sites called origins . Replication origins have been well studied in prokaryotes . However, the study of eukaryotic chromosomal origins has lagged behind, because until recently there has been no method for reliably determining the identity and location of origins from eukaryotic chromosomes . Here, we review a technique we developed with the yeast Saccharomyces cerevisiae that allows both the mapping of replication origins and an assessment of their activity . Two-dimensional agarose gel electrophoresis and Southern hybridization with total genomic DNA are used to determine whether a particular restriction fragment acquires the branched structure diagnostic of replication initiation . The technique has been used to localize origins in yeast chromosomes and assess their initiation efficiency . In some cases, origin activation is dependent upon the surrounding context . The technique is also being applied to a variety of eukaryotic organisms. Biochimie, 1991 Jul-Aug, 73(7-8), 1007 - 19 Eukaryotic initiation factors eIF-2 and eIF-3: interactions, structure and localization in ribosomal initiation complexes; Bommer UA et al.; More than ten different protein factors are involved in initiation of protein synthesis in eukaryotes . For binding of initiator tRNA and mRNA to the 40S ribosomal subunit, the initiation factors eIF-2 and eIF-3 are particularly important . They consist of several different subunits and form stable complexes with the 40S ribosomal subunit . The location of eIF-2 and eIF-3 in these complexes as well as the interactions of the individual components have been analyzed by biochemical methods and electron microscopy . The results obtained are summarized in this article, and a model is derived describing the spatial arrangement of eIF-2 and eIF-3 together with initiator tRNA and mRNA on the 40S subunit . Conclusions on the location of functionally important sites of eukaryotic small ribosomal subunits are discussed with regard to the respective location of these sites in the prokaryotic counterpart. Biotechniques, 1991 Jul, 11(1), 94 - 101 Simultaneous and rapid isolation of bacterial and eukaryotic DNA and RNA: a new approach for isolating DNA; Majumdar D et al.; A very simple procedure for the simultaneous preparation of genomic DNA and total RNA is described . The procedure is essentially the same for eukaryotes and prokaryotes except for the lysis buffer and can be used for small or large numbers of cells . Mammalian cells are lysed in sodium dodecyl sulfate and bacterial cells are lysed in Triton X-100, both in the presence of EDTA . RNA is obtained in the aqueous phase after phenol (acidic pH):chloroform:isoamyl alcohol extraction . DNA is eluted out of the organic phase (and the interface) into the aqueous phase by increasing the pH with highly basic 1 M Tris solution . The method is extremely rapid for small or large numbers of cells, and several large samples can be processed in one day . The qualities of both nucleic acids are excellent and the yield is high. Res Microbiol, 1991 Jul-Aug, 142(6), 689 - 700 Transposition in prokaryotes: transposon Tn501; Brown NL et al.; Bacteria contain a large number of transposable elements that can be categorized in four major groups according to their mechanisms of transposition . These are: class I: insertion sequences (IS) and compound transposons (with IS sequences at their termini) which usually require only one protein for transposition to occur (e.g . Tn10); class II: complex transposons and insertion sequences with short inverted repeats in which transposition is replicative and requires two gene products (e.g . Tn3); class III: transposable bacteriophage (e.g . Mu) . The fourth group consists of the transposons and IS of variable mechanism, which do not fall into the above classes (e.g . Tn7) . We have studied the mechanism of transposition of Tn501 and Tn21, closely-related class II mercury-resistance transposons, which transpose via a cointegrate intermediate . By using genetic methods, we have shown that the region of the 989 amino acid transposase between amino acids 57 and 186 determines the specificity for recognition of the 38-bp terminal inverted repeats of the transposon in normal transposition and for replicon fusion catalysed by a single transposon terminus . The Tn501 transposase has been over-expressed and is functional in vivo, raising the frequency of transposition approximately 10(4)-fold. Biochem Biophys Res Commun, 1991 Jun 28, 177(3), 1076 - 81 Is there methylmalonyl CoA mutase in Aspergillus nidulans? Ledley FD, Crane AM, Klish KT, May GS. In most animal species and many prokaryotes, methylmalonyl CoA mutase catalyzes isomerization between methylmalonyl CoA and succinyl CoA using adenosylcobalamin as a cofactor . We describe the absence of this enzyme in Aspergillus nidulans based on the absence of enzyme activity in vitro and the failure to metabolize methylmalonate or grow in media containing this organic acid as the sole carbon source . These data contrast previous assumptions that propionate may be metabolized through propionyl CoA and methylmalonyl CoA to the TCA cycle in this organism . This is consistent with the separate evolution of these pathways in animals and lower eukaryotes due to the distinct endosymbiotic origin of their mitochondria. Gene, 1991 Jun 15, 102(1), 27 - 32 Homology between proteins controlling Streptomyces fradiae tylosin resistance and ATP-binding transport; Rosteck PR Jr et al.; A tylosin(Ty)-producing strain of Streptomyces fradiae contains at least three genes, tlrA, tlrB, tlrC, specifying resistance to Ty (TyR) . The complete nucleotide sequence of the TyR-encoding gene, tlrC, and the transcription start point of the gene were determined . The sequence contains an open reading frame coding for a protein of 548 amino acids (aa) with an Mr of 59129 . The TlrC protein was identified by expression of the cloned gene by in vitro coupled transcription and translation in cell-free extracts derived from Streptomyces lividans . The N- and C-terminal halves of TlrC share extensive homology, suggesting that the protein evolved through tandem gene duplication . Each half of the deduced TlrC aa sequence also shows significant homology to numerous eukaryotic and prokaryotic membrane-associated, active-transport protein subunits . The homologous proteins include examples from the systems responsible for efflux of cytotoxic drugs from multidrug-resistant human cells and for export of hemolysin from Escherichia coli . The greatest similarity to TlrC is in regions containing the ATP-binding sites found in these proteins . These results suggest a role for the tlrC gene product as part of a multiple component, ATP-dependent transport system for the active excretion of Ty from the producing organism. Carbohydr Res, 1991 Jun 25, 213, 19 - 25 The effects of N-glycosylation on the lectin activity of recombinant ricin B chain; Richardson PT et al.; Soluble, biologically-active recombinant ricin B chain has been produced by expressing B chain-encoding DNA in heterologous eukaryotic or prokaryotic hosts . N-Glycosylated recombinant ricin B chain expressed in Xenopus oocytes bound to both immobilized asialofetuin and immobilized lactose . Non-glycosylated ricin B chain expressed in either E . coli or in tunicamycin-treated oocytes did not bind to immobilized lactose . However, it did bind to asialofetuin, and increasing concentrations of free lactose did not reduce this asialofetuin binding dramatically, in contrast to the effect of free lactose on the binding of either glycosylated recombinant B chain or native ricin B chain. Proc R Soc Lond B Biol Sci, 1991 Jun 22, 244(1311), 207 - 10 Frameshifting by eukaryotic ribosomes during expression of Escherichia coli release factor 2; Donly BC et al.; Normal translation of the gene for E . coli release factor 2 (RF-2) is characterized by a +1 frameshift event that occurs with 30-50% efficiency . Frameshifting on synthetic RF-2 mRNA by eukaryotic ribosomes has also been observed, even though they lack the capability to interact with the frameshift signal in the same manner as prokaryotic ribosomes . We have mutagenized the sequence of the RF-2 gene to eliminate the need for a frameshift, thereby allowing frameshifting efficiency to be measured by direct comparison of RF-2 production from the mutant with production from the wild-type . Measurements using this approach confirm that frameshifting by rabbit reticulocyte lysate ribosomes occurs at the frameshift region, but with a limited efficiency of approximately 0.4%. Biochim Biophys Acta, 1991 Jun 19, 1084(1), 35 - 40 A two component-type cytochrome P-450 monooxygenase system in a prokaryote that catalyzes hydroxylation of ML-236B to pravastatin, a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase; Serizawa N et al.; Pravastatin (CS-514) is a tissue selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), a key enzyme in cholesterol biosynthesis . This compound is obtained by hydroxylation of ML-236B (mevastatin) in Streptomyces carbophilus catalyzed by a cytochrome P-450sca monooxygenase system . NADH-cytochrome P-450 reductase was purified to homogeneity from S . carbophilus as a single polypeptide chain with a molecular weight of 51 kDa, and reconstituted the hydroxylation in vitro with cytochrome P-450sca, NADH and O2 . This protein contained FAD and FMN molecule . The FMN molecule was easily dissociated from the reductase, and had a Kd value of 5 x 10(-5) M . The cytochrome P-450sca monooxygenase system was present in the soluble fraction and consisted of only two components, cytochrome P-450sca and flavoprotein . Our results constitute the demonstration of a two component-type cytochrome P-450 system in a prokaryote. Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5433 - 7 Insertional mutagenesis and illegitimate recombination in mycobacteria; Kalpana GV et al.; Mycobacteria, particularly Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium, are major pathogens of man . Although insertional mutagenesis has been an invaluable genetic tool for analyzing the mechanisms of microbial pathogenesis, it has not yet been possible to apply it to the mycobacteria . To overcome intrinsic difficulties in directly manipulating the genetics of slow-growing mycobacteria, including M . tuberculosis and bacille Calmette-Guerin (BCG) vaccine strains, we developed a system for random shuttle mutagenesis . A genomic library of Mycobacterium smegmatis was subjected to transposon mutagenesis with Tn5 seq1, a derivative of Tn5, in Escherichia coli and these transposon-containing recombinant plasmids were reintroduced into mycobacterial chromosomes by homologous recombination . This system has allowed us to isolate several random auxotrophic mutants of M . smegmatis . To extend this strategy to M . tuberculosis and BCG, targeted mutagenesis was performed using a cloned BCG methionine gene that was subjected to Tn5 seq1 mutagenesis in E . coli and reintroduced into the mycobacteria . Surprisingly for prokaryotes, both BCG and M . tuberculosis were found to incorporate linear DNA fragments into illegitimate sites throughout the mycobacterial genomes at a frequency of 10(-5) to 10(-4) relative to the number of transformants obtained with autonomously replicating vectors . Thus the efficient illegitimate recombination of linear DNA fragments provides the basis for an insertional mutagenesis system for M . tuberculosis and BCG. Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5197 - 201 A cluster of transcribed sequences between the Pb and Ob genes of the murine major histocompatibility complex; Cho S et al.; The region of the murine major histocompatibility complex (MHC) between the Pb (A beta 3) and Ob (A beta 2) genes controls the expression of an intracellular complex named the LMP (low molecular weight polypeptide) complex . DNA probes for at least seven different genes mapping to this region were isolated . These hybridize to a minimum of eight different transcripts ranging from approximately 1.3 to 3.7 kilobases (kb) . The deduced amino acid sequences of the corresponding cDNAs indicate that three of these genes are new members of the MHC class II gene family . These genes are transcribed in a tissue-specific pattern similar to that of the traditional class II genes . Two of the remaining four genes, HAM1 and HAM2, are homologous to one another and to a family of eukaryotic and prokaryotic transport proteins and may be involved in antigen processing . The tissue distribution of HAM1 transcripts is consistent with its proposed role in class I-restricted antigen processing, whereas HAM2 transcription appears more restricted and may be involved in antigen processing for class II-restricted T cells . The HAM2 gene may produce two differentially spliced transcripts . The identity of the remaining two genes is not known . Analyses of transcript sizes, tissue distribution, sequence, and genetic mapping data suggest that none of these genes code for LMP antigens. J Biol Chem, 1991 Jun 5, 266(16), 10392 - 9 Structural and functional properties of thesaurin a (42Sp50), the major protein of the 42 S particles present in Xenopus laevis previtellogenic oocytes; Viel A et al.; Thesaurin a is one of two protein components of a 42 S ribonucleoprotein particle that is very abundant in previtellogenic oocytes of Xenopus laevis . The primary function of the 42 S particle is the long-term storage of 5 S RNA and aminoacyl-tRNA . Thesaurin a is homologous to eukaryotic elongation factor 1 alpha (EF-1 alpha) and to prokaryotic elongation factor Tu (EF-Tu) . Sequence comparison with EF-1 alpha and EF-Tu of different species indicates that thesaurin a is rather distantly related to all eukaryotic elongation factors . In spite of this, the secondary structure of thesaurin a, deduced from hydrophobic cluster analysis, is remarkably similar to that of EF-1 alpha and EF-Tu . The binding and catalytic properties of thesaurin a are also similar but not identical to those of EF-1 alpha . Like EF-1 alpha, purified thesaurin a binds tRNA, GDP, and GTP . Unlike EF-1 alpha, thesaurin a binds discharged tRNA more tightly than charged tRNA, and GTP more tightly than GDP . Thesaurin a also hydrolyzes GTP and catalyzes the mRNA-dependent binding of aminoacyl-tRNA to 80 S ribosomes . The functional properties of the 42 S particle are in general agreement with those of purified thesaurin a . In particular, the 42 S particle contains GTP and efficiently transfers aminoacyl-tRNA to 80 S ribosomes without addition of exogenous elongation factor. J Steroid Biochem Mol Biol, 1991 Jun, 38(6), 787 - 94 20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: 3 alpha/beta-hydroxysteroid dehydrogenase activities catalyzed by highly purified enzyme; Ohno S et al.; Pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) has also 3 alpha- and 3 beta-HSD (3 alpha/beta-HSD) activities . The purified 20 beta-HSD preparation from neonatal pig testes could catalyze the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) in the presence of beta-NADPH to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol at the ratio of 4:3, and the specific 3 alpha/beta-HSD activity of 20 beta-HSD for 5 alpha-DHT was about 10 or 15 times larger than the 20 beta-HSD activities for 17 alpha-hydroxypregn-4-ene-3,20-dione (17 alpha-hydroxyprogesterone) or progesterone, respectively . The result indicates that the testicular 20 beta-HSD has high 3 alpha(axial, 3R)- and 3 beta(equatorial, 3S)-HSD activity . The testicular 20 beta-HSD could catalyze the reversible conversion of various 5 alpha- or 5 beta-dihydrosteroids which have a 3-carbonyl or 3-hydroxyl group with beta-NADP(H) as the preferred cofactor . The enzyme transferred the 4-proS hydrogen of NADPH to the 5 alpha-DHT for both 3 alpha- and 3 beta-hydroxylation and it was the same as the 20 beta-hydroxylation of 17 alpha-hydroxyprogesterone . Although the 3 alpha/beta-HSD activity has been known to be present in 3 alpha,20 beta-HSD of Streptomyces hydrogenans, the enzymological properties for 3 alpha/beta-HSD activity catalyzed by testicular 20 beta-HSD were different from the properties for 3 alpha/beta-HSD activity catalyzed by prokaryotic 3 alpha, 20 beta-HSD with respect to the specificity of the catalytic reaction and the cofactor requirement. Mutat Res, 1991 Jun, 260(2), 145 - 52 Genotoxicity of the boldine aporphine alkaloid in prokaryotic and eukaryotic organisms; Moreno PR et al.; The aporphine alkaloid boldine, present in Peumus boldus (boldo-do-Chile) widely used all over the world, was tested for the presence of genotoxic, mutagenic and recombinogenic activities in microorganisms . This alkaloid did not show genotoxic activity with or without metabolic activation in the SOS chromotest and Ames tester strains TA100, TA98 and TA102 . It was not able to induce point and frameshift mutations in haploid Saccharomyces cerevisiae cells . However, mitotic recombinational events such as crossing-over and gene conversion were weakly induced in diploid yeast cells by this alkaloid . Also, boldine was able to induce weakly cytoplasmic 'petite' mutation in haploid yeast cells. EMBO J, 1991 Jun, 10(6), 1481 - 91 Exceptional codon recognition by the glutamine tRNAs in Saccharomyces cerevisiae; Edelman I et al.; Recently, it was shown that wild-type glutamine tRNAs in yeast cause low-level nonsense suppression that can be enhanced by increasing glutamine tRNA gene copy number . In order to investigate glutamine tRNA behavior further, anticodon mutations that confer nonsense suppression were identified in yeast sup70 gene, which codes for glutamine tRNA(CAG) . In this study we show that suppressors derived by mutation severely limit growth such that suppressor-bearing spores germinate but arrest cell division at approximately the 50 cell stage . Analysis of a sup70 deletion was used to establish that growth limitation results from loss of wild-type glutamine tRNA(CAG) function . By exploiting the growth inhibition of sup70 alleles, some exceptional codon recognition properties of glutamine tRNAs were revealed . Our results indicate that amber suppressor glutamine tRNA(UAG) can translate 5'-CAG-3' glutamine codons with low efficiency in the presence of an A/C mismatch at the first position of the codon, suggesting that reading may occur at a low level by a two-out-of-three reading mechanism . In addition, when glutamine tRNA(CAA) is over-expressed in vivo, it translates 5'-CAG-3' codons using a mechanism that resembles prokaryotic-like U/G wobble, which normally does not occur in yeast . Our studies also suggest that the yeast glutamine tRNA suppressors could potentially be exploited to express ciliated protozoan genes that normally contain internal 5'-UAG-3' and 5'-UAA-3' codons. Immunol Rev, 1991 Jun, 121, 67 - 90 Heat-shock protein 60: implications for pathogenesis of and protection against bacterial infections; Kaufmann SH et al.; In this review we have focused on antigenic features of hsp 60 related to: its ubiquitous distribution in the biosphere; its extraordinary homology among various bacteria; its high conservation from prokaryotic to eukaryotic cells; and its abundant expression under stress situations occurring during infection . These unique features make hsp 60 an excellent candidate antigen relevant to protection and pathogenesis of bacterial infections and, perhaps in a broader sense, to surveillance and autoimmunity . We will briefly discuss these possibilities in the following . Acquired resistance . If we assume that bacterial organisms contain some thousand different proteins which all represent potential antigens, the frequency of T cells with specificity for mycobacterial hsp 60 appears surprisingly high . Although, during the course of infection, high levels of hsp may be induced in bacteria, mere abundance appears to be an important though insufficient explanation . In addition, constant boosting by similar hsp 60 cognates from various microbes with which humans come into contact may contribute to dominance . This could easily explain the occurrence of hsp 60-specific T cells in healthy individuals with no clinical history of mycobacterial infections . Involvement of more sophisticated mechanisms, such as the affinity of hsp to other proteins, cannot be excluded (Flynn et al . 1989) . Yet dominance does not necessarily mean protection and definite proof that hsp are protective antigens is lacking . Perhaps the immune response against epitopes shared by various mycobacterial pathogens represents a first line of defence preceding a more specific immune response . Such broadly reactive antigens would not qualify as prime candidates for vaccine design . Immunesurveillance . T cells with specificity for epitopes shared by bacterial and human hsp 60 are readily demonstrable and stressed host cells are recognized by hsp 60-specific T cells . Such T lymphocytes are endowed with the capacity to identify host cells stressed by a variety of assaults such as inflammation, infection, trauma, or transformation . Although it has been claimed that hsp-reactive gamma/delta T cells are particularly destined for such surveillance functions (Born et al . 1990, Asarnow et al . 1988), alpha/beta T cells could also participate . Pathogenesis . The mechanisms causing pathogenesis should be similar to those underlying protection and surveillance . In the former case bacterial hsp would be responsible for both induction of immunity and expression of pathogenic reactions; in the latter case an immune response stimulated by conserved regions of bacterial hsp 60 would be converted against a host-derived cognate.(ABSTRACT TRUNCATED AT 400 WORDS) Arch Biochem Biophys, 1991 Jun, 287(2), 234 - 9 Role of aspartate-37 in determining cofactor specificity and binding in rat liver dihydropteridine reductase; Matthews DA et al.; Full-length rat dihydropteridine reductase (DHPR) cDNAs have been combined with a prokaryotic expression vector and introduced into Escherichia coli . Transformed bacteria express dihydropteridine reductase immunoreactive proteins and demonstrate conversion of quinonoid dihydropteridines to their tetrahydro forms . Several recombinant enzymes have been purified to homogeneity and biochemical studies have been carried out comparing their properties with those exhibited by the rat liver enzyme . The optimal reaction conditions, kinetic constants, and stability are similar for the recombinant and naturally occurring enzyme . The results indicate that the nonmutant recombinant rat DHPR is an authentic replica of the natural protein and that the characteristics of DHPR activity are determined by a single gene product and do not require specific modification via the eukaryotic cell . In addition to the wild type, three specific mutagenic forms of the reductase, A-6-V, W-104-F, and D-37-I, and an additional abbreviated structure have also been formed . Each of the products exhibits reductase activity, although they show varied affinities for their cofactor, NADH, and less stability to chromatography, dialysis, and concentration than the wild-type enzyme . The N-terminal sequence contains a classic NADH binding region between amino acids 9 and 36, and Asp 37 is essential for binding the cofactor as is shown by the approximately 20-fold increase in dissociation constant for the D-37-I mutant and diminished kcat (approximately 43 s-1 compared to 156 s-1 for the wild-type enzyme) . The results indicate that the DHPR cofactor binding site is similar to typical dinucleotide requiring dehydrogenases such as lactic acid and liver alcohol dehydrogenase. Curr Genet, 1991 Jun, 19(6), 503 - 7 Evidence for multiple xenogenous origins of plastids: comparison of psbA-genes with a xanthophyte sequence; Scherer S et al.; When only plastidic features are considered, it is difficult to distinguish between monophyletic and polyphyletic xenogenous origins of plastids . We suggest that a direct comparison of nuclear and plastidic sequence-similarity pattern will help to solve this problem . The D1 amino acid sequence of six major groups of photosynthetic eukaryotes and of the two groups of photosynthetic prokaryotes are now available, including the psbA-gene product from Bumilleriopsis filiformis, which is the first molecular sequence reported for a xanthophycean alga . Evidence is provided for an independent and polyphyletic origin of plastids from five out of the six major taxa of photosynthetic eukaryotes . This conclusion is reached by comparing a plastid-based pattern of D1 similarity with a nucleus-based similarity pattern published recently . Furthermore, the availability of D1 sequences from five eukaryotic algae led to a re-evaluation of the taxonomic position of Prochlorothrix. J Virol, 1991 Jun, 65(6), 2884 - 94 Identification, characterization, and sequence analysis of a cDNA encoding a phosphoprotein of human herpesvirus 6; Chang CK et al.; Human herpesvirus 6 (HHV-6)-specific monoclonal antibody (Mab) 9A5D12 reacted with the nucleus of HHV-6 strain GS-infected cells and immunoprecipitated a phosphorylated polypeptide with an approximate size of 41 kDa, designated HHV-6 P41 . A 110-kDa polypeptide was also immunoprecipitated by the MAb . These polypeptides were synthesized early in infection, and the synthesis was greatly reduced by phosphonoacetic acid . Polypeptides with identical sizes were recognized by the MAb from cells infected with an additional eight HHV-6 strains . A 2.1-kb cDNA insert was identified from an HHV-6(GS) cDNA library constructed in the lambda gt11 expression system by using MAb 9A5D12 . This cDNA insert hybridized specifically with viral DNA from HHV-6 strains GS and Z-29 and with two predominant transcripts with approximate sizes of 2.5 and 1.2 kb from infected cells . The reactivity of the MAb with a fusion protein expressed in the prokaryotic vector suggested that the cDNA encodes a 62- to 66-kDa protein . Analysis of the nucleotide sequence of the cDNA insert revealed a 623-amino-acid-residue single open reading frame of 1,871 nucleotides, with an open 5' end . The predicted polypeptide is highly basic and contains a long stretch of highly hydrophobic residues localized to the carboxy terminus . The amino-terminal half of the predicted HHV-6 protein from the cDNA shows significant homology with the UL44 gene product of human cytomegalovirus, coding for the ICP36 family of early-late-class phosphoproteins . Two TATA boxes are located at nucleotide positions 668 and 722 of the cDNA . In vitro translation of RNA transcribed in vitro from the cDNA resulted in the synthesis of a 41-kDa polypeptide only . This polypeptide was readily immunoprecipitated by MAb 9A5D12, and its partial peptide map was identical to that of the 41-kDa polypeptide detected in infected cells . Together, these results indicate that the HHV-6 P41 is encoded within a gene coding for a larger protein. Virology, 1991 Jun, 182(2), 644 - 54 Antibodies to human papillomavirus type-16 in human sera as revealed by the use of prokaryotically expressed viral gene products; Kochel HG et al.; Open reading frames of human papillomaviruses were expressed in Escherichia coli as beta-galactosidase fusion proteins . These bacterially derived papillomaviral gene products were used to examine sera from 67 women (63 healthy subjects, 4 patients with genital carcinoma) for antibodies to papillomavirus type-16 antigens (E1, E2, E4, E5, E6, E7, L1, L2) and the L2 proteins of HPV-6b and HPV-18 by Western-blot analysis . The serologic data were compared with cytological findings classified according to Papanicolaou and with nucleic acid hybridization data from cervical smears of the same individuals . Twenty-three of the normal individuals showed antibodies exclusively directed against L2 gene products; whereas in the sera from the four genital cancer patients, antibodies to the early gene products E4 and/or E7 could be detected . In one case these antibodies were found to be combined with antibodies to L2 of HPV-16 and -18 and in another case with those to E1 and E2 of HPV-16 . In none of the sera examined could antibodies to L1, E5 or E6 be identified . Three of the antibody positive normal women were found to be also positive for HPV-16/18 DNA, while all of the 40 seronegative women were HPV-16/18 DNA negative . These data indicate that serology may be a valuable means to study the epidemiology of genital human papillomavirus infection. Hua Xi Yi Ke Da Xue Xue Bao, 1991 Jun, 22(2), 140 - 3 {Studies on the mutagenicity of vesnarinone}; Hu H et al.; The mutagenicity of domestic Vesnarinone (OPC-8212) was studied by Ames test, micronucleus test of NIH mouse bone marrow and chromosome aberration assay in CHL cells . Negative results were obtained, which suggested that OPC-8212 did not induce prokaryotic cell gene mutation or chromosome damage both in vitro and in vivo. Biochimie, 1991 Jun, 73(6), 739 - 55 The assembly of prokaryotic ribosomes; Nierhaus KH; The targets of in vivo studies of the ribosomal assembly process are mainly the events of rRNA processing, whereas in vitro studies (total reconstitution) focus on principles of the assembly process such as assembly-initiation proteins, rate-limiting steps and a detailed sequence of assembly reactions (assembly map) . The success of in vitro analyses is particularly remarkable in view of ionic and temperature requirements of the total reconstitution which differ significantly from the in vivo conditions . Features of the in vivo assembly are surveyed, however, the focal point is a description of experimental strategies and results concerning the in vitro assembly of ribosomes. DNA Cell Biol, 1991 Jun, 10(5), 319 - 28 cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues; Barr PJ et al.; A cDNA encoding the human fur gene product was isolated from a human hepatoma cell line . The cDNA encodes a protein with significant amino acid sequence identity to the prokaryotic subtilisin family of serine proteases . More extensive sequence identity was found when the protein was compared with eukaryotic proteases such as PRB1 of Saccharomyces cerevisiae, and with PC2 and PC3, the only other known mammalian subtilisin-like proteases . In contrast to these proteins, however, the fur gene product shares a more extensive topographic and functional homology with the KEX2 endoprotease of S . cerevisiae . Each protease contains a signal peptide, a glycosylated extra cytoplasmic domain, a hydrophobic membrane-spanning region, and a short, hydrophilic "tail" sequence . As with KEX2, the expressed human protease was shown to cleave mammalian proproteins at their paired basic amino acid processing sites . We have, therefore, proposed the function-based acronym PACE (paired basic amino acid cleaving enzyme) for this prototypic mammalian proprotein processing enzyme. J Bacteriol, 1991 Jun, 173(12), 3807 - 13 Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase; Holmes ML et al.; We report here on advances made in the construction of plasmid shuttle vectors suitable for genetic manipulations in both Escherichia coli and halobacteria . Starting with a 20.4-kb construct, pMDS1, new vectors were engineered which were considerably smaller yet retained several alternative cloning sites . A restriction barrier observed when plasmid DNA was transferred into Haloferax volcanii cells was found to operate via adenine methylation, resulting in a 10(3) drop in transformation efficiency and the loss of most constructs by incorporation of the resistance marker into the chromosome . Passing shuttle vectors through E . coli dam mutants effectively avoided this barrier . Deletion analysis revealed that the gene(s) for autonomous replication of pHK2 (the plasmid endogenous to Haloferax strain Aa2.2 and used in the construction of pMDS1) was located within a 4.2-kb SmaI-KpnI fragment . Convenient restriction sites were identified near the termini of the novobiocin resistance determinant (gyrB), allowing the removal of flanking sequences (including gyrA) . These deletions did not appear to significantly affect transformation efficiencies or the novobiocin resistance phenotype of halobacterial transformants . Northern blot hybridization with strand- and gene-specific probes identified a single gyrB-gyrA transcript of 4.7 kb . This is the first demonstration in prokaryotes that the two subunits of DNA gyrase may be cotranscribed. Proc Natl Acad Sci U S A, 1991 Jun 1, 88(11), 5001 - 5 From one gene to two proteins: the biogenesis of cytochromes b and c1 in Bradyrhizobium japonicum; Thony-Meyer L et al.; Genes coding for polyproteins that are cleaved posttranslationally into two or more functional proteins are rarely found in prokaryotes . One example concerns the biogenesis of the Bradyrhizobium japonicum cytochromes b and c1, two of the three constituent subunits of ubiquinol-cytochrome-c reductase (ubiquinol:ferricytochrome-c oxidoreductase, EC 1.10.2.2); the respective apoproteins for these subunits are encoded by the 5' and 3' halves of a single gene, fbcH . These two halves are linked by an extra piece of DNA encoding a characteristic signal peptide for protein translocation across the cytoplasmic membrane . Processing of the fbcH gene product is shown to occur at a typical signal peptidase recognition site . This reaction is reminiscent of that catalyzed by the regular bacterial signal peptidase that normally cleaves off presequences from the N termini of translocated proteins . Mutational alteration of the signal peptidase recognition site within FbcH results in the appearance of an uncleaved bc1 fusion protein in the membrane . Additionally, a functional heme-binding site in the apocytochrome c1 section of FbcH is shown to be a necessary prerequisite for the formation of the bc1 complex. Gene, 1991 May 30, 101(2), 267 - 71 Isolation and characterization of biologically active murine interleukin-6 produced in Escherichia coli; Grenett HE et al.; Interleukin-6 (IL-6) is a multi-functional cytokine produced and secreted by several different cell types, including those of the immune system . A cDNA coding for the mature murine IL-6 (mIL-6), which extends from amino acid (aa) 25 through 211, was cloned into a prokaryotic vector and then expressed in Escherichia coli . The recombinant mIL-6 (remIL-6) was isolated from bacterial inclusion bodies by solubilization in 4 M guanidine hydrochloride followed by gel-filtration chromatography . The protein was refolded to an active conformation by dialysis against 25 mM Na . acetate pH 5.5 . A final step of purification and concentration on a cation exchange resin yielded pure and biologically active remIL-6 . The purified preparation had the expected aa composition, as confirmed by aa analysis and pI of 7.0-7.1 . The biological activity of the recombinant protein was measured in two systems; a proliferation assay employing 7TD1 cells, and a fibrinogen biosynthesis assay employing primary rat hepatocytes . Both assay systems demonstrated that the remIL-6 was active in the range of 10(8) units/mg, which is similar to that estimated for native cytokine . Antibodies raised in rabbits against remIL-6 neutralized the biological activity of both recombinant and native IL-6. J Biol Chem, 1991 May 25, 266(15), 9367 - 73 Expression of cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase in Escherichia coli . Differences in kinetic mechanisms; Kim H et al.; The cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase (E3) were expressed in Escherichia coli using a lambda PL promoter-driven prokaryotic expression vector . The expressed proteins in total cell extracts were identified by Western blot analysis using anti-pig heart E3 antibody and also by measurement of E3 activity . Most of the expressed human E3 polypeptides (five bands) were found in the insoluble pellet while primarily full-length mature E3 was found in the soluble fraction . About 2% of the total soluble protein was mature human E3 when expressed in wild type E . coli AR120 . Since wild type E . coli has its own endogenous E3 activity, the expression of human E3 was performed in a pyruvate dehydrogenase complex-deficient strain of E . coli, JRG1342 . The expressed recombinant human E3s in JRG1342 were purified to near homogeneity . The amino-terminal amino acid sequence analysis revealed that the recombinant mature E3 had an expected sequence while the recombinant precursor E3 lost 19 amino acid residues of its 35-amino acid leader sequence presumably due to a proteolytic cleavage . The recombinant mature E3 displayed comparable kinetic properties to those reported for highly purified mammalian E3s . The truncated precursor E3 showed about half of the mature E3 activity . The double-reciprocal plot for the mature E3 in the direction of NAD+ reduction showed parallel lines (ping-pong mechanism) while that for the truncated precursor E3 displayed intersecting lines (sequential mechanism) . In the direction of NADH oxidation, the kinetic mechanisms of both E3s were apparently a ping-pong mechanism . These kinetic results showed that the partial 16-amino acid extension in the leader sequence changed the kinetic mechanism of human E3 so that it resembled that of glutathione reductase. J Biol Chem, 1991 May 25, 266(15), 9740 - 5 Purification of glutamyl-tRNA reductase from Synechocystis sp . PCC 6803; Rieble S et al.; delta-Aminolevulinic acid is the universal precursor for all tetrapyrroles including hemes, chlorophylls, and bilins . In plants, algae, cyanobacteria, and many other bacteria, delta-aminolevulinic acid is synthesized from glutamate in a reaction sequence that requires three enzymes, ATP, NADPH, and tRNA(Glu) . The three enzymes have been characterized as glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase . All three enzymes have been separated and partially characterized from plants and algae . In prokaryotic phototrophs, only the glutamyl-tRNA synthetase and glutamate-1-semialdehyde aminotransferase have been decribed . We report here the purification and some properties of the glutamyl-tRNA reductase from extracts of the unicellular cyanobacterium, Synechocystis sp . PCC 6803 . The glutamyl-tRNA reductase has been purified over 370-fold to apparent homogeneity . Its native molecular mass was determined to be 350 kDa by glycerol density gradient centrifugation, and its subunit size was estimated to be 39 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The N-terminal amino acid sequence was determined for 42 residues . Much higher activity occurred with NADPH than with NADH as the reduced pyridine nucleotide substrate . Half-maximal rates occurred at 5 microM NADPH, whereas saturation was not reached even at 10 mM NADH . Purified Synechocystis glutamyl-tRNA reductase was inhibited 50% by 5 microM heme . Activity was unaffected by 10 microM 3-amino-2,3-dihydrobenzoic acid . No flavin, pyridine nucleotide, or other light-absorbing prosthetic group was detected on the purified enzyme . The catalytic turnover number of purified Synechocystis glutamyl-tRNA reductase is comparable to those of prokaryotic and plastidic glutamyl-tRNA synthetases. Biochim Biophys Acta, 1991 May 23, 1058(1), 56 - 60 Primary structure diversity of prokaryotic diheme cytochromes c; Van Beeumen J; The primary structure of five diheme cytochromes from photosynthetic bacteria recently determined in our laboratory lead to the first insights in the structural diversity of this type of cytochrome . A schematic overview is given, relating these structures to the four diheme cytochrome sequences already available . The comparison reveals unexpected homologies. Biochim Biophys Acta, 1991 May 23, 1058(1), 38 - 41 Bacterial 4-alpha-helical bundle cytochromes; Moore GR; The biological functions of cytochrome c' and bacterioferritin, both haemoproteins with a common 4-alpha-helical bundle structure, are discussed and an example given of one of Kamen's laws, namely: comparative studies of prokaryotic cytochromes and their eukaryotic counterparts are useful . In the present case, the comparison is between bacterioferritin and its animal counterpart, haemoferritin. Biochim Biophys Acta, 1991 May 23, 1058(1), 21 - 4 Some recent advances relating to prokaryotic cytochrome c reductases and cytochrome c oxidases; Gennis RB; Prokaryotic systems provide excellent experimental opportunities for exploring structure/function relationships for the complex, membrane-bound, multisubunit enzymes responsible for the reduction and subsequent oxidation of c-type cytochromes in respiratory or photosynthetic electron transport chains . Two points are made in this mini-review: (1) The eukaryotic and prokaryotic aa3-type cytochrome c oxidases are members of an apparently large superfamily of structurally related respiratory oxidases . This superfamily displays considerable variation in terms of the heme prosthetic groups (a or b) as well as the substrate oxidized (quinol or cytochrome c) . The relationships among these enzymes help to facilitate explorations of how they work . (2) Molecular biology techniques can be used to generate intact, redox-active, water-soluble domains of membrane-bound subunits . These soluble domains can be used for detailed examination, including obtaining high resolution structure by NMR techniques or by X-ray crystallography . This approach is being used to study the soluble heme-binding domain of cytochrome c1 from the bc1 complex of Rhodobacter sphaeroides. Proc R Soc Lond B Biol Sci, 1991 May 22, 244(1310), 117 - 21 Possible intermediate steps in the evolution of a prokaryotic developmental system; Errington J; Sigma factors (sigma) are transcription factors that operate global switches in gene expression in prokaryotes . They work by directing core RNA polymerase to specific cis-acting promoter sequences; each sigma has a cognate class of promoters with specific sequence characteristics . In Bacillus subtilis four different sigma factors have been implicated in the regulation of gene expression during spore formation, which is a simple differentiation system involving two cell types . In this review I show how the modern developmental system may have arisen from a primitive organism that used only two sigma factors, by a series of steps involving gene duplication and divergence . The increasing sophistication of eukaryotic developmental systems may reflect similar evolutionary processes. Gene, 1991 May 15, 101(1), 9 - 14 Escherichia coli thymidine kinase: nucleotide sequence of the gene and relationships to other thymidine kinases; Bockamp EO et al.; The thymidine kinase (TK)-encoding gene (tdk) of Escherichia coli is located at min 27 of the E . coli genetic map . Sequence analysis of this region revealed an open reading frame of 205 codons . Identification of this region as the E . coli tdk gene was confirmed by its similarity to other TK-encoding genes . The E . coli amino acid (aa) sequence showed significant similarity to the corresponding TK polypeptides of vertebrates and large DNA viruses, but showed no similarity to known herpes virus TK enzymes . Mapping of highly conserved positions among all sequences indicates the importance of these residues for catalytic activity and may facilitate further functional studies . Using a distance matrix method, the evolutionary relationships among the TK aa sequence of poxviruses, eukaryotes and prokaryotes were analyzed and a potential phylogenetic tree was established. Proc Natl Acad Sci U S A, 1991 May 15, 88(10), 4066 - 70 iciA, an Escherichia coli gene encoding a specific inhibitor of chromosomal initiation of replication in vitro; Thony B et al.; The gene encoding the protein that binds the three 13-mers in the origin (oriC) of Escherichia coli to block initiation of replication in vitro has been cloned, sequenced, and overexpressed . The gene possesses an open reading frame for 297 amino acids (mass of 33,471 Da) . The protein has a motif for DNA-binding (helix-turn-helix) and has homology to a diverse set of prokaryotic regulatory proteins, known as the LysR family . The protein, previously referred to as the 33-kDa protein, has been named IciA (for inhibitor of chromosome initiation) . The iciA gene is at 62.8 min on the chromosomal map . Cells with enhanced levels of the protein grow at a normal rate but generally exhibit a pronounced lag upon transfer to a fresh medium. Mol Cell Biochem, 1991 May 29-Jun 12, 104(1-2), 119 - 26 Initiation and regulation mechanisms of ribosomal RNA transcription in the eukaryote Acanthamoeba castellanii; Paule MR et al.; Acanthamoeba rRNA transcription involves the binding of a transcription initiation factor (TIF) to the core promoter of rDNA to form the preinitiation complex . This complex is formed in the absence of RNA polymerase I, and persists for multiple rounds of initiation . Polymerase I next binds to form the initiation complex . This binding is DNA sequence-independent, and is directed by protein-protein contacts with TIF . DNA melting occurs in a separate step . In contrast to most prokaryotic transcription, melting occurs only following nucleotide addition and beta-gamma hydrolysis of ATP is not required as for polymerase II . Growth-dependent regulation of rRNA transcription is accomplished by modification of RNA polymerase I . The inactive form of polymerase (PolE) is unable to bind to the promoter and has altered heat stability . PolE is still active in elongation; thus, the modification affects the polymerase site involved in TIF contact . Modification of a polymerases I and III common subunit has been detected leading to the suggestion that transcription of stable RNAs of the ribosome might be co-regulated by this mechanism. J Bacteriol, 1991 May, 173(10), 3117 - 27 Nucleotide sequence of the Rhodobacter capsulatus fruK gene, which encodes fructose-1-phosphate kinase: evidence for a kinase superfamily including both phosphofructokinases of Escherichia coli; Wu LF et al.; The fruK gene encoding fructose-1-phosphate kinase (FruK), located within the fructose (fru)-catabolic operon of Rhodobacter capsulatus, was sequenced . FruK of R . capsulatus (316 amino acids; molecular weight = 31,232) is the same size as and is homologous to FruK of Escherichia coli, phosphofructokinase B (PfkB) of E . coli, phosphotagatokinase of Staphylococcus aureus, and ribokinase of E . coli . These proteins therefore make up a family of homologous proteins, termed the PfkB family . A phylogenetic tree for this new family was constructed . Sequence comparisons plus chemical inactivation studies suggested the lack of involvement of specific residues in catalysis . Although the Rhodobacter FruK differed markedly from the other enzymes within the PfkB family with respect to amino acid composition, these enzymes exhibited similar predicted secondary structural features . A large internal segment of the Rhodobacter FruK was found to be similar in sequence to the domain bearing the sugar bisphosphate-binding region of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase of plants and bacteria . Proteins of the PfkB family did not exhibit statistically significant sequence identity with PfkA of E . coli . PfkA, however, is homologous to other prokaryotic and eukaryotic ATP- and PPi-dependent Pfks (the PfkA family) . These eukaryotic, ATP-dependent enzymes each consist of a homotetramer (mammalian) or a heterooctamer (yeasts), with each subunit containing an internal duplication of the size of the entire PfkA protein of E . coli . In some of these enzymes, additional domains are present . A phylogenetic tree was constructed for the PfkA family and revealed that the bacterial enzymes closely resemble the N-terminal domains of the eukaryotic enzyme subunits whereas the C-terminal domains have diverged more extensively . The PPi-dependent Pfk of potato is only distantly related to the ATP-dependent enzymes . On the basis of their similar functions, sizes, predicted secondary structures, and sequences, we suggest that the PfkA and PfkB families share a common evolutionary origin. Nucleic Acids Res, 1991 May 11, 19(9), 2261 - 5 Streptomycin inhibits splicing of group I introns by competition with the guanosine substrate; von Ahsen U et al.; Streptomycin is an aminocyclitol glycoside antibiotic, which interferes with prokaryotic protein synthesis by interacting with the ribosomal RNA . We report here that streptomycin is also able to inhibit self splicing of the group I intron of the thymidylate synthase gene of phage T4 . The inhibition is kinetically competitive with the substrate guanosine . Streptomycin and guanosine have in common a guanidino group, which has been shown to undergo hydrogen bonds with the ribozyme (Bass & Cech, Biochemistry, 25, 1986, 4473) . The inhibitory effect of streptomycin extends to other group I introns, but does not affect group II introns . Mutating the bulged nucleotide in the conserved P7 secondary structure element of the td intron alters the affinity of the ribozyme for both guanosine and streptomycin . Myomycin, an antibiotic with similar effects on protein synthesis as streptomycin, is also able to inhibit splicing . In contrast, bluensomycin, which is structurally related to streptomycin, but contains only one guanidino group does not inhibit splicing . We discuss these findings in support of an evolutionary model that stresses the antiquity of antibiotics (J . Davies, Molecular Microbiology 4, 1990, 1227). Science, 1991 May 10, 252(5007), 817 - 24 A new cofactor in a prokaryotic enzyme: tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase; McIntire WS et al.; Methylamine dehydrogenase (MADH), an alpha 2 beta 2 enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small beta subunit through two amino acyl residues . A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by the Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues . This info