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| J Biol Chem, 1996 Jan 19, 271(3), 1424 - 9 Reverse phosphotransfer from OmpR to EnvZ in a kinase-/phosphatase+ mutant of EnvZ (EnvZ.N347D), a bifunctional signal transducer of Escherichia coli; Dutta R et al.; EnvZ of Escherichia coli is a transmembrane histidine kinase belonging to the family of two-component signal transducing systems prevalent in prokaryotes and recently discovered in eukaryotes . In response to changes in medium osmolarity EnvZ regulates the level of phosphorylated OmpR, its conjugate response-regulating transcription factor for ompF and ompC genes . EnvZ has dual opposing enzymatic activities; OmpR-phosphorylase (kinase) and phospho-OmpR-dephosphorylase (phosphatase) . The osmotic signal is proposed to regulate the ratio of the kinase to the phosphatase activities of EnvZ to modulate the level of OmpR phosphorylation . In this work we used a COOH-terminal fragment of a previously identified kinase-/phosphatase+ EnvZ mutant (EnvZ-N347D) to demonstrate that the phosphoryl group on phospho-OmpR is transferred back to EnvZ to the same histidine residue (His243) that is utilized for the autokinase reaction by the wild type protein . Phospho-EnvZ-N347D thus formed could also transfer its phosphoryl group back to OmpR . The phosphotransfer reaction from phospho-OmpR to EnvZ.N347D was inhibited by ADP while Mg2+ ions stimulated the dephosphorylation reaction, resulting in release of inorganic phosphate . These results indicate that the energy levels of phosphoryl groups on OmpR and EnvZ are very similar and that the phosphatase reaction in the EnvZ.N347D mutant involves a reversal of the phosphotransfer reaction from EnvZ to OmpR using the identical His243 residue. Eur J Biochem, 1996 Jan 15, 235(1-2), 289 - 96 Purification and characterization of mitochondrial ribonuclease P from Aspergillus nidulans; Lee YC et al.; Mitochondrial ribonuclease (RNase) P from Aspergillus nidulans was purified to near homogeneity using whole-cell extract as the starting material . A 4400-fold purification with a yield of 5.2% was achieved by ammonium sulfate fractionation, heat treatment, and five types of column chromatography, including tRNA-affinity column chromatography . This enzyme, which has a molecular mass of 232 kDa determined by glycerol gradient sedimentation analysis, appears to be composed of seven polypeptides and an RNA moiety . These seven polypeptides consistently copurified with the RNase P activity through two ion-exchange chromatography columns and in a glycerol gradient . As judged by nuclease sensitivity, the enzyme requires an RNA component for its activity . The 3'-end-labeled RNAs that copurified with the enzyme displayed identical sequences but had variable lengths for the 5' end, indicating that they originated from a common RNA molecule, the putative RNA component of RNase P . The purified enzyme cleaved mitochondrial precursor tRNAHis, resulting in an 8-bp acceptor stem . This implies that the purified RNase P is a mitochondrial enzyme and that an additional guanylate residue (at position -1) of tRNAHis in A . nidulans mitochondria is generated by a mode that is analogous to the generation of their counterparts in prokaryotes and chloroplasts. Eur J Biochem, 1996 Jan 15, 235(1-2), 159 - 66 Major structural differences between pokeweed antiviral protein and ricin A-chain do not account for their differing ribosome specificity; Chaddock JA et al.; Pokeweed antiviral protein (PAP) and the A-chain of ricin (RTA) are two members of a family of ribosome-inactivating proteins (RIPS) that are characterised by their ability to catalytically depurinate eukaryotic ribosomes, a modification that makes the ribosomes incapable of protein synthesis . In contrast to RTA, PAP can also inactivate prokaryotic ribosomes . In order to investigate the reason for this differing ribosome specificity, a series of PAP/RTA hybrid proteins was prepared to test for their ability to depurinate prokaryotic and eukaryotic ribosomes . Information from the X-ray structures of RTA and PAP was used to design gross polypeptide switches and specific peptide insertions . Initial gross polypeptide swaps created hybrids that had altered ribosome inactivation properties . Preliminary results suggest that the carboxy-terminus of the RIPs (PAP 219-262) does not contribute to ribosome recognition, whereas polypeptide swaps in the amino-terminal half of the proteins did affect ribosome inactivation . Structural examination identified three loop regions that were different in both structure and composition within the amino-terminal region . Directed substitution of RTA sequences into PAP at these sites, however, had little effect on the ribosome inactivation characteristics of the mutant PAPs, suggesting that the loops were not crucial for prokaryotic ribosome recognition . On the basis of these results we have identified regions of RIP primary sequence that may be important in ribosome recognition . The implications of this work are discussed. Eur J Biochem, 1996 Jan 15, 235(1-2), 103 - 13 A novel dystrophin/utrophin-associated protein is an enzymatically inactive member of the phosphoglucomutase superfamily; Moiseeva EP et al.; A 60-kDa protein localised in adherens-type cellular junctions, and previously called aciculin, has been found to interact with the cytoskeletal proteins dystrophin and utrophin {Belkin, A . M . & Burridge, K . (1995) J . Biol . Chem . 270, 6328-6337} . In this study, we report the complete sequence of this protein, and show that it is a novel member of the phosphoglucomutase (PGM) family of proteins . The PGM-related protein (PGM-RP), which contains 506 amino acids (55.6 kDa), is smaller than PGM1 (566 amino acids, 61 kDa) . The active site consensus sequences of prokaryotic and eukaryotic mutases are not conserved in PGM-RP, a finding consistent with the lack of enzymatic activity of PGM-RP in vitro, and the absence of a phosphorylated intermediate in vivo . The organisation of the PGM-RP gene is essentially identical to that of PGM1 . We propose that the PGM-RP gene, which we have mapped to human chromosome 9qcen-q13, evolved from the PGM1 gene, and encodes a protein with a structural rather than an enzymatic role . PGM-RP is expressed predominantly in muscle with the highest levels in smooth muscle . The significance of the interaction between dystrophin/utrophin and an increasing number of cytoplasmic proteins including PGM-RP remains to be explored. FEBS Lett, 1996 Jan 15, 378(3), 240 - 4 Purification and characterization of the acetate forming enzyme, acetyl-CoA synthetase (ADP-forming) from the amitochondriate protist, Giardia lamblia; Sanchez LB et al.; Giardia lamblia, an amitochondriate eukaryote, contains acetyl-CoA synthetase (ADP-forming), an enzyme known only from one other eukaryote (Entamoeba histolytica) and a few anaerobic prokaryotes . The enzyme has been purified about 350-fold . The activity in the direction of acetate formation was dependent on ADP and inorganic phosphate . The reverse reaction could not be detected . Succinyl-CoA, propionyl-CoA and dADP were utilized with lower efficiency . The enzyme did not utilize AMP plus PPi thus differs from the broadly distributed acetyl-CoA synthetase (AMP-forming) . The enzyme is responsible for acetate production accompanied by ATP generation, thus plays an important role in G . lamblia metabolism. Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 336 - 41 Control of cell division in Escherichia coli: regulation of transcription of ftsQA involves both rpoS and SdiA-mediated autoinduction; Sitnikov DM et al.; The conditioning of culture medium by the production of growth-regulatory substances is a well-established phenomenon with eukaryotic cells . It has recently been shown that many prokaryotes are also capable of modulating growth, and in some cases sensing cell density, by production of extracellular signaling molecules, thereby allowing single celled prokaryotes to function in some respects as multicellular organisms . As Escherichia coli shifts from exponential growth to stationary growth, many changes occur, including cell division leading to formation of short minicells and expression of numerous genes not expressed in exponential phase . An understanding of the coordination between the morphological changes associated with cell division and the physiological and metabolic changes is of fundamental importance to understanding regulation of the prokaryotic cell cycle . The ftsQA genes, which encode functions required for cell division in E . coli, are regulated by promoters P1 and P2, located upstream of the ftsQ gene . The P1 promoter is rpoS-stimulated and the second, P2, is regulated by a member of the LuxR subfamily of transcriptional activators, SdiA, exhibiting features characteristic of an autoinduction (quorum sensing) mechanism . The activity of SdiA is potentiated by N-acyl-homoserine lactones, which are the autoinducers of luciferase synthesis in luminous marine bacteria as well as of pathogenesis functions in several pathogenic bacteria . A compound(s) produced by E . coli itself during growth in Luria Broth stimulates transcription from P2 in an SdiA-dependent process . Another substance(s) enhances transcription of rpoS and (perhaps indirectly) of ftsQA via promoter P1 . It appears that this bimodal control mechanism may comprise a fail-safe system, such that transcription of the ftsQA genes may be properly regulated under a variety of different environmental and physiological conditions. Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 106 - 10 A two-subunit type I DNA topoisomerase (reverse gyrase) from an extreme hyperthermophile; Krah R et al.; A recently described reverse gyrase from the hyperthermophilic methanogen Methanopyrus kandleri is the only known example of a heterodimeric type I topoisomerase . The enzyme is made up of a 42-kDa subunit which covalently interacts with DNA (RgyA) and a 138-kDa subunit which binds ATP (RgyB) . We have now cloned and sequenced the genes for both subunits of this enzyme . Surprisingly, the universally conserved type I topoisomerase domain {Lima, C . D., Wang, J . C . & Mondragon, A . (1994) Nature (London) 367, 138-146} which has been found as a contiguous polypeptide in the prokaryotes and eukaryotes is shared between the protomers . The subdomain with the active-site tyrosine is entirely within RgyA, whereas the subdomain implicated in noncovalent binding of the cleaved DNA strand is contained entirely in RgyB . The appearance of this unique structure in a highly conserved enzyme family supports the hypothesis that the methanogens branched from other prokaryotes and eukaryotes very early in evolution. Ciba Found Symp, 1996, 202, 1 - 10; discussion 11-8 Hyperthermophiles in the history of life; Stetter KO; Prokaryotes requiring extremely high growth temperatures (optimum 80-110 degrees C) have recently been isolated from water-containing terrestrial, subterranean and submarine high temperature environments . These hyperthermophiles consist of primary producers and consumers of organic matter, forming unique high temperature ecosystems . Surprisingly, within the 16S rRNA-based phylogenetic tree, hyperthermophiles occupy all the shortest and deepest branches closest to the root . Therefore, they appear to be the most primitive extant organisms . Most of them (the primary producers) are able to grow chemolithoautotrophically, using CO2 as sole carbon source and inorganic energy sources, suggesting a hyperthermophilic autotrophic common ancestor . They gain energy from various kinds of respiration . Molecular hydrogen and reduced sulfur compounds serve as electron donors while CO2, oxidized sulfur compounds, NO3- and O2 (only rarely) serve as electron acceptors . Growth demands of hyperthermophiles fit the scenario of a hot volcanism-dominated primitive Earth . Similar anaerobic chemolithoautotrophic hyperthermophiles, completely independent of a sun, could even exist on other planets provided that active volcanism and liquid water were present. Hist Philos Life Sci, 1996, 18(2), 163 - 93 Escherichia coli as a model system with which to study cell differentiation; Thieffry D; This article concerns the elaboration of epigenetic models for differentiation . I discuss how results and conclusions arising from studies of prokaryotes were extrapolated to explain differentiation during metazoan development . In this respect, I focus on the presentation of a multi-stable biochemical model by Delbruck in 1949, and on a series of works dealing with enzyme adaptation in Escherichia coli that culminated in Jacob and Monod's operon model . These influential contributions are discussed in the context of debates on nuclear versus cytoplasmic heredity, on the regulation of gene expression, as well as on the mechanisms at the basis of cell differentiation. Genes Cells, 1996 Jan, 1(1), 101 - 13 Clamp loading, unloading and intrinsic stability of the PCNA, beta and gp45 sliding clamps of human, E . coli and T4 replicases; Yao N et al.; BACKGROUND: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein . This protein ("sliding clamp') encircles DNA and tethers the catalytic unit to the template . Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states . The Escherichia coli clamp (the beta subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers . The clamp can not assemble itself on DNA . Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template . This study compares properties of the human PCNA clamp with those of E . coli and T4 phage . RESULTS: The PCNA ring is a stable trimer down to a concentration below 100 nM (Kd approximately 21 nM) . On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 approximately 24 min) . beta is more stable in solution (Kd < 60 PM) and on DNA (t1/2 approximately 1 h) than PCNA which may be explained by its simpler oligomeric state . The T4 gp45 clamp is a much less stable trimer than PCNA (Kd approximately 250 nM) and requires association with the polymerase to stabilize it on DNA as observed previously . The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance . However, the greater stability of the PCNA and beta clamps on DNA necessitates an active process for their removal . The clamp loaders (RFC and gamma complex) were also capable of unloading their respective clamps from DNA in the presence of ATP . CONCLUSIONS: The stability of the different clamps in solution correlates with their stability on DNA . Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA . The stability of the PCNA and beta clamps predicts they will require an unloading factor to recycle them on and off DNA during replication . The clamp loaders of PCNA and beta double as clamp unloaders presumably for the purpose of clamp recycling. Appl Theor Electrophor, 1996, 6(1), 43 - 7 Effect of methylation on the electrophoretic mobility of chromosomal DNA in pulsed-field agarose gels; Xydas S et al.; Factors other than molecular weight are known to affect DNA electrophoretic mobility . DNA methylation has been found to affect the curvature of DNA, causing anomalous mobility in polyacrylamide gels; the effect of methylation on the mobility of large DNA molecules in agarose gels was unknown . Chromosomal DNA from Mycoplasma capricolum, a wall-less prokaryote which has a low intrinsic methylation rate, was methylated in agarose blocks by SssI methylase, a de novo methylase with a CpG recognition sequence . (A surprising finding was that SssI methylase altered the structure of InCert, but not SeaKem Gold, agarose.) Restriction enzyme analysis was used to estimate the extent of CpG methylation . DNA methylation was found to have no effect on the electrophoretic mobility of full-length chromosomal DNA (1,120 kbp) in agarose gels . Therefore, methylation is not a source of error in PFGE-based size estimation for chromosomal DNA molecules less than 1.12 Mbp in agarose gels. Crit Rev Microbiol, 1996, 22(4), 295 - 314 Antioxidant defense mechanisms in parasitic protozoa; Mehlotra RK; Many of the parasitic protozoa, such as Entamoeba histolytica, Giardia, Trypanosoma, Leishmania, and Plasmodium, are considered to be anaerobes because they can be grown in vitro only under conditions of reduced oxygen tension . However, these parasitic protozoa have been found to be aerotolerant or microaerophilic, and also to consume oxygen to a certain extent . Furthermore, these organisms are highly susceptible to exogenous reactive oxygen species, such as hydrogen peroxide . They must, therefore, detoxify both oxygen and free radical products of enzymatic reactions . However, they lack some or all of the usual antioxidant defense mechanisms present in aerobic or other aerotolerant cells, such as catalase, superoxide dismutase, reduced glutathione, and the glutathione-recycling enzymes glutathione peroxidase and glutathione reductase . Instead, they possess alternative mechanisms for detoxification similar to those known to exist in certain prokaryotes . Although the functional aspects of these alternative mechanisms are yet to be understood completely, they could provide new insights into the biochemical peculiarities of these enigmatic pathogens. DNA Seq, 1996, 6(6), 351 - 5 Cloning, nucleotide sequence, and functional expression of the Escherichia coli enolase (eno) gene in a temperature-sensitive eno mutant strain; Klein M et al.; The entire Escherichia coli eno gene was cloned by functional complementation of a newly isolated temperature-sensitive enolase mutant and its nucleotide sequence determined . The deduced amino acid sequence is homologous to other known prokaryotic or eukaryotic enolases and amino acid residues, assumed to be involved in substrate or cofactor binding and catalysis, were found to be strictly conserved among all enolase proteins . Expression of the eno gene under the control of the lac promoter/operator resulted in an IPTG-inducible production of enzymatically active enolase in wild-type and enolase mutant strains. Annu Rev Genet, 1996, 30, 465 - 506 Proteases and their targets in Escherichia coli; Gottesman S; Proteolysis in Escherichia coli serves to rid the cell of abnormal and misfolded proteins and to limit the time and amounts of availability of critical regulatory proteins . Most intracellular proteolysis is initiated by energy-dependent proteases, including Lon, ClpXP, and HflB; HflB is the only essential E . coli protease . The ATPase domains of these proteases mediate substrate recognition . Recognition elements in target are not well defined, but are probably not specific amino acid sequences . Naturally unstable protein substrates include the regulatory sigma factors for heat shock and stationary phase gene expression, sigma 32 and RpoS . Other cellular proteins serve as environmental sensors that modulate the availability of the unstable proteins to the proteases, resulting in rapid changes in sigma factor levels and therefore in gene transcription . Many of the specific proteases found in E . coli are well-conserved in both prokaryotes and eukaryotes, and serve critical functions in developmental systems. Annu Rev Genet, 1996, 30, 35 - 57 Control of transcription termination in prokaryotes; Henkin TM; A growing number of genetic systems have been shown to be controlled at the level of premature termination of transcription . Genes in this class contain transcription termination signals in the region upstream of the coding sequence . The activity of these regulatory termination signals is controlled through a variety of mechanisms . These include modification of RNA polymerase to a terminator-resistant, or terminator-prone form, and alterations in the structure of the nascent transcript, to determine whether the stem-loop structure of an intrinsic terminator or an alternate antiterminator is formed . Structural alterations in the transcript can be controlled by the kinetics of translation of the RNA, by binding of specific regulatory proteins, and by mRNA-tRNA interactions . This review describes a number of variations on the termination control theme that have been uncovered in prokaryotes. Cytogenet Cell Genet, 1996, 72(2-3), 242 - 5 Cloning, expression and chromosomal mapping of a novel cyclophilin-related gene (PPIL1) from human fetal brain; Ozaki K et al.; We isolated a human cDNA clone encoding a novel protein homologous to cyclophilins, specific cellular targets of cyclosporin A, which are conserved in species ranging from human to prokaryotes . This cDNA, designated hCyPX, contained an open reading frame of 498 nucleotides encoding 166 amino acids . Computer analysis indicated that its predicted amino acid sequence had 41.6%, 40.4%, and 39.2% homology to those of human, bovine, and Drosophila cyclophilins, respectively . Northern blot analysis indicated ubiquitous expression in adult human tissues, but most abundant expression in heart . Fluorescence in situ hybridization to human metaphase chromosomes localized this gene (PPIL1, peptidylprolyl isomerase {cyclophilin}-like 1) to chromosome bands 2p23.3-->p23.1. Annu Rev Cell Dev Biol, 1996, 12, 1 - 26 Import and routing of nucleus-encoded chloroplast proteins; Cline K et al.; Most chloroplast proteins are nuclear encoded, synthesized as larger precursor proteins in the cytosol, posttranslationally imported into the organelle, and routed to one of six different compartments . Import across the outer and inner envelope membranes into the stroma is the major means for entry of proteins destined for the stroma, the thylakoid membrane, and the thylakoid lumen . Recent investigations have identified several unique protein components of the envelope translocation machinery . These include two GTP-binding proteins that appear to participate in the early events of import and probably regulate precursor recognition and advancement into the translocon . Localization of imported precursor proteins to the thylakoid membrane and thylakoid lumen is accomplished by four distinct mechanisms; two are homologous to bacterial and endoplasmic reticulum protein transport systems, one appears unique, and the last may be a spontaneous mechanism . Thus chloroplast protein targeting is a unique and surprisingly complex process . The presence of GTP-binding proteins in the envelope translocation machinery indicates a different precursor recognition process than is present in mitochondria . Mechanisms for thylakoid protein localization are in part derived from the prokaryotic endosymbiont, but are more unusual and diverse than expected. Crit Rev Eukaryot Gene Expr, 1996, 6(4), 377 - 89 Two-stage gene regulation of the superoxide stress response soxRS system in Escherichia coli; Nunoshiba T; All organisms have adapted to environmental changes by acquiring various functions controlled by gene regulation . In bacteria, a number of specific responses have been found to confer cell survival in various nutrient-limited conditions, and under physiological stresses such as high or low temperature, extreme pH, radiation, and oxidation (for review, see Neidhardt et al., 1987) . In this article, I introduce an Escherichia coli (E . coli) global response induced by superoxide stress, the soxRS regulon . The functions controlled by this system consist of a wide variety of enzymes such as manganese-containing SOD (Mn-SOD); glucose 6-phosphate dehydrogenase (G6PD), the DNA repair enzyme endonuclease IV, fumarase C, NADPH:ferredoxin oxidoreductase, and aconitase . This response is positively regulated by a two-stage control system in which SoxR iron-sulfur protein senses exposure to superoxide and nitric oxide, and then activates transcription of the soxS gene, whose product stimulates the expression of the regulon genes . Our recent finding indicates that soxS transcription is initiated in a manner dependent on the rpoS gene encoding RNA polymerase sigma factor, theta s, in response to entering the stationary phase of growth . With this information, mechanisms for prokaryotic coordinating gene expression in response to superoxide stress and in stationary phase are discussed. Crit Rev Eukaryot Gene Expr, 1996, 6(4), 299 - 343 Circadian rhythms and cancer chemotherapy; Wood PA et al.; Temporal coordination of biologic processes with an approximately 24-h cycle (circadian) is common throughout the animal and plant kingdom and even in some prokaryotic organisms . In all organisms studied, the capability to keep biologic time is an inherited characteristic located intracellularly . These biological clocks anticipate and get the organism ready for regular environmental changes . This indicates both the ubiquity and the weight of the selective environmental pressure to keep time accurately . Several molecular strategies for biologic time keeping have apparently arisen independently several times throughout evolution . The anatomic, biochemical, and molecular mechanisms of the clock are in the process of being defined . This temporal organization at the cellular, organ, and organismic levels results in predictable differences in the capacity of plants, animals, and human beings to respond to therapeutic interventions administered at different times throughout important biologic cycles (e.g., circadian timed therapy) . In the treatment of the cancer bearing host, circadian timing of surgery, anticancer drugs, radiation therapy, and biologic agents can result in improved toxicity profiles, enhanced tumor control, and improved host survival . The routine clinical application of such principles is facilitated by the availability of programmable drug delivery devices . Rhythm frequency ranges other than 24-h (e.g., low frequency: menstrual; high frequency: 10 to 120 min) may also be important to understanding health and disease and to designing successful therapy in diseases as diverse as cancer, infertility, and diabetes. Biochimie, 1996, 78(7), 590 - 6 RNAs mediating cotranslational insertion of selenocysteine in eukaryotic selenoproteins; Hubert N et al.; Selenocysteine, a selenium-containing analog of cysteine, is found in the prokaryotic and eukaryotic kingdoms in active sites of enzymes involved in oxidation-reduction reactions . Its biosynthesis and cotranslational insertion into selenoproteins is performed by an outstanding mechanism, implying the participation of several gene products . The tRNA(Sec) is one of these . In eukaryotes, its transcription mode by RNA polymerase III differs from that of classical tRNA genes, both at the level of the promoter elements and transcription factors involved . In addition, enhanced transcription is afforded by a newly characterized zinc finger activator . Not only transcription of the gene, but also the tRNA(Sec) itself is atypical since its 2D and 3D structures exhibit features which set it apart from classical tRNAs . Decoding of eukaryotic selenocysteine UGA codons requires a stem-loop structure in the 3'UTR of mRNAs, the selenocysteine insertion sequence (SECIS) element . Structure probing and sequence comparisons led us to propose a 2D structure model for the SECIS element, containing a novel RNA motif composed of four consecutive non-Watson-Crick base-pairs . A 3D model, rationalizing the accessibility data, was elaborated by computer modeling . It yields indicative or suggestive evidence for the role that could play some conserved residues and/or structural features in SECIS function . These might act as signals for interaction with SBP, the SECIS binding protein that we have characterized. Biochimie, 1996, 78(7), 577 - 89 Messenger RNA translation in prokaryotes: GTPase centers associated with translational factors; Laalami S et al.; During the decoding of messenger RNA, each step of the translational cycle requires the intervention of protein factors and the hydrolysis of one or more GTP molecule(s) . Of the prokaryotic translational factors, IF2, EF-Tu, SELB, EF-G and RF3 are GTP-binding proteins . In this review we summarize the latest findings on the structures and the roles of these GTPases in the translational process. Yi Chuan Xue Bao, 1996, 23(3), 183 - 9 {Re-analysis of DNA sequence data from a dinosaur egg fossil unearthed in Xixia of Henan Province}; Wang H; To identify the real source of two putative dinosaur 18S rDNA sequences of DA18S1 and DA18S7 cloned from a dinosaur egg fossil unearthed in Xixia of Henan Province, China, sequence homology searching was performed on the INTERNET by programs of the BLAST server and FASTA server . Neither of the two sequences was found to be similar with prokaryotic sequences . The base identity between DA18S7 with several plant 18S rDNA sequences is among 89.0-92.4%, much higher than that with 18S rDNAs of other organisms . DA18S1 has larger similarities with fungal, invertebrate and plant 18S rDNAs than with the vertebrate sequences . Molecular phylogeny analysis of DA18S1 indicated that DA18S1 clusters with fungal 18S rDNAs . These results show that DA18S1 and DA18S7 are of fungal and plant origin, respectively. Cytogenet Cell Genet, 1996, 74(3), 157 - 60 Chromosome microdissection: a brief overview; Cannizzaro LA; Chromosome microdissection arose as a means of facilitating long range physical mapping of chromosome regions involved in either a genetic or malignant disorder . However, with the rapid development of improved techniques for mapping and sequencing the human genome, microdissection is considered by many investigators to be a cumbersome and time consuming procedure . Nonetheless, based on the impressive number of informative diagnostic DNA markers that are now available as a result of this technology, microdissection still must be considered one of the most rapid and direct methods available for generating new DNA markers from any chromosome region, irrespective of its sequence composition . In addition, it remains an important means to dissect DNA markers from any organism, eukaryotic and prokaryotic, and has resulted in generating disease associated DNA sequences from both human and animal genomes . Recently, microdissection of single cells has emerged as a viable alternative for isolating pure populations of specific cell types, especially tumor cells, which can then be studied without background contamination from any other cellular constituents . This overview will provide a glimpse into the present applications of the microdissection technology, as well as the importance this technology will have for future exploration into the human genome. J Basic Microbiol, 1996, 36(5), 341 - 9 Properties of lysophospholipase in Mycobacterium leprae; Prabhakaran K et al.; Lysophospholipids are key intermediates in the metabolism of phospholipids . Cytoplasmic membranes of both eukaryotes and prokaryotes are made of phospholipid bilayers . Phospholipases are activated during phagocytosis . Lysophospholipids generated by phospholipase A2 or A1 degrade cell membranes and can cause cell lysis . An active lysophospholipase, that hydrolyzes lysophospholipids, was detected by the radioisotope technique in Mycobacterium leprae . About two-thirds of the enzyme was particulate and one-third cytoplasmic . Optimum activity was at 37 degrees C, and at pH 6.0 . Temperatures above 70 degrees C completely inactivated the enzyme . The compound AACOCF3, a trifluromethylketone analog of arachiodonic acid, inhibited the activity; the inhibition appeared to be of the uncompetetive type . The K(m) of the enzyme was 2.5 x 10(-4)M, suggesting a fairly strong affinity for the substrate . Lysophospholipids have been shown to be microbicidal to invading organisms . Possession of lysophospholipase by M . leprae is apparently one of the methods by which the bacilli overcome the defense mechanisms of the host. Annu Rev Microbiol, 1996, 50, 401 - 29 Towards a unified evolutionary genetics of microorganisms; Tibayrenc M; I propose here that evolutionary genetics, apart from improving our basic knowledge of the taxonomy and evolution of microbes (either eukaryotes or prokaryotes), can also greatly contribute to applied research in microbiology . Evolutionary genetics provides convenient guidelines for better interpreting genetic and molecular data dealing with microorganisms . The three main potential applications of evolutionary genetics in microbiology are (a) epidemiological follow-up (with the necessity of evaluating the stability of microbial genotypes over space and time); (b) taxonomy in the broad sense (better definition and sharper delimitation of presently described taxa, research of hidden genetic subdivisions); and (c) evaluation of the impact of the genetic diversity of microbes on their relevant properties (pathogenicity, resistance to drugs, etc) . At present, two main kinds of population structure can be distinguished in natural microbial populations: (a) species that are not subdivided into discrete phylogenetic lineages (panmictic species or basically sexual species with occasional bouts of short-term clonality fall into this category); (b) species that are strongly subdivided by either cryptic speciation or clonal evolution . Improvements in available statistical methods are required to refine these distinctions and to better quantify the actual impact of gene exchange in natural microbial populations . Moreover, a codified selection of markers with appropriate molecular clocks (in other words: adapted levels of resolution) is sorely needed to answer distinct questions that address different scales of time and space: experimental, epidemic, and evolutionary . The problems raised by natural genetic diversity are very similar for all microbial species, in terms of both basic and applied science . Despite this fact, a regrettable compartmentalization among specialists has hampered progress in this field . I propose a synthetic approach, relying on the statistical improvements and technical standardizations called for above, to settle a unified evolutionary genetics of microorganisms, valid whatever the species studied, whether eukaryotic (parasitic protozoa and fungi) or prokaryotic (bacteria) . Apart from benefits for basic evolutionary research, the anticipated payoff from this synthetic approach is to render routine and common-place the use of microbial evolutionary genetics in the fields of epidemiology, medicine, and agronomy. Annu Rev Microbiol, 1996, 50, 317 - 48 What size should a bacterium be? A question of scale; Koch AL; There are living prokaryotes (Bacteria and Archaea) that have cell sizes that range from 0.02-400 microns3 . Over this tremendous range, various abilities to cope with the environment are needed . This review attempts to formulate some of the problems and some of the solutions . The smallest size for a free-living organism is suggested to be largely set by the catalytic efficiency of enzymes and protein synthetic machinery . Because of fluctuations in the environment, cells must maintain machinery to cope with various catastrophes; these mechanisms increase the minimum size of the cell . On the other hand, the largest cell is reasonably assumed to be limited by the ability of diffusion to bring nutrients to the appropriate part of the cell and to dispose of waste products . To explore the limitation imposed by diffusion, analysis is developed of diffusion processes through stirred and unstirred media, diffusion through media that contains obstacles, and the effect of size and shape. Proc Int Conf Intell Syst Mol Biol, 1996, 4, 182 - 91 Characterization of prokaryotic and eukaryotic promoters using hidden Markov models; Pedersen AG et al.; In this paper we utilize hidden Markov models (HMMs) and information theory to analyze prokaryotic and eukaryotic promoters . We perform this analysis with special emphasis on the fact that promoters are divided into a number of different classes, depending on which polymerase-associated factors that bind to them . We find that HMMs trained on such subclasses of Escherichia coli promoters (specifically, the so-called sigma 70 and sigma 54 classes) give an excellent classification of unknown promoters with respect to sigma-class . HMMs trained on eukaryotic sequences from human genes also model nicely all the essential well known signals, in addition to a potentially new signal upstream of the TATA-box . We furthermore employ a novel technique for automatically discovering different classes in the input data (the promoters) using a system of self-organizing parallel HMMs . These self-organizing HMMs have at the same time the ability to find clusters and the ability to model the sequential structure in the input data . This is highly relevant in situations where the variance in the data is high, as is the case for the subclass structure in for example promoter sequences. EXS, 1996, 77, 239 - 54 Transcriptional regulators of oxidative stress-inducible genes in prokaryotes and eukaryotes; Storz G et al.; It appears that redox regulation is an important mechanism for the control of transcription factor activation . The role of oxidation-reduction is probably determined in part by the structure of the transcription factors . For example, the presence of cysteine residues within the DNA binding sites may sensitize a transcription factor to ROS . The ROS-mediated regulation of transcription factors is specific, some ROS are more efficient than other ROS in activating defined regulators . While the protective antioxidant responses induced by ROS in prokaryotes and eukaryotes are rather conserved (for example, SOD, HSP...), the regulators for these genes do not appear to be conserved . Further studies designed to fully characterize these regulators and understand the subtle mechanisms involved in redox gene regulation are ongoing, and should provide the theoretical basis for clinical approaches using antioxidant therapies in human diseases in which oxidative stress is implicated. EXS, 1996, 77, 221 - 35 SOS response as an adaptive response to DNA damage in prokaryotes; Shinagawa H; Escherichia coli possesses an elaborate adaptive mechanism called the "SOS response" to cope with various types of DNA damage . More than 20 SOS genes, most of which are known to be involved in the functions that promote the survival of DNA-damaged cells, are induced by treatments that damage DNA or inhibit DNA synthesis . All the SOS genes share similar sequences in the regulatory regions called the "SOS box", to which LexA repressor binds to repress the transcription in the absence of DNA damage . The SOS signal appears to be the single-stranded DNA produced in vicinity of DNA damage, to which RecA protein binds to be activated as a coprotease . The activated RecA promotes autocleavage of LexA protein by allosteric interaction, which activates the latent serine protease activity of LexA . The induced products of the SOS genes repair DNA lesions by various mechanisms, including recombination, excision repair and error-prone repair, and as the consequence, the SOS signal in the cell decreases and the repression of the SOS genes is restored. EXS, 1996, 77, 97 - 117 Protein disulfide isomerase: a multifunctional protein of the endoplasmic reticulum; Luz JM et al.; Protein disulfide isomerase (PDI) is a resident enzyme of the endoplasmic reticulum (ER) that was discovered over three decades ago . Contemporary biochemical and molecular biology techniques have revealed that it is present in all eukaryotic cells studied and retained in the ER via a -KDEL or -HDEL sequence at its C-terminus . However, evidence is accumulating that in certain cell types, PDI can be found in other subcellular compartments, despite possessing an intact retention sequence . A wide range of studies has established that in presence of a redox pair, PDI acts catalytically to both form and reduce disulfide bonds, therefore acting as a disulfide isomerase . Recent studies have focused on the mechanism of the isomerization process and the precise role of the two active site sequences (-CGHC-) in the process . In addition, prokaryotes have been shown to possess a set of proteins that function in a similar fashion, being able to generate disulfide bonds on polypeptides translocated into the periplasmic space . Following the recent discovery that PDI binds peptides, coupled with earlier findings that PDI is a subunit of at least two enzymatic complexes (prolyl 4-hydroxylase and microsomal triglyceride transfer protein), it seems that it may serve functions other than merely that of a disulfide isomerase . In fact, it is now clear that PDI can facilitate protein folding independently of its disulfide isomerase activity . A major challenge for the future is to define mechanistically how it accomplishes isomerization and the relationship between this process and the protein folding steps that culminate in the final, fully mature protein. EXS, 1996, 77, 3 - 24 Normal protein folding machinery; Hartman D et al.; A highly conserved protein folding machine has been maintained in the cytosol of both prokaryotic and eukaryotic organisms and in eukaryotic mitochondria . Homologous components of this machinery have also been identified in other organelles such as the endoplasmic reticulum in which HSP70 and DnaJ-like homologs reside . The high degree of conservation presumably reflects the proficiency with which these molecules have evolved to mediate the folding of proteins to their native functional states. Connect Tissue Res, 1996, 34(1), 1 - 9 Engineering, expression and renaturation of targeted TGF-beta fusion proteins; Tuan TL et al.; This study reports the expression, purification, and renaturation of biologically active Transforming Growth Factor-beta 1 (TGF-beta 1) fusion proteins from Escherichia coli (E . coli) . A prokaryotic expression vector was engineered to produce tripartite fusion proteins consisting of (i) a purification tag, (ii) a protease-sensitive linker/collagen binding domain, and (iii) a cDNA sequence encoding the active fragment of human TGF-beta 1 . The expressed fusion proteins TGF-B1-F1 and TGF-B1-F2, located in inclusion bodies, were solubilized with 8 M urea and renatured using a glutathione redox-coupled system and protracted dialysis under several experimental conditions . The purification of the recombinant proteins was achieved by binding the His-tag of the fusion proteins on a Ni-NTA metal chelate column . The biological activity of the recombinant growth factor was demonstrated by its ability to inhibit mink lung (Mv1Lu) cell proliferation and/or to stimulate proliferation of NIH-3T3 mouse fibroblasts, where purified human platelet TGF-beta 1 served as a positive control . Purified TGF-B1-F1 and TGF-B1-F2 (collagen-binding) constructs exhibited anti-proliferative activities comparable to purified platelet TGF-beta 1, but at lower specific activities . Binding of the renatured TGF-B1-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G15 column . The high affinity binding was also demonstrated by the binding of 3H-collagen to the TGF-B1-F2 protein immobilized on a Ni-NTA column . The TGF-B1-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration . Taken together, these results demonstrate that biologically active TGF-beta 1 fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for its high-yield production, purification, and renaturation from microorganisms . Furthermore, these results support the concept that auxiliary domains may be used to modulate and/or target TGF-beta 1 for specific applications. Hum Mutat, 1996, 7(3), 187 - 92 Molecular basis of congenital erythropoietic porphyria: mutations in the human uroporphyrinogen III synthase gene; Xu W et al.; Congenital erythropoietic porphyria (CEP) is an autosomal recessive inborn error of metabolism that results from the markedly deficient activity of the fourth enzyme in the heme biosynthetic pathway, uroporphyrinogen III synthase (URO-synthase) . To date, 17 mutations have been described including 11 missense, one nonsense, two mRNA splicing defects, one deletion and two coding region insertions . Most mutations have been identified in one or a few unrelated families with the exception of C73R and L4F which occurred in 29.6% and 9.3% of the 54 mutant alleles studied, respectively . Interestingly, analysis of the mutant alleles identified only 83% of the causative mutations, suggesting that about 20% of the mutations causing CEP lie elsewhere in the gene . Of note, mutation V82F, resulting from a G to T transversion of the last nucleotide of exon 4, caused both a missense mutation and an aberrantly spliced RNA transcript . Prokaryotic expression of the mutant URO-synthase alleles identified those with significant residual activity, thereby permitting genotype/phenotype predictions for this clinically heterogeneous disease. Biosci Biotechnol Biochem, 1996 Jan, 60(1), 137 - 8 Desulfation of tyrosine-O-sulfated peptides by some eukaryotic sulfatases; Suiko M et al.; Three mammalian and eight non-mammalian arylsulfatases were investigated for their activities toward tyrosine-O-sulfate (TyrS) in peptides . None of the mammalian arylsulfatases exhibited detectable activities toward TyrS-containing peptides . Of the non-mammalian arylsulfatases tested, Types VII, VIII, and H-1, 2, and 5 displayed strong activity on endo-TyrS residues . The prokaryotic sulfatase, Type VI, was active only on free TyrS and N-terminal TyrS of Leu-enkephalin . All the sulfatases were active on p-nitrophenyl sulfate and p-nitrocatechol sulfate. Prog Nucleic Acid Res Mol Biol, 1996, 52, 89 - 122 Nutritional and hormonal regulation of expression of the gene for malic enzyme; Goodridge AG et al.; We have provided a historical and personal description of the analysis of physiological and molecular mechanisms by which diet and hormones regulate the activity of hepatic malic enzyme . For the most part, our analyses have been reductionist in approach, striving for increasingly simpler systems in which we can ask more direct questions about the molecular nature of the signaling pathways that regulate the activity of malic enzyme . The reductionist approaches that were so successful at analyzing molecular mechanisms in cells in culture may now provide the means to analyze more definitively questions about the physiological mechanisms involved in nutritional regulation of gene expression . In addition to physiological questions, however, there are still many aspects of the molecular mechanisms that have not been elucidated . Despite considerable effort from many laboratories, the molecular mechanisms by which T3 regulates transcription are not clear . Similarly, the molecular details for the mechanisms by which glucagon, insulin, glucocorticoids, and fatty acids regulate gene expression remain to be determined . The role of fatty acids is particularly interesting because it may provide a model for mechanisms by which genes are regulated by metabolic intermediates; this is a form of transcriptional regulation widely used by prokaryotic organisms and extensively analyzed in prokaryotic systems, but poorly understood in higher eukaryotes . At any specific time, there is, of course, only one rate of transcription for each copy of the malic-enzyme gene in a cell . Our long-term objective is to understand how signals from all of the relevant regulatory pathways are integrated to bring about that rate. Microsc Res Tech, 1996 Jan 1, 33(1), 47 - 72 Microscale physiological and ecological studies of aquatic cyanobacteria: macroscale implications; Paerl HW; Cyanobacteria have had a profound and unparalleled biogeochemical impact on the earth's biosphere . As the first oxygenic phototrophs, cyanobacteria were responsible for the transition from anaerobic to aerobic life . Ironically, molecular oxygen (O2) is inhibitory to critical components of cyanobacterial metabolism, including photosynthesis and nitrogen fixation . Cyanobacteria have developed a great variety of biochemical, structural, and biotic adaptations ensuring optimal growth and proliferation in diverse oxic environments to counter this difficult situation . Structurally, cyanobacteria reveal remarkable diversity, including the formation of highly differentiated, O2-deplete cells (heterocysts), multicellularity as trichomes, and aggregates, that, among N2-fixing genera, facilitate division of labor between aerobic and anaerobic processes . Cyanobacteria enjoy unique consortial and symbiotic associations with other microorganisms, higher plants, and animals, in which O2 consumption is closely coupled in time and space to its production . Because as prokaryotes they are devoid of O2-consuming organelles (e.g., mitochondria), cyanobacteria have developed alternative strategies for locally protecting O2-sensitive processes, including consortial relationships with other microorganisms . Specific organic compounds released by cyanobacteria are capable of chemotactically attracting bacterial consorts, which in turn attach to the host cyanobacteria, consume O2, and recycle inorganic nutrients within the cyanobacterial "phycosphere." Multicellularity and aggregation lead to localized O2 gradients and hypoxic/anoxic microzones in which O2-sensitive processes can coexist . Microscale partitioning of O2-producing and O2-inhibited processes promotes contiguous and effective metabolite and nutrient exchange between these processes in oxygenated waters, representing a bulk of the world's oceanic and freshwater ecosystems. Microsc Res Tech, 1996 Jan 1, 33(1), 23 - 31 Filamentous sulfide-oxidizing bacteria at hydrocarbon seeps of the Gulf of Mexico; Larkin JM et al.; Mats consisting of the large sulfide-oxidizing bacterium, Beggiatoa, were collected from the sediment/water interface at several locations in the Gulf of Mexico . The collection sites were associated with the presence of petroleum hydrocarbons or the microbial breakdown products of the hydrocarbons . The morphologies of the mats varied with the nature of the underlying sediments, and some mats were pigmented either yellow or orange instead of the usual white . At one site, beggiatoas were found that had a diameter of nearly 200 mu m, making them the largest prokaryotic organism known . In filaments with a diameter of over approximately 10 mu m the cytoplasm was restricted to a thin layer immediately underlying the cell membrane, and the majority of the cell consisted of a vacuole with unknown contents . Beggiatoa filaments often rotated as they moved by gliding . Parallel rows of 15 nm diameter pores were found on the surface of the beggiatoas . The pores may have been wound in a spiral fashion around the cell . These pores may be involved in the gliding motility of the bacteria by the motion imparted by the excretion of slime through the pores . Several structures with the typical morphology of prokaryotic cells but lacking a cell wall were found within the vacuolar and cytoplasmic portions of the hollow beggiatoas . Some of these internal "symbionts" ultrastructurally resembled methanotrophic bacteria like those that have been seen in animals taken from vent areas . Other symbionts ultrastructurally resembled autotrophic bacteria with carboxysome-like structures . These internal symbionts may enable the Beggiatoa to grow in different environments on different carbon sources . They also provide important evidence for the endosymbiotic theory of the evolution of internal organelles of eukaryotic organisms. Planta, 1996, 199(4), 520 - 7 Regulation and molecular structure of a circadian oscillating protein located in the cell membrane of the prokaryote Synechococcus RF-1; Chen HM et al.; When a light/light-adapted culture of Synechococcus RF-1 is exposed to a diurnal light/dark regimen, the synthesis of more than ten of its polypeptides is known to become entrained to a circadian oscillating pattern which persists for some time under free-running conditions . One of the circadian oscillating polypeptides, COP23, was found to be located in the cell membrane . The rate of COP23 synthesis is controlled at the transcription level . In addition to the protein synthesis rate, the content of COP23 also exhibited a circadian rhythm . Pulse labeling with {35S}methionine revealed that COP23 was relatively stable in an arrhythmic culture . However, the exposure of Synechococcus RF-1 to a light/dark regimen induced not only a circadian synthesis rhythm, but also a rapid degradation of COP23 protein at a defined period of time . The induction of rapid protein degradation was prevented by the presence of chloramphenicol . The gene encoding the COP23 polypeptide has been cloned and sequenced . The amino acid sequence derived from the open-reading frame revealed that a signal peptide (28 amino acids) does not appear to be part of the mature COP23 . The mature COP23 does not have a membrane-associated segment, and it is suggested to be a peripheral molecule . With respect to their DNA base sequence and protein amino acid sequence, none of the proteins documented in the EMBL and PC/Gene data bases are significantly homologous with the COP23 molecule. Annu Rev Biochem, 1996, 65, 83 - 100 Selenocysteine; Stadtman TC; Selenocysteine is recognized as the 21st amino acid in ribosome-mediated protein synthesis and its specific incorporation is directed by the UGA codon . Unique tRNAs that have complementary UCA anticodons are aminoacylated with serine, the seryl-tRNA is converted to selenocysteyl-tRNA and the latter binds specifically to a special elongation factor and is delivered to the ribosome . Recognition elements within the mRNAs are essential for translation of UGA as selenocysteine . A reactive oxygen-labile compound, selenophosphate, is the selenium donor required for synthesis of selenocysteyl-tRNA . Selenophosphate synthetase, which forms selenophosphate from selenide and ATP, is found in various prokaryotes, eukaryotes, and archaebacteria . The distribution and properties of selenocysteine-containing enzymes and proteins that have been discovered to date are discussed . Artificial selenoenzymes such as selenosubtilisin have been produced by chemical modification . Genetic engineering techniques also have been used to replace cysteine residues in proteins with selenocysteine . The mechanistic roles of selenocysteine residues in the glutathione peroxidase family of enzymes, the 5' deiodinases, formate dehydrogenases, glycine reductase, and a few hydrogenases are discussed . In some cases a marked decrease in catalytic activity of an enzyme is observed when a selenocysteine residue is replaced with cysteine . This substitution caused complete loss of glycine reductase selenoprotein A activity. Annu Rev Biochem, 1996, 65, 43 - 81 DNA excision repair; Sancar A; In nucleotide excision repair DNA damage is removed through incision of the damaged strand on both sides of the lesion, followed by repair synthesis, which fills the gap using the intact strand as a template, and finally ligation . In prokaryotes the damaged base is removed in a 12-13 nucleotide (nt)-long oligomer; in eukaryotes including humans the damage is excised in a 24-32 nt-long fragment . Excision in Escherichia coli is accomplished by three proteins designated UvrA, UvrB, and UvrC . In humans, by contrast, 16 polypeptides including seven xeroderma pigmentosum (XP) proteins, the trimeric replication protein A {RPA, human single-stranded DNA binding protein (HSSB)}, and the multisubunit (7-10) general transcription factor TFIIH are required for the dual incisions . Transcribed strands are specifically targeted for excision repair by a transcription-repair coupling factor both in E . coli and in humans . In humans, excision repair is an important defense mechanism against the two major carcinogens, sunlight and cigarette smoke . Individuals defective in excision repair exhibit a high incidence of cancer while individuals with a defect in coupling transcription to repair suffer from neurological and skeletal abnormalities. Annu Rev Biochem, 1996, 65, 15 - 42 Relationships between DNA repair and transcription; Friedberg EC; Multiple relationships have been noted between DNA repair and transcription in both prokaryotic and eukaryotic cells . First, in both prokaryotes and eukaryotes nucleotide excision repair of the template strand of transcriptionally active regions of the genome is faster than in the coding strand . In prokaryotes the biochemical basis for this kinetic difference appears to be related to the specific coupling of repair to arrested transcription by RNA polymerase . The biochemical basis for strand-specific repair in eukaryotes is unknown . Second, in eukaryotes some or all of the subunits of transcription factor IIH (TFIIH) are required for nucleotide excision repair . The biological significance of this dual function of TFIIH proteins is not obvious . Finally, there are indications that the genes CSA and CSB, which are implicated in the human hereditary disease Cockayne syndrome, may have a role in transcription. Pathol Biol (Paris), 1996 Jan, 44(1), 29 - 35 Reactive oxygen intermediates as second messengers of a general pathogen response; Baeuerle PA et al.; Oxygen and derived ROIs became a threat for all organisms more than two billion years ago . Both prokaryotic and higher eukaryotic cells are able to alter their genetic programme in response to changes in the intracellular levels of reactive oxygen intermediates (ROIs) . In bacteria and yeast, this response leads to the new synthesis of proteins that protect the induced cells from the consequences of oxidative damage, such as DNA strand breaks, lipid peroxidation and oxidative damage of proteins, thereby ensuring growth in a prooxidant environment . In higher eukaryotic cells, oxidative stress can be the consequence of reoxygenation of ischemic tissus or of exposure to environmental hazards . Intriguingly, multicellular organisms have also evolved cellular mechanisms to actively produce ROIs . In one case, the reactive compounds are needed as weapons against invading microorganisms . Granulocytes, neutrophils and macrophages have specialized in releasing of large amounts of H2O2 and superoxide . However, many other cell types can also inducibly produce ROIs but in amounts insufficient to threat microorganisms . There is increasing evidence that the small increases in ROI levels fulfil a role as second messengers . We propose that these pandemic pathogens have been conserved throughout evolution as universal pathogen messengers turning on genes with important functions in the immune response and cell proliferation . The higher eukaryotic transcription factor NF-kappa B will be described as a protein which is activated by ROIs under a great variety of pathogenic conditions and initiates the new expression of genes with important roles in inflammatory, immune and acute phase responses. Gene, 1996, 173(1 Spec No), 39 - 44 Deletion mapping of the Aequorea victoria green fluorescent protein; Dopf J et al.; Aequorea victoria green fluorescent protein (GFP) is a promising fluorescent marker which is active in a diverse array of prokaryotic and eukaryotic organisms . A key feature underlying the versatility of GFP is its capacity to undergo heterocyclic chromophore formation by cyclization of a tripeptide present in its primary sequence and thereby acquiring fluorescent activity in a variety of intracellular environments . In order to define further the primary structure requirements for chromophore formation and fluorescence in GFP, a series of N- and C-terminal GFP deletion variant expression vectors were created using the polymerase chain reaction . Scanning spectrofluorometric analyses of crude soluble protein extracts derived from eleven GFP expression constructs revealed that amino acid (aa) residues 2-232, of a total of 238 aa in the native protein, were required for the characteristic emission and absorption spectra of native GFP . Heterocyclic chromophore formation was assayed by comparing the absorption spectrum of GFP deletion variants over the 300-500-nm range to the absorption spectra of full-length GFP and GFP deletion variants missing the chromophore substrate domain from the primary sequence . GFP deletion variants lacking fluorescent activity showed no evidence of heterocyclic ring structure formation when the soluble extracts of their bacterial expression hosts were studied at pH 7.9 . These observations suggest that the primary structure requirements for the fluorescent activity of GFP are relatively extensive and are compatible with the view that much of the primary structure serves an autocatalytic function. Prog Nucleic Acid Res Mol Biol, 1996, 53, 219 - 48 Eukaryotic gene expression: metabolite control by amino acids; Laine RO et al.; Our understanding of the metabolite control in mammalian cells lags far behind that in prokaryotes . This is particularly true for amino-acid-dependent gene expression . Few proteins have been identified for which synthesis is selectively regulated by amino-acid availability, and the mechanisms for control of transcription and translation in response to changes in amino-acid availability have not yet been elucidated . The intimate relationship between amino-acid supply and the fundamental cellular process of protein synthesis makes amino-acid-dependent control of gene expression particularly important . Future studies should provide important insight into amino-acid and other nutrient signaling pathways, and their impact on cellular growth and metabolism. Crit Rev Biotechnol, 1996, 16(2), 157 - 83 Luminescence-based systems for detection of bacteria in the environment; Prosser JI et al.; The development of techniques for detection and tracking of microorganisms in natural environments has been accelerated by the requirement for assessment of the risks associated with environmental release of genetically engineered microbial inocula . Molecular marker systems are particularly appropriate for such studies and luminescence-based markers have the broadest range of applications, involving the introduction of prokaryotic (lux) or eukaryotic (luc) genes for the enzyme luciferase . Lux or luc genes can be detected on the basis of unique DNA sequences by gene probing and PCR amplification, but the major advantage of luminescence-based systems is the ability to detect light emitted by marked organisms or by luciferase activity in cell-free extracts . Luminescent colonies can be detected by eye, providing distinction from colonies of indigenous organisms, and the sensitivity of plate counting can be increased greatly by CCD imaging . Single cells or microcolonies of luminescent organisms can also be detected in environmental samples by CCD image-enhanced microscopy, facilitating study of their spatial distribution . The metabolic activity of luminescence-marked populations can be quantified by luminometry and does not require extraction of cells or laboratory growth . Metabolic activity, and potential activity, of marked organisms therefore can be measured during colonization of soil particles and plant material in real time without disturbing the colonization process . In comparison with traditional activity techniques, luminometry provides significant increases in sensitivity, accuracy, and, most importantly, selectivity, as activity can be measured in the presence of indigenous microbial communities . The sensitivity, speed, and convenience of luminescence measurements make this a powerful technique that is being applied to the study of an increasingly wide range of ecological problems . These include microbial survival and recovery, microbial predation, plant pathogenicity, phylloplane and rhizosphere colonization and reporting of gene expression in environmental samples. Adv Biochem Eng Biotechnol, 1996, 54, 31 - 74 Analysis and modeling of substrate uptake and product release by prokaryotic and eukaryotic cells; Kramer R; Translocation of molecules and ions across cell membranes is an important step for a complete description of the metabolic network in terms of kinetics, energetics and control . With a few exceptions, most molecules cross the permeability barrier of the membrane with the aid of membrane-embedded carrier proteins . Uptake of nutrients (carbon, energy and nitrogen sources as well as supplements) and excretion of the majority of products are thus carrier-mediated transport processes . Consequently, they are characterized by particular kinetic properties of the respective carrier systems, they depend on energy sources (driving forces) which must be provided by the cell, and they are subject to regulation both on the level of activity and expression . They are thus fully integrated into the functional and regulatory networks of the cell . Structural (primary structure, conformation and topology) and functional properties (kinetics, energetics and regulation) of the different classes of carrier systems from both prokaryotic and eukaryotic membranes are summarized . The methodical requirements for a quantitative measurement of their function and possible pitfalls in transport studies are described, both for determination using isolated cells and for analysis in a bioreactor . The significance of transport reactions for biotechnological processes in general and for metabolic design in particular is discussed, with respect to nutrient uptake, product excretion and the occurrence of energy wasting combinations of transport reactions (futile cycles) . Some examples are given where transport reactions have been incorporated into modeling approaches with respect to metabolic control, to flux analysis, to kinetic properties and to energetic demands. Can J Microbiol, 1996 Jan, 42(1), 46 - 59 UV photoaffinity labeling of Tn3 transposase--DNA complexes: identification of DNA binding domains; Gottlieb GS et al.; The prokaryotic transposon Tn3 requires the transposase protein, as well as the cis-acting terminal inverted repeats (IRs), for transposition . The first step in the transposition process requires transposase binding to the IRs, as well as target site selection for element insertion . The primary aim of this study is to define the relationship between the structure of Tn3 transposase and its DNA binding functions . We have defined, by UV cross-linking, two broad regions of transposase that interact with DNA: a 70-kDa N-terminal domain and a 30-kDa C-terminal domain . The 70-kDa N-terminal domain encompasses the IR sequence specific binding domain, as well as a nonspecific DNA binding domain that has been previously described . We have also defined, by UV cross-linking, a region in the nonspecific DNA binding domain centered at amino acids 376 and 381 that is in contact with DNA . We have used site-directed mutagenesis of amino acids 376 and 381 to help delineate the function of this region of the transposase protein . Mutations in this region reduce transposition frequency to 30-40% of the wild type . These mutations reduce nonspecific DNA binding three- to four-fold but do not appear to affect specific binding to the IR . Transposition immunity is unaffected by mutations in the nonspecific DNA binding domain . This suggests that this region may be involved in target site selection. Nucleic Acids Res, 1996 Jan 1, 24(1), 73 - 5 Small RNA database; Gu J et al.; The small RNA database is a compilation of all the small size RNA sequences available to date from prokaryotic and eukaryotic organisms . About 500 small RNA sequences are in our database currently . The sources of individual RNAs and their GenBank accession numbers are also included . The small RNA database can be accessed through the World Wide Web(WWW) . Our WWW URL is html . The new small RNA sequences published since our last compilation are listed in this paper. Physiol Rev, 1996 Jan, 76(1), 31 - 47 Molecular mechanisms of iron uptake in eukaryotes; de Silva DM et al.; Iron serves essential functions in both prokaryotes and eukaryotes, and cells have highly specialized mechanisms for acquiring and handling this metal . The primary mechanism by which the concentration of iron in biologic systems is controlled is through the regulation of iron uptake . Although the role of transferrin in mammalian iron homeostasis has been well characterized, the study of genetic disorders of iron metabolism has revealed other, transferrin-independent, mechanisms by which cells can acquire iron . In an attempt to understand how eukaryotic systems take up this essential element, investigators have begun studying the simple eukaryote Saccharomyces cerevisiae . Several genes have been identified and cloned that act in concert to allow iron acquisition from the environment . Some of these genes appear to have functional homologues in human systems . This review focuses on the recent developments in understanding eukaryotic iron uptake with an emphasis on the genetic and molecular characterization of these systems in both cultured mammalian cells and S . cerevisiae . An unexpected connection between iron and copper homeostasis has been revealed by recent genetic studies, which confirm biologic observations made several decades ago. Infect Immun, 1996 Jan, 64(1), 262 - 8 A glyceraldehyde-3-phosphate dehydrogenase homolog in Borrelia burgdorferi and Borrelia hermsii; Anda P et al.; A polyreactive monoclonal antibody recognized a 38.5-kDa polypeptide with amino-terminal sequence identity to conserved regions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Borrelia burgdorferi, the Lyme disease agent, and Borrelia hermsii, an agent of American relapsing fever . This monoclonal antibody also recognized GAPDH from other pathogenic spirochetes and other prokaryotes and eukaryotes as well . GAPDH activity was detected in sonicates of both B . burgdorferi and B . hermsii but not in live, intact organisms, indicating the possibility of a subsurface localization for the Borrelia GAPDH activity . Degenerate primers constructed from highly conserved regions of gapdh of other prokaryotes successfully amplified this gene homolog in both B . burgdorferi and B . hermsii . Nuclei acid and deduced amino acid sequence analysis of the 838-bp probes for each borrelia indicated 93.9% identity between B . burgdorferi and B . hermsii at the amino acid level . Amino acid identities of B . burgdorferi and B . hermsii with Bacillus stearothermophilus were 59.2% and 58.8% respectively . Southern hybridization studies indicated that the gene encoding GAPDH is located on the chromosome of each borrella . In other bacterial species, GAPDH has other functions in addition to its traditional enzymatic role in the glycolytic pathway . GAPDH may play a similar role in borrelias. Arch Biochem Biophys, 1996 Jan 1, 325(1), 1 - 7 A second molybdoprotein aldehyde dehydrogenase from Amycolatopsis methanolica NCIB 11946; Kim SW et al.; Methanol-grown Amycolatopsis methanolica NCIB 11946 contains a molybdoprotein dehydrogenase, active with aldehydes and formate esters as substrates and with Wurster's blue as electron acceptor, the so-called formate ester dehydrogenase (FEDH) (van Ophem et al., 1992, Eur . J . Biochem . 206, 519-525) . It appears now that another molybdoprotein dehydrogenase is present in this organism . This enzyme, indicated here as dye-linked aldehyde dehydrogenase (DL-AlDH), has the same set of cofactors and converts the same type of substrates but with different specificity, and uses 2,6-dichlorophenol-indophenol as sole artificial electron acceptor for those conversions . The enzymes also differ in their quaternary structure, FEDH having an alpha, beta, gamma and DL-AlDH having an alpha, beta, gamma 2 composition . Furthermore, differences exist with respect to the sizes and the N-terminal amino acid sequences of their subunits, indicating that the enzymes derive from different genes . However, neither their substrate specificity nor their induction pattern give a clear indication for distinct physiological roles . Just like other bacterial molybdoprotein dehydrogenases, DL-AlDH consists of three different subunits (87, 35, and 17 kDa) and contains FAD, molybdopterin-cytosine-dinucleotide cofactor, Fe, and acid-labile sulfide in a molar ratio of 1:1:4:4 . Although eukaryotic xanthine oxidase and dehydrogenase differ from these prokaryotic dehydrogenases in size and number of their subunits, certain stretches of amino acid sequences show similarity and the magnetic coupling between the Mo and the {2Fe-2S}-1 cluster in DL-AlDH and bovine milk xanthine oxidase is of the same magnitude . In view of this similarity, the topology of the cofactors in the active site of this type of molybdoproteins might be conserved among enzymes from prokaryotic as well as eukaryotic organisms. J Bacteriol, 1996 Jan, 178(2), 332 - 9 Primary structure of cyanelle peptidoglycan of Cyanophora paradoxa: a prokaryotic cell wall as part of an organelle envelope; Pfanzagl B et al.; The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa is thought to be a relic of their cyanobacterial ancestors . The separation of muropeptides by gel filtration and reverse-phase high-performance liquid chromatography revealed four different muropeptide monomers . A number of muropeptides were identical in retention behavior to muropeptides of Escherichia coli, while others had remarkably long retention times with respect to their sizes, as indicated by gel filtration . Molecular mass determination by plasma desorption and matrix-assisted laser desorption ionization mass spectrometry showed that these unusual muropeptides had molecular masses greater by 112 Da or a multiple thereof than those of ones common to both species . Fast atom bombardment-tandem mass spectrometry of these reduced muropeptide monomers allowed the localization of the modification to D-glutamic acid . High-resolution fast atom bombardment-mass spectrometry and amino acid analysis revealed N-acetylputrescine to be the substituent (E . Pittenauer, E . R . Schmid, G . Allmaier, B . Pfanzagl, W . Loffelhardt, C . Quintela, M . A . de Pedro, and W . Stanek, Biol . Mass Spectrom . 22:524-536, 1993) . In addition to the 4 monomers already known, 8 dimers, 11 trimers, and 6 tetramers were characterized . An average glycan chain length of 51 disaccharide units was determined by the transfer of {U-14C}galactose to the terminal N-acetylglucosamine residues of cyanelle peptidoglycan . The muropeptide pattern is discussed with respect to peptidoglycan biosynthesis and processing. J Bacteriol, 1996 Jan, 178(1), 136 - 42 Cloning, purification, and properties of a phosphotyrosine protein phosphatase from Streptomyces coelicolor A3(2); Li Y et al.; We describe the isolation and characterization of a gene (ptpA) from Streptomyces coelicolor A3(2) that codes for a protein with a deduced M(r) of 17,690 containing significant amino acid sequence identity with mammalian and prokaryotic small, acidic phosphotyrosine protein phosphatases (PTPases) . After expression of S . coelicolor ptpA in Escherichia coli with a pT7-7-based vector system, PtpA was purified to homogeneity as a fusion protein containing five extra amino acids . The purified fusion enzyme catalyzed the removal of phosphate from p-nitrophenylphosphate (PNPP), phosphotyrosine (PY), and a commercial phosphopeptide containing a single phosphotyrosine residue but did not cleave phosphoserine or phosphothreonine . The pH optima for PNPP and PY hydrolysis by PtpA were 6.0 and 6.5, respectively . The Km values for hydrolysis of PNPP and PY by PtpA were 0.75 mM (pH 6.0, 37 degrees C) and 2.7 mM (pH 6.5, 37 degrees C), respectively . Hydrolysis of PNPP by S . coelicolor PtpA were 0.75 mM (pH 6.0, 37 degrees C) and 2.7 mM (pH 6.5, 37 degrees C), respectively . Hydrolysis of PNPP by S . coelicolor PtpA was competitively inhibited by dephostatin with a Ki of 1.64 microM; the known PTPase inhibitors phenylarsine oxide, sodium vanadate, and iodoacetate also inhibited enzyme activity . Apparent homologs of ptpA were detected in other streptomycetes by Southern hybridization; the biological functions of PtpA and its putative homologs in streptomycetes are not yet known. Mol Cell Biol, 1996 Jan, 16(1), 130 - 4 A newly formed telomere in Ascaris suum does not exert a telomere position effect on a nearby gene; Huang YJ et al.; During the process of chromatin diminution in Ascaris suum (formerly named Ascaris lumbricoides var . suum), developmentally regulated chromosomal fragmentation and new telomere addition occur within specific chromosomal breakage regions (CBRs) . The DNA sequences flanking one of these CBRs (CBR-1) were analyzed, and two protein-encoding genes were found on either side . The noneliminated gene, agp-1, whose AUG start codon is located within approximately 2 kb of the boundary of CBR-1, encodes a putative GTP-binding protein which is structurally related to eukaryotic and prokaryotic elongation factors . Northern (RNA) blot analyses revealed that transcripts of this gene are present at all developmental stages, suggesting that the massive chromosomal rearrangements associated with the process of chromatin diminution have no influence on agp-1 expression . This demonstrates that addition of new telomeres in CBR-1 does not result in a telomeric position effect, a phenomenon previously described in Saccharomyces cerevisiae. Gene, 1995 Dec 29, 167(1-2), 203 - 7 Arabidopsis expresses two genes that encode polypeptides similar to the yeast RNA polymerase I and III AC40 subunit; Ulmasov T et al.; A 40-kDa subunit in eukaryotic RNA polymerases (Pol) I and III (e.g., yeast yAC40) is related in a part of its aa sequence to the alpha subunit of prokaryotic Pol and to a 35-44-kDa subunit in Pol II (e.g., yeast yB44) . We have cloned two cDNAs, AtRPAC42 and AtRPAC43, from an Arabidopsis thaliana (At) (ecotype Columbia) lambda Yes expression library that encode Pol I and III subunits related to yAC40 . The aa sequences derived from the cDNA clones were found to be 72% identical to each other and 40% identical to yeast Pol I and III subunits yAC40, but only 30% identical to yeast Pol II subunit yB44 . While most other nuclear Pol genes identified to date are single-copy genes, two genes encode 42 and 43-kDa subunits of At Pol I and/or III . A 42-kDa subunit with identical mobility in SDS-PAGE to the aAC42 in vitro translated subunit is found in Pol III purified from At suspension culture cells. J Biol Chem, 1995 Dec 29, 270(52), 31077 - 82 Site-directed mutagenesis of evolutionary conserved carboxylic amino acids in the chitosanase from Streptomyces sp . N174 reveals two residues essential for catalysis; Boucher I et al.; The comparison of four sequences of prokaryotic chitosanases, belonging to the family 46 of glycosyl hydrolases, revealed a conserved N-terminal module of 50 residues, including five invariant carboxylic residues . To verify if some of these residues are important for catalytic activity in the chitosanase from Streptomyces sp . N174, these 5 residues were replaced by site-directed mutagenesis . Substitutions of Glu-22 or Asp-40 with sterically conservative (E22Q, D40N) or functionally conservative (E22D, D40E) residues reduced drastically specific activity and kcat, while Km was only slightly changed . The other residues examined, Asp-6, Glu-36, and Asp-37, retained significant activity after mutation . Circular dichroism studies of the mutant chitosanases confirmed that the observed effects are not due to changes in secondary structure . These results suggested that Glu-22 and Asp-40 are directly involved in the catalytic center of the chitosanase and the other residues are not essential for catalytic activity. J Biol Chem, 1995 Dec 29, 270(52), 31065 - 70 Rhodoquinone and complex II of the electron transport chain in anaerobically functioning eukaryotes; Van Hellemond JJ et al.; Many anaerobically functioning eukaryotes have an anaerobic energy metabolism in which fumarate is reduced to succinate . This reduction of fumarate is the opposite reaction to succinate oxidation catalyzed by succinate-ubiquinone oxidoreductase, complex II of the aerobic respiratory chain . Prokaryotes are known to contain two distinct enzyme complexes and distinct quinones, menaquinone and ubiquinone (Q), for the reduction of fumarate and the oxidation of succinate, respectively . Parasitic helminths are also known to contain two different quinones, Q and rhodoquinone (RQ) . This report demonstrates that RQ was present in all examined eukaryotes that reduce fumarate during anoxia, not only in parasitic helminths, but also in freshwater snails, mussels, lugworms, and oysters . It was shown that the measured RQ/Q ratio correlated with the importance of fumarate reduction in vivo . This is the first demonstration of the role of RQ in eukaryotes, other than parasitic helminths . Furthermore, throughout the development of the liver fluke Fasciola hepatica, a strong correlation was found between the quinone composition and the type of metabolism: the amount of Q was correlated with the use of the aerobic respiratory chain, and the amount of RQ with the use of fumarate reduction . It can be concluded that RQ is an essential component for fumarate reduction in eukaryotes, in contrast to prokaryotes, which use menaquinone in this process . Analyses of enzyme kinetics, as well as the known differences in primary structures of prokaryotic and eukaryotic complexes that reduce fumarate, support the idea that fumarate-reducing eukaryotes possess an enzyme complex for the reduction of fumarate, structurally related to the succinate dehydrogenase-type complex II, but with the functional characteristics of the prokaryotic fumarate reductases. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12495 - 9 Intron phase correlations and the evolution of the intron/exon structure of genes; Long M et al.; Two issues in the evolution of the intron/exon structure of genes are the role of exon shuffling and the origin of introns . Using a large data base of eukaryotic intron-containing genes, we have found that there are correlations between intron phases leading to an excess of symmetric exons and symmetric exon sets . We interpret these excesses as manifestations of exon shuffling and make a conservative estimate that at least 19% of the exons in the data base were involved in exon shuffling, suggesting an important role for exon shuffling in evolution . Furthermore, these excesses of symmetric exons appear also in those regions of eukaryotic genes that are homologous to prokaryotic genes: the ancient conserved regions . This last fact cannot be explained in terms of the insertional theory of introns but rather supports the concept that some of the introns were ancient, the exon theory of genes. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12357 - 61 Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases; Li X et al.; We previously characterized a methionine aminopeptidase (EC 3.4.11.18; Met-AP1; also called peptidase M) in Saccharomyces cerevisiae, which differs from its prokaryotic homologues in that it (i) contains an N-terminal zinc-finger domain and (ii) does not produce lethality when disrupted, although it does slow growth dramatically; it is encoded by a gene called MAP1 . Here we describe a second methionine aminopeptidase (Met-AP2) in S . cerevisiae, encoded by MAP2, which was cloned as a suppressor of the slow-growth phenotype of the map1 null strain . The DNA sequence of MAP2 encodes a protein of 421 amino acids that shows 22% identity with the sequence of yeast Met-AP1 . Surprisingly, comparison with sequences in the GenBank data base showed that the product of MAP2 has even greater homology (55% identity) with rat p67, which was characterized as an initiation factor 2-associated protein but not yet shown to have Met-AP activity . Transformants of map1 null cells expressing MAP2 in a high-copy-number plasmid contained 3- to 12-fold increases in Met-AP activity on different peptide substrates . The epitope-tagged suppressor gene product was purified by immunoaffinity chromatography and shown to contain Met-AP activity . To evaluate the physiological significance of Met-AP2, the MAP2 gene was deleted from wild-type and map1 null yeast strains . The map2 null strain, like the map1 null strain, is viable but with a slower growth rate . The map1, map2 double-null strains are nonviable . Thus, removal of N-terminal methionine is an essential function in yeast, as in prokaryotes, but yeast require two methionine aminopeptidases to provide the essential function which can only be partially provided by Met-AP1 or Met-AP2 alone. J Biol Chem, 1995 Dec 15, 270(50), 29889 - 93 A base substitution within the GTPase-associated domain of mammalian 28 S ribosomal RNA causes high thiostrepton accessibility; Uchiumi T et al.; A molecular basis for the insensitivity of eukaryotic ribosomes to the antibiotic thiostrepton was investigated using synthetic 100-nucleotide-long fragments covering the GTPase domain of 23/28 S rRNA . Filter binding assay showed no detectable binding of the rat RNA to thiostrepton, but the binding capacity was markedly increased by base substitution of G1878 to A at the position corresponding to 1067 of Escherichia coli 23 S rRNA . The association constant (K alpha) for the rat A 1878 mutant was 0.60 x 10(6) M-1, which was comparable with that of the E . coli RNA (K alpha = 1.1 x 10(6) M-1) . This suggests that the eukaryotic G 1878 participates in the resistance for thiostrepton . On the other hand, the RNA fragments of the two species had a similar binding capacity for E . coli ribosomal protein L11 and its mammalian homologue L12 . Gel electrophoresis under a high ionic condition, however, revealed a difference between the two proteins . E . coli L11 formed stable complexes with both the E . coli RNA and the rat A 1878 mutant RNA in the presence of thiostrepton, while rat L12 failed to exhibit such complex formation . This suggests that the eukaryotic L12 protein may also be an element giving the resistance for thiostrepton . These results are discussed in terms of preserved three-dimensional conformation of the RNA backbone between prokaryotes and higher eukaryotes. Science, 1995 Dec 15, 270(5243), 1807 - 9 An ethylene-inducible component of signal transduction encoded by never-ripe; Wilkinson JQ et al.; The ripening-impaired tomato mutant Never-ripe (Nr) is insensitive to the plant hormone ethylene . The gene that cosegregates with the Nr locus encodes a protein with homology to the Arabidopsis ethylene receptor ETR1 but is lacking the response regulator domain found in ETR1 and related prokaryotic two-component signal transducers . A single amino acid change in the sensor domain confers ethylene insensitivity when expressed in transgenic tomato plants . Modulation of NR gene expression during fruit ripening controls response to the hormone ethylene. Biochim Biophys Acta, 1995 Dec 7, 1259(3), 305 - 12 Molecular cloning and expression of rat hepatic neutral cholesteryl ester hydrolase; Ghosh S et al.; The 1923 bp cDNA for rat hepatic cholesteryl ester hydrolase (CEH) was cloned by screening a lambda gt11 expression library with an oligonucleotide containing the consensus active site sequence for cholesteryl esterases . Expression of a fusion protein, cross-reacting with antibody to the purified liver CEH, was demonstrated by Western blot analysis . The cDNA was sequenced and found to have only 44% homology with pancreatic CEH . Although unique, the cDNA sequence exhibited much greater overall homology with liver carboxylesterases, in both coding and 5'/3' non-coding regions . In Northern blot analysis, the cDNA hybridized with a single band from liver mRNA but not with pancreatic mRNA . The 1.7 kb coding sequence, predicting a 62 kDa protein, was cloned into an Escherichia coli expression system with an inducible promoter and into COS-7 cells . Both expression systems produced a protein which comigrated with liver CEH (66 kDa) on SDS-PAGE and immunoreacted with antibodies to liver CEH on Western blots . Whereas the prokaryotic system produced an inactive protein, expression in COS-7 cells was accompanied by a 5-fold increase in CEH activity and a corresponding increase in immunoreactive protein. Biochim Biophys Acta, 1995 Dec 6, 1253(2), 208 - 14 Functional analysis of E . coli threonine dehydrogenase by means of mutant isolation and characterization; Chen YW et al.; The oxidation of L-threonine to 2-amino-ketobutyrate, as catalyzed by L-threonine dehydrogenase, is the first step in the major pathway for threonine catabolism in both eukaryotes and prokaryotes . Threonine dehydrogenase of E . coli has considerable amino-acid sequence homology with a number of Zn(2+)-containing, medium-chain alcohol dehydrogenases . In order to further explore structure/function interrelationships of E . coli threonine dehydrogenase, 35 alleles of tdh that imparted a no-growth or slow-growth phenotype on appropriate indicator media were isolated after mutagenesis with hydroxylamine . Within this collection, 14 mutants had single amino-acid changes that were divided into 4 groups: (a) amino-acid changes associated with proposed ligands to Zn2+; (b) a substitution of one of several conserved glycine residues; (c) mutations at the substrate or coenzyme binding site; (d) alterations that resulted in a change of charge near the active site . These findings uncover previously unidentified amino-acid residues that are important for threonine dehydrogenase catalysis and also indicate that the three-dimensional structure of tetrameric E . coli threonine dehydrogenase has considerable similarity with the dimeric horse liver alcohol dehydrogenase. Protein Eng, 1995 Dec, 8(12), 1259 - 66 Residues important for the function of a multihelical DNA binding domain in the new transcription factor family of Cam and Tet repressors; Aramaki H et al.; We report that some prokaryotic repressors including CamR and TetR belong to the same family . CamR and TetR bind to DNA using a multihelical DNA binding domain (DBD) at the N-termini of the proteins, while the C-termini are important for regulating the DNA binding in a manner dependent on their co-factors (camphor for CamR, tetracycline for TetR) . In all, 11 important amino acid positions have been identified in the CamR DBD by the systematic substitution of residues by Ala . Of the 11 positions, 10 are either buried in the core, and thus important for creating the hydrophobic environment, or exposed on the surface, and thus important for binding to DNA . The eleventh residue, Gly, seems to be important for a loop structure . The DNA binding mode of this type of DBD and a general mechanism of regulating their DNA binding are discussed in reference to the crystal structure of TetR {Hinrichs et al., (1994) Science, 264, 418-420}. J Gen Virol, 1995 Dec, 76 ( Pt 12), 3229 - 32 Replication slippage in the evolution of potyviruses; Hancock JM et al.; Recently published evidence for sequence repetition in potyvirus genomes prompted us to analyse the published complete genome sequences and coat protein gene sequences of viruses of this family for evidence of replication slippage . Five of nine complete genomic sequences and 17 of 32 coat protein genes had significant sequence repetitions . Most of these were in coat protein genes, although the 5' region of the turnip mosaic virus genome also showed evidence of slippage . The results suggest that replication slippage may be involved in the evolution of viruses, as well as prokaryotes and eukaryotes, and that slippage can occur in both RNA and DNA when it is being replicated. Mol Microbiol, 1995 Dec, 18(5), 933 - 41 Inducible transposition in Streptomyces lividans of insertion sequence IS6100 from Mycobacterium fortuitum; Smith B et al.; Transposition of IS6100, originally isolated as part of the compound transposon Tn610 from Mycobacterium fortuitum, was tested in the related actinomycete Streptomyces lividans . Cointegrate formation was observed, as expected for this IS6-related element, and involved apparent random integration of the temperature-sensitive vector carrying IS6100 and concomitant duplication of the insertion sequence . This establishes that a single copy of the insertion sequence can promote transposition and is a precedent for the functioning of a heterologous transposable element in Streptomyces . Transposition could be induced 100-fold by external transcription emanating from a copy of the thiostrepton-inducible promoter ptipA located outwith the insertion sequence and resulting in overexpression of the transposase gene . Thus, in contrast to other prokaryotic transposable elements, IS6100 appears to have no effective means of protecting itself from external activation. Mol Microbiol, 1995 Dec, 18(5), 831 - 40 A TnphoA insertion within the Bradyrhizobium japonicum sipS gene, homologous to prokaryotic signal peptidases, results in extensive changes in the expression of PBM-specific nodulins of infected soybean (Glycine max) cells; Muller P et al.; Bradyrhizobium japonicum mutant 132 was obtained by random TnphoA mutagenesis of strain 110spc4 . A 6.5 kb BamHI kanamycin-resistance-coding DNA fragment of mutant 132 was used as a hybridization probe to clone the corresponding wild-type fragment . DNA sequence analysis of a 3213 bp BamHI-ClaI fragment revealed that three open reading frames (ORFs) were encoded in the same orientation . Based on sequence similarities to other proteins in the database, the second ORF was called sipS (signal peptidase) . The TnphoA insertion in mutant 132 was found to be in frame near the 3' end of sipS . The resulting SipS-PhoA hybrid polypeptide was shown to be expressed in free-living B . japonicum and in Escherichia coli cultures . An immunoblot analysis with a polyclonal antibody directed against the alkaline phosphatase revealed the appearance of a weak signal of a 70 kDa polypeptide both in B . japonicum and in E . coli, in agreement with the calculated size of the hybrid polypeptide . A much stronger 52 kDa band was also detected . Mutant 132 was specifically disturbed in the interaction with soybean (Glycine max) when the bacteria were released from the infection threads . The bacteroids were not stably maintained within the symbiosome . Numerous vesicles were found in the plant cytosol, which finally resulted in the formation of large vacuoles within the infected nodule cells . Immunohistochemical analyses with antibodies directed against nodulins of the peribacteroid membrane indicated a lower expression of these proteins . The mutant phenotype was genetically complemented by a 4.4 kb BamHI fragment including sipS . Subfragments thereof also complemented a temperature-sensitive E . coli lepB mutant, demonstrating that the B . japonicum fragment was functionally replacing Lepts in E . coli . Genetic studies suggested that the three genes are organized in one common operon which is expressed from a promoter upstream of the sequenced region . Inactivation of the gene downstream of sipS did not result in a detectable phenotype. Curr Opin Genet Dev, 1995 Dec, 5(6), 779 - 85 Evolution of ATP-binding cassette transporter genes; Dean M et al.; The transport of molecules across lipid membranes is an essential function of all living organisms . One of the families of genes that have evolved to carry out this function is that which encodes the ATP-binding cassette proteins . These molecules use active transport to pump specific molecules across membranes, and the genes that encode them are found in abundance in the genomes of both prokaryotes and eukaryotes . By using gene disruption techniques and by studying homologous genes in model organisms, significant progress has been made during the last few years in evaluating the physiological functions of ABC proteins in higher eukaryotes. Indian J Biochem Biophys, 1995 Dec, 32(6), 417 - 23 Contextual constraints in the choice of synonymous codons; Kolaskar AS et al.; From EMBL Nucleotide Sequence Database, protein coding sequences of all E . coli and its DNA phages, were extracted using our computer programme . Same programme has been used to form a database of sequence of oligonucleotides of length 18 nucleotides on both sides of each of the 61 codons . From analysis of this database and study of variations in twist parameter (Tw) values, as an indicator of sequence dependent variations in B-DNA helix, a method is developed to fix the codon among the set of synonymous codons . The accuracy of the method was checked on enlarged data set by adding data from more prokaryotes . Our method assign the codon 85-90% times correctly if the selection has to be made between codons having different sequence in terms of R and Y . The accuracy of the method is somewhat lower when choice of the codon has to be made between codons having same codes in terms of R and Y . This study points out that the major factors which decide the choice of a codon from a set of synonymous codons are contextual constraints arising from flanking regions. J Clin Invest, 1995 Dec, 96(6), 2569 - 77 Autoantibodies against heat shock protein 60 mediate endothelial cytotoxicity; Schett G et al.; Stress or heat shock proteins (hsp) are a family of approximately two dozen proteins with a high degree of amino acid sequence homology between different species, ranging from prokaryotes to humans, and are representative of a generalized response to environmental and metabolic stressors . Our previous studies showed increased expression of human hsp60 on endothelial cells of arterial intima with atherosclerotic lesions, and elevated levels of serum antibodies (Ab) against hsp65/60 in subjects with carotid atherosclerosis . To investigate the possible involvement of anti-hsp65/60 Ab in endothelial injury, specific hsp-Ab were isolated from human high titer sera by affinity chromatography and probed on heat-shock human umbilical vein endothelial cells . Purified human anti-hsp65/60 Ab reacted specifically with mycobacterial hsp65, human hsp60, and a 60-kD protein band of heat-shocked endothelial cells . High levels of hsp60 mRNA expression in endothelial cells were found between 4 and 12 h after 30 min treatment at 42 degrees C . In immunofluorescence tests, positive staining of heat-stressed endothelial cells was observed not only in the cytoplasm but also on the cell surface . Furthermore, only heat-stressed, but not untreated, Cr-labeled endothelial cells were lysed by anti-hsp65/60 Ab in the presence of complement (complement-mediated cytotoxicity) or peripheral blood mononuclear cells (antibody-dependent cellular cytotoxicity) . Control Abs, including human anti-hsp65/60 low titer antiserum, human Ig fraction deprived of hsp65/60 Ab, and mAbs to Factor VIII, alpha-actin, hsp70, and CD3 showed no cytotoxic effect . In conclusion, human serum anti-hsp65 antibodies act as autoantibodies reacting with hsp60 on stressed endothelial cells and are able to mediate endothelial cytotoxicity . Thus, a humoral immune reaction to hsp60 may play an important role in the pathogenesis of atherosclerosis. Plant Mol Biol, 1995 Dec, 29(6), 1235 - 52 Increased transcript levels of a methionine synthase during adhesion-induced activation of Chlamydomonas reinhardtii gametes; Kurvari V et al.; Chlamydomonas gametes of opposite mating types interact through flagellar adhesion molecules called agglutinins leading to a signal transduction cascade that induces cell wall loss and activation of mating structures along with other cellular responses that ultimately result in zygote formation . To identify molecules involved in these complex cellular events, we have employed subtractive and differential hybridization with cDNA from mt+ gametes activated for fertilization and non-signaling, vegetative (non-gametic) cells . We identified 55 cDNA clones whose transcripts were regulated in activated gametes . Here we report the molecular cloning and characterization of the complementary DNA (cDNA) for one clone whose transcripts in activated gametes were several-fold higher than in normal gametes . Regulation of the transcript was not related simply to protein synthesis because it was not increased in cells synthesizing new cell wall proteins . The cDNA contained a single open reading frame (ORF) of 815 amino acids encoding a polypeptide of calculated relative mass of 87 kDa . Database search analysis and sequence alignment indicated that the deduced amino acid sequence exhibited 42% identity and 62% similarity to a class of prokaryotic methyl transferases (5-methyltetrahydrofolate-homocysteine methyl transferase; EC 2.1.1.14) known to be involved in the terminal step of de novo biosynthesis of methionine . This enzyme catalyzes transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine resulting in methionine formation . Affinity-purified polyclonal antibodies raised against a bacterially produced GST-fusion protein identified a 85 kDa soluble protein in Chlamydomonas gametes . Southern blot hybridization indicated that the enzyme is encoded by a single-copy gene . The evidence presented in this paper raises the possibility that, in addition to its participation in de novo biosynthesis and regeneration of methionine, Chlamydomonas methionine synthase may play a role in adhesion-induced events during fertilization. Mol Mar Biol Biotechnol, 1995 Dec, 4(4), 275 - 83 Future of biotechnology-based control of disease in marine invertebrates; Mialhe E et al.; Infectious disease is the single most devastating problem in mollusc and shrimp aquaculture . Pathogens causing the greatest problems have been identified as viruses, prokaryotes, and protozoans . Two approaches employing methods of biotechnology have been proposed to prevent, manage, and control mollusc and shrimp diseases . The first is development of a diagnostic scheme for detection and identification of pathogens, using molecular probes . This offers the opportunity for prophylactic measures to be taken . Molecular probes have been prepared for the major pathogens of molluscs, but in the case of shrimp pathogens, only a few are available . Monoclonal antibodies have also been prepared and are used in immunodiagnosis, e.g., immunofluorescence detection . Such diagnostic tools are relatively new to aquaculture, but have enormous potential . A second approach to the control of disease in marine invertebrates, notably shrimp, involves use of genetically transformed strains resistant to specific pathogens . Pathogen-resistant transgenic animals have been developed, but such research has only just begun for molluscs and shrimp . Transfection methods applied to mollusc and shrimp embryos have been successful, with preliminary data showing efficiency of heterologous promoters in controlling expression of reporter genes . Other transformation systems also show promise, including transposable elements and densoviruses. Biophys Chem, 1995 Dec, 57(1), 71 - 92 Condensation and cohesion of lambda DNA in cell extracts and other media: implications for the structure and function of DNA in prokaryotes; Murphy LD et al.; DNA added to concentrated extracts of Escherichia coli undergoes a reversible transition to a readily-sedimentable ('condensed') form . The transition occurs over a relatively small increment in extract concentration . The extract appears to play two roles in this transition, supplying both DNA-binding protein(s) and a crowded environment that increases protein binding and favors compact DNA conformations . The two roles of the extract are suggested by properties of fractions prepared by absorption of extracts with DNA-cellulose . The DNA-binding fraction and the DNA-nonbinding fractions from these columns are separately poorer condensing agents than the original extract, but when rejoined are similar to the original extract in the amount required for condensation . The dual role for the extract is supported by model studies of condensation with combinations of purified DNA-binding materials (protein HU or spermidine) and concentrated solutions of crowding agents (albumin or polyethylene glycol 8000); in each case, crowding agents and DNA-binding materials jointly reduce the amounts of each other required for condensation . The condensation reaction as studied in extracts or in the purified systems may be a useful approach to the forces which stabilize the compact form of DNA within the bacterial nucleoid . The effect of condensation on the reactivity of the DNA was measured by changes in the rate of cohesion between duplex DNA molecules bearing the complementary single-strand termini of lambda DNA . Condensation caused large increases in the rates of cohesion of both lambda DNA and of restriction fragments of lambda DNA bearing the cohesive termini . Cohesion products of lambda DNA made in vitro are a mixture of linear and circular aggregates, whereas those made in vivo are cyclic monomers . We suggest a simple mechanism based upon condensation at the site of viral injection which may explain this discrepancy. Microbiol Rev, 1995 Dec, 59(4), 604 - 22 Nitrogen control in bacteria; Merrick MJ et al.; Nitrogen metabolism in prokaryotes involves the coordinated expression of a large number of enzymes concerned with both utilization of extracellular nitrogen sources and intracellular biosynthesis of nitrogen-containing compounds . The control of this expression is determined by the availability of fixed nitrogen to the cell and is effected by complex regulatory networks involving regulation at both the transcriptional and posttranslational levels . While the most detailed studies to date have been carried out with enteric bacteria, there is a considerable body of evidence to show that the nitrogen regulation (ntr) systems described in the enterics extend to many other genera . Furthermore, as the range of bacteria in which the phenomenon of nitrogen control is examined is being extended, new regulatory mechanisms are also being discovered . In this review, we have attempted to summarize recent research in prokaryotic nitrogen control; to show the ubiquity of the ntr system, at least in gram-negative organisms; and to identify those areas and groups of organisms about which there is much still to learn. J Bacteriol, 1995 Dec, 177(24), 7150 - 4 MlpA, a lipoprotein required for normal development of Myxococcus xanthus; Hanlon WA et al.; The mlpA gene encoding a 236-residue polypeptide has been identified immediately downstream of the oar gene of Myxococcus xanthus (M . Martinez-Canamero, J . Munoz-Dorado, E . Farez-Vidal, M . Inouye, and S . Inouye, J . Bacteriol . 175:4756-4763, 1993) . The amino-terminal 21 residues of MlpA encode a typical prokaryotic signal sequence with a putative lipoprotein cleavage site . When expressed in Escherichia coli in the presence of {2-3H}glycerol, 3H-labeled MlpA had a molecular mass of 33 kDa and was found to be associated with the membrane fraction . Globomycin, an inhibitor of signal peptidase II, caused a shift in the mobility of E . coli-expressed MlpA to 35 kDa . Subsequently, a mlpA disruption strain (oar+) was constructed and found to have delayed fruiting body formation (by approximately 36 h), with significantly larger fruiting bodies being produced compared with those of the wild-type strain . Nevertheless, spore yields for the two strains were identical after 120 h of development . These data indicate that MlpA, the lipoprotein identified in M . xanthus, is required for normal fruiting body formation. J Infect Dis, 1995 Dec, 172(6), 1431 - 6 Epitope type-specific IgG responses to capsid proteins VP1 and VP2 of human parvovirus B19; Soderlund M et al.; Temporal reactivities of IgG towards native and linear antigenic determinants in assembled capsids or isolated structural proteins of human parvovirus B19 were measured by an epitope type-specific IgG EIA and by immunoblots . Antigens used were baculovirus-expressed B19 capsids composed of the proteins VP1 and VP2 in their native proportion, VP2 alone, or a prokaryotic VP1 fusion protein . Follow-up sera after primary infection were compared with samples from previously infected persons . The IgG responses during acute and early convalescence phases were directed towards both conformational and linear epitopes of VP2 . The antibodies against the linear VP2 epitopes disappeared abruptly within 6 months; however, the conformational VP2 antibodies persisted . The epitope type-specific IgG reactivity of VP1 was strikingly different from that of VP2 . On the basis of these results, two novel tests were developed for patient diagnosis . Both tests are suitable for verifying the time of human parvovirus infection. Genes Dev, 1995 Dec 1, 9(23), 2903 - 10 The tetratricopeptide repeats of Ssn6 interact with the homeo domain of alpha 2; Smith RL et al.; The tetratricopeptide repeat (TPR) is a 34-amino-acid degenerate sequence motif that is found in a large variety of proteins, both prokaryotic and eukaryotic . TPRs are usually found in tandem arrays of up to 16 copies . In this paper we identify a direct interaction between the TPRs of Ssn6, a general transcriptional repressor, and alpha 2, a cell-type regulator in Saccharomyces cerevisiae . Five of the Ssn6 TPRs were tested individually, and all were found to interact specifically with alpha 2 . These results suggest a model for TPR-protein interactions and for the role that a tandem array of TPRs may have in mediating transcriptional repression. Mol Cell Endocrinol, 1995 Nov 30, 115(1), 1 - 11 Preparation and characterization of recombinant prolactin receptor extracellular domain from rat; Sandowski Y et al.; Complementary (c)DNA of the extracellular domain of rat prolactin receptor (rPRLR-ECD) was cloned in the prokaryotic expression vector pTrc99A, and expressed in Escherichia coli following induction with isopropyl-b-D-thiogalactopyranoside . The expressed rPRLR-ECD protein, contained within the refractile body pellet was solubilized in 4.5 M urea, refolded and purified on a Q-Sepharose column by stepwise elution with NaCl . Only approximately 10% of the expressed protein refolded as a monomeric fraction, yielding 5-6 mg/l of induced culture . The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent, and by chromatography on a Superdex column . Its molecular mass, determined by SDS-PAGE in the absence of reducing agent, was 28 kDa, and by gel filtration, 25.6 kDa . Binding experiments indicated high affinity for bovine placental lactogen (bPL) and human growth hormone (hGH) as compared to ovine (o) or rat PRLs . Gel filtration was used to determine the stoichiometry of rPRLR-ECD's interaction with these hormones . At a 5 microM initial concentration of the hormones, formation of 2:1 (ECD:ligand) complexes was detected with bPL, hGH and oPRL whereas only 1:1 complex was formed with rPRL . Dilution (25-fold) of these complexes did not affect the stoichiometry with bPL, whereas with hGH a clear tendency towards dissociation of the initial 2:1 complex to 1:1 complex was observed . This tendency was even stronger in the case of oPRL . Although all four hormones exhibited nearly identical activities in the Nb2-11C lymphoma cell bioassay, the ability of the purified rat or rabbit PRLR-ECD to inhibit hormonal mitogenic activity generally reflected their affinity for the respective hormones . In view of these and former results, we suggest that unlike in the GH:GHR-ECD interaction, the inability of lactogenic hormones to form a 1:2 complex with soluble recombinant PRLR-ECDs does not necessarily predicts lack of biological activity. Nucleic Acids Res, 1995 Nov 25, 23(22), 4712 - 6 5' contexts of Escherichia coli and human termination codons are similar; Arkov AL et al.; The nearest 5' context of 2559 human stop codons was analysed in comparison with the same context of stop-like codons (UGG, UGC, UGU, CGA for UGA; CAA, UAU, UAC for UAA; and UGG, UAU, UAC, CAG for UAG) . The non-random distribution of some nucleotides upstream of the stop codons was observed . For instance, uridine is over-represented in position -3 upstream of UAG . Several codons were shown to be over-represented immediately upstream of the stop codons: UUU(Phe), AGC(Ser), and the Lys and Ala codon families before UGA; AAG(Lys), GCG(Ala), and the Ser and Leu codon families before UAA; and UCA(Ser), AUG(Met), and the Phe codon family before UAG . In contrast, the Thr and Gly codon families were under-represented before UGA, while ACC(Thr) and the Gly codon family were under-represented before UAG and UAA respectively . In an earlier study, uridine was shown to be over-represented in position -3 before UGA in Escherichia coli {Arkov,A.L., Korolev,S.V . and Kisselev,L.L . (1993) Nucleic Acids Res., 21,2891-2897} . In that study, the codons for Lys, Phe and Ser were shown to be over-represented immediately upstream of E . coli stop codons . Consequently, E . coli and human termination codons have similar 5' contexts . The present study suggests that the 5' context of stop codons may modulate the efficiency of peptide chain termination and (or) stop codon readthrough in higher eukaryotes, and that the mechanisms of such a modulation in prokaryotes and higher eukaryotes may be very similar. J Mol Biol, 1995 Nov 24, 254(2), 247 - 59 Solution structure of the ribosome-binding domain of E . coli translation initiation factor IF3 . Homology with the U1A protein of the eukaryotic spliceosome; Garcia C et al.; Initiation of translation in prokaryotes requires the formation of a complex between the messenger RNA, the 30 S ribosomal subunit and the initiator tRNA(fMet) . Initiation factor IF3 binds to the 30 S ribosomal subunit and proof-reads the initiation complex, thereby ensuring the accuracy of this step . IF3 also plays a pleiotropic role in the regulation of translation, as a result of differential influences exerted on the levels of the initiation of translation of genes or groups of genes . IF3 is composed of two independent domain or roughly identical sizes . We have expressed and purified the C-terminal domain of E . coli IF3 and shown that it retains both the 30 S particle binding and 70 S ribosome dissociating activities of the native protein . We have obtained 1H and 15N NMR resonance assignments and its 3D solution structure was calculated using 551 restraints . It is composed of a mixed beta-sheet backed by two alpha-helices . It shows a striking resemblance to the U1A small nuclear ribonucleoprotein structure, which binds to the U1 snRNA in the eukaryotic spliceosome . This suggests a convergent evolution process for these two proteins that are associated with ribonucleoproteic complexes. Proc Natl Acad Sci U S A, 1995 Nov 21, 92(24), 11317 - 21 Inferring phylogenies from DNA sequences of unequal base compositions; Galtier N et al.; A new method for computing evolutionary distances between DNA sequences is proposed . Contrasting with classical methods, the underlying model does not assume that sequence base compositions (A, C, G, and T contents) are at equilibrium, thus allowing unequal base compositions among compared sequences . This makes the method more efficient than the usual ones in recovering phylogenetic trees from sequence data when base composition is heterogeneous within the data set, as we show by using both simulated and empirical data . When applied to small-subunit ribosomal RNA sequences from several prokaryotic or eukaryotic organisms, this method provides evidence for an early divergence of the microsporidian Vairimorpha necatrix in the eukaryotic lineage. Proc Natl Acad Sci U S A, 1995 Nov 21, 92(24), 11019 - 23 Mismatch repair in Escherichia coli enhances instability of (CTG)n triplet repeats from human hereditary diseases; Jaworski A et al.; Long CTG triplet repeats which are associated with several human hereditary neuromuscular disease genes are stabilized in ColE1-derived plasmids in Escherichia coli containing mutations in the methyl-directed mismatch repair genes (mutS, mutL, or mutH) . When plasmids containing (CTG)180 were grown for about 100 generations in mutS, mutL, or mutH strains, 60-85% of the plasmids contained a full-length repeat, whereas in the parent strain only about 20% of the plasmids contained the full-length repeat . The deletions occur only in the (CTG)180 insert, not in DNA flanking the repeat . While many products of the deletions are heterogeneous in length, preferential deletion products of about 140, 100, 60, and 20 repeats were observed . We propose that the E . coli mismatch repair proteins recognize three-base loops formed during replication and then generate long single-stranded gaps where stable hairpin structures may form which can be bypassed by DNA polymerase during the resynthesis of duplex DNA . Similar studies were conducted with plasmids containing CGG repeats; no stabilization of these triplets was found in the mismatch repair mutants . Since prokaryotic and human mismatch repair proteins are similar, and since several carcinoma cell lines which are defective in mismatch repair show instability of simple DNA microsatellites, these mechanistic investigations in a bacterial cell may provide insights into the molecular basis for some human genetic diseases. J Biol Chem, 1995 Nov 17, 270(46), 27876 - 9 Systematic introduction of proline in a eukaryotic signal sequence suggests asymmetry within the hydrophobic core; Ryan P et al.; The hydrophobic core or h region of both prokaryotic and eukaryotic signal sequences is the predominant structural domain that controls the efficiency of protein translocation across membranes . Characteristically, hydrophobic cores appear to assume alpha-helical conformations, and studies in prokaryotes have indicated that this conformation is necessary for efficient signal sequence function . To address the conformational constraints of a eukaryotic signal sequence, we have introduced a single proline in almost each position of the signal sequence hydrophobic core of glycoprotein C (gC) of the swine herpesvirus, pseudorabies virus . When the resulting mutant virus strains were used to infect cells, we found that substitution of proline at certain positions affected gC translocation greater than its introduction at other sites within the hydrophobic core . The observed positional effects did not completely correlate with reductions in overall hydrophobicity or linear position within the hydrophobic core . Rather, it appeared that one face of the gC signal sequence alpha-helix is far more sensitive to proline disruption than the other, potentially indicating a functional asymmetry. J Biol Chem, 1995 Nov 10, 270(45), 27277 - 82 Identification, isolation, and cloning of a Bacillus thuringiensis CryIAc toxin-binding protein from the midgut of the lepidopteran insect Heliothis virescens; Gill SS et al.; Bacillus thuringiensis toxins are insecticidal to a variety of insect species . The selectivity of the toxins produced by these bacteria is dependent on both the toxin structure and the receptor sites that are present in different insect species . One of these toxins, CryIAc, is highly insecticidal to the noctuid pest Heliothis virescens . Using toxin overlay assay, a 120-kDa glycoprotein was identified as a toxin-binding protein . This protein was partially purified, its N-terminal sequence was determined, and the full-length cDNA encoding this protein was isolated from a H . virescens midgut library . The B . thuringiensis toxin-binding protein, BTBP1, has high homology to aminopeptidase N from eukaryotes and prokaryotes. J Biol Chem, 1995 Nov 10, 270(45), 27079 - 86 Topogenesis of cytochrome oxidase subunit II . Mechanisms of protein export from the mitochondrial matrix; Herrmann JM et al.; Cytochrome c oxidase subunit II (COXII) in yeast mitochondria is synthesized as a precursor (preCOXII) and is sorted across the inner membrane, whereby both N and C termini become exposed to the intermembrane space . We describe here how this process can be experimentally dissected into a number of distinct stages . Our results demonstrate that the translation of COXII is not obligatorily coupled to translocation . Insertion into the inner membrane and export of the N- and C-terminal domains require an energized inner membrane . The export of COXII is independent of both maturation by the Imp1p protease and assembly into the cytochrome c oxidase complex . When linked to a mitochondrial matrix-targeting sequence, the N-terminal portion of preCOXII (fused to mouse dihydrofolate reductase) can be imported into the mitochondrial matrix . Following accumulation in the matrix, this chimeric protein can become exported across the inner membrane, delivering the N terminus into the intermembrane space where it undergoes processing by the Imp1p protease . This export process displays a number of similarities to bacterial protein export and supports the view that the principles of sorting are conserved from prokaryotes to eukaryotic organelles. Biochim Biophys Acta, 1995 Nov 7, 1264(2), 155 - 8 A secY homologous gene in the crenarchaeon Sulfolobus acidocaldarius; Kath T et al.; The nucleotide sequence of an open reading frame, located upstream of the gene for adenylate kinase, was determined in the thermoacidophile crenarchaeon Sulfolobus acidocaldarius . Data bank searches identified the sequence as a secY homologous gene . The DNA derived protein sequence of total 463 amino acids contains 10 hydrophobic domains . A sequence alignment with other prokaryotic and eukaryotic secY sequences reveals significant homology, but the secY primary sequence of S . acidocaldarius shows only a low degree of similarity with the secY counterparts of the euryarchaea Methanococcus vannielii and Haloarcula marismortui . A transcription analysis indicates, that the secY gene is cotranscribed with the gene coding for adenylate kinase. Mol Microbiol, 1995 Nov, 18(4), 649 - 60 Mycoplasma hyorhinis vlp gene transcription: critical role in phase variation and expression of surface lipoproteins; Citti C et al.; Mycoplasma hyorhinis contains clustered vlp genes encoding variable lipoproteins (Vlps), the major coat proteins and surface antigens of this wall-less prokaryotic pathogen . vlp genes are subject to discrete, high frequency mutations independently affecting the size or the expression of variant Vlp products . Change in Vlp size occurs by mutations altering the number of tandem intragenic repeats at the 3' end of each single-copy vlp gene . In this report, phase-variant Vlp expression is shown to result from altered vlp gene transcription . vlpA, vlpB and vlpC transcripts were monitored in a clonal lineage selected to display various Vlp phenotypes . Each vlp gene was expressed as a distinct transcript, which was subject to drastic ON/OFF switches associated with random insertion/ deletion mutations in a homopolymeric tract of adenine residues in the promoter region of all vlp genes . Unexpectedly, the level of vlp transcripts appeared to depend on the length of the corresponding genes in the ON configuration . Higher proportional levels of shorter vlp transcripts were shown to reflect a greater abundance of short Vlp lipoproteins present in L-{35S}-cysteine-labelled membrane protein preparations . The vlp cluster provides a heritable, and highly mutable, locus for the generation of surface diversity through random promoter mutations affecting the expression of genes, whose products also vary in length and abundance, by virtue of separate mutations in structural regions of the genes. Med Hypotheses, 1995 Nov, 45(5), 476 - 80 Tyrosine phosphorylation as a possible regulatory mechanism in the expression of human immunodeficiency virus genes; Nandi J et al.; Phosphorylation of proteins on serine, threonine and tyrosine is one of the significant regulatory mechanisms in gene expression and post-translational modifications in both eukaryotes and prokaryotes . Protein tyrosine phosphorylation in particular is implicated in cell proliferation, differentiation and certain pathological modifications including transformation . The overall protein tyrosine phosphorylation is modulated by protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) . There are several viruses known to contain PTK and PTPs . A computer-based protein sequence search using the FAST P programme was used to investigate whether, theoretically, a sequence for a putative protein tyrosine phosphatase is present in the genomic sequence of the human immunodeficiency virus (HIV) . A conserved motif GXGXXG characteristic of both PTK and PTP was found at the 5' LTR region of the HIV genome . Interesting sequence similarities with regulatory proteins of other retroviruses, viz . VPx of HIV-2 and X-protein of HTLV-1, and some transforming proteins were also observed . The implication of the possible phosphorylation event in association with the HIV regulatory proteins tat, rev and nef in AIDS-related malignancies is discussed. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1113 - 22 Regulation of translation termination: conserved structural motifs in bacterial and eukaryotic polypeptide release factors; Nakamura Y et al.; Translation termination requires codon-dependent polypeptide release factors . The mechanism of stop codon recognition by release factors is unknown and holds considerable interest since it entails protein-RNA recognition rather than the well-understood mRNA-tRNA interaction in codon-anticodon pairing . Bacteria have two codon-specific release factors and our picture of prokaryotic translation is changing because a third factor, which stimulates the other two, has now been found . Moreover, a highly conserved eukaryotic protein family possessing properties of polypeptide release factor has now been sought . This review summarizes our current understanding of the structural and functional organization of release factors as well as our recent findings of highly conserved structural motifs in bacterial and eukaryotic polypeptide release factors. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1095 - 103 Translational termination efficiency in both bacteria and mammals is regulated by the base following the stop codon; Tate WP et al.; The translational stop signal and polypeptide release factor (RF) complexed with Escherichia coli ribosomes have been shown to be in close physical contact by site-directed photochemical cross-linking experiments . The RF has a protease-sensitive site in a highly conserved exposed loop that is proposed to interact with the peptidyltransferase center of the ribosome . Loss of peptidyl-tRNA hydrolysis activity and enhanced codon-ribosome binding by the cleaved RF is consistent with a model whereby the RF spans the decoding and peptidyltransferase centers of the ribosome with domains of the RF linked by conformational coupling . The cross-link between the stop signal and RF at the ribosomal decoding site is influenced by the base following the termination codon . This base determines the efficiency with which the stop signal is decoded by the RF in both mammalian and bacterial systems in vivo . The wide range of efficiencies correlates with the frequency with which the signals occur at natural termination sites, with rarely used weak signals often found at recoding sites and strong signals found in highly expressed genes . Stop signals are found at some recoding sites in viruses where -1 frame-shifting occurs, but the generally accepted mechanism of simultaneous slippage from the A and P sites does not explain their presence here . The HIV-1 gag-pol-1 frame shifting site has been used to show that stop signals significantly influence frame-shifting efficiency on prokaryotic ribosomes by a RF-mediated mechanism . These data can be explained by an E/P site simultaneous slippage mechanism whereby the stop codon actually enters the ribosomal A site and can influence the event. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1079 - 86 Termination of translation in eukaryotes; Kisselev LL et al.; Termination of translation is governed in ribosomes by polypeptide chain release factors (pRF and eRF in prokaryotes and eukaryotes, respectively) . In prokaryotes, three pRF have been indentified and sequenced, while in eukaryotes, only a single eRF has been identified to date . Recently, we have characterized a highly conserved protein family called eRF1 . At least, human and Xenopus laevis proteins from this family are active as eRFs in the in vitro assay with any of the three stop codons . No structural similarity has been revealed between any of the three pRFs and eRF1 family . Furthermore, GTP-binding motifs have not been revealed, although translation termination in eukaryotes is a GTP-dependent process . We have demonstrated that in eukaryotes a second eRF exists in addition to eRF1, called eRF3 . The eRF3 family has two features in common: presence of GTP-binding motifs and high conservation of the C-terminal domain structure . The C-terminal domain of the X . laevis eRF3 has no RF activity although it stimulates the eRF1 activity considerably at low concentration of the stop codons, conferring GTP dependence to the termination reaction . Without eRF3, the eRF1 activity is entirely GTP independent . Some features of X . laevis eRF3 (C-terminal domain) resemble those of pRF3 . The newly identified eRF1 and eRF3 are structurally conserved and distinct from the respective pRF1/2 and pRF3 proteins, pointing to the possibility of different evolution of translation termination machinery in prokaryotes and eukaryotes . Bipartition of the translation termination apparatus probably provides high rate and accuracy of translation termination. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 979 - 86 Structures of prokaryotic ribosomal proteins: implications for RNA binding and evolution; Ramakrishnan V et al.; After a long hiatus, the pace of determination of the structures of ribosomal proteins has accelerated dramatically . We discuss here the structures of five ribosomal proteins from Bacillus stearothermophilus: S5, S17, L6, L9, and L14 . These structures represent several new motifs . Each of these structures has revealed new insights, and we have developed criteria for recognizing RNA-binding regions of each protein and correlating the structures with such properties as antibiotic resistance . The information here should also prove invaluable in an eventual high-resolution picture of the intact ribosome. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 915 - 24 The pseudouridine residues of ribosomal RNA; Ofengand J et al.; Pseudouridine (psi), the most common single modified nucleoside in ribosomal RNA, has been positioned in the small subunit (SSU) and large subunit (LSU) RNAs of a number of representative species . Most of the information has been obtained by application of a rapid primed reverse transcriptase sequencing technique . The locations of these psi residues have been compared . Many sites for psi are the same among species, but others are distinct . In general, the percentage psi in multicellular eukaryotes is greater than in prokaryotes . In LSU RNA, the psi residues are strongly clustered in three domains, all of which are near or connected to the peptidyl transferase center . There is no apparent clustering of psi in SSU RNA . The psi sites in LSU RNA overlap those for the methylated nucleosides, but this is not the case in SSU RNA . There are 265 psi sites known to nucleotide resolution, of which 246 are in defined secondary structures, and 112 of these are in nonidentical structural contexts . All 246 psi sites can be classified into five structural types . Two Escherichia coli psi synthases have been cloned and characterized, one for psi 516 in SSU RNA and one for psi 746 in LSU RNA . The psi 746 synthase recognizes free RNA, but the psi 516 enzyme requires an intermediate RNP particle . Possible functional roles for psi in the ribosome are discussed. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 887 - 97 A proposal for the conformation of loop E in Escherichia coli 5S rRNA; Dallas A et al.; A proposal is advanced for the conformation of the loop E region of prokaryotic 5S rRNAs based on spectroscopic data obtained from pAD3 RNA, a construct that includes helix IV, helix V, and loops D and E from Escherichia coli 5S rRNA . Even though loop E juxtaposes bases that cannot form Watson-Crick base pairs, it resembles an A-form double helix; its nucleotides relate to each other spectroscopically in a helix-like way and are in the anti conformation . The ends of loop E, which is palindromic, have the same conformation . Working in from either end towards the center of the loop, a closing GC is followed by a side-by-side GA and then by a reversed Hoogsteen AU, a pattern resembling that found at one end of eukaryotic loop E . The center of the loop consists of three nucleotide pairs, which appear to be an asymmetric GG pair, a Watson-Crick-like AG, and a GU stabilized by a single hydrogen bond. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 845 - 58 Small nucleolar RNA; Gerbi SA; A growing list of small nucleolar RNAs (snoRNAs) has been characterized in eukaryotes . They are transcribed by RNA polymerase II or III; some snoRNAs are encoded in the introns of other genes . The nonintronic polymerase II transcribed snoRNAs receive a trimethylguanosine cap, probably in the nucleus, and move to the nucleolus . snoRNAs are complexed with proteins, sometimes including fibrillarin . Localization and maintenance in the nucleolus of some snoRNAs requires the presence of initial precursor rRNA (pre-rRNA) . Many snoRNAs have conserved sequence boxes C and D and a 3' terminal stem; the role of these features are discussed . Functional assays done for a few snoRNAs indicate their roles in rRNA processing for cleavage of the external and internal transcribed spacers (ETS and ITS) . U3 is the most abundant snoRNA and is needed for cleavage of ETS1 and ITS1; experimental results on U3 binding sites in pre-rRNA are reviewed . 18S rRNA production also needs U14, U22, and snR30 snoRNAs, whereas U8 snoRNA is needed for 5.8S and 28S rRNA production . Other snoRNAs that are complementary to 18S or 28S rRNA might act as chaperones to mediate RNA folding . Whether snoRNAs join together in a large rRNA processing complex (the "processome") is not yet clear . It has been hypothesized that such complexes could anchor the ends of loops in pre-rRNA containing 18S or 28S rRNA, thereby replacing base-paired stems found in pre-rRNA of prokaryotes. Mikrobiologiia, 1995 Nov-Dec, 64(6), 756 - 61 {Protective and reactive effect of a cellular extract from propionic acid bacteria on Candida guilliermondii and Escherichia coli, inactivated by ultraviolet radiation}; Fraikin GIa et al.; Dialyzed soluble proteins of Propionibacterium freudenreichii subsp . shermanii cell extract (dialyzate) were found to exert a protective and reactivating effect on Candida guilliermondii and Escherichia coli cells inactivated by UV light of C and B ranges . Reactivation occurred when dialyzate was added to irradiated bacterial suspensions immediately after irradiation or 15 min afterwards . Some common features of dialyzate effect on irradiated prokaryotic and eukaryotic cells were established . No reactivation was observed when the cells were irradiated with the light of visible (400-600 nm) or entire optical (> 290 nm) ranges . The possible mechanisms of a reactivating effect of dialyzate are discussed. Biotechniques, 1995 Nov, 19(5), 784 - 6, 788-90 Identification and isolation of human epithelial cell colonies that express specific gene products; Gibson-D'Ambrosio RE et al.; A new and simple method for the identification and isolation of human cell clones expressing specific gene products is presented . This technique is analogous to the colony blotting techniques described for prokaryotes . In this technique, human epithelial cells are grown in tissue-culture dishes, and cells from the colonies on the dish are transferred in situ to a high-density cationized quaternary amine-charged nylon membrane . The membrane can then be processed multiple times, using antibody and/or nucleic acid probes, for the identification of those cells producing the desired protein, mRNA or DNA sequence . Once identified, the desired cell colony is isolated for expansion, direct DNA sequencing and/or cell function . We demonstrate the potential of the technique by identifying and isolating colonies of replicating normal human liver hepatocytes producing albumin and keratin. Arch Microbiol, 1995 Nov, 164(5), 309 - 23 Genetics of gliding motility and development in Myxococcus xanthus; Hartzell PL et al.; Successful development in multicellular eukaryotes requires cell-cell communication and the coordinated spatial and temporal movements of cells . The complex array of networks required to bring eukaryotic development to fruition can be modeled by the development of the simpler prokaryote Myxococcus xanthus . As part of its life cycle, M . xanthus forms multicellular fruiting bodies containing differentiated cells . Analysis of the genes essential for M . xanthus development is possible because strains with mutations that block development can be maintained in the vegetative state . Development in M . xanthus is induced by starvation, and early events in development suggest that signaling stages have evolved to monitor the metabolic state of the developing cell . In the absence of these signals, which include amino acids, alpha-keto acids, and other intermediary metabolites, the ability of cells to differentiate into myxospores is impaired . Mutations that block genes controlling gliding motility disrupt the morphogenesis of fruiting bodies and sporogenesis in surprising ways . In this review, we present data that encourage future genetic and biochemical studies of the relationships between motility, cell-cell signaling, and development in M . xanthus. Plant Physiol, 1995 Nov, 109(3), 797 - 802 Formation of the ferritin iron mineral occurs in plastids; Waldo GS et al.; Ferritin in plants is a nuclear-encoded, multisubunit protein found in plastids; an N-terminal transit peptide targets the protein to the plastid, but the site for formation of the ferritin Fe mineral is unknown . In biology, ferritin is required to concentrate Fe to levels needed by cells (approximately 10(-7) M), far above the solubility of the free ion (10(-18) M); the protein directs the reversible phase transition of the hydrated metal ion in solution to hydrated Fe-oxo mineral . Low phosphate characterizes the solid-phase Fe mineral in the center of ferritin of the cytosolic animal ferritin, but high phosphate is the hallmark of Fe mineral in prokaryotic ferritin and plant (pea {Pisum sativum L.} seed) ferritin . Earlier studies using x-ray absorption spectroscopy showed that high concentrations of phosphate present during ferritin mineralization in vivo altered the local structure of Fe in the ferritin mineral so that it mimicked the prokaryotic type, whether the protein was from animals or bacteria . The use of x-ray absorption spectroscopy to analyze the Fe environment in pea-seed ferritin now shows that the natural ferritin mineral in plants has an Fe-P interaction at 3.26A, similar to that of bacterial ferritin; phosphate also prevented formation of the longer Fe-Fe interactions at 3.5A found in animal ferritins or in pea-seed ferritin reconstituted without phosphate . Such results indicate that ferritin mineralization occurs in the plastid, where the phosphate content is higher; a corollary is the existence of a plastid Fe uptake system to allow the concentration of Fe in the ferritin mineral. Microbiology, 1995 Nov, 141 ( Pt 11), 3005 - 14 Expression studies on four members of the pMGA multigene family in Mycoplasma gallisepticum S6; Glew MD et al.; A large family of related genes known as pMGA exists in the avian pathogen Mycoplasma gallisepticum but only a single member of this family was previously found to be expressed in one strain of this bacterium . In this work two unrelated strains of M . gallisepticum were also shown by amino-terminal sequencing to express a unique pMGA polypeptide in both cases . To investigate pMGA gene selection in M . gallisepticum, mRNA expression was analysed in M . gallisepticum strain 56 using reverse transcription-PCR (RT-PCR) and Northern blot techniques with probes for several members of the pMGA multigene family . It was shown that the pMGA message is 2.2 kb in size and is monocistronic . RT-PCR detected four different pMGA mRNA molecules but their relative yields were significantly affected by magnesium concentration . By quantitative Northern analysis, the relative abundances of the four pMGA mRNAs in M . gallisepticum S6 total RNA was determined: the pMGA1.1 mRNA predominated {1.88 ng (micrograms total RNA)-1} but at least three other pMGA genes were found to be transcribed but at much lower levels (20 to 40-fold lower) . The pMGA1.1 mRNA is expressed at a level five times higher than the tuf gene, known to be one of the most abundantly expressed proteins in the prokaryotic cell . The start point of transcription for pMGA1.1 was determined and probable promoter assigned . From these data it appears likely that transcriptional control plays a major role in the selection of pMGA gene expression in the M . gallisepticum cell. Microbiology, 1995 Nov, 141 ( Pt 11), 2861 - 71 Molecular cloning of a Coxiella burnetii gene encoding a macrophage infectivity potentiator (Mip) analogue; Mo YY et al.; The gene encoding a protein that reacted with antibodies specific for Legionella pneumophila macrophage infectivity potentiator (LpMip) was cloned from Coxiella burnetii, the obligate intracellular rickettsia that causes Q fever in humans . Nucleotide sequencing analysis revealed an ORF encoding a gene product of 230 amino acids with a molecular mass of 25.5 kDa and a predicted pI of 10.7 . The predicted amino acid sequence from the ORF shows similarity with Mip/Mip-like proteins of Legionella (46%) and Chlamydia (30%) . Moreover, like LpMip, the amino acid sequence of the C terminus of this protein has over 35% identity to prokaryotic and eukaryotic FK506-binding proteins (FKBPs) that belong to a superfamily of immunophilins and are peptidyl-prolyl cis-trans isomerases (PPIases) . When overproduced in Escherichia coli, the C . burnetii protein also exhibited PPIase activity . Taken together, these results demonstrate that C . burnetii encodes a Mip analogue (CbMip) . A putative leader peptide at the N terminus of CbMip was detected by computer analysis . Furthermore, TnphoA mutagenesis demonstrated that in E . coli CbMip was secreted . In view of the role of Mip/Mip-like proteins in the pathogenesis of Legionella and Chlamydia, CbMip may be a C . burnetii virulence factor. Bioessays, 1995 Nov, 17(11), 959 - 65 Stress signaling in yeast; Ruis H et al.; In the yeast Saccharomyces cerevisiae three positive transcriptional control elements are activated by stress conditions: heat shock elements (HSEs), stress response elements (STREs) and AP-1 responsive elements (AREs) . HSEs bind heat shock transcription factor (HSF), which is activated by stress conditions causing accumulation of abnormal proteins . STREs mediate transcriptional activation by multiple stress conditions . They are controlled by high osmolarity via the HOG signal pathway, which comprises a MAP kinase module and a two-component system homologous to prokaryotic signal transducers . AREs bind the transcription factor Yap1p . The three types of control elements seem to have overlapping, but distinct functions . Some stress proteins encoded by HSE-regulated genes are necessary for growth of yeast under moderate stress, products of STRE-activated genes appear to be important for survival under severe stress and ARE-controlled genes may mainly function during oxidative stress and in the response to toxic conditions, such as caused by heavy metal ions. Appl Environ Microbiol, 1995 Nov, 61(11), 4074 - 82 In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities; Hodson RE et al.; Obtaining information on the genetic capabilities and phylogenetic affinities of individual prokaryotic cells within natural communities is a high priority in the fields of microbial ecology, microbial biogeochemistry, and applied microbiology, among others . A method for prokaryotic in situ PCR (PI-PCR), a technique which will allow single cells within complex mixtures to be identified and characterized genetically, is presented here . The method involves amplification of specific nuclei acid sequences inside intact prokaryotic cells followed by color or fluorescence detection of the localized PCR product via bright-field or epifluorescence microscopy . Prokaryotic DNA and mRNA were both used successfully as targets for PI-PCR . We demonstrate the use of PI-PCR to identify nahA-positive cells in mixtures of bacterial isolates and in model marine bacterial communities. J Bacteriol, 1995 Nov, 177(21), 6211 - 22 A new Escherichia coli cell division gene, ftsK; Begg KJ et al.; A mutation in a newly discovered Escherichia coli cell division gene, ftsK, causes a temperature-sensitive late-stage block in division but does not affect chromosome replication or segregation . This defect is specifically suppressed by deletion of dacA, coding for the peptidoglycan DD-carboxypeptidase, PBP 5 . FtsK is a large polypeptide (147 kDa) consisting of an N-terminal domain with several predicted membrane-spanning regions, a proline-glutamine-rich domain, and a C-terminal domain with a nucleotide-binding consensus sequence . FtsK has extensive sequence identity with a family of proteins from a wide variety of prokaryotes and plasmids . The plasmid proteins are required for intercellular DNA transfer, and one of the bacterial proteins (the SpoIIIE protein of Bacillus subtilis) has also been implicated in intracellular chromosomal DNA transfer. EMBO J, 1995 Nov 1, 14(21), 5170 - 8 A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans; Schrempf H et al.; We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of an up to now unique prokaryotic potassium ion channel (KcsA) . It has a rectifying current-voltage relationship and displays subconductance states, the largest of which amounts to A approximately equal to 90 pS . The channel is blocked by Cs- ions and gating requires the presence of Mg2+ ions . The kcsA gene has been identified in the gram-positive soil bacterium Streptomyces lividans . It encodes a predicted 17.6 kDa protein with two potential membrane-spanning helices linked by a central domain which shares a high degree of similarity with the H5 segment conserved among eukaryotic ion channels . Multiple alignments of deduced amino acids suggest that the novel channel has the closest kinship to the S5, H5 and S6 regions of voltage-gated K+ channel families, mainly to the subfamily represented by the Shaker protein from Drosophila melanogaster . Moreover, KcsA is most distantly related to eukaryotic inwardly rectifying channels with two putative predicted transmembrane segments. Biochem J, 1995 Nov 1, 311 ( Pt 3), 717 - 21 Human diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase is a member of the MutT family of nucleotide pyrophosphatases; Thorne NM et al.; The cDNA and derived amino acid sequence of human diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase have been determined with the aid of the GenBank Expressed Sequence Tag database . This enzyme possesses a modification of the MutT sequence motif found in certain nucleotide pyrophosphatases . It is unrelated to the enzymes of diadenosine tetraphosphate catabolism found in prokaryotes and fungi. Gene, 1995 Oct 27, 164(2), 341 - 5 A high-level prokaryotic expression system: synthesis of human interleukin 1 alpha and its receptor antagonist; Birikh KR et al.; Synthetic intronless genes, coding for human interleukin 1 alpha (IL 1 alpha) and interleukin 1 receptor antagonist (IL1ra), have been expressed efficiently in a specially designed prokaryotic vector, pGMCE (a pGEM1 derivative), where the target gene forms the second part of a two-cistron system . The first part of the system is a translation enhancer-containing mini-cistron, whose termination codon overlaps the start codon of the target gene . In the case of the IL1 alpha gene, the high expression level is largely due to the direct efficient translation initiation at the second cistron, whereas with the IL1ra gene in the same system, the proximal translation initiation region (TIR) provides a high level of coupled expression of the target gene . Thus, pGMCE is a potentially versatile vector for direct prokaryotic expression. Gene, 1995 Oct 27, 164(2), 203 - 9 A human antibody specific for hepatitis C virus core protein: synthesis in a bacterial system and characterization; Esposito G et al.; The cDNA coding for the Fab fragment of the human B12.F8 antibody (Ab), directed against the putative nucleocapsid component (core protein) of hepatitis C virus (HCV), was cloned in the prokaryotic phagemid vector, pHEN-1, to obtain its expression in Escherichia coli . The functionality and specificity of the recombinant Ab, called B12Fab, were examined by Western blot and ELISA using recombinant HCV core protein as antigen . The specificity of B12Fab was further confirmed by ELISA with the 33-mer peptide epitope recognized by the original whole B12.F8 Ab . By immunofluorescence, the recombinant B12Fab was shown to recognize HCV core protein produced in cells transfected with HCV cDNA, indicating that the recombinant B12Fab is suitable as a diagnostic tool for tissue localization of the virus . The B12Fab also functioned when displayed on phage particles, providing the basis for future experiments of in vitro affinity maturation and selection of mutants . The variable chain coding regions of the recombinant B12Fab clone were sequenced and the V-gene usage was determined by comparison with the V kappa and VH germline sequences . The B12Fab V kappa chain belongs to the subgroup II and shows the highest degree of homology with the A3 germline gene, whereas the sequence of the VH chain is strictly related to that of the Humhv3019b18 gene of the VH3 family . These results are, to our knowledge, the first report of molecular cloning and characterization of a functional human Ab specific for an HCV antigen. Mol Gen Genet, 1995 Oct 25, 248(6), 657 - 67 Arabidopsis thaliana tryptophan synthase alpha: gene cloning, expression, and subunit interaction; Radwanski ER et al.; The tryptophan synthase alpha subunit catalyzes the conversion of indole-3-glycerolphosphate to indole, the penultimate reaction in the biosynthesis of the essential amino acid tryptophan . A cDNA encoding Arabidopsis thaliana tryptophan synthase alpha(TSA1) was isolated by complementation of an Escherichia coli delta trpA mutation and by polymerase chain reaction amplification from a cDNA library using degenerate primers . A TSA1 genomic clone was also isolated and 5 kb of the DNA sequence determined . A single sequence in the Arabidopsis genome with homology to the TSA1 cDNA was detected by high-stringency genomic Southern blot hybridization . In contrast under hybridization conditions of reduced stringency, one or two additional homologous sequences were observed . A 1.4 kb transcript was detected in wild-type RNA with the TSA1 cDNA as a probe . Several lines of evidence, including immunoaffinity chromatography, suggest that the active A . thaliana tryptophan synthase enzyme consists of a heterosubunit complex, presumably analogous to the prokaryotic alpha 2 beta 2 complex . Immunoblot analysis indicated that the plant alpha and beta subunits are present throughout development. Biochemistry, 1995 Oct 24, 34(42), 13818 - 24 Acholeplasma laidlawii B membranes contain a lipid (glycerylphosphoryldiglucosyldiacylglycerol) which forms micelles rather than lamellar or reversed phases when dispersed in water; Lewis RN et al.; It has been proposed that each of the lipids from the Acholeplasma laidlawii membrane prefers to form either a lamellar or a reversed cubic or hexagonal phase when dispersed in excess water at physiologically relevant temperatures and ionic strengths . In this study, we have reinvestigated the thermotropic phase behavior of all the major membrane lipids from A . laidlawii B membranes derived from cells grown in equimolar palmitic and elaidic acids . We confirm that phosphatidylglycerol (PG) and diglucosyldiacylglycerol (DGDG) from such membranes do indeed form only lamellar phases over the temperature range 5-80 degrees C . We also confirm that the monoglucosyldiacylglycerol (MGDG) and acyl polyprenyl glucoside (APG) exist in lamellar phases at lower temperatures but do form reversed phases at higher temperatures . However, we present here optical, differential scanning calorimetric, quasielastic light scattering, and 2H- and 31P-nuclear magnetic resonance spectroscopic evidence indicating that one lipid component of the A . laidlawii B membrane, namely, glycerylphosphoryldiglucosyldiacylglycerol (GPDGDG), actually forms normal micellar rather than lamellar or reversed phases when dispersed in excess water at physiological temperatures . To the best of our knowledge, this is the first demonstration of the existence of a micellar phase-preferring lipid in a prokaryotic cell membrane, and only the second demonstration of the existence of a micellar phase-forming lipid in any biological membrane . We also show that GPDGDG levels change greatly depending on the fatty acid composition of the membrane lipids of this organism.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1995 Oct 20, 253(2), 228 - 42 Single base-pair precision and structural rigidity in a small IHF-induced DNA loop; Nunes-Duby SE et al.; The prokaryotic integration host factor (IHF) is a DNA-bending protein that binds to specific DNA sites as a heterodimer . Genetic and mutational analyses have previously identified asymmetric protein-DNA contacts by the individual subunits . By exploiting the unique sequence and positional context of one IHF binding site, H' in Lambda attachment sites (att sites), we have identified a symmetry element of binding and have localized the functional bend center to the center of this symmetry . A shift of the H' bend center by a single base-pair to the right or to the left within the very tight loop formed with Lambda integrase (Int) and IHF in att-site "intasomes" severely reduces recombination . This suggests that a precise, but wrongly positioned, DNA bend within a loop of constant length negatively influences the juxtaposition or "phasing" of the core-type and arm-type Int binding sites by differentially affecting the length of each leg of the loop . Furthermore, ten base-pair insertions within this loop that should not interfere with correct helical phasing are sensed in a position-dependent manner . Distal insertions abolish recombination, whereas proximal or double insertions (in both legs of the loop) are well tolerated. J Biol Chem, 1995 Oct 20, 270(42), 25286 - 90 The interaction of Escherichia coli topoisomerase IV with DNA; Peng H et al.; The two type II topoisomerases in Escherichia coli, DNA gyrase and topoisomerase (Topo) IV, share considerable amino acid sequence similarity, yet they have distinctive topoisomerization activities . Only DNA gyrase can supercoil relaxed DNA, whereas during oriC DNA replication in vitro, only Topo IV can support the final stages of replication, processing of the late intermediate and decatenation of the daughter molecules . In order to develop an understanding for the basis of the differential activities of these two enzymes, we have initiated a characterization of Topo IV binding to DNA . We find that unlike gyrase, Topo IV neither constrains DNA in a positive supercoil when it binds nor protects a 150-base pair region of DNA from digestion with micro-coccal nuclease . Consistent with this, DNase I footprinting experiments showed that Topo IV protected a 34-base pair region roughly centered about the topoisomerase-induced cleavage site . In addition, Topo IV preferentially bound supercoiled rather than relaxed DNA . Thus, the DNA binding characteristics of Topo IV are more akin to those of the type II eukaryotic enzymes rather than those of its prokaryotic partner. Gene, 1995 Oct 16, 164(1), 75 - 9 Phage RNA polymerase vectors that allow efficient gene expression in both prokaryotic and eukaryotic cells; He B et al.; We have developed expression vectors that direct the synthesis of proteins from a common set of signals in both prokaryotic and eukaryotic cells . To allow transcription from a common promoter the vectors rely upon a phage RNA polymerase (RNAP) . To direct initiation of translation to the same start codon the vectors utilize an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) that has been modified to include a prokaryotic ribosome-binding site (RBS) at an appropriate distance upstream from the desired start codon . These vectors provide levels of expression in eukaryotic cells that exceed those of a conventional RNAP-II-based system by 7-fold, and expression in bacterial cells at levels comparable to other phage RNAP-based systems . Inclusion of a lac repressor and a phage promoter/lac operator fusion element allows tight regulation . Cotransfection of eukaryotic cells with the expression vector and a vector that encodes the phage RNAP provides high-level transient expression without the need to construct specialized stable cell lines. Gene, 1995 Oct 16, 164(1), 71 - 4 A novel repetitive sequence lies near the gene encoding a cytosine methyltransferase in the cyanobacterium Dactylococcopsis salina; Yebra MJ et al.; An unusual cluster of tandemly repeated DNA sequences (TRS) was found downstream from the gene encoding DsaV methyltransferase, the DNA modification enzyme in the DsaV restriction-modification system found in a strain of Dactylococcopsis salina (Ds) . The repeat unit is about 32-bp long and is present 13 times in the cluster . Each repeat unit can be divided into two distinct parts based on the level of sequence conservation and evolution . Hybridization of Ds DNA with a probe specific for this cluster revealed that there were at least two additional sites within the genome with similar TRS . The TRS units are localized in one region of the Ds genome . They do not share significant sequence similarity with other TRS found in prokaryotes. Gene, 1995 Oct 16, 164(1), 45 - 7 pALEX, a dual-tag prokaryotic expression vector for the purification of full-length proteins; Panagiotidis CA et al.; pALEX, a prokaryotic expression vector, was constructed in which the multiple cloning site (MCS, polylinker) is flanked by sequences encoding glutathione S-transferase (GST) at the 5' end and a His6 residue tag at the 3' end . Open reading frames cloned into this vector can direct production of fusion proteins with GST at their N terminus and a His6 tag at their C terminus . This allows for the purification of full-size fusion proteins by a sequential two-step procedure on glutathione-agarose and Ni(2+)-agarose columns. Genes Dev, 1995 Oct 15, 9(20), 2556 - 67 Enhanced and coordinated processing of synapsed viral DNA ends by retroviral integrases in vitro; Kukolj G et al.; We have designed novel substrates to investigate the first step in retroviral integration: the site-specific processing of two nucleotides from the 3' ends of viral DNA . The substrates consist of short duplex oligodeoxynucleotides whose sequences match those of the U3 and U5 ends of viral DNA but are covalently synapsed across the termini by short, single-strand nucleotide linkers . We show here that the optimal separation between termini in a synapsed-end substrate for avian sarcoma/leukosis virus (ASV) IN is 2 nucleotides . This places the two conserved 5'-CA-3' processing sites 6 nucleotides apart, a separation equal to the staggered cut in target DNA produced by this enzyme during the subsequent joining reaction . Based on estimates of initial reaction rates, this synapsed-end substrate is processed by IN at > 10-fold higher efficiency than observed with an equivalent mixture of U3 and U5 single-end (uncoupled) substrates . Enhanced processing is maintained at low IN concentrations, suggesting that the synapsed-end substrate may facilitate enzyme multimerization . Enhanced processing by HIV-1 IN, which produces a 5-bp stagger during integration, was observed with a synapsed-end substrate in which the separation between processing sites was 5 nucleotides . These observations provide estimates of the distances between active sites in the multimeric IN-DNA complexes of ASV and HIV-1 . Our results also show that processing of paired U3 and U5 ends need not be coupled temporally . Finally, we observed that substrates that paired a wild-type with a mutated terminus were cleaved poorly at both ends . Thus, in vitro processing of the synapsed-end substrates requires specific recognition of the sequences at both ends . These findings provide new insights into the mechanism of integrative recombination by retroviral integrases and, by extension, other prokaryotic and eukaryotic transposases that are related to the viral enzymes. Biochem J, 1995 Oct 15, 311 ( Pt 2), 417 - 24 Protochlorophyllide reductase in photosynthetic prokaryotes and its role in chlorophyll synthesis; Rowe JD et al.; 1 . DNA sequences hybridizing with the wheat protochlorophyllide reductase gene have been detected during genomic Southern blots of various cyanobacterial DNA samples . No such hybridization was observed with DNA from photosynthetic bacteria . 2 . A fragment amplified from Phormidium laminosum DNA has been characterized and shown to be 73% similar to the corresponding wheat sequence . At the protein level the similarity is 91% . When used as a probe for Southern blotting the Phormidium DNA fragment confirmed the authenticity of some of the original signals obtained with the wheat probe . 3 . Peptides of molecular mass 36, 30 and 60 kDa are immunodetected by a wheat reductase antibody during Western blotting of Phormidium preparations . These are purported to correspond to the cyanobacterial mature reductase protein, a stable proteolytic fragment and soluble dimeric forms of the latter respectively . 4 . Adaptation of Phormidium to growth in red light (delta > 670 nm) or darkness led to no significant changes in the total level of immunodetected peptides or protochlorophyllide within the cells . 5 . The specific activity of the reductase in Phormidium membranes has been tentatively estimated as 0.5 unit/mg of protein, a value comparable with that found in preparations from mature chloroplasts of higher plants. J Biol Chem, 1995 Oct 13, 270(41), 23930 - 3 Streptolydigin resistance can be conferred by alterations to either the beta or beta' subunits of Bacillus subtilis RNA polymerase; Yang X et al.; Rifampicin and streptolydigin are antibiotics which inhibit prokaryotic RNA polymerase at the initiation and elongation steps, respectively . In Escherichia coli, resistance to each antibiotic results from alterations in the beta subunit of the core enzyme . However, in Bacillus subtilis, reconstitution studies found rifampicin resistance (RifR) associated with the beta subunit and streptolydigin resistance (StlR) with beta' . To understand the basis of bacterial StlR, we isolated the B . subtilis rpoC gene, which encodes a 1,199-residue product that is 53% identical to E . coli beta' . Two spontaneous StlR mutants carried the same D796G substitution in rpoC, and this substitution alone was sufficient to confer StlR in vivo . D796 falls within Region F, which is conserved among the largest subunits of prokaryotic and eukaryotic RNA polymerases . Among eukaryotes, alterations in Region F promote resistance to alpha-amanitin, a toxin which inhibits transcription elongation; among prokaryotes, alterations in Region F cause aberrant termination . To determine whether alterations in the beta subunit of B . subtilis could also confer StlR, we made three StlR substitutions (A499V, G500R, and E502V) in the rif region of rpoB . Together these results suggest that beta and beta' interact to form an Stl binding site, and that this site is important for transcription elongation. Proc Natl Acad Sci U S A, 1995 Oct 10, 92(21), 9662 - 6 Calculating the probability of multitaxon evolutionary trees: bootstrappers Gambit; Lake JA; The reconstruction of multitaxon trees from molecular sequences is confounded by the variety of algorithms and criteria used to evaluate trees, making it difficult to compare the results of different analyses . A global method of multitaxon phylogenetic reconstruction described here, Bootstrappers Gambit, can be used with any four-taxon algorithm, including distance, maximum likelihood, and parsimony methods . It incorporates a Bayesian-Jeffreys'-bootstrap analysis to provide a uniform probability-based criterion for comparing the results from diverse algorithms . To examine the usefulness of the method, the origin of the eukaryotes has been investigated by the analysis of ribosomal small subunit RNA sequences . Three common algorithms (paralinear distances, Jukes-Cantor distances, and Kimura distances) support the eocyte topology, whereas one (maximum parsimony) supports the archaebacterial topology, suggesting that the eocyte prokaryotes are the closest prokaryotic relatives of the eukaryotes. J Theor Biol, 1995 Oct 7, 176(3), 403 - 10 The origin of synergistic symbiosis; Frank SA; A dominant theme in the history of life has been the evolutionary innovations of cooperative symbioses: the first genomes near the origin of life, integrated prokaryotic cells, the complex symbiotic communities that evolved into modern eukaryotic cells, lichens, mycorrhizae, and so on . In this paper, a model of cooperative symbiosis that shows a threshold condition for the evolution of cooperation is analyzed . The threshold is not easily passed, but cooperative evolution proceeds rapidly once a symbiosis overcomes the threshold . In the model presented here, each species has genetic variability for a symbiotic trait . The trait imposes a reproductive cost on its bearer but enhances the reproduction of its partner species . For example, in the origin of genetic systems, the trait may cause biochemical synergism for the rate of replication of primitive RNA strands as in Eigen and Schuster's hypercycle model . Models of growth are contrasted with synergism, which are most appropriate for the evolution of genetic systems and for mutualisms such as lichens, with the strategic and psychological applications of the Prisoner's Dilemma model. J Biol Chem, 1995 Oct 6, 270(40), 23838 - 44 Studies on the influence of cytosine methylation on DNA recombination and end-joining in mammalian cells; Liang F et al.; To test the influence of cytosine methylation on homologous recombination and the rejoining of DNA double strand breaks in mammalian cells, we developed a sensitive and quantitative assay system using extrachromosomal substrates . First, methylation was introduced into substrates in vitro with the prokaryotic SssI methylase, which specifically methylates the C-5 position of cytosine bases within CpG dinucleotides, mimicking the mammalian DNA methyltransferase . Next, methylated substrates were incubated in mammalian cells for a sufficient length of time to recombine or rejoin prior to substrate recovery . Results from bacterial transformation of the substrates and from direct Southern analysis demonstrate that cytosine methylation has no detectable effect on either DNA end-joining or homologous recombination . Thus, the components of the protein machinery involved in these complex processes are unaffected by the major DNA modification in mammalian cells . These results leave open the possibility that methylation may modulate the accessibility of these components to chromosomal DNA by altering local chromatin structure. J Biol Chem, 1995 Oct 6, 270(40), 23801 - 7 The Saccharomyces cerevisiae RIB4 gene codes for 6,7-dimethyl-8-ribityllumazine synthase involved in riboflavin biosynthesis . Molecular characterization of the gene and purification of the encoded protein; Garcia-Ramirez JJ et al.; 6,7-Dimethyl-8-ribityllumazine, the immediate biosynthetic precursor of riboflavin, is synthesized by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate . The gene coding for 6,7-dimethyl-8-ribityllumazine synthase in Saccharomyces cerevisiae (RIB4) has been cloned by functional complementation of a mutant accumulating 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which can grow on riboflavin- or diacetyl- but not on 3,4-dihydroxy-2-butanone-supplemented media . Gene disruption of the chromosomal copy of RIB4 led to riboflavin auxotrophy and loss of enzyme activity . Nucleotide sequencing revealed a 169-base pair open reading frame encoding a 18.6-kDa protein . Hybridization analysis indicated that RIB4 is a single copy gene located on the left arm of chromosome XV . Overexpression of the RIB4 coding sequence in yeast cells under the control of the strong TEF1 promoter allowed ready purification of 6,7-dimethyl-8-ribityllumazine synthase to apparent homogeneity by a simple procedure . Initial structural characterization of 6,7-dimethyl-8-ribityllumazine synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a pentamer of identical 16.8-kDa subunits . The derived amino acid sequence of RIB4 shows extensive homology to the sequences of the beta subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes. J Biol Chem, 1995 Oct 6, 270(40), 23275 - 81 Sec-dependent thylakoid protein translocation . Delta pH requirement is dictated by passenger protein and ATP concentration; Mant A et al.; A Sec-type system is responsible for the translocation of a subset of proteins across the thylakoid membrane in higher plant chloroplasts . Previous studies have suggested that the thylakoidal delta pH plays a minor role in this translocation mechanism, but we show here that it can be essential for the translocation process, depending on the identity of the passenger protein and the concentration of ATP . Studies using chimeric proteins show that, whereas the presequence dictates the translocation pathway, the delta pH requirement is dictated exclusively by the passenger protein; some passenger proteins are virtually delta pH-independent whereas others are absolutely dependent . delta pH requirement is not related to charge characteristics of the passenger proteins, ruling out an electrophoretic effect . Analysis of the 33-kDa photosystem II protein reveals an inverse relationship between delta pH requirement and ATP concentration; import into isolated thylakoids is inhibited 14-fold by nigericin at moderate ATP concentrations, and totally inhibited when the ATP concentration is reduced to 2 microM . The results indicate that the roles of the delta pH and ATP overlap and suggest that the delta pH may be obligatory when the passenger protein is abnormally difficult to translocate, possibly due to the folding of the polypeptide chain . We compare the energetics of this system with those of prokaryotic systems from which the chloroplast system is believed to have evolved. Cell, 1995 Oct 6, 83(1), 147 - 55 Mechanism of corepressor-mediated specific DNA binding by the purine repressor; Schumacher MA et al.; The modulation of the affinity of DNA-binding proteins by small molecule effectors for cognate DNA sites is common to both prokaryotes and eukaryotes . However, the mechanisms by which effector binding to one domain affects DNA binding by a distal domain are poorly understood structurally . In initial studies to provide insight into the mechanism of effector-modulated DNA binding of the lactose repressor family, we determined the crystal structure of the purine repressor bound to a corepressor and purF operator . To extend our understanding, we have determined the structure of the corepressor-free corepressor-binding domain of the purine repressor at 2.2 A resolution . In the unliganded state, structural changes in the corepressor-binding pocket cause each subunit to rotate open by as much as 23 degrees, the consequences of which are the disengagement of the minor groove-binding hinge helices and repressor-DNA dissociation. Cell, 1995 Oct 6, 83(1), 137 - 46 A mammalian RNA polymerase II holoenzyme containing all components required for promoter-specific transcription initiation; Ossipow V et al.; The protein kinase MO15/CDK7 has recently been shown to be associated with the general transcription factor TFIIH and to be capable of phosphorylating the RNA polymerase II carboxy-terminal domain . Here, we show that a monoclonal MO15/CDK7 antibody coimmunoprecipitates, from a rat liver nuclear extract, all components of the RNA polymerase II transcription apparatus required for initiation at the albumin and adenovirus major late promoters . The immunoprecipitate includes RNA polymerase II, TFIID, TFIIB, TFIIH, TFIIF, and TFIIE, but is devoid of transcriptional activator proteins, such as HNF1, HNF4, and C/EBP alpha . The finding of an autonomously initiating RNA polymerase II holoenzyme in mammalian cells suggests conceptual similarities between transcription initiation in prokaryotes and eukaryotes. Biochemistry, 1995 Oct 3, 34(39), 12489 - 95 Species-specific microhelix aminoacylation by a eukaryotic pathogen tRNA synthetase dependent on a single base pair; Quinn CL et al.; We report here that tyrosyl-tRNA synthetase from the eukaryotic pathogen Pneumocystis carinii is a 370 amino acid polypeptide with characteristic elements of a class I aminoacyl-tRNA synthetase and aligns with the prokaryotic tyrosyl-tRNA synthetases in the class-defining active site region, including the tRNA acceptor helix-binding region . The expressed enzyme is a dimer that aminoacylates yeast tRNA but not Escherichia coli tRNA(Tyr) . Like most tRNAs, prokaryotic tyrosine tRNAs have a G1.C72 base pair at the ends of their respective acceptor helices . However, the eukaryote cytoplasmic tyrosine tRNAs have an uncommon C1.G72 base pair . We show that P . carinii tyrosyl-tRNA synthetase charges a seven base pair hairpin microhelix (microhelixTyr) whose sequence is derived from the acceptor stem of yeast cytoplasmic tRNATyr . In contrast, the enzyme does not charge E . coli microhelixTyr . Changing the C1.G72 of yeast microhelixTyr to G1.C72 abolishes charging by the P . carinii tyrosyl-tRNA synthetase . Conversely, we found that E . coli tyrosyl-tRNA synthetase can charge an E . coli microhelixTyr and that charging is sensitive to having a G1.C72 rather than a C1.G72 base pair . The results demonstrate that the common structural framework of homologous tRNA synthetases has the capacity to coadapt to a transversion in a critical acceptor helix base pair and that this coadaptation can account for species-selective microhelix aminoacylation . We propose that species-selective acceptor helix recognition can be used as a conceptual basis for species-specific inhibitors of tRNA synthetases. FEBS Lett, 1995 Oct 2, 373(1), 5 - 9 Cloning and sequencing of a human thioredoxin reductase; Gasdaska PY et al.; The DNA sequence encoding human placental thioredoxin reductase has been determined . Of the 3826 base pairs sequenced, 1650 base pairs were in an open reading frame encoding a mature protein with 495 amino acids and a calculated molecular mass of 54,171 . Sequence analysis showed strong similarity to glutathione reductases and other NADPH-dependent reductases . Human thioredoxin reductase contains the redox-active cysteines in the putative FAD binding domain and has a dimer interface domain not previously seen with prokaryote and lower eukaryote thioredoxin reductases. Curr Opin Genet Dev, 1995 Oct, 5(5), 662 - 8 Molecular control of circadian rhythms; Rosbash M; Circadian rhythms are virtually ubiquitous in eukaryotes and have been shown to exist even in some prokaryotes . The generally accepted view is that these rhythms are generated by an endogenous clock . Recent progress, especially in the Drosophila, Neurospora and mouse systems, has revealed new clock components and mechanisms . These include the mouse clock gene, the Drosophila timeless gene, and the role of light in Neurospora. Mol Microbiol, 1995 Oct, 18(1), 89 - 99 Tissue-specific glycogen branching isoenzymes in a multicellular prokaryote, Streptomyces coelicolor A3(2); Bruton CJ et al.; In the overtly differentiated colonies of Streptomyces coelicolor A3(2), discrete phases of glycogen synthesis are found at the vegetative/aerial mycelium boundary (phase I) and in the immature spore chains at aerial hyphal tips (phase II) . We have characterized two S . coelicolor glgB genes encoding glycogen branching enzyme, which are well separated in the genome . Disruption of glgBl led to the formation of abnormal polyglucan deposits at phase I, with phase II remaining normal, whereas disruption of glgBII interfered specifically with phase II deposits, and not with those of phase I . Thus, each branching enzyme isoform is involved in a different phase of glycogen synthesis . This situation contrasts with that in simple bacteria, which typically have a single set of enzymes for glycogen metabolism, and more closely resembles that in plants. Can J Microbiol, 1995 Oct, 41(10), 869 - 76 DNA sequence and transcriptional characterization of a beta-glucanase gene (celB) from Ruminococcus flavefaciens FD-1; Vercoe PE et al.; The recombinant clone pBAW101 (in pBluescript SK-) contains the celB endoglucanase gene from Ruminococcus flavefaciens FD-1 . Subcloning indicated that the endoglucanase activity expressed was present within a 2.4-kb insert (pBAW104) . The nucleotide sequence of the celB gene was determined, and upon analysis, revealed an open reading frame of 1943 nucleotides that encodes a polypeptide of 632 amino acids with a molecular weight of 69,414 . A putative Shine-Dalgarno sequence was identified 6 bp upstream from the translation start site . The N-terminal 32 amino acid residues were typical of prokaryotic signal sequences . Hydrophobic cluster analysis (HCA) and DNA alignment of CelB to other published beta-glucanase polypeptide sequences in GenBank indicate that CelB belongs in HCA cellulase family 44 . Primer extension analyses were performed using RNA isolated from R . flavefaciens grown on cellulose and cellobiose, and from Escherichia coli containing the plasmid clone pBAW104 . Transcription is initiated at different sites in E . coli and R . flavefaciens . In the case of R . flavefaciens transcription is initiated at a C residue (nucleotides 329), 221 bp upstream from the translation start site . There were no regions resembling E . coli sigma 70-like promoter sequences present upstream from this putative transcription initiation site . In contrast, numerous transcription initiation sites were identified when RNA from E . coli was used in the primer extension analyses. J Biochem (Tokyo), 1995 Oct, 118(4), 738 - 44 The primary structure of pepstatin-insensitive carboxyl proteinase produced by Pseudomonas sp . No . 101; Hayashi K et al.; A unique carboxyl proteinase {EC 3.4.23.33} from Pseudomonas sp . No . 101 is the first example of a prokaryotic enzyme which is insensitive to the classical inhibitor, pepstatin . The primary structure of the proteinase was determined by conventional methods . Pseudomonas carboxyl proteinase consists of 370 amino acid residues with one disulfide bond . This enzyme has no homologous sequence with any other known carboxyl proteinase, including carboxyl proteinase B from Scytalidium lignicolum, which is a pepstatin-insensitive carboxyl proteinase . In addition, Pseudomonas carboxyl proteinase lacks the Asp*-Thr-Gly, Glu*-Thr-Gly, and Asp*-Thr-Ser-Gly (*indicates the catalytic residue) sequences which are known as the motif sequences around a pair of catalytic residues in carboxyl proteinases reported so far . The results strongly indicate that Pseudomonas carboxyl proteinase is a new type of carboxyl proteinase. J Biochem (Tokyo), 1995 Oct, 118(4), 671 - 8 Biogenesis of novel quinone coenzymes; Tanizawa K; Recently, two novel quinonoid coenzymes, 2,4,5-trihydroxyphenylalanine quinone (topa quinone; TPQ) and tryptophan tryptophylquinone (TTQ), were identified in copper-containing amine oxidase and methylamine dehydrogenase, respectively . Unlike the formerly known quinonoid coenzyme, pyrroloquinoline quinone (PQQ), which is non-covalently bound to several prokaryotic dehydrogenases and produced through its own biosynthetic pathway, each of TPQ and TTQ is bound covalently to the polypeptide chain as an integral amino acid residue and encoded by a codon for a normal (unmodified) amino acid in the gene . Thus, these coenzymes must be generated through post-translational modification of the precursor amino acid; for TPQ, oxidation of a specific tyrosine occurring in the consensus Asn-Tyr-Asp/Glu sequence, and for TTQ, oxidation of a specific tryptophan and cross-linking with another tryptophan separated by 50 residues in the same polypeptide chain . We recently demonstrated that, using the inactive precursor forms of bacterial copper amine oxidases, TPQ is generated through self-processing of the protein with the participation of the bound copper ions . On the other hand, the absence of a prosthetic metal ion in methylamine dehydrogenase as well as its existence in the periplasm renders TTQ biogenesis more complicated, likely requiring an external enzymatic system(s). Antonie Van Leeuwenhoek, 1995 Oct, 68(3), 209 - 14 Purification and properties of urease from Sporobolomyces roseus; Jahns T; Urease (EC 3.5.1.5) catalyses the hydrolysis of urea to ammonia and carbon dioxide . The enzyme from Sporobolomyces roseus was enriched 780-fold and purified to apparent homogeneity using heat treatment, ion exchange chromatography on Q-Sepharose fast flow, hydrophobic interaction chromatography on Phenyl-Sepharose, size exclusion chromatography on Sephacryl S 300 HR, and ion exchange chromatography on MonoQ . Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of subunits with a molecular weight of 90 (+/- 4) kDa . The M(r) of the native enzyme was estimated by size exclusion chromatography to be 340 (+/- 30) kDa, suggesting a tetrameric structure different from other ureases isolated so far from both prokaryotes and eukaryotes . The enzyme was heat-stable, showing no loss of activity after incubation at 70 degrees C for 15 min . The highest urease activities were observed after growth on media containing urea as the sole source of nitrogen. Trends Biochem Sci, 1995 Oct, 20(10), 397 - 401 Mismatch repair: mechanisms and relationship to cancer susceptibility; Kolodner RD; DNA mismatch-repair systems exist that repair mispaired bases formed during DNA replication, genetic recombination and as a result of damage to DNA . Some components of these systems are conserved in prokaryotes and eukaryotes . Genetic defects in mismatch-repair genes play an important role in common cancer-susceptibility syndromes and sporadic cancers. J Gen Virol, 1995 Oct, 76 ( Pt 10), 2519 - 27 Complete genomic sequence of the fish rhabdovirus infectious haematopoietic necrosis virus; Schutze H et al.; The complete nucleotide sequence of the genome of the fish rhabdovirus infectious haematopoietic necrosis virus (IHNV) has been determined after cDNA cloning of the viral genomic RNA . Sequence analysis showed the presence of six open reading frames encoding the nucleoprotein N, the matrix proteins M1 and M2, the glycoprotein G, a so-called non-structural protein NV, and the RNA polymerase L . The genome organization is 3'N-M1-M2-G-NV-L 5' . The extreme 5' and 3' ends of the genome were sequenced after RNA ligation or RACE . Prokaryotic expression products of the open reading frames predicted to encode the matrix proteins M1 and M2, the glycoprotein G and the NV protein reacted with rabbit anti-IHNV serum thereby confirming their identity . This is the first complete nucleotide sequence of a fish rhabdovirus . Knowledge of the complete sequence is an essential prerequisite for future manipulation of the genome and also serves to provide gene- and protein specific reagents for use in further examination of the replication of the fish rhabdoviruses. J Bacteriol, 1995 Oct, 177(20), 5846 - 52 Gliding movements in Myxococcus xanthus; Spormann AM et al.; Prokaryotic gliding motility is described as the movement of a cell on a solid surface in the direction of the cell's long axis, but its mechanics are unknown . To investigate the basis of gliding, movements of individual Myxococcus xanthus cells were monitored by employing a video microscopy method by which displacements as small as 0.03 micron could be detected and speeds as low as 1 micron/min could be resolved . Single cells were observed to glide with speeds varying between 1 and 20 microns/min . We found that speed variation was due to differences in distance between the moving cell and the nearest cell . Cells separated by less than one cell diameter (0.5 micron) moved with an average speed of 5.0 micron/min, whereas cells separated by more than 0.5 micron glided with an average speed of 3.8 microns/min . The power to glide was found to be carried separately at both ends of a cell. J Bacteriol, 1995 Oct, 177(20), 5740 - 7 Nucleotide sequence, transcriptional analysis, and glucose regulation of the phenoxazinone synthase gene (phsA) from Streptomyces antibioticus; Hsieh CJ et al.; The nucleotide sequence of a 2.3-kb SphI fragment containing the structural gene (phsA) for phenoxazinone synthase (PHS) of Streptomyces antibioticus was determined . The sequence was found to contain an open reading frame (ORF) with a G+C content of 71.5% oriented in the direction of transcription that was confirmed by primer extension . The ORF encodes a protein with an M(r) of 70,223 consisting of 642 amino acids and is preceded by a potential ribosome-binding site . The codon usage pattern is in agreement with the general pattern for streptomycete genes, with a 92.5 mol% G+C content in the third position . The N-terminal sequence of the mature PHS subunit corresponds exactly to that predicted from the nucleotide sequence . Neither ATG nor GTG initiator codons were identified for the protein . However, a TTG codon was located near the amino terminus of the mature protein and is a good candidate for the initiator codon . The transcriptional start point of phsA was located 36 bp upstream of the start codon by primer extension . The -10 region of the putative promoter showed some similarity to the consensus sequence for the major class of prokaryotic promoters, but the -35 region was less similar . Comparison of the primary amino acid sequence of PHS of S . antibioticus with other amino acid sequences indicated that PHS is a blue copper protein with copper binding domains in the N-terminal and C-terminal regions of the polypeptide chain . A BsrBI fragment containing the promoter region of phsA and a portion of the ORF was shown to promote xylE expression when cloned in the streptomycete promoter probe vector pIJ2843 . This phsA promoter-dependent xylE expression could be repressed by glucose in S . antibioticus when the organism was grown on glucose or galactose plus glucose . Thus, the cloned promoter region appears to contain the sequences responsible for catabolite repression of PHS production. Comp Biochem Physiol A Physiol, 1995 Oct, 112(2), 247 - 63 gamma-Aminobutyric acid (GABA) metabolism in mammalian neural and nonneural tissues; Tillakaratne NJ et al.; 4-Aminobutyric acid (GABA), a major inhibitory neurotransmitter of mammalian central nervous system, is found in a wide range of organisms, from prokaryotes to vertebrates . GABA is widely distributed in nonneural tissue including peripheral nervous and endocrine systems . GABA acts on GABAA and GABAB receptors . GABAA receptors are ligand-gated chloride channels modulated by a variety of drugs . GABAB receptors are essentially presynaptic, usually coupled to potassium or calcium channels, and they function via a GTP binding protein . In neural and nonneural tissues, GABA is metabolized by three enzymes--glutamic acid decarboxylase (GAD), which produces GABA from glutamic acid, and the catabolic enzymes GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) . Production of succinic acid by SSADH allows entry of the GABA carbon skeleton into the tricarboxylic acid cycle . Alternate sources of GABA include putrescine, spermine, spermidine and ornithine, which produce GABA via deamination and decarboxylation reactions, while L-glutamine is an additional source of glutamic acid via deamination . GAD from mammalian brain occurs in two molecular forms, GAD65 and GAD67 (referring to subunit relative molecular weight (Mr) in kilodaltons) . These different forms of GAD are the product of different genes, differing in nucleotide sequence, immunoreactivity and subcellular localization . The presence and characteristics of GAD have been investigated in a wide variety of nonneural tissues including liver, kidney, pancreas, testis, ova, oviduct, adrenal, sympathetic ganglia, gastrointestinal tract and circulating erythrocytes . In some tissues, one form (GAD65 or GAD67) predominates . GABA-T has been located in most of the same tissues, primarily through histochemical and/or immunochemical methods; GABA-T is also present in a variety of circulating cells, including platelets and lymphocytes . SSADH, the final enzyme GABA catabolism, has been detected in some of the tissues in which GAD and GABA-T have been identified, although the presence of this enzyme has not been in mammalian pancreas, ova, oviduct, testis or sympathetic ganglia. Plant Cell, 1995 Oct, 7(10), 1713 - 22 The stroma of higher plant plastids contain ClpP and ClpC, functional homologs of Escherichia coli ClpP and ClpA: an archetypal two-component ATP-dependent protease; Shanklin J et al.; A cDNA representing the plastid-encoded homolog of the prokaryotic ATP-dependent protease ClpP was amplified by reverse transcription-polymerase chain reaction, cloned, and sequenced . ClpP and a previously isolated cDNA designated ClpC, encoding an ATPase related to proteins encoded by the ClpA/B gene family, were expressed in Escherichia coli . Antibodies directed against these recombinant proteins recognized proteins in a wide variety of organisms . N-terminal analysis of the Clp protein isolated from crude leaf extracts showed that the N-terminal methionine is absent from ClpP and that the transit peptide is cleaved from ClpC . A combination of chloroplast subfractionation and immunolocalization showed that in Arabidopsis, ClpP and ClpC localize to the stroma of the plastid . Immunoblot analyses indicated that ClpP and ClpC are constitutively expressed in all tissues of Arabidopsis at levels equivalent to those of E . coli ClpP and ClpA . ClpP, immunopurified from tobacco extracts, hydrolyzed N-succinyl-Leu-Tyr-amidomethylcoumarin, a substrate of E . coli ClpP . Purified recombinant ClpC facilitated the degradation of 3H-methylcasein by E . coli ClpP in an ATP-dependent fashion . This demonstrates that ClpC is a functional homolog of E . coli ClpA and not of ClpB or ClpX . These data represent the only in vitro demonstration of the activity of a specific ATP-dependent chloroplast protease reported to date. Plant Mol Biol, 1995 Oct, 29(2), 353 - 65 Molecular comparison of carbonic anhydrase from Flaveria species demonstrating different photosynthetic pathways; Ludwig M et al.; During the evolution of C4 plants from C3 plants, both the function and intracellular location of carbonic anhydrase (CA) have changed . To determine whether these changes are due to changes at the molecular level, we have studied the cDNA sequences and the expression of CA from Flaveria species demonstrating different photosynthetic pathways . In leaf extracts from F . bidentis (C4), F . brownii (C4-like), F . linearis (C3-C4) and F . pringlei (C3), two polypeptides of M(r) 31 kDa and 35 kDa cross-reacted with anti-spinach CA antibodies . However, the relative labelling intensities of the two polypeptides differed depending on the species . Northern blot analysis indicated at least two CA transcripts are present in each Flaveria species with sizes ranging from 1.1 to 1.6 kb . Carbonic anhydrase cDNAs from all four Flaveria species studied encode an open reading frame for a polypeptide of 35-36 kDa . The amino acid sequences deduced from all four Flaveria cDNAs share at least 70% homology with the sequences of other dicot CAs . The F . bidentis (C4) CA sequence was found to be the least similar of the Flaveria proteins and, as most of the sequence dissimilarity was found in the first third of the CA molecule, these differences may be involved in the intracellular targeting of CA . A neighbour-joining tree inferred from CA amino acid sequences showed that the Flaveria CAs cluster with other dicot CAs forming a group distinct from those of monocot CAs and prokaryotic and Chlamydomonas periplasmic CAs. Genes Dev, 1995 Oct 1, 9(19), 2399 - 408 Disassembly of the Mu transposase tetramer by the ClpX chaperone; Levchenko I et al.; Mu transposition is promoted by an extremely stable complex containing a tetramer of the transposase (MuA) bound to the recombining DNA . Here we purify the Escherichia coli ClpX protein, a member of a family of multimeric ATPases present in prokaryotes and eukaryotes (the Clp family), on the basis of its ability to remove the transposase from the DNA after recombination . Previously, ClpX has been shown to function with the ClpP peptidase in protein turnover . However, neither ClpP nor any other protease is required for disassembly of the transposase . The released MuA is not modified extensively, degraded, or irreversibly denatured, and is able to perform another round of recombination in vitro . We conclude that ClpX catalyzes the ATP-dependent release of MuA by promoting a transient conformational change in the protein and, therefore, can be considered a molecular chaperone . ClpX is important at the transition between the recombination and DNA replication steps of transposition in vitro; this function probably corresponds to the essential contribution of ClpX for Mu growth . Deletion analysis reveals that the sequence at the carboxyl terminus of MuA is important for disassembly by ClpX and can target MuA for degradation by ClpXP in vitro . These data contribute to the emerging picture that members of the Clp family are chaperones specifically suited for disaggregating proteins and are able to function with or without a collaborating protease. Int J Biochem Cell Biol, 1995 Oct, 27(10), 1033 - 41 Analysis by limited proteolysis of domain organization and GSH-site arrangement of bacterial glutathione transferase B1-1; Aceto A et al.; Limited proteolysis method has been used to study the structure-function relationship of bacterial glutathione transferase (GSTB1-1) . In absence of three-dimensional structural data of prokaryote GST, the results represent the first information concerning the G-site and domains organization of GSTB1-1 . The tryptic cleavages occur mainly at the peptide bonds Lys35-Lys36 and Phe43-Leu44, generating two major molecular species of 20-kDa, 3-kDa and traces of 10-kDa . 1-chloro-2,4-dinitrobenzene favoured the proteolysis of the 20-kDa fragment markedly enhancing the production of the 10-kDa peptide by cleaving the chemical bonds Lys87-Ala88 and Arg91-Tyr92 . The tryptic cleavage sites of GSTB1-1 was found to be located close to those previously found for the mammalian GSTP1-1 isozyme . It was concluded that despite their low sequence homology (18%), GSTB1-1 and GSTP1-1 displayed similar structural features in their G-site regions and probably a common organization in structural domains. Philos Trans R Soc Lond B Biol Sci, 1995 Sep 29, 349(1329), 249 - 53 Transcriptional noise and the evolution of gene number; Bird A et al.; Several proposals are made to explain the apparent increase in complexity of certain lineages during evolution . The proposals (not made in this order) are: (1) that gene number is a valid measure of biological complexity; (2) that gene number has not increased continuously during evolution, but has risen in discrete steps; (3) that two of the biggest steps occurred at the transition from prokaryotes to eukaryotes and the transition from invertebrates to vertebrates; (4) that these steps were made possible by 'systemic' changes in the way that genetic information is managed in the genome; (5) that the ability to silence inappropriate promoters is the primary limitation on gene number; (6) that the invention of nucleosomes (and perhaps the nuclear membrane) facilitated the evolution of eukaryotes from prokaryotic ancestors; (7) that the spread of low density methylation throughout the genome facilitated the evolution of vertebrates from invertebrate ancestors. J Biol Chem, 1995 Sep 29, 270(39), 23097 - 103 Stationary phase expression of a novel Escherichia coli outer membrane lipoprotein and its relationship with mammalian apolipoprotein D . Implications for the origin of lipocalins; Bishop RE et al.; We report a novel outer membrane lipoprotein of Escherichia coli . DNA sequencing between ampC and sugE at the 94.5 min region of the E . coli chromosome revealed an open reading frame specifying 177 amino acid residues . Primer extension analysis demonstrated that the promoter is activated at the transition between exponential and stationary growth phases under control of the rpoS sigma factor gene, and this was confirmed in vivo by monitoring expression of beta-galactosidase activity from a lacZ translational fusion . The amino acid sequence exhibited 31% identity with human apolipoprotein D (apoD), which is a component of plasma high density lipoprotein and belongs to the eukaryotic family of lipocalins . The bacterial lipocalin (Blc) contained a short deletion of 7 amino acid residues corresponding to a hydrophobic surface loop that is thought to facilitate the physical interaction between apoD and high density lipoprotein . However, Blc exhibited a typical prokaryotic lipoprotein signal peptide at its amino terminus . Overexpression, membrane fractionation, and metabolic labeling with {3H}palmitate demonstrated that Blc is indeed a globomycin-sensitive outer membrane lipoprotein . Blc represents the first bacterial member of the family of lipocalins and may serve a starvation response function in E . coli. J Biol Chem, 1995 Sep 29, 270(39), 22669 - 72 A novel function of Escherichia coli chaperone DnaJ . Protein-disulfide isomerase; de Crouy-Chanel A et al.; Molecular chaperones, protein-disulfide isomerases, and peptidyl prolyl cis-trans isomerases assist protein folding in both prokaryotes and eukaryotes . The DnaJ protein of Escherichia coli and the DnaJ-like proteins of eukaryotes are known as molecular chaperones and specific regulators of DnaK-like proteins and are involved in protein folding and renaturation after stress . In this study we show that DnaJ, like thioredoxin, protein-disulfide isomerase, and DsbA, possesses an active dithiol/disulfide group and catalyzes protein disulfide formation (oxidative renaturation of reduced RNase), reduction (reduction of insulin disulfides), and isomerization (refolding of randomly oxidized RNase) . These results suggest that, in addition to its known function as a chaperone, DnaJ might be involved in controlling the redox state of cytoplasmic, membrane, or exported proteins. Proc Natl Acad Sci U S A, 1995 Sep 26, 92(20), 9392 - 6 The amino-terminal domain of the prokaryotic enhancer-binding protein XylR is a specific intramolecular repressor; Perez-Martin J et al.; The mechanism under which the signal-reception amino-terminal portion (A domain) of the prokaryotic enhancer-binding protein XylR controls the activity of the regulator has been investigated through complementation tests in vivo, in which the various protein segments were produced as independent polypeptides . Separate expression of the A domain repressed the otherwise constitutive activity of a truncated derivative of XylR deleted of its A domain (XylR delta A) . Such inhibition was not released by m-xylene, the natural inducer of the system . Repression caused by the A domain was specific for XylR because it did not affect activation of the sigma 54 promoter PnifH by a derivative of its cognate regulator, NifA, deleted of its own A domain . The A domain was also unable to repress the activity of a NifA-XylR hybrid protein resulting from fusing two-thirds of the central domain of NifA to the carboxyl-terminal third of XylR, which includes its DNA-binding domain . The inhibitory effect caused by the A domain of XylR on XylR delta A seems, therefore, to result from specific interactions in trans between the two truncated proteins and not from mere hindering of an activating surface. Biochemistry, 1995 Sep 26, 34(38), 12265 - 75 Structural characterization of the active site of Brucella abortus Cu-Zn superoxide dismutase: a 15N and 1H NMR investigation; Chen YL et al.; Prokaryotic Cu-Zn superoxide dismutases (SODs) are rare and poorly characterized compared to their eukaryotic counterparts . To better characterize the structure of the prokaryotic enzyme, an NMR investigation of Brucella abortus Cu-Zn SOD in the reduced form was undertaken . The enzyme studied was a recombinant form, expressed in Escherichia coli . The enzyme initially lacked a full complement of Cu and Zn ion . After demetallation and remetallation with a stoichiometric amount of Cu and Zn ion, the specific activity of the recombinant B . abortus Cu-Zn SOD was comparable to the specific activity of the bovine enzyme . The 15N and 1H resonances of seven active site histidine imidazole rings were assigned using two-dimensional NMR methods . A self-consistent set of nuclear Overhauser effects between imidazole ring protons was observed, which was in agreement with the predictions of a model based on the X-ray crystallographic structure of the oxidized bovine enzyme (Tainer, J.A., Getzoff, E . D., Beem, K . M., Richardson, J.S., & Richardson, D.C . (1982) J . Mol . Biol . 160, 181-217) . These observations strongly suggest that the structure of the active site of the prokaryotic enzyme is similar to that of the eukaryotic enzyme . Differences in the observed and predicted nuclear Overhauser effects could be ascribed to differences in the oxidation state of the Cu ion (Cu(I) in the reduced B . abortus enzyme and Cu(II) in the oxidized bovine enzyme), as much as they could to the different origins of the enzymes . The NMR data were also compared to a similar 1H NMR study of the human enzyme (Bertini, I., Capozzi, F., Luchinat, C., Piccioli, M., & Viezzoli, M . S . (1991) Eur . J . Biochem . 197, 691-697) . The pattern of nuclear Overhauser effects and the chemical shifts of corresponding resonances were very similar in 1H NMR spectra of the human and B . abortus enzymes . Significant differences in the chemical shifts or exchange behavior of a few resonances indicated differences in the environments of several histidines in the active sites of reduced B . abortus and human Cu-Zn SODs . This is consistent with the presence of a number of insertions and deletions in the loop regions that make up the active site as indicated by amino acid sequence alignment studies . The tautomeric and protonation states of the active site histidines were also determined in this study, and the results were in agreement with previous studies . The resonances of nitrogen atoms coordinated to metal ions were found to fall between those of protonated and unprotonated nitrogens on histidine imidazoles. Biochem Biophys Res Commun, 1995 Sep 25, 214(3), 1254 - 9 Improved recombinant tandem expression of translation initiation factor IF2 in RNASE E deficient E . coli cells; Mortensen KK et al.; The prokaryotic translation initiation factor IF2 exists in a varying number of nested forms in different species . In E . coli three natural forms exist, IF2 alpha, IF2 beta and IF2 gamma differing only in the N-terminal: IF2 beta and IF2 gamma lack 158 and 165 amino acid residues, respectively, as compared to IF2 alpha . We have earlier shown that the smaller forms of IF2 are not the result of a specific proteolysis of IF2 alpha, but produced from individual translation initiation sites in the mRNA . However it has not been known whether the expression in E . coli of IF2 beta and IF2 gamma is dependent on or related to a posttranscriptional processing of the polycistronic nusA operon, containing infB, the gene for IF2 . Here we have used S1 mapping to study the existence of such mRNA processing in the region between the initiation sites for IF2 alpha and IF2 beta/IF2 gamma . The results show a Ribonuclease E cleavage site at position +200 in the infB mRNA between the translation initiation sites . However, studies of the overexpression of the different forms of IF2 show that the relative expression of IF2 alpha and IF2 beta/IF2 gamma is independent of RNase E activity . Thus E . coli exhibits a true tandem translation of intact infB mRNA with multiple in-frame translation initiation sites resulting in gene products of different sizes . An additional observation is a significant increase in the level of overexpression of IF2 in cells devoid of RNase E activity . We conclude that due to lack of RNase E activity, the amount of plasmid-transcribed infB mRNA available for translation is accumulated, resulting in an elevated amount of recombinant IF2 . This observation may have a more general application within the field of recombinant protein production and expression efficiency. J Immunol Methods, 1995 Sep 25, 185(2), 237 - 44 Versatile E . coli thioredoxin specific monoclonal antibodies afford convenient analysis and purification of prokaryote expressed soluble fusion protein; Dickason RR et al.; A recently developed E . coli thioredoxin (Trx) gene fusion expression system has circumvented the difficulties associated with inclusion body formation . Although ample quantities of soluble recombinant protein can be expressed using this system, no universal means of quantifying or purifying the fusion product exists . To facilitate the study of Trx fusion proteins, anti-E . coli Trx monoclonal antibodies (mAb) were generated . Two distinct Trx epitopes were defined by competitive ELISA . Both mAb were capable of detecting Trx fusion proteins by sandwich ELISA, and by immunoblot analysis under reducing and non-reducing conditions . In addition, these mAb enabled purification of Trx fusion proteins by immunoprecipitation, as well as affinity chromatography . This report provides the first description of anti-Trx antibodies . These reagents represent a major advance in the isolation and analysis of prokaryote expressed recombinant Trx fusion proteins. Nucleic Acids Res, 1995 Sep 25, 23(18), 3613 - 20 Structural and functional similarities of prokaryotic and eukaryotic DNA polymerase sliding clamps; Kelman Z et al.; The remarkable processivity of cellular replicative DNA polymerases derive their tight grip to DNA from a ring-shaped protein that encircles DNA and tethers the polymerase to the chromosome . The crystal structures of prototypical 'sliding clamps' of prokaryotes (beta subunit) and eukaryotes (PCNA) are ring shaped proteins for encircling DNA . Although beta is a dimer and PCNA is a trimer, their structures are nearly superimposable . Even though they are not hexamers, the sliding clamps have a pseudo 6-fold symmetry resulting from three globular domains comprising each beta monomer and two domains comprising each PCNA monomer . These domains have the same chain fold and are nearly identical in three-dimensions . The amino acid sequences of 11 beta and 13 PCNA proteins from different organisms have been aligned and studied to gain further insight into the relation between the structure and function of these sliding clamps . Furthermore, a putative embryonic form of PCNA is the size of beta and thus may encircle DNA as a dimer like the prokaryotic clamps. Gene, 1995 Sep 22, 163(1), 161 - 2 Sequence and transcriptional analysis of the genes encoding the class-II topoisomerase of Mycoplasma gallisepticum; Forsyth MH et al.; The gyrAB genes encoding the entire B and a portion of the A subunit of DNA gyrase (E.C . 5.99.1.3) from Mycoplasma gallisepticum (Mg), strain S6, were cloned and sequenced . These gyrAB genes are co-transcribed as a single, polycistronic mRNA transcript . The Mg gyrB appears unique among prokaryotic gyrB in its use of GUG as a start codon. Biochim Biophys Acta, 1995 Sep 19, 1263(3), 201 - 11 Repair of base alkylation damage in targeted restriction endonuclease sequences of plasmid DNA; Musarrat J et al.; Sequence specific ethylation damage and repair of ethyl-adducts in selected restriction endonuclease recognition sites within p220-ras plasmid DNA was assessed by a modified Southern blotting coupled immunoprobing technique . In situ UV irradiation of DNA in gels clearly ameliorated the immunodetection of minute amounts of facultative fragments generated due to inhibition of enzyme cleavage site by covalent alkylation modification of the cognate sites . Specific and quantitative localization of induced facultative fragments was achieved in as low as 1 ng of DNA digest corresponding to a peak intensity below 0.1 absorbance unit upon laser scanning . An ENU dose dependent increase in the intensity of representative 7.1 and 7.7 kb facultative fragments was observed as a result of cleavage block at EcoRI (G/ATTC) and BamHI (G/GATCC) restriction endonuclease sites, respectively . To determine the repair in prokaryotic cells, the half-life of repairable alkyl-adducts was assessed in plasmid DNA established in various Escherichia coli strains as a function of post-treatment incubation time in the recovery medium . The repair is indicated by the gradual disappearance of the 7.1, 7.7, 11.9 and 5.5 kb facultative fragments within the wild-type and mutant E . coli strains . The ethyl-adducts within EcoRI and BamHI restriction sites were effectively lost from the target DNA in repair-proficient E . coli with an estimated t1/2 of approximately 40 min . However, decreased overall rate and at least 2.2-times lesser extent of repair was observed in the repair-deficient (ada+ogt-) and (ada-ogt+) cells . No measurable repair was noticed in alkyltransferase defective double mutant (ada-ogt-) even after 2 h of post-treatment incubation . The repair of ethyl-adducts at NotI site (GC/GGCCGC) in 5.5 kb facultative fragment occurred at a relatively faster rate (t1/2 of 27 min) in wild-type bacteria . A 1.5-fold slower repair of ethyl-adducts in BamHI and EcoRI sequences containing G/G and A/G at their cleavage sites was observed compared to C/G in NotI sequence . These results demonstrate the regioselective induction of alkyl-adducts in ethylated DNA and their differential repair in E . coli due to varied efficiency of the repair enzymes for promutagenic DNA base lesions present in different sequence context. J Biol Chem, 1995 Sep 15, 270(37), 21659 - 64 Cloning and functional characterization of human selenophosphate synthetase, an essential component of selenoprotein synthesis; Low SC et al.; Selenocysteine is co-translationally incorporated into prokaryotic and eukaryotic selenoproteins at in-frame UGA codons . However, the only component of the eukaryotic selenocysteine incorporation machinery identified to date is the selenocysteine-specific tRNA(Sec) . In prokaryotes, selenocysteine is synthesized from seryl-tRNA(Sec) and the active selenium donor, selenophosphate . Selenophosphate is synthesized from selenide and ATP by the selD gene product, selenophosphate synthetase, and is required for selenocysteine synthesis and incorporation into bacterial selenoproteins . We have now cloned human selD and shown that transfection of the human selD cDNA into mammalian cells results in increased selenium labeling of a mammalian selenoprotein, type 1 iodothyronine deiodinase . Despite significant differences between the mechanisms of selenoprotein synthesis in prokaryotes and eukaryotes, human selD weakly complements a bacterial selD mutation, partially restoring selenium incorporation into bacterial selenoproteins . Human selenophosphate synthetase has only 32% homology with the bacterial protein, although a highly homologous region that has similarity to a consensus ATP/GTP binding domain has been identified . Point mutations within this region result in decreased incorporation of selenium into type 1 iodothyronine deiodinase in all but one case . Further analysis revealed that reduced selenium labeling was due to altered ATP binding properties of the mutant selenophosphate synthetases. Yeast, 1995 Sep 15, 11(11), 1061 - 7 Sequence analysis of a 9873 bp fragment of the left arm of yeast chromosome XV that contains the ARG8 and CDC33 genes, a putative riboflavin synthase beta chain gene, and four new open reading frames; Casas C et al.; The DNA sequence of a 9873 bp fragment located near the left telomere of chromosome XV has been determined . Sequence analysis reveals seven open reading frames . One is the ARG8 gene coding for N-acetylornithine aminotransferase . Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein . The open reading frame AOE169 can be considered as the putative gene for the Saccharomyces cerevisiae riboflavin synthase beta chain, since its translation product shows strong homology with four prokaryotic riboflavin synthase beta chains. Proc Natl Acad Sci U S A, 1995 Sep 12, 92(19), 8975 - 9 Transfection and continuous expression of heterologous genes in the protozoan parasite Entamoeba histolytica; Hamann L et al.; To provide tools for functional molecular genetics of the protozoan parasite Entamoeba histolytica, we investigated the use of the prokaryotic neomycin phosphotransferase (NEO) gene as a selectable marker for the transfection of the parasite . An Escherichia coli-derived plasmid vector was constructed (pA5'A3'NEO) containing the NEO coding region flanked by untranslated 5' and 3' sequences of an Ent . histolytica actin gene . Preceding experiments had revealed that amoebae are highly sensitive to the neomycin analogue G418 and do not survive in the presence of as little as 2 micrograms/ml . Transfection of circular pA5'A3'NEO via electroporation resulted in Ent . histolytica trophozoites resistant to G418 up to 100 micrograms/ml . DNA and RNA analyses of resistant cells indicated that (i) the transfected DNA was not integrated into the amoeba genome but was segregated episomally, (ii) in the amoebae, the plasmid replicated autonomously, (iii) the copy number of the plasmid and the expression of NEO-specific RNA were proportional to the amount of G418 used for selection, and (iv) under continuous selection, the plasmid was propagated over an observation period of 6 months . Moreover, the plasmid could be recloned into E . coli and was found to be unrearranged . To investigate the use of pA5'A3'NEO to coexpress other genes in Ent . histolytica, a second marker, the prokaryotic chloramphenicol acetyltransferase (CAT) gene under control of an Ent . histolytica lectin gene promoter was introduced into the plasmid . Transfection of the amoebae with this construct also conferred G418 resistance and, in addition, allowed continuous expression of CAT activity in quantities corresponding to the amount of G418 used for selection . When selection was discontinued, transfected plasmids were lost as indicated by an exponential decline of CAT activity in trophozoite extracts. Biochemistry, 1995 Sep 5, 34(35), 11204 - 10 Residues in a class I tRNA synthetase which determine selectivity of amino acid recognition in the context of tRNA; Schmidt E et al.; Certain aminoacyl-tRNA synthetases discriminate between closely similar amino acids by hydrolytic editing reactions in the presence of their cognate tRNA . An example is the class I isoleucyl-tRNA synthetase . We recently showed that a mutation which eliminates discrimination between isoleucine (Ile) and valine (Val) in the initial amino acid binding and activation steps had little effect on the hydrolytic editing of activated valine in the presence of isoleucine tRNA (tRNA(Ile)) . The results showed that initial amino acid binding and discrimination are functionally independent of tRNA-dependent amino acid discrimination . In this work, we cross-linked (to isoleucyl-tRNA synthetase) a reactive analog of valine misacylated onto tRNA(Ile) . Mutation of specific residues within a peptide segment identified by the cross-linking analysis severely affected discrimination of Val-tRNA(Ile) versus Ile-tRNA(Ile) . The mutationally sensitive residues are part of an insertion into the catalytic domain and are themselves completely conserved among all known prokaryotic and eukaryotic sequences of the enzyme. Nutrition, 1995 Sep-Oct, 11(5 Suppl), 653 - 6 Isolation and characterization of the genes of pathogenic mycobacteria that express antigens for T cell reactivity; Mustafa AS; Tuberculosis and leprosy are caused by Mycobacterium tuberculosis and Mycobacterium leprae, respectively . Identification and characterization of the genes expressing proteins that stimulate Th1-type cells is required to understand the mechanisms involved in protection from mycobacterial diseases . We isolated several recombinant genes from recombinant DNA libraries that expressed antigens recognized by well-characterized monoclonal antibodies . We first used these isolated recombinants to show that many of these genes expressed antigens with T cell reactivity . Most of the genes in the lambda gt11 system were truncated at the amino terminus . Full-length genes were isolated from libraries in other systems and sequenced . Four of the full-length genes were homologous to heat shock proteins of eukaryotes and prokaryotes . We also screened the recombinant DNA libraries directly with T cell probes for antigens that may not be recognized by antibodies . We isolated one gene expressing an epitope recognized by T cells reactive with M . tuberculosis, M . leprae, and M . bovis BCG but not with other mycobacteria . All recombinant proteins were presented to T cells in association with multiple HLA-DR molecules . The responding T cells were the Th1 type with long-lasting memory . The protein products of these genes, either by themselves or expressed in suitable vaccines, may be used to protect against tuberculosis and leprosy. Biochem Mol Biol Int, 1995 Sep, 37(1), 39 - 44 Rat monoamine oxidase B expressed in Escherichia coli has a covalently-bound FAD; Hirashiki I et al.; Rat liver monoamine oxidase B (MAO B) was expressed in E . coli as catalytically active form, though inclusion bodies of the enzyme were also formed as a major protein in the cell . The active form of the recombinant MAO B exhibited similar properties as rat liver enzyme and localized in membrane of the bacteria . Covalent attachment of FAD to polypeptide chain of the recombinant enzyme was revealed by a labeling experiment with {3H}-pargyline, an irreversible mechanism-based inhibitor, indicating that the covalent linkage of FAD to the apoprotein was formed even in the prokaryotic cell . This observation suggests autocatalytic formation of the linkage in MAO B. J Neurosci Res, 1995 Sep 1, 42(1), 9 - 20 Several extracellular domains of the neural cell adhesion molecule L1 are involved in homophilic interactions; Holm J et al.; The neural cell adhesion molecule L1 is a multidomain protein that plays important roles in cell adhesion, migration, and neurite outgrowth . It can interact with itself by a self-binding, i.e., homophilic adhesion mechanism (Kadmon et al.: J Cell Biol 110: 193-208, 1990a) . To determine the domains of L1 involved in homophilic binding, we have generated protein fragments of L1 in a prokaryotic and a eukaryotic expression system and used these covalently coupled to fluorescent microspheres to quantify aggregation between them by cytofluorometric analysis . Protein fragments containing the first and second Ig-like domains and the third fibronectin type III homologous repeat showed avid self-binding . Ig-like domains III and IV also showed some self-binding, whereas Ig-like domains V and VI and fibronectin type III homologous repeats 1 and 2 as well as 4 and 5 were less or not active . Binding between different domains was also observed: fibronectin type III homologous repeats 4 and 5 interacted with Ig-like domains I and II, and fibronectin type III homologous repeats 3-5 interacted with all Ig-like domains . These results were confirmed by experiments testing the binding of fragment-conjugated microspheres to substrate-coated L1 or to cell surface-expressed L1 on cultured neurons . Binding of L1 to itself was interfered with by all protein fragments tested, suggesting that also less avidly binding domains of L1 contribute to homophilic binding . These observations indicate prominent functional roles of both Ig-like domains and fibronectin type III homologous repeats in homophilic binding of L1. J Bacteriol, 1995 Sep, 177(17), 5179 - 85 The glycyl-tRNA synthetase of Chlamydia trachomatis; Wagar EA et al.; Aminoacyl-tRNA synthetases specifically charge tRNAs with their cognate amino acids . A prototype for the most complex aminoacyl-tRNA synthetases is the four-subunit glycyl-tRNA synthetase from Escherichia coli, encoded by two open reading frames . We examined the glycyl-tRNA synthetase gene from Chlamydia trachomatis, a genetically isolated bacterium, and identified only a single open reading frame for the chlamydial homolog (glyQS) . This is the first report of a prokaryotic glycyl-tRNA synthetase encoded by a single gene. J Cell Biol, 1995 Sep, 130(5), 1171 - 9 A 39-kD DNA-binding protein from mouse brain stimulates transcription of myelin basic protein gene in oligodendrocytic cells; Haas S et al.; The MB1 regulatory sequence of the myelin basic protein (MBP) gene spanning between nucleotides -14 to -50 with respect to the transcription start site is critical for cell type-specific transcription of the MBP gene, which encodes the major protein component of myelin sheath in cells derived from the central nervous system (CNS) . This regulatory sequence has the ability to interact with a developmentally controlled DNA-binding protein from mouse brain that stimulates transcription of MBP promoter in an in vitro system (Haas, S., J . Gordon, and K . Khalili . 1993 . Mol . Cell . Biol . 13:3103-3112) . Here, we report the purification of a 39-kD protein from mouse brain tissue at the peak of myelination and MBP production that binds to the MB1 regulatory motif . Following partial amino acid sequence analysis, we have identified a complementary DNA encoding a 39-kD DNA-binding protein called pur alpha . Expression of pur alpha cDNA in the prokaryotic and eukaryotic cells resulted in the synthesis of a protein with characteristics similar to the purified brain-derived 39-kD protein in band shift competition assays . Cotransfection of the recombinant pur alpha expressor plasmid with MBP promoter construct indicated that Pur alpha stimulates transcription of the MBP promoter in oligodendrocytic cells, and that the nucleotide sequence required for binding of the 39-kD Pur alpha to DNA within the MB1 region is crucial for this activity . Moreover, transient expression of Pur alpha caused elevation in the level of endogenous MBP RNA in oligodendrocytic cells . Thus, Pur alpha, a sequence-specific DNA-binding protein upon binding to MB1 regulatory region may play a significant role in determining the cell type-specific expression of MBP in brain. J Biol Chem, 1995 Sep 1, 270(35), 20359 - 64 A mutation in yeast TOP2 homologous to a quinolone-resistant mutation in bacteria . Mutation of the amino acid homologous to Ser83 of Escherichia coli gyrA alters sensitivity to eukaryotic topoisomerase inhibitors; Hsiung Y et al.; In prokaryotic type II topoisomerases (DNA gyrases), mutations that result in resistance to quinolones frequently occur at Ser83 or Ser84 of the gyrA subunit . Mutations to Trp, Ala, and Leu have been identified, all of which confer high levels of quinolone resistance . Extensive segments of DNA gyrase are homologous to eukaryotic topoisomerase II, and Ser741 of yeast TOP2 is homologous to Ser83 of prokaryotic DNA gyrA . Introduction of the Ser741-->Trp mutation into yeast TOP2 confers resistance to 6,8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl- 4-quinolone-3-carboxylic acid (CP-115,953), a fluoroquinolone with substantial activity against eukaryotic topoisomerase II, whereas changing Ser741 to either Leu or Ala does not change sensitivity to quinolones . Interestingly, Ser741-->Trp in the yeast TOP2 also confers hypersensitivity to etoposide . Sensitivity to intercalating anti-topoisomerase II agents such as amsacrine is not changed by any of the three mutations . The topoisomerase II protein carrying the Ser741-->Trp mutation was overexpressed and purified . The purified mutant enzyme had enhanced levels of etoposide stabilized covalent complex as compared with the wild type enzyme and reduced cleavage with CP-115,953 . Unlike the wild type enzyme, etoposide-stabilized cleavage is not readily reversible by heat . We suggest that Ser741 is near a binding site for both quinolones and etoposide and that the Ser741-->Trp mutation leads to a more stable ternary complex between etoposide, DNA, and the mutant enzyme. Mol Cell Biol, 1995 Sep, 15(9), 4867 - 72 Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia; Yu DC et al.; Giardia lamblia, a prevalent human pathogen and one of the lineages that branched earliest from prokaryotes, can be infected with a double-stranded RNA virus, giardiavirus (GLV) . The 6,277-bp viral genome has been previously cloned (A.L . Wang, H.-M . Yang, K.A . Shen, and C.C . Wang, Proc . Natl . Acad . Sci . USA 90:8595-8599, 1993; C.-H . Wu, C.C . Wang, H.M . Yang, and A.L . Wang, Gene, in press) and was converted to a transfection vector for G . lamblia in the present study . By flanking the firefly luciferase gene with the 5' and 3' untranslated regions (UTRs) of the GLV genome, transcript of the construct was synthesized in vitro with T7 polymerase and used to transfect G . lamblia WB trophozoites already infected with GLV (WBI) . Optimal electroporation conditions used for the transfection were set at 1,000 V/cm and 500 microF, which resulted in expression of significant luciferase activity up to 120 h after electroporation . Furthermore, the mRNA and the antisense RNA of the luciferase gene were both detected by reverse transcription and PCR from 6 to 120 h postelectroporation, whereas no antisense RNA of luciferase was observed in the electroporated virus-free Giardia WB trophozoites . The mRNA of luciferase was detectable in the virus-free trophozoites by reverse transcription and PCR only up to 20 h after the electroporation, indicating that the introduced mRNA was replicated only by the viral RNA-dependent RNA polymerase inside the WBI cells . This expression of luciferase was dependent on the presence of UTRs on both ends of the viral genome transcript, including a putative packaging site that was apparently indispensable for luciferase expression . This is the first time that a viral vector in the form of mRNA URTs has been successfully used in transfecting a protozoan. Infect Immun, 1995 Sep, 63(9), 3665 - 73 Interaction of Listeria monocytogenes with mouse dendritic cells; Guzman CA et al.; In this study, the interaction of murine dendritic cells with Listeria monocytogenes was investigated . Dendritic cells are efficient antigen-presenting cells, play a key role in the immune response, and are capable of migrating over substantial distances between sites of infection and lymphoid tissues . L . monocytogenes EGD invaded dendritic cells, escaped from phagosomes into the cytoplasm, and there directed actin nucleation, polymerization, and polarization in a typical fashion, thereby achieving intracellular movement and cell-to-cell spread . The internalization process appears to be independent of the inl locus . Interestingly, an intact microtubular function was essential for efficient uptake, whereas in a previous report, microtubule disruption did not affect bacterial spread in Caco-2 cells . The results obtained also suggest that L . monocytogenes binds to glycosylated receptors of dendritic cells . Uptake of Listeria cells was mediated by a protein kinase-dependent transducing phosphorylation signal that induces the actin polymerization-dependent phagocytic process . To achieve efficient uptake, de novo protein synthesis of eukaryotic and prokaryotic cells is also required . Despite the killing of dendritic cells, wild-type bacteria were found to persist in small numbers in some cells for at least 24 h . When different isogenic mutants of the EGD strain were analyzed for their capability to interact with dendritic cells, it was observed that some virulence-attenuated mutants (i.e., prfA and delta hly) persisted in large numbers for even longer times . Invasion of dendritic cells by L . monocytogenes, which in turn could result in either cell death or persistent infection, might have an important role in the pathogenesis of listeriosis, leading to impaired immune responses with inefficient bacterial clearance and/or promoting bacterial spread. J Invertebr Pathol, 1995 Sep, 66(2), 111 - 20 Association of prokaryotes with symptomatic appearance of withering syndrome in black abalone Haliotis cracherodii; Gardner GR et al.; Withering syndrome (WS) is an epizootic fatal wasting disease that is devastating California Channel Island populations of black abalone Haliotis cracherodii . Our studies suggest a strong pathogen-disease association . The pathogen is an intracellular prokaryote that infects epithelial cells lining the gut and enzyme secreting cells of the digestive diverticula . It multiplies by binary fission in round to oval, basophilic, membrane-bound colonies teeming in the cytoplasm . Infection of the digestive diverticula is accompanied by a complete loss of digestive enzyme granules and metaplasia of enzyme secretory cells to a morphology similar to epithelium lining the gut . Extensive infection of digestive diverticular cells and the resultant deficiency in digestive enzymes correlates to the degree of pedal muscle atrophy and the severity of signs associated with WS . Electron microscopically the intracellular pathogen is a rod-shaped, ribosome-rich, gram-negative, prokaryote with a trilaminar cell wall consistent with the order Rickettsiales . Microbiological and protozoological methods produced no patterns that implicated other types of microbes . Chemical analysis of tissue from animals from a population with WS did not support an association between WS and environmental pollutant exposure to polycyclic aromatic hydrocarbons, polychlorinated biphenyls, or chlorinated pesticides. J Antibiot (Tokyo), 1995 Sep, 48(9), 997 - 1003 Bacillaene, a novel inhibitor of procaryotic protein synthesis produced by Bacillus subtilis: production, taxonomy, isolation, physico-chemical characterization and biological activity; Patel PS et al.; Bacillaene, a novel polyene antibiotic, was discovered and isolated from fermentation broths of a strain of Bacillus subtilis . The novel antibiotic has a nominal molecular weight of 580 and an empirical formula of C35H48O7 . Bacillaene is active against a broad spectrum of bacteria in agar-plate diffusion assays . Studies in vitro indicate that the antibiotic inhibits prokaryotic protein synthesis but not eukaryotic protein synthesis . Cell survival studies performed with strains of Escherichia coli indicate that the antibiotic is a bacteriostatic agent. Microbiol Rev, 1995 Sep, 59(3), 506 - 31 Stress-induced transcriptional activation; Mager WH et al.; Living cells, both prokaryotic and eukaryotic, employ specific sensory and signalling systems to obtain and transmit information from their environment in order to adjust cellular metabolism, growth, and development to environmental alterations . Among external factors that trigger such molecular communications are nutrients, ions, drugs and other compounds, and physical parameters such as temperature and pressure . One could consider stress imposed on cells as any disturbance of the normal growth condition and even as any deviation from optimal growth circumstances . It may be worthwhile to distinguish specific and general stress circumstances . Reasoning from this angle, the extensively studied response to heat stress on the one hand is a specific response of cells challenged with supra-optimal temperatures . This response makes use of the sophisticated chaperoning mechanisms playing a role during normal protein folding and turnover . The response is aimed primarily at protection and repair of cellular components and partly at acquisition of heat tolerance . In addition, heat stress conditions induce a general response, in common with other metabolically adverse circumstances leading to physiological perturbations, such as oxidative stress or osmostress . Furthermore, it is obvious that limitation of essential nutrients, such as glucose or amino acids for yeasts, leads to such a metabolic response . The purpose of the general response may be to promote rapid recovery from the stressful condition and resumption of normal growth . This review focuses on the changes in gene expression that occur when cells are challenged by stress, with major emphasis on the transcription factors involved, their cognate promoter elements, and the modulation of their activity upon stress signal transduction . With respect to heat shock-induced changes, a wealth of information on both prokaryotic and eukaryotic organisms, including yeasts, is available . As far as the concept of the general (metabolic) stress response is concerned, major attention will be paid to Saccharomyces cerevisiae. J Allergy Clin Immunol, 1995 Sep, 96(3), 395 - 402 Type I skin reactivity to native and recombinant phospholipase A2 from honeybee venom is similar; Muller UR et al.; Phospholipase A2 is the major allergen in honeybee venom . Recombinant phospholipase A2 was produced in prokaryotes and tested for its biologic activity by intracutaneous skin testing with serial 10-fold dilutions in comparison with native and deglycosylated phospholipase A2 in patients allergic to bee venom . Linear regressions of the log of the wheal area versus the log of the allergen concentration were calculated for all allergens in each patient . The relative allergenic potency of the various preparations was analyzed by comparing the linear regressions . Native phospholipase A2 was about 10 times more potent than whole bee venom . None of 58 patients allergic to bee venom was missed by testing with native phospholipase A2 alone . This allergen and deglycosylated native phospholipase A2 resulted in similar skin reactions, indicating that the sugar residues were of little relevance for IgE-binding in the patients tested . Native phospholipase A2 also had relative potency similar to that of recombinant refolded phospholipase A2, whereas recombinant nonrefolded phospholipase A2 had almost no biologic activity in skin testing . These results demonstrate in vivo activity of the recombinant bee venom allergen phospholipase A2 . Although correct refolding is a prerequisite for type I skin reactivity, glycosylation seems to be less important. EMBO J, 1995 Sep 1, 14(17), 4365 - 73 The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae; Stansfield I et al.; The product of the yeast SUP45 gene (Sup45p) is highly homologous to the Xenopus eukaryote release factor 1 (eRF1), which has release factor activity in vitro . We show, using the two-hybrid system, that in Saccharomyces cerevisiae Sup45p and the product of the SUP35 gene (Sup35p) interact in vivo . The ability of Sup45p C-terminally tagged with (His)6 to specifically precipitate Sup35p from a cell lysate was used to confirm this interaction in vitro . Although overexpression of either the SUP45 or SUP35 genes alone did not reduce the efficiency of codon-specific tRNA nonsense suppression, the simultaneous overexpression of both the SUP35 and SUP45 genes in nonsense suppressor tRNA-containing strains produced an antisuppressor phenotype . These data are consistent with Sup35p and Sup45p forming a complex with release factor properties . Furthermore, overexpression of either Xenopus or human eRF1 (SUP45) genes also resulted in anti-suppression only if that strain was also overexpressing the yeast SUP35 gene . Antisuppression is a characteristic phenotype associated with overexpression of both prokaryote and mitochondrial release factors . We propose that Sup45p and Sup35p interact to form a release factor complex in yeast and that Sup35p, which has GTP binding sequence motifs in its C-terminal domain, provides the GTP hydrolytic activity which is a demonstrated requirement of the eukaryote translation termination reaction. J Bacteriol, 1995 Sep, 177(18), 5342 - 5 Properties of peptide chain release factor 2 from Streptomyces coelicolor A3(2): conserved primary structure but no frameshift regulation; Ogawara H et al.; A gene was cloned from Streptomyces coelicolor A3(2) . It encodes a protein of 368 amino acid residues with a high degree of similarity to prokaryotic release factor 2 . However, it has neither an internal stop codon nor the Shine-Dalgarno-like sequence immediately upstream of the assumed frameshift position . The gene is expressed and functional in Escherichia coli as peptide chain release factor 2 . The transcription start site is at or adjacent to the translational start site . The size of the mRNA detected by hybridization suggests that the gene (prfB) is monocistronic in S . coelicolor A3(2) . However, about 80 bp upstream of the gene there is an operon which is composed of two genes encoding eukaryotic-type serine/threonine kinases. J Infect Dis, 1995 Sep, 172(3), 785 - 93 Peptide from a prokaryotic adhesin blocks leukocyte migration in vitro and in vivo; Rozdzinski E et al.; The integrin CD11b/CD18 promotes leukocyte extravasation during inflammation . Filamentous hemagglutinin (FHA) of Bordetella pertussis binds to CD11b/CD18, raising the possibility that peptides derived from FHA might inhibit leukocyte migration . The Arg-Gly-Asp (RGD) sequence of FHA has been suggested to modulate binding of ligands to CD11b/CD18 . Peptides derived from this region inhibited adherence and transendothelial migration of neutrophils in vitro and prevented recruitment of leukocytes into the cerebrospinal fluid in an experimental model of meningitis in rabbits . The mechanism of the antiinflammatory effect may involve modulation of the activity of CD11b/CD18 through peptide interaction with the leukocyte response integrin/integrin-associated protein complex. Microbiology, 1995 Sep, 141 ( Pt 9), 2063 - 70 L-arginine transport and metabolism in Giardia intestinalis support its position as a transition between the prokaryotic and eukaryotic kingdoms; Knodler LA et al.; Arginine is metabolized by the arginine dihydrolase pathway in Giardia intestinalis trophozoites and is an important metabolic fuel for this parasite . Radiolabelled arginine was used to characterize the transport of arginine into Giardia intestinalis trophozoites . The transporter had a high affinity for arginine (Km 15 microM) and a high activity {Vmax 76 nmol min-1 (mg protein)-1 at 25 degrees C} . Substrate specificity studies indicated an absolute requirement for the alpha-amino and carboxyl groups, but a tolerance for some substitutions in the guanidino group . The use of non-metabolized arginine analogues in combination with HPLC amino acid analysis of intra- and extracellular pools demonstrated that the arginine transporter is an arginine-ornithine antiport . Investigations of the first step of arginine metabolism, involving arginine deiminase, revealed a relatively high affinity for arginine (Km 0.16 mM) and a large maximal velocity {Vmax 550 nmol min-1 (mg protein)-1 at 37 degrees C} . Substrate specificity studies showed that the arginine deiminase had a characteristically different substrate recognition profile to that of the arginine transporter . Overall, the combination of the transporter and the deiminase result in very low intracellular arginine concentrations and their properties are consistent with the rapid transport of arginine for metabolism via the arginine dihydrolase pathway. Proc Natl Acad Sci U S A, 1995 Aug 29, 92(18), 8140 - 4 Forced evolution of glutathione S-transferase to create a more efficient drug detoxication enzyme; Gulick AM et al.; Glutathione S-transferases (EC 2.5.1.18) in mammalian cells catalyze the conjugation, and thus, the detoxication of a structurally diverse group of electrophilic environmental carcinogens and alkylating drugs, including the antineoplastic nitrogen mustards . We proposed that structural alteration of the nonspecific electrophile-binding site would produce mutant enzymes with increased efficiency for detoxication of a single drug and that these mutants could serve as useful somatic transgenes to protect healthy human cells against single alkylating agents used in cancer chemotherapy protocols . Random mutagenesis of three regions (residues 9-14, 102-112, and 210-220), which together compose the glutathione S-transferase electrophile-binding site, followed by selection of Escherichia coli expressing the enzyme library with the nitrogen mustard mechlorethamine (20-500 microM), yielded mutant enzymes that showed significant improvement in catalytic efficiency for mechlorethamine conjugation (up to 15-fold increase in kcat and up to 6-fold increase in kcat/Km) and that confer up to 31-fold resistance, which is 9-fold greater drug resistance than that conferred by the wild-type enzyme . The results suggest a general strategy for modification of drug- and carcinogen-metabolizing enzymes to achieve desired resistance in both prokaryotic and eukaryotic plant and animal cells. Biochemistry, 1995 Aug 29, 34(34), 10879 - 85 Construction, cloning, and expression of synthetic genes encoding spider dragline silk; Prince JT et al.; Synthetic genes encoding recombinant spider silk proteins have been constructed, cloned, and expressed . Protein sequences were derived from Nephila clavipes dragline silk proteins and reverse-translated to the corresponding DNA sequences . Codon selection was chosen to maximize expression levels in Escherichia coli . DNA "monomer" sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy . Multimers were cloned into a prokaryotic expression vector and the encoded silk proteins were expressed in E . coli upon induction with IPTG . Four multimer, ranging in size from 14.7 to 41.3 kDa, were chosen for detailed analysis . These proteins were isolated by immobilized metal affinity chromatography and purified using reverse-phase HPLC . The composition and identity of the purified proteins were confirmed by amino acid composition analysis, N-terminal sequencing, laser desorption mass spectroscopy, and Western analysis using antibodies reactive to native spider dragline silk . Circular dichroism measurements indicate that the synthetic spider silks have substantial beta-sheet structure. J Mol Biol, 1995 Aug 25, 251(4), 533 - 49 The PS-IAA4/5-like family of early auxin-inducible mRNAs in Arabidopsis thaliana; Abel S et al.; The plant hormone auxin transcriptionally activates early genes . We have isolated a 14-member family of DNA sequences complementary to indoleacetic acid (IAA)-inducible transcripts in Arabidopsis thaliana . The corresponding genes, IAA1 to IAA14, are homologs of PS-IAA4/5 and PS-IAA6 from pea, Aux22 and Aux28 from soybean, ARG3 and ARG4 from mungbean, and AtAux2-11 and AtAux2-27 from Arabidopsis . The members of the family are differentially expressed in mature Arabidopsis plants . Characterization of IAA gene expression in etiolated seedlings demonstrates specificity for auxin inducibility . The response of most family members to IAA is rapid (within 4 to 30 minutes) and insensitive to cycloheximide . Cycloheximide alone induces all the early genes . Auxin-induction of two late genes, IAA7 and IAA8, is inhibited by cycloheximide, indicating requirement of protein synthesis for their activation . All IAA genes display a biphasic dose response that is optimal at 10 microM IAA . However, individual genes respond differentially between 10 nM and 5 microM IAA . Expression of all genes is defective in the Arabidopsis auxin-resistant mutant lines axr1, axr2 and aux1 . The encoded polypeptides share four conserved domains, and seven invariant residues in the intervening regions . The spacers vary considerably in length, rendering the calculated molecular mass of IAA proteins to range from 19 kDa to 36 kDa . Overall sequence identity between members of the family is highly variable (36 to 87%) . Their most significant structural features are functional nuclear transport signals, and a putative beta alpha alpha-fold whose modeled three dimensional structure appears to be compatible with the prokaryotic beta-ribbon DNA recognition motif . The data suggest that auxin induces in a differential and hierarchical fashion a large family of early genes that encode a structurally diverse class of nuclear proteins . These proteins are proposed to mediate tissue-specific and cell-type restricted responses to the hormone during plant growth and development. J Biol Chem, 1995 Aug 25, 270(34), 19861 - 7 Expression and characterization of branched-chain alpha-ketoacid dehydrogenase kinase from the rat . Is it a histidine-protein kinase? Davie JR, Wynn RM, Meng M, Huang YS, Aalund G, Chuang DT, Lau KS. The recombinant rat branched-chain alpha-ketoacid dehydrogenase kinase has been amplified from rat kidney cDNA, based on the previously reported rat cDNA sequence (Popov, K . M., Zhao, Y., Shimomura, Y., Kuntz, M . J., and Harris, R . A . (1992) J . Biol . Chem . 267, 13127-13130) . This kinase was expressed in Escherichia coli as a fusion protein with bacterial maltose-binding protein (MBP) . Expression was improved by overexpression of chaperonins GroEL and GroES . The MBP-kinase, when reconstituted with lipoylated recombinant E2 (dihydrolipoyl transacylase), catalyzed phosphorylation of recombinant E1 (branched-chain alpha-ketoacid decarboxylase) with a kcat of 28.5 nmol of phosphate/min/nmol of MBP-kinase at 25 degrees C . Recombinant MBP-kinase alone demonstrated a slow rate of autophosphorylation with a kcat of 3.25 pmol of phosphate/min/nmol of kinase at 25 degrees C . Serine 22 of the kinase was identified as the possible site of autophosphorylation by Edman microsequencing analysis . Autophosphorylated kinase cannot transfer phosphate to E1, indicating that autophosphorylation of kinase is not an intermediate in ATP-dependent phosphorylation of E1 . Therefore, despite the reported sequence similarity to prokaryotic histidine protein kinases, the mitochondrial rat branched-chain alpha-ketoacid dehydrogenase kinase apparently does not phosphorylate E1 via a histidine-mediated phosphotransfer reaction . Significant corrections to the published cDNA sequence of rat branched-chain alpha-ketoacid dehydrogenase kinase are included. J Theor Biol, 1995 Aug 21, 175(4), 533 - 44 Analytical solutions of the dinucleotide probability after and before random mutations; Arques DG et al.; The mutation process is a classical evolutionary genetic process mainly based on the (random) substitutions of one base (A = Adenine, C = Cytosine, G = Guanine, T = Thymine) for another . Two analytical solutions derived here allow us to analyse in genes the occurrence probabilities of motifs (e.g . dinucleotides) after substitutions (in the evolutionary sense: from the past to the present) and, unexpectedly, also before substitutions (after back substitutions, in the inverse evolutionary sense: from the present to the past) . We generalize on the alphabet {A, C, G, T} of the analytical solutions and of the properties derived on the alphabet {R, Y} (R = purine = A or G, Y = pyrimidine = C or T) . Application of the theory is based on the analytical solution giving the probabilities of the 16 dinucleotides AA, ..., TT in the protein (coding) genes of (nuclear) eukaryotes, viruses and prokaryotes and in (eukaryotic) introns after back substitutions (called primitive genes) . After back substitutions, four of 16 dinucleotides--CG, TA, GT and AC--occur with low probabilities in each of these four primitive gene populations, except for CG in the primitive prokaryotic protein genes . In the primitive eukaryotic protein genes, the dinucleotide AT has also a significant low probability . We present the properties of the two analytical solutions, and the functions which may have these five dinucleotides in primitive genes are described in terms of biological signals. EMBO J, 1995 Aug 15, 14(16), 4065 - 72 Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3; Zhouravleva G et al.; Termination of translation in higher organisms is a GTP-dependent process . However, in the structure of the single polypeptide chain release factor known so far (eRF1) there are no GTP binding motifs . Moreover, in prokaryotes, a GTP binding protein, RF3, stimulates translation termination . From these observations we proposed that a second eRF should exist, conferring GTP dependence for translation termination . Here, we have shown that the newly sequenced GTP binding Sup35-like protein from Xenopus laevis, termed eRF3, exhibits in vitro three important functional properties: (i) although being inactive as an eRF on its own, it greatly stimulates eRF1 activity in the presence of GTP and low concentrations of stop codons, resembling the properties of prokaryotic RF3; (ii) it binds and probably hydrolyses GTP; and (iii) it binds to eRF1 . The structure of the C-domain of the X.laevis eRF3 protein is highly conserved with other Sup35-like proteins, as was also shown earlier for the eRF1 protein family . From these and our previous data, we propose that yeast Sup45 and Sup35 proteins belonging to eRF1 and eRF3 protein families respectively are also yeast termination factors . The absence of structural resemblance of eRF1 and eRF3 to prokaryotic RF1/2 and RF3 respectively, may point to the different evolutionary origin of the translation termination machinery in eukaryotes and prokaryotes . It is proposed that a quaternary complex composed of eRF1, eRF3, GTP and a stop codon of the mRNA is involved in termination of polypeptide synthesis in ribosomes. EMBO J, 1995 Aug 15, 14(16), 3855 - 63 Crystal structure of the phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with myo-inositol; Heinz DW et al.; Phosphatidylinositol (PI), once regarded as an obscure component of membranes, is now recognized as an important reservoir of second messenger precursors and as an anchor for membrane enzymes . PI-specific phospholipase C (PI-PLC) is the enzyme that cleaves PI, invoking numerous cellular responses . The crystal structure of PI-PLC from Bacillus cereus (EC 3.1.4.10) has been solved at 2.6 A resolution and refined to a crystallographic R factor of 18.7% . The structure consists of an imperfect (beta alpha)8-barrel similar to that first observed for triose phosphate isomerase and does not resemble any other known phospholipase structure . The active site of the enzyme has been identified by determining the structure of PI-PLC in complex with its inhibitor, myo-inositol, at 2.6 A resolution (R factor = 19.5%) . This substrate-like inhibitor interacts with a number of residues highly conserved among prokaryotic PI-PLCs . Residues His32 and His82, which are also conserved between prokaryotic and eukaryotic PI-PLCs, most likely act as general base and acid respectively in a catalytic mechanism analogous to that observed for ribonucleases. Genes Dev, 1995 Aug 15, 9(16), 2042 - 52 The bacterial enhancer-binding protein NTRC is a molecular machine: ATP hydrolysis is coupled to transcriptional activation; Wedel A et al.; NTRC is a prokaryotic enhancer-binding protein that activates transcription by sigma 54-holoenzyme . NTRC has an ATPase activity that is required for transcriptional activation, specifically for isomerization of closed complexes between sigma 54-holoenzyme and a promoter to open complexes . In the absence of ATP hydrolysis, there is known to be a kinetic barrier to open complex formation (i.e., the reaction proceeds so slowly that the polymerase synthesizes essentially no transcripts even from a supercoiled template) . We show here that open complex formation is also thermodynamically unfavorable . In the absence of ATP hydrolysis the position of equilibrium between closed and open complexes favors the closed ones . Use of linear templates with a region of heteroduplex around the transcriptional start site--"preopened" templates--does not bypass the requirement for either NTRC or ATP hydrolysis, providing evidence that the rate-limiting step in open complex formation does not lie in DNA strand denaturation per se . These results are in contrast to recent findings regarding the ATP requirement for initiation of transcription by eukaryotic RNA polymerase II; in the latter case, the ATP requirement is circumvented by use of a supercoiled plasmid template or a preopened linear template. Proc Natl Acad Sci U S A, 1995 Aug 15, 92(17), 7814 - 8 The two single-strand cleavages at each end of Tn10 occur in a specific order during transposition; Bolland S et al.; During Tn10 transposition, the element is excised from the donor site by double-strand cleavages at the two transposon ends . Double-strand cleavage is a central step in the nonreplicative transposition reaction of many transposons in both prokaryotes and eukaryotes . Evidence is presented to show that the Tn10 double-strand cut is made by an ordered, sequential cleavage of the two strands . The transferred strand is cut first, and then the nontransferred strand is cleaved . The single-strand nicked intermediate is seen to accumulate when Mn2+ is substituted for Mg2+ in the reaction or when certain mutant transposases are used . The fact that the transferred strand is cleaved before the non-transferred strand implies that the order of strand cleavages is not the determining factor that precludes a replicative mechanism of transposition. Proc Natl Acad Sci U S A, 1995 Aug 15, 92(17), 7774 - 8 Molecular cloning of insect pro-phenol oxidase: a copper-containing protein homologous to arthropod hemocyanin; Kawabata T et al.; Pro-phenol oxidase {pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)} is present in the hemolymph plasma of the silkworm Bombyx mori . Pro-PO is a heterodimeric protein synthesized by hemocytes . A specific serine proteinase activates both subunits through a limited proteolysis . The amino acid sequences of both subunits were deduced from their respective cDNAs; amino acid sequence homology between the subunits was 51% . The deduced amino acid sequences revealed domains highly homologous to the copper-binding site sequences (copper-binding sites A and B) of arthropod hemocyanins . The overall sequence homology between silkworm pro-PO and arthropod hemocyanins ranged from 29 to 39% . Phenol oxidases from prokaryotes, fungi, and vertebrates have sequences homologous to only the copper-binding site B of arthropod hemocyanins . Thus, silkworm pro-PO DNA described here appears distinctive and more closely related to arthropod hemocyanins . The pro-PO-activating serine proteinase was shown to hydrolyze peptide bonds at the carboxyl side of arginine in the sequence-Asn-49-Arg-50-Phe-51-Gly-52- of both subunits . Amino groups of N termini of both subunits were indicated to be N-acetylated . The cDNAs of both pro-PO subunits lacked signal peptide sequences . This result supports our contention that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins. Proc Natl Acad Sci U S A, 1995 Aug 15, 92(17), 7714 - 8 Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes; Arfin SM et al.; Using partial amino acid sequence data derived from porcine methionyl aminopeptidase (MetAP; methionine aminopeptidase, peptidase M; EC 3.4.11.18), a full-length clone of the homologous human enzyme has been obtained . The cDNA sequence contains 2569 nt with a single open reading frame corresponding to a protein of 478 amino acids . The C-terminal portion representing the catalytic domain shows limited identity with MetAP sequences from various prokaryotes and yeast, while the N terminus is rich in charged amino acids, including extended strings of basic and acidic residues . These highly polar stretches likely result in the spuriously high observed molecular mass (67 kDa) . This cDNA sequence is highly similar to a rat protein, termed p67, which was identified as an inhibitor of phosphorylation of initiation factor eIF2 alpha and was previously predicted to be a metallopeptidase based on limited sequence homology . Model building established that human MetAP (p67) could be readily accommodated into the Escherichia coli MetAP structure and that the Co2+ ligands were fully preserved . However, human MetAP was found to be much more similar to a yeast open reading frame that differed markedly from the previously reported yeast MetAP . A similar partial sequence from Methanothermus fervidus suggests that this p67-like sequence is also found in prokaryotes . These findings suggest that there are two cobalt-dependent MetAP families, presently composed of the prokaryote and yeast sequences (and represented by the E . coli structure) (type I), on the one hand, and by human MetAP, the yeast open reading frame, and the partial prokaryotic sequence (type II), on the other. Biochemistry, 1995 Aug 15, 34(32), 10146 - 52 Identification of the active site catalytic residue in human isovaleryl-CoA dehydrogenase; Mohsen AW et al.; Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric flavoenzyme which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA . E376 of pig medium chain acyl-CoA dehydrogenase (MCAD), a homologous enzyme, has been identified as the active site catalytic residue . Amino acid sequence alignment shows that A375 is the corresponding residue in human IVD . Using the atomic coordinates determined for MCAD, molecular modeling suggests that E254 is the substituting catalytic residue in IVD . To substantiate the importance of this residue for enzyme function, cDNAs for the wild-type human IVD and E254G, E254D, E254Q, and E254G/A375E mutant IVDs were constructed and cloned into a prokaryotic expression vector . The proteins were synthesized in Escherichia coli and purified, and their properties were examined . The catalytic activity of the recombinant wild-type IVD was the highest in the presence of isovaleryl-CoA, and its UV/visible light spectrum in the presence of isovaleryl-CoA showed quenching of its characteristic absorption in the 445-nm region and appearance of absorption at 600 nm . The E254G and E254Q mutant IVDs had no detectable enzymatic activity, and isovaleryl-CoA did not induce quenching of the absorption in the 445-nm region or the appearance of absorption at 600 nm . The E254D mutant IVD had residual activity for isovaleryl-CoA, and its spectrum was altered compared to that of the wild type . The E254G/A375E mutant IVD exhibited catalytic activity toward isovaleryl-CoA, and its spectrum in the absence or presence of the substrate was similar to that of the wild-type IVD.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1995 Aug 14, 370(1-2), 23 - 6 Class III alcohol dehydrogenase from Saccharomyces cerevisiae: structural and enzymatic features differ toward the human/mammalian forms in a manner consistent with functional needs in formaldehyde detoxication; Fernandez MR et al.; Alcohol dehydrogenase class III (glutathione-dependent formaldehyde dehydrogenase) from Saccharomyces cerevisiae was purified and analyzed structurally and enzymatically . The corresponding gene was also analyzed after cloning from a yeast genome library by screening with a probe prepared through PCR amplification . As with class III alcohol dehydrogenase from other sources, the yeast protein was obtained in two active forms, deduced to reflect different adducts/modifications . Protein analysis established N-terminal and C-terminal positions, showing different and specific patterns in protein start positions between the human/mammalian, yeast, and prokaryotic forms . Km values with formaldehyde differ consistently, being about 10-fold higher in the yeast than the human/mammalian enzymes, but compensated for by similar changes in kcat values . This is compatible with the different functional needs, emphasizing low formaldehyde concentration in the animal cells but efficient formaldehyde elimination in the microorganisms . This supports a general role of the enzyme in formaldehyde detoxication rather than in long-chain alcohol turnover. J Biol Chem, 1995 Aug 4, 270(31), 18543 - 50 Mammalian DNA (cytosine-5-)-methyltransferase expressed in Escherichia coli, purified and characterized; Tollefsbol TO et al.; Besides modulating specific DNA-protein interactions, methylated cytosine, frequently referred to as the fifth base of the genome, also influences DNA structure, recombination, transposition, repair, transcription, imprinting, and mutagenesis . DNA (cytosine-5-)-methyltransferase catalyzes cytosine methylation in eukaryotes . We have cloned and expressed this enzyme in Escherichia coli, purified it to apparent homogeneity, characterized its properties, and we have shown that it hemimethylates DNA . The cDNA for murine maintenance methyltransferase was reconstructed and cloned for direct expression in native form . Immunoblotting revealed a unique protein (M(r) = 190,000) not present in control cells . The mostly soluble overexpressed protein was purified by DEAE, Sephadex, and DNA cellulose chromatography . Peak methylating activity correlated with methyltransferase immunoblots . The purified enzyme preferentially transferred radioactive methyl moieties to hemimethylated DNA in assays and on autoradiograms . All of the examined properties of the purified recombinant DNA methyltransferase are consistent with the enzyme purified from mammalian cells . Further characterization revealed enhanced in vitro methylation of premethylated oligodeoxynucleotides . The cloning of hemimethyltransferase in E . coli should allow facilitated structure-function mutational analysis of this enzyme, studies of its biological effects in prokaryotes, and potential large scale methyltransferase production for crystallography, and it may have broad applications in maintaining the native methylated state of cloned DNA. J Biol Chem, 1995 Aug 4, 270(31), 18341 - 6 Isolation and characterization of a cDNA encoding the SecA protein from spinach chloroplasts . Evidence for azide resistance of Sec-dependent protein translocation across thylakoid membranes in spinach; Berghofer J et al.; Thylakoid membranes of chloroplasts in higher plants harbor different pathways for the translocation of proteins . One of these routes is related to the prokaryotic Sec pathway, which mediates the secretion of particular proteins into the periplasmic space and involves the SecA protein as an essential component . We have isolated a full size cDNA of 3739 nucleotides encoding the SecA homologue from spinach . It contains an open reading frame of 1036 codons corresponding to a polypeptide with a calculated mass of 117 kDa . The deduced amino acid sequence shows between 43 and 49% identity to SecA proteins from bacteria and lower algae and 62% identity to SecA of the cyanobacterium Synechococcus sp . PCC7942 . Compared with the Escherichia coli protein, spinach SecA carries an amino-terminal extension of approximately 80 residues . In organello experiments performed with the protein made in vitro by transcription of the cDNA and cell-free translation of the resulting RNA showed that this extension comprises a transit peptide that mediates the import of the protein into the chloroplast . The processed product of approximately 107 kDa accumulates predominantly in the stroma and to a lower extent associates with the thylakoid membrane . Comparably to E . coli, in which SecA activity can be inhibited by sodium azide, thylakoid translocation of a subset of lumenal proteins is sensitive to sodium azide in pea but not in spinach chloroplasts, suggesting that the latter contain an azide-resistant SecA variant. J Natl Cancer Inst, 1995 Aug 2, 87(15), 1114 - 25 Hereditary nonpolyposis colorectal cancer: the syndrome, the genes, and historical perspectives; Marra G et al.; Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disorder characterized by the occurrence within a family of multiple cases of colorectal cancer in the absence of gastrointestinal polyposis . The prevalence of this syndrome is not yet clear, but it may account for 1%-5% of all colorectal cancers . Prior to the identification of the genetic basis of this syndrome, the disease was recognized by the familial aggregation of colorectal cancers that had an early age of onset, an excess of proximally located, and often multiple, primary tumors, and an excess occurrence of cancers in certain other organs . The recent description of an abnormality called "microsatellite instability," present in almost all cancers from HNPCC patients and in about 12%-15% of sporadic cases, led to a series of discoveries that linked this type of genomic instability to a defect in the DNA mismatch repair (MMR) system . Independent investigators have identified four HNPCC genes: hMSH2 (a homologue of the prokaryotic DNA MMR gene MutS) and hMLH1, hPMS1, and hPMS2 (all homologues of the prokaryotic DNA MMR gene MutL) . Mutations in each of the four genes have been found in the germline cells of HNPCC families . A major target for research in this area is the development of clinically practical screening tests for the genetic carrier state of HNPCC. Yeast, 1995 Aug, 11(10), 975 - 86 A 29.425 kb segment on the left arm of yeast chromosome XV contains more than twice as many unknown as known open reading frames; Zumstein E et al.; The nucleotide sequence of a 29.425 kb fragment localized on the left arm of chromosome XV from Saccharomyces cerevisiae has been determined . The sequence contains 13 open reading frames (ORFs) of which four encode the known genes ADH1, COQ3, MSH2 and RCF4 . Predictions are made concerning the functions of the unknown ORFs . Some of the ORFs contain sequences similar to expressed sequence tags (EST) found in the database made available by TIGR . In particular, the highly expressed ADH1 gene is represented in this database by no less than 20 EST sequences . Two ARS sequences and a putative functional GCN4 motif have also been detected . One ORF (O0953) containing nine putative transmembrane segments is similar to a hypothetical membrane protein of Arabidopsis thaliana . Characteristic features of the other ORFs include ATP/GTP binding sites, a fungal Zn(2)-Cys(6) binuclear centre, an endoplasmic reticulum targeting sequence, a beta-transducin repeat signature and in two instances, good similarity to the prokaryotic lipoprotein signal peptide motif. Bioessays, 1995 Aug, 17(8), 713 - 9 Structure and function of apurinic/apyrimidinic endonucleases; Barzilay G et al.; The DNA of all species is constantly under threat from both endogenous and exogenous factors, which damage its chemical structure . Probably the most common lesion that arises in cellular DNA is the loss of a base to generate an abasic site, which is usually referred to as an apurinic or apyrimidinic (AP) site . Since these lesions are potentially both cytotoxic and mutagenic, cells of all organisms express dedicated repair enzymes, termed AP endonucleases, to counteract their damaging effects . Indeed, many organisms consider it necessary to express two or more of these lesion-specific endonucleases, underscoring the requirement that exists to remove AP sites for the maintenance of genome integrity and cell viability . Most AP endonucleases are very versatile enzymes, capable of performing numerous additional repair roles . In this article, we review the AP endonuclease class of repair enzymes, with emphasis on the evolutionary conservation of structural features, not only between prokaryotic and eukaryotic homologues, but also between these enzymes and the RNase H domain of one class of reverse transcriptase. FASEB J, 1995 Aug, 9(11), 1034 - 42 Hypermodified bases in DNA; Gommers-Ampt JH et al.; Modified DNA bases are widespread in nature . They can be found in eukaryotes, prokaryotes, and bacteriophages . They may completely replace the standard base or replace only a small fraction . Their substituents vary from simple methyl or hydroxy groups to large moieties like amino acids and multiply hexosylated side chains . This review gives an overview of the modified DNA bases identified thus far, with emphasis on the "very unusual" or hypermodified DNA bases . Although these have been detected mainly in bacteriophages, recent work has shown the presence of a novel hypermodified DNA base in the eukaryote Trypanosoma brucei . We speculate on the biosynthesis and function of this novel base beta-D-glucosyl-hydroxymethyluracil. Virology, 1995 Aug 1, 211(1), 307 - 11 Herpes simplex virus type 1 (HSV-1) uracil-DNA glycosylase: functional expression in Escherichia coli, biochemical characterization, and selective inhibition by 6-(p-n-octylanilino)uracil; Argnani R et al.; The Herpes simplex virus type 1 (HSV-1) uracil-DNA glycosylase (UDG) is encoded by the UL2 gene . The translation from the first putative start codon of UL2 predicts a polypeptide of 334 residues, while the translation from the second start codon predicts a polypeptide of 244 residues . We have cloned and expressed the two forms of UDG, by means of the prokaryotic expression vector pMAL-c2, and both of them were enzymatically active . Furthermore, the enzymatic properties of the recombinant UDGs and of the enzyme purified from HSV-1-infected cells were similar . The two UDG polypeptides have molecular weights of 27 and 37 kDa, respectively . The 37-kDa form of recombinant UDG is consistent with the reported molecular mass of 37 kDa for the enzyme purified from HSV-1-infected cells . Both recombinant UDGs were as sensitive as UDG purified from HSV-1-infected cells to 6-(p-n-octylanilino)uracil, the most potent of a series of uracil analogs that inhibit the viral enzyme. Virology, 1995 Aug 1, 211(1), 285 - 9 The activation domain of simian immunodeficiency virus SIVmac239 Rev protein is structurally and functionally analogous to the HIV-1 Rev activation domain; Berchtold S et al.; The Rev proteins of primate immunodeficiency viruses are essential transactivators for the switch from early to late phase in the viral replication cycle . By mutational analysis, a putative activation domain (AD) has been assigned to the carboxy-terminus . This leucine-rich stretch of amino acids proved to be essential for the transactivating properties of HIV-1 Rev . Some mutants in the AD transdominantly inhibit the function of wild-type Rev protein very efficiently . We identified a similar domain structure for SIVmac239 Rev by sequence comparison and in vitro mutagenesis . The leucine/isoleucine residues of the SIVmac239 Rev activation domain appeared to be of similar importance for function . The mutants of these residues in the SIV AD displayed a dominant negative phenotype on both HIV-1 and SIVmac 239 rev-responsive elements (RRE) . The prokaryotically expressed wild-type and mutant proteins were analyzed for RNA-binding properties in a gel-shift assay in vitro . This assay revealed a similar binding pattern of wild-type and transdominant proteins on either RRE. Proc Natl Acad Sci U S A, 1995 Aug 1, 92(16), 7535 - 9 Binding of the transcription activator NRI (NTRC) to a supercoiled DNA segment imitates association with the natural enhancer: an electron microscopic investigation; Revet B et al.; Electron microscopic visualization indicates that the transcription activator NRI (NTRC) binds with exceptional selectivity and efficiency to a sequence-induced superhelical (spiral) segment inserted upstream of the glnA promoter, accounting for its observed ability to substitute for the natural glnA enhancer . The cooperative binding of NRI to the spiral insert leads to protein oligomerization which, at higher concentration, promotes selective coating of the entire superhelical segment with protein . Localization of NRI at apical loops is observed with negatively supercoiled plasmid DNA . With a linear plasmid, bending of DNA is observed . We confirm that NRI is a DNA-bending protein, consistent with its high affinity for spiral DNA . These results prove that spiral DNA without any homology to the NRI-binding sequence site can substitute for the glnA enhancer by promoting cooperative activator binding to DNA and facilitating protein oligomerization . Similar mechanisms might apply to other prokaryotic and eukaryotic activator proteins that share the ability to bend DNA and act efficiently as multimers. J Gen Virol, 1995 Aug, 76 ( Pt 8), 2091 - 6 Characterization of the small open reading frame on genome segment A of infectious pancreatic necrosis virus; Heppell J et al.; The genome of infectious pancreatic necrosis virus (IPNV) is composed of two segments of dsRNA . The larger segment contains a small ORF partly overlapping the 5' end of the polyprotein reading frame . Yet very little is known about this possible new gene, which presumably codes for a 17 kDa polypeptide (VP5) . The region of the viral genome which encompasses the small ORF was reverse-transcribed and amplified by PCR before cloning and sequencing . Analysis of the sequences obtained from five different virus strains revealed that the small ORF is not found on one of them, and that it is truncated on two others . Moreover, the deduced amino acid sequences did not appear to be well conserved . Despite the large variations between IPNV strains at the genomic level, all predicted VP5 are arginine-rich basic polypeptides . To verify whether the small ORF is translated into protein in fish cells, the 17 kDa polypeptide of the VR-299 strain was expressed as fusion protein in a prokaryotic expression vector and used to produce a specific antiserum . This antiserum reacted with concentrated virus in an immunodot assay indicating that VP5 is synthesized in infected cells, but probably only in small quantities . When tested with 12 other IPNV strains, results were less conclusive than those obtained with strain VR-299 . Nevertheless, three of the 12 viruses gave a clearly negative signal in the immunodot assay, suggesting that possibly more than one viral strain lacks the small ORF. J Gen Virol, 1995 Aug, 76 ( Pt 8), 1937 - 43 Identification and characterization of the frog virus 3 DNA methyltransferase gene; Kaur K et al.; Cytosine DNA methyltransferases (MTases) first recognize specific nucleotide sequences and then transfer a methyl group from S-adenosylmethionine to cytosine . This division of function is reflected in five highly conserved motifs shared by cytosine MTases . The region containing the first four motifs is responsible for the catalytic function whereas the region containing the fifth motif V provides specificity of binding to DNA . In at least one case, two separate proteins, one containing the first four motifs and the second containing the last motif combine to provide full functional activity . In the frog virus 3 (FV3) genome we have identified an open reading frame (ORF) whose deduced amino acid (aa) sequence contains motifs characteristic of prokaryotic as well as eukaryotic MTases . The ORF consists of 642 bp which codes for a protein of 214 aa with a predicted molecular mass of 24.8 kDa . This ORF contains the first four highly conserved motifs of cytosine MTases but the fifth motif, responsible for DNA binding specificity, is missing . Presumably, FV3 MTase is composed of two subunits . Northern blot analysis showed that the putative MTase ORF is transcribed into two transcripts belonging to the delayed-early class of FV3 messages . These two transcripts appear to be initiated at two different start sites but terminate in the same 3' region of the gene . The transcription start sites are not preceded by any known promoter sequences, but two regions of hyphenated dyad symmetry are present at the 3' end of the message . A protein with a molecular mass of approximately 28 kDa was synthesized by a rabbit reticulocyte lysate programmed with capped runoff transcripts from the cloned gene, suggesting that the ORF can be transcribed into a message coding for a viral protein . Overall, our results suggest that we have identified a gene for a subunit of MTase in the FV3 genome. J Bacteriol, 1995 Aug, 177(15), 4544 - 8 Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3(2); Bedford DJ et al.; The multifunctional 6-methylsalicylic acid synthase gene from Penicillium patulum was engineered for regulated expression in Streptomyces coelicolor . Production of significant amounts of 6-methylsalicylic acid by the recombinant strain was proven by nuclear magnetic resonance spectroscopy . These results suggest that it is possible to harness the molecular diversity of eukaryotic polyketide pathways by heterologous expression of biosynthetic genes in an easily manipulated model bacterial host in which prokaryotic aromatic and modular polyketide synthase genes are already expressed and recombined. Infect Immun, 1995 Aug, 63(8), 2811 - 7 Borrelia burgdorferi shows specificity of binding to glycosphingolipids; Backenson PB et al.; Live but not fixed or heat-killed Borrelia burgdorferi bound to galactocerebroside, lactosylceramide, and ceramide trihexoside . In addition, this organism bound to the disialoganglioside GD1a and the trisialoganglioside GT1b but not to gangliosides GM1, GD1b, GM2, and GM3 and not to asialo GM1 . This adhesion pattern confirmed earlier findings of binding to galactocerebroside and places this organism within a prokaryotic group which binds to lactosylceramide . The binding to GD1a and GT1b, both of which carry terminal as well as multiple sialic acids, indicates that B . burgdorferi can show specificity of binding within a group of acidic gangliosides . Adhesion could not be inhibited by several concentrations of sugars and sialic acid, indicating more complex binding requirements than for terminal carbohydrates alone . Low-passage strains adhered to the four substrates in greater numbers than strains in culture for long periods of time . OspB mutants in general bound better or at least equally well to several of the glycosphingolipids, and preincubation of substrates with soluble recombinant and affinity-purified Osp did not inhibitor or weakly inhibited the binding of the organisms . These findings suggest that outer surface lipoproteins A and B are not directly involved in adhesion to glycosphingolipids. Proc Natl Acad Sci U S A, 1995 Aug 1, 92(16), 7232 - 6 Dominance of one bacterial phylotype at a Mid-Atlantic Ridge hydrothermal vent site; Polz MF et al.; Microbial community structure in natural environments has remained largely unexplored yet is generally considered to be complex . It is shown here that in a Mid-Atlantic Ridge hydrothermal vent habitat, where food webs depend on prokaryotic primary production, the surface microbial community consists largely of only one bacterial phylogenetic type (phylotype) as indicated by the dominance of a single 16S rRNA sequence . The main part of its population occurs as an ectosymbiont on the dominant animals, the shrimp Rimicaris exoculata, where it grows as a monoculture within the carapace and on the extremities . However, the same bacteria are also the major microbial component of the free-living substrate community . Phylogenetically, this type forms a distinct branch within the epsilon-Proteobacteria . This is different from all previously studied chemoautotrophic endo- and ectosymbioses from hydrothermal vents and other sulfidic habitats in which all the bacterial members cluster within the gamma-Proteobacteria. Vet Microbiol, 1995 Aug, 45(4), 363 - 70 Genetic and antigenic characterization of caev (caprine arthritis-encephalitis virus) recombinant transmembrane protein; Rosati S et al.; The env gene fragment of an Italian strain of Caprine Arthritis Encephalitis virus (CAEV) coding for the hydrophilic region of transmembrane protein was amplified, cloned and expressed in prokaryotic system as fusion protein with glutathione-S-transferase . Sequence analysis revealed 63 to 66% amino acid homology, when compared with three ovine lentiviruses and 83% when compared with one caprine lentivirus . The recombinant transmembrane protein was efficiently expressed, purified under denaturing conditions and used as antigen in western blotting and ELISA . Sera from clinically diseased goats strongly reacted in western blotting and naturally infected animals seroconverted between 20 and 33 weeks of age . An indirect ELISA performed with this antigen showed improved sensitivity in comparison with agar gel immunodiffusion test . Our results confirm that transmembrane protein is an important immunological marker in CAEV infection and its use as antigen may enhance the validity of serological diagnosis of Caprine Arthritis Encephalitis. Gene, 1995 Jul 28, 160(2), 263 - 7 High-level expression of an altered cDNA encoding human isovaleryl-CoA dehydrogenase in Escherichia coli; Mohsen AW et al.; Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway . The cDNA encoding the mature human IVD polypeptide was cloned in a prokaryotic expression vector, but the level of expression in Escherichia coli was extremely low and attempts to purify the enzyme to homogeneity were unsuccessful . To enhance expression, the nucleotide sequence of 22 codons within the 111-bp region at the 5'-end of the cDNA was altered to accommodate E . coli codon usage without altering the amino-acid coding sequence . The altered IVD cDNA was synthesized by PCR, using a primer containing the desired modifications . Following overnight induction of the E . coli transformed with this cDNA, the enzyme was purified to homogeneity using diethylaminoethyl agarose and high-pressure ceramic hydroxyapatite resins . IVD activity was increased 165-fold in the crude extract of cells containing the modified cDNA, as compared to that containing the wild-type cDNA. Gene, 1995 Jul 28, 160(2), 173 - 8 Expression of the measles virus nucleoprotein gene in Escherichia coli and assembly of nucleocapsid-like structures; Warnes A et al.; To investigate the use of fusion systems to aid the purification of recombinant proteins for structure/function studies and potential uses as diagnostic reagents, the measles virus (MV) gene encoding the nucleoprotein was cloned and expressed in Escherichia coli in three forms: as a full-length intact protein and as two fusion proteins . Expression of the intact N gene under the control of the tac promoter in the pTrc99c plasmid produced a protein of the correct size (60 kDa) which represented approx . 4% of the total cellular protein, and was recognised by known measles positive human sera . 'Herringbone' structures characteristic of paramyxovirus nucleocapsids (NuC) were identified in fractured cells examined by electron microscopy . The production of NuC-like structures in a prokaryotic cell indicates folding of the nucleoprotein can occur in the absence of MV genomic RNA, other MV-encoded gene products and eukaryotic cell proteins or RNA, to produce structures which are morphologically and antigenically similar to those seen in virus-infected cells . Conversely, synthesis of N protein as a fusion protein with either E . coli beta-galactosidase or the E . coli maltose-binding protein resulted in the production of fused proteins which could not be assembled into NuC-like structures or readily used as diagnostic reagents . However, the ability of MV N protein to form NuC-like structures in E . coli will facilitate structure/function and mutational analysis of the NuC protein. J Biol Chem, 1995 Jul 28, 270(30), 17664 - 7 A SecY homolog in Arabidopsis thaliana . Sequence of a full-length cDNA clone and import of the precursor protein into chloroplasts; Laidler V et al.; Proteins are translocated across the thylakoid membrane by two distinct pathways in higher plant chloroplasts, one of which is related to prokaryotic Sec-dependent translocation mechanisms . SecY is an essential, hydrophobic component of the membrane-bound translocase complex in bacteria, and we report here the nucleotide sequence of a full-length cDNA encoding a homolog of SecY from Arabidopsis thaliana . The predicted protein of 551 residues includes an amino-terminal extension of approximately 120 residues when compared with other SecY proteins . The deduced sequence of the mature protein, cpSecY, is 41% identical with SecY from Synechococcus and 33% identical with the Escherichia coli protein . The extension serves to target the protein into chloroplasts; transcription-translation of the cDNA yields a 58-kDa precursor protein which is imported into pea chloroplasts, processed to a product of 46 kDa, and targeted into the thylakoid membrane. Cell, 1995 Jul 28, 82(2), 279 - 86 Histidine phosphorylation of P-selectin upon stimulation of human platelets: a novel pathway for activation-dependent signal transduction; Crovello CS et al.; Transient phosphorylation of histidine characterizes the two-component systems in prokaryotes that control important physiological functions, but analogous events have not been implicated in signal transduction in mammalian cells . To explore histidine phosphorylation during activation of human cells, stimulated platelets were analyzed for the formation of protein phosphohistidine in a model system employing P-selectin . P-selectin, a leukocyte adhesion molecule, undergoes rapid phosphorylation and selective dephosphorylation of tyrosine, serine, and threonine . We now establish that phosphorylation following platelet activation with thrombin or collagen generates phosphohistidine at histidines on the cytoplasmic tail of P-selectin . With thrombin stimulation, the kinetics of phosphohistidine appearance and disappearance of P-selectin are very rapid . Platelets exhibit a novel ligand-induced signaling pathway to generate phosphohistidine . These results provide direct biochemical evidence for the induction of rapid and reversible histidine phosphorylation in mammalian cells upon cell activation and represent a novel paradigm for mammalian cell signaling. Biochim Biophys Acta, 1995 Jul 26, 1237(2), 189 - 92 A Synechococcus gene encoding a putative pore-forming intrinsic membrane protein; Kashiwagi S et al.; A cyanobacterium, Synechococcus species PCC7942, has a gene encoding a copper-transporting P-type ATPase, which is located in the thylakoid membrane . At the 5'-upstream of this ATPase gene, we identified another gene, which was supposed to be implicated in a copper-transport process . This novel gene was found to encode a putative pore-forming membrane protein that belongs to a growing family of homologous intrinsic membrane proteins (the MIP family of proteins), which include the major intrinsic protein (MIP) from animal lens fibre junction membranes, the tonoplast intrinsic protein (TIP) from vacuolar membranes of higher plants, and the Escherichia coli glycerol facilitator (GlpF) in the cytoplasmic membrane . The deduced product, named SmpX (Synechococcus membrane protein), is highly homologous throughout its entire sequence to these intrinsic membrane proteins which were postulated to be pore-forming proteins involved in a variety of transport processes . The primary amino acid sequence of SmpX shares all properties characteristic for members of the MIP family . SmpX is more similar to the eukaryotic members (e.g., nodulin-26 from soybean) than to the prokaryotic ones. J Biol Chem, 1995 Jul 21, 270(29), 17311 - 6 Evaluation of cysteine 266 of human 3-hydroxy-3-methylglutaryl-CoA lyase as a catalytic residue; Roberts JR et al.; The role of cysteine 266 in human 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, a residue that is homologous to a cysteine mapped to the active site of prokaryotic HMG-CoA lyase by protein chemistry approaches, has been investigated by site-directed mutagenesis . Both the wild-type human enzyme and a C323S variant, in which a regulatory sulfhydryl has been eliminated without any negative effect on catalytic activity (Roberts, J . R., Narasimhan, C., Hruz, P . W., Mitchell, G . A., and Miziorko, H . M . (1994) J . Biol . Chem . 269, 17841-17846), were used as models . Mutant enzymes C266A, C266A/C323S, C266S, and C266S/C323S were overexpressed in Escherichia coli and purified to homogeneity . In all cases, kinetic characterization indicated that the Km value for HMG-CoA was not substantially different from the value measured using wild-type human lyase, suggesting that no serious structural perturbation occurs upon replacing Cys-266 . A dissociable divalent cation (Mn2+ or Mg2+), which is required for activity in both native and C323S enzymes, is also an essential component for activity in each of the Cys-266 mutants . The structural integrity of the human mutants was further indicated by Mn2+ binding studies, which demonstrate similarities not only in the activator cation binding stoichiometries, but also in the KD values for Mn2+ as determined for wild-type and mutant C266A or C266S proteins . Purified C266A and C266A/C323S mutants both displayed approximately 1.3 x 10(4)-fold diminution in specific activity, while the kcat value was diminished in both C266S and C266S/C323S by approximately 9.9 x 10(2)-fold . This large diminution in catalytic efficiency in enzyme variants that display no substantial structural perturbations is in accord with an active-site assignment to Cys-266 and qualifies its sulfhydryl group for consideration as a component of the catalytic apparatus. Nature, 1995 Jul 20, 376(6537), 225 - 9 Targeting of non-Ig sequences in place of the V segment by somatic hypermutation; Yelamos J et al.; Affinity maturation of antibodies is characterized by localized hypermutation of the DNA around the V segment . Here we show, using mice containing single or multiple transgene constructs, that an immunoglobulin V kappa segment can be replaced by human beta-globin or prokaryotic neo or gpt genes without affecting the rate of hypermutation; the V gene itself is not necessary for recruiting hypermutation . The ability to target hypermutation to heterologous genes in vivo could find more general applications in biology. Mol Cell Biochem, 1995 Jul 19, 148(2), 165 - 81 Structures of small subunit ribosomal RNAs in situ from Escherichia coli and Thermomyces lanuginosus; Beniac DR et al.; Small ribosomal subunits from the prokaryote Escherichia coli and the eukaryote Thermomyces lanuginosus were imaged electron spectroscopically, and single particle analysis used to yield three-dimensional reconstructions of the net phosphorus distribution representing the nucleic acid (RNA) backbone . This direct approach showed both ribosomal RNAs to have a three domain structure and other characteristic morphological features . The eukaryotic small ribosomal subunit had a prominent bill present in the head domain, while the prokaryotic subunit had a small vestigial bill . Both ribosomal subunits contained a thick 'collar' central domain which correlates to the site of the evolutionarily conserved ribosomal RNA core, and the location of the majority of ribosomal RNA bases that have been implicated in translation . The reconstruction of the prokaryotic subunit had a prominent protrusion extending from the collar, forming a channel approximately 1.5 nm wide and potentially representing a 'bridge' to the large subunit in the intact monosome . The basal domain of the prokaryotic ribosomal subunit was protein free . In this region of the eukaryotic subunit, there were two basal lobes composed of ribosomal RNA, consistent with previous hypotheses that this is a site for the 'non-conserved core' ribosomal RNA. EMBO J, 1995 Jul 17, 14(14), 3563 - 71 NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5; Bycroft M et al.; The double-stranded RNA binding domain (dsRBD) is an approximately 65 amino acid motif that is found in a variety of proteins that interact with double-stranded (ds) RNA, such as Escherichia coli RNase III and the dsRNA-dependent kinase, PKR . Drosophila staufen protein contains five copies of this motif, and the third of these binds dsRNA in vitro . Using multinuclear/multidimensional NMR methods, we have determined that staufen dsRBD3 forms a compact protein domain with an alpha-beta-beta-beta-alpha structure in which the two alpha-helices lie on one face of a three-stranded anti-parallel beta-sheet . This structure is very similar to that of the N-terminal domain of a prokaryotic ribosomal protein S5 . Furthermore, the consensus derived from all known S5p family sequences shares several conserved residues with the dsRBD consensus sequence, indicating that the two domains share a common evolutionary origin . Using in vitro mutagenesis, we have identified several surface residues which are important for the RNA binding of the dsRBD, and these all lie on the same side of the domain . Two residues that are essential for RNA binding, F32 and K50, are also conserved in the S5 protein family, suggesting that the two domains interact with RNA in a similar way. EMBO J, 1995 Jul 17, 14(14), 3445 - 51 Conservative sorting of F0-ATPase subunit 9: export from matrix requires delta pH across inner membrane and matrix ATP; Rojo EE et al.; In an attempt to understand the mechanisms of sorting of mitochondrial inner membrane proteins, we have analyzed the import of subunit 9 of the mitochondrial F1F0-ATPase (Su9) from Neurospora crassa, an integral inner membrane protein . A chimeric protein was used consisting of the presequence and the first transmembrane domain of Su9 fused to mouse dihydrofolate reductase (preSu9(1-112)-DHFR) . This protein attains the correct topology across the inner membrane (Nout-Cin) following import . The transmembrane domain becomes first completely imported into the matrix, where after processing of the presequence, it mediates membrane insertion and export of the N-terminal tail . Import and export steps can be experimentally dissected into two distinct events . Translocation of the N-terminal hydrophilic tail out of the matrix was blocked when the presequence was not processed, indicating an important role of the sequences and charges flanking the hydrophobic domain . Furthermore, export was supported by a delta pH and required matrix ATP hydrolysis . Thus the hydrophobic transmembrane domain operates as a membrane insertion signal and not as a stop-transfer signal . Our findings suggest that several aspects of this sorting process have been conserved from their prokaryotic ancestors. Nucleic Acids Res, 1995 Jul 11, 23(13), 2526 - 30 Disruption of the Crithidia fasciculata RNH1 gene results in the loss of two active forms of ribonuclease H; Ray DS et al.; Both prokaryotic and eukaryotic cells contain multiple forms of ribonuclease H, a ribonuclease that specifically degrades the RNA strand of RNA-DNA hybrids and which has been implicated in the processing of initiator RNAs and in the removal of RNA primers from Okazaki fragments . The Crithidia fasciculata RNH1 gene encodes an RNase H and was shown to be a single-copy gene in this diploid trypanosomatid . The RNH1 gene has been disrupted by targeted gene disruption using hygromycin or G418 drug-resistance cassettes . Major active forms of RNase H (38 and 45 kDa) were observed on activity gels of extracts of wild-type cells or cells in which one allele of RNH1 was disrupted . Both the 38 and 45 kDa activities were absent in extracts of cells in which both alleles of RNH1 were disrupted indicating that both forms of the C.fasciculata RNase H are encoded by the RNH1 gene. Biochemistry, 1995 Jul 11, 34(27), 8904 - 13 Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli; Wrzesinski J et al.; Pseudouridine (psi) is commonly found in both small and large subunit ribosomal RNAs of prokaryotes and eukaryotes . In Escherichia coli small subunit RNA, there is only one psi, at position 516, in a region of the RNA known to be involved in codon recognition {Bakin et al . (1994) Nucleic Acids Res . 22, 3681-3684} . To assess the function of this single psi residue, the enzyme catalyzing its formation was purified and cloned . The enzyme contains 231 amino acids and has a calculated molecular mass of 25,836 Da . It converts U516 in E . coli 16S RNA transcripts into psi but does not modify any other position in this RNA . It does not react with free unmodified 16S RNA at all, and only poorly with 30S particles containing unmodified RNA . The preferred substrate is an RNA fragment from residues 1 to 678 which has been complexed with 30S ribosomal proteins . The yield varied from 0.6 to 1.0 mol of psi/mol of RNA, depending on the preparation . Free RNA(1-678) was inactive, as was RNA(1-526) and the RNP particle made from it . 23S RNA and tRNAVal transcripts were also inactive . These results suggest that psi formation in vivo occurs at an intermediate stage of 30S assembly . The gene is located at 47.1 min immediately 5' to, and oriented in the same direction as, the bicyclomycin resistance gene . The gene was cloned behind a (His)6 leader for affinity purification . Virtually all of the overexpressed protein was found in inclusion bodies but could be purified to homogeneity on a Ni2+(-) containing resin . Over 200 mg of pure protein could be obtained from a liter of cell culture . Amino acid sequence comparison revealed the existence of a gene in Bacillus subtilis with a similar sequence, and psi sequence analysis established that B . subtilis has the equivalent of psi 516 in its small subunit rRNA . On the other hand, no common sequence motifs could be detected among this enzyme and the two tRNA psi synthases which have been cloned up to now. Gene, 1995 Jul 4, 160(1), 105 - 10 Isolation and characterization of ten mutator alleles of the mitochondrial DNA polymerase-encoding MIP1 gene from Saccharomyces cerevisiae; Hu JP et al.; Ten mutator alleles of MIP1, the gene encoding mitochondrial (mt) DNA polymerase, have been isolated after in vitro random mutagenesis . Five mutations causing a 100-400-fold increase in the frequency of erythromycin-resistant (ErR) mt mutants in yeast mapped to the 3'-5' exonuclease (Exo) domain, and mainly to the three conserved motifs Exo1, Exo2 and Exo3 of this domain, highlighting the importance of proofreading in accurate mt DNA replication . The essential role of the invariant glutamate at the Exo1 site was confirmed and the participation of four amino acids (aa) in the 3'-5' Exo function revealed . Another mutation that is located between the Exo1 and Exo2 sites produced an extremely strong mutator phenotype associated with impaired DNA replication, but could be assigned neither to a conserved aa nor to a conserved portion of the 3'-5' exonuclease domain . The importance of the polymerization domain in accurate mt DNA replication was pointed out by three mutator mutations . Two of these severely impaired mt DNA replication and were assigned to a subdomain of the polymerase which probably corresponds to the 'fingers' module of the Klenow (large) fragment of Escherichia coli DNA polymerase I (PolIk) . The third, which did not alter the efficiency of DNA replication, was located at the active center of the polymerization reaction . Finally, the mutation, R1001I, mapped to the C-terminal part of the MIP1 protein which has no counterpart in prokaryotic DNA polymerases. Int J Syst Bacteriol, 1995 Jul, 45(3), 595 - 9 Intraspecific variation in small-subunit rRNA sequences in GenBank: why single sequences may not adequately represent prokaryotic taxa; Clayton RA et al.; Small-subunit rRNA (SSU rRNA) sequencing is a powerful tool to detect, identify, and classify prokaryotic organisms, and there is currently an explosion of SSU rRNA sequencing in the microbiology community . We report unexpectedly high levels of intraspecific variation (within and between strains) of prokaryote SSU rRNA sequences deposited in GenBank . A total of 82% of the prokaryote species with two published SSU rRNA sequences had more variable positions than a 0.1% random sequencing error would predict, and 48% of these sequence pairs had more variable positions than predicted by a 1.0% random sequencing error . Other sources of sequence variability must account for some of this intraspecific variation . Given these results, phylogenetic studies and biodiversity estimates obtained by using prokaryotic SSU rRNA sequences cannot proceed under the assumption that rRNA sequences of single operons from single isolates adequately represent their taxa . Sequencing SSU rRNA molecules from multiple operons and multiple isolates is highly recommended to obtain meaningful phylogenetic hypotheses, as is careful attention to accurate strain identification. Curr Genet, 1995 Jul, 28(2), 128 - 30 Identification and characterization of the Arabidopsis thaliana chloroplast DNA region containing the genes psbA, trnH and rps19'; Liere K et al.; A 1887-nucleotide chloroplast-DNA region from Arabidopsis thaliana was analyzed . It contains the conserved genes psbA for the precursor of the D1 reaction-centre protein of photosystem II, trnH for tRNAHis, and rps19' for the 6.8-kDa protein of the small ribosomal subunit . Northern hybridization and RNase protection experiments suggest co-transcription of a minor RNA fraction over the full lengths of psbA and the preceding trnK-UUU gene, but not including downstream trnH sequences . In front of the mapped 5' end of the major 1.2-kb psbA transcript is a DNA region that shows the typical architecture of a psbA promoter, consisting of the prokaryotic-type '-35' and '-10' elements as well as the eukaryotic-type 'TATA' motif . The common 3' end of psbA transcripts seems to be located immediately after a stem-loop structure downstream from the coding region. Immunol Lett, 1995 Jul-Aug, 47(1-2), 93 - 6 Identification of natural resistance-associated macrophage protein in peripheral blood lymphocytes; Kishi F et al.; Natural resistance-associated macrophage protein gene (Nramp) was isolated from the gene locus Lsh/Ity/Bcg, which regulates macrophage activation for antimicrobial activity against intracellular pathogens . The deduced protein sequence encodes an integral membrane protein that has structural homology with known prokaryotic and eukaryotic transport systems . In the present study, a polyclonal antibody was raised with the synthetic peptide of the carboxy-terminal 17 amino acids of human Nramp . The protein product of the gene is apparently present in human peripheral blood lymphocyte (PBL) as a 60 kD a protein recognized by the antibody, which is consistent with the calculated molecular mass . Bacterial lipopolysaccharide or interferon-gamma did not appear to stimulate the level of Nramp expression in PBL. Biochem Mol Biol Int, 1995 Jul, 36(4), 705 - 13 Sequencing of RAPD fragments amplified from the genome of the prokaryote Prochlorococcus marinus (prochlorophyta); Lorenz M et al.; DNA fingerprint patterns from the chlorophyll a and b containing prokaryote Prochlorococcus marinus were generated with the RAPD technique using two primers derived from repetitive sequence motifs, {(GATA)4 and M13} and a random primer (OPB-10} . Five RAPD fragments were reamplified, cloned and sequenced . The clones M13/1300 and OPB-10/1100 contained open reading frames, whereas the (GATA)4 fragments were interrupted by stop codons in all frames indicating their noncoding function and possessed a high AT score of 63% and 71%, respectively . With the two (GATA)4 clones and the M13/300 clone strain-specific signals were obtained in a Southern blot analysis of various Prochlorococcus strains . Clones M13/1300 and OPB-10/1100, containing the ORFs, produced RFLPs between the strains analyzed . All RAPD fragments are represented as single copy in the genome of Prochlorococcus. Infect Immun, 1995 Jul, 63(7), 2424 - 34 Membrane topology of Borrelia burgdorferi and Treponema pallidum lipoproteins; Jones JD et al.; A critical issue regarding the molecular architectures of Treponema pallidum and Borrelia burgdorferi, the agents of venereal syphilis and Lyme disease, respectively, concerns the membrane topologies of their major lipoprotein immunogens . A related question is whether these lipid-modified membrane proteins form intramembranous particles during freeze fracture electron microscopy . To address these issues, native borrelial and treponemal lipoproteins were reconstituted into liposomes of diverse composition . The importance of the covalently associated lipids for membrane association of lipoproteins was revealed by the observation that nonlipidated recombinant forms of both B . burgdorferi OspA and the T . pallidum 47-kDa immunogen (Tpp47) showed very weak or no binding to model bilayer vesicles . In contrast to control liposomes reconstituted with bacteriorhodopsin or bovine rhodopsin, two well-characterized transmembrane proteins, none of the lipoprotein-liposomes contained particles when examined by freeze fracture electron microscopy . To extend these findings to prokaryotic lipoproteins with relatively amphiphilic polypeptides, similar experiments were conducted with a recombinant nonlipidated form of Escherichia coli TraT, a lipoprotein which has putative transmembrane domains . The nonlipidated TraT oligomers bound vesicles derived from E . coli lipids but, surprisingly, did not form particles in the freeze-fractured liposomes . These findings support (i) a proposed topology of spirochetal lipoproteins in which the polypeptide is extrinsic to the membrane surface and (ii) the contention that particles visualized in freeze-fractured spirochetal membranes represent poorly characterized transmembrane proteins. FEMS Microbiol Rev, 1995 Jul, 16(4), 309 - 21 Signal transduction in bacteria: phospho-neural network(s) in Escherichia coli? Hellingwerf KJ, Postma PW, Tommassen J, Westerhoff HV. The molecular basis of many forms of signal transfer in living organisms is provided via the transient phosphorylation of regulatory proteins by transfer of phosphoryl groups between these proteins . The dominant form of signal transduction in prokaryotic microorganisms proceeds via so-called two-component regulatory systems . These systems constitute phosphoryl transfer pathways, consisting of two or more components . Most of these pathways are linear, but some converge and some are divergent . The molecular properties of some of the well-characterised representatives of two-component systems comply with the requirements to be put upon the elements of a neural network: they function as logical operators and show the phenomenon of autoamplification . Because there are many phosphoryl transfer pathways in parallel and because there also appears to be cross-talk between these pathways, the total of all two-component regulatory systems in a single prokaryotic cell may show the typical characteristics of a 'phospho-neural network' . This may well lead to signal amplification, associative responses and memory effects, characteristics which are typical for neural networks . One of the main challenges in molecular microbial physiology is to determine the extent of the connectivity of the constituting elements of this presumed 'phospho-neural network', and to outline the extent of intelligence-like behaviour this network can generate . Escherichia coli is the organism of choice for this characterization. Trends Biotechnol, 1995 Jul, 13(7), 264 - 9 Signal transduction and secondary metabolism: prospects for controlling productivity; Beppu T; Evidence is accumulating that demonstrates the key roles played by diffusible molecules in regulating cellular differentiation, even among prokaryotic microorganisms . This is exemplified by A-factor and its analogues, which act as autoregulators for morphological differentiation and secondary metabolism in Streptomyces . The identification of a specific receptor for A-factor and an A-factor-controlled promoter sequence in S . griseus indicate the close similarity of this system to eukaryotic hormonal control . The involvement of prokaryotic homologues of the eukaryotic Ser/Thr-kinases in the regulation of differentiation processes seems to be another characteristic feature of this group of bacteria . Recent evidence for the presence of these molecular signalling systems in Streptomyces is reviewed, along with the inherent implications. Bioessays, 1995 Jul, 17(7), 597 - 607 The significances of bacterial colony patterns; Shapiro JA; Bacteria do many things as organized populations . We have recently learned much about the molecular basis of intercellular communication among prokaryotes . Colonies display bacterial capacities for multicellular coordination which can be useful in nature where bacteria predominantly grow as films, chains, mats and colonies . E . coli colonies are organized into differentiated non-clonal populations and undergo complex morphogenesis . Multicellularity regulates many aspects of bacterial physiology, including DNA rearrangement systems . In some bacterial species, colony development involves swarming (active migration of cell groups) . Swarm colony development displays precise geometrical controls and periodic phenomena . Motile E . coli cells in semi-solid media form organized patterns due to chemotactic autoaggregation . On poor media, B . subtilis forms branched colonies using group motility and long-range chemical signalling . The significances of bacterial colony patterns thus reside in a deeper understanding of prokaryotic biology and evolution and in experimental systems for studying self-organization and morphogenesis. Eur J Immunol, 1995 Jul, 25(7), 2052 - 8 Human V gamma 9-V delta 2 cells are stimulated in a cross-reactive fashion by a variety of phosphorylated metabolites; Burk MR et al.; Many different pathogens stimulate cells bearing the V gamma 9-V delta 2 T cell receptor (TCR), which represent the most abundant population of human gamma delta cells . The antigens responsible for the stimulation of these gamma delta cells are not well characterized . Here, we describe six non-peptidic molecules which share this property: isopentenylpyrophosphate, dimethylallylpyrophosphate, 2,3-diphosphoglyceric acid, glycerol-3-phosphoric acid, xylose-1-phosphate, and ribose-1-phosphate . All these molecules are naturally occurring metabolites in prokaryotic and eukaryotic cells, and stimulate freshly isolated gamma delta cells from peripheral blood of different donors as well as established gamma delta clones . Comparison of their structure with that of similar but inactive molecules showed that both the number and position of the phosphate groups, as well as the residues connected with the carbon backbone are required for stimulation . The CD3-TCR complex is involved in cell triggering as shown by inhibition with anti-CD3 Fab fragments . However, all gamma delta clones were broadly cross-reactive and we could not isolate cells specific for only one ligand . The capacity of this frequent subset of gamma delta cells to recognize common bacterial metabolites confers the advantage to react rapidly to different invading pathogens. Mol Biol Cell, 1995 Jul, 6(7), 759 - 75 Human RNA polymerase II subunit hsRPB7 functions in yeast and influences stress survival and cell morphology; Khazak V et al.; Using a screen to identify human genes that promote pseudohyphal conversion in Saccharomyces cerevisiae, we obtained a cDNA encoding hsRPB7, a human homologue of the seventh largest subunit of yeast RNA polymerase II (RPB7) . Overexpression of yeast RPB7 in a comparable strain background caused more pronounced cell elongation than overexpression of hsRPB7 . hsRPB7 sequence and function are strongly conserved with its yeast counterpart because its expression can rescue deletion of the essential RPB7 gene at moderate temperatures . Further, immuno-precipitation of RNA polymerase II from yeast cells containing hsRPB7 revealed that the hsRPB7 assembles the complete set of 11 other yeast subunits . However, at temperature extremes and during maintenance at stationary phase, hsRPB7-containing yeast cells lose viability rapidly, stress-sensitive phenotypes reminiscent of those associated with deletion of the RPB4 subunit with which RPB7 normally complexes . Two-hybrid analysis revealed that although hsRPB7 and RPB4 interact, the association is of lower affinity than the RPB4-RPB7 interaction, providing a probable mechanism for the failure of hsRPB7 to fully function in yeast cells at high and low temperatures . Finally, surprisingly, hsRPB7 RNA in human cells is expressed in a tissue-specific pattern that differs from that of the RNA polymerase II largest subunit, implying a potential regulatory role for hsRPB7 . Taken together, these results suggest that some RPB7 functions may be analogous to those possessed by the stress-specific prokaryotic sigma factor rpoS. J Biol Chem, 1995 Jun 30, 270(26), 15479 - 84 Calf thymus Hsc70 protein protects and reactivates prokaryotic and eukaryotic enzymes; Ziemienowicz A et al.; The heat-shock 70 protein (Hsp70) chaperone family is very conserved and its prokaryotic homologue, the DnaK protein, is assumed to form one of the cellular systems for the prevention and restoration of heat-induced protein denaturation . By using anti-DnaK antibodies we purified the DnaK homologue heat-shock cognate protein (Hsc70) from calf thymus to apparent homogeneity . This protein was classified as an eukaryotic Hsc70, since (i) monoclonal antibodies against eukaryotic Hsc70 recognized it, (ii) its amino-terminal sequence showed strong homology to Hsp70s from eukaryotes and, (iii) it had an intrinsic weak ATPase activity that was stimulated by various peptide substrates . We show that this calf thymus Hsc70 protein protected calf thymus DNA polymerases alpha and epsilon as well as Escherichia coli DNA polymerase III and RNA polymerase from heat inactivation and could reactivate these heat-inactivated enzymes in an ATP-hydrolysis dependent manner, likely leading to the dissociation of aggregates formed during heat inactivation . In contrast to this, DnaK protein was exclusively able to protect and to reactivate the enzymes from E.coli but not from eukaryotic cells . Finally, the addition of calf thymus DnaJ co-chaperone homologue reduced the amount of Hsc70 required for reactivation at least 10-fold. Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 6012 - 6 PXA1, a possible Saccharomyces cerevisiae ortholog of the human adrenoleukodystrophy gene; Shani N et al.; The adrenoleukodystrophy protein (ALDp) is an ATP-binding cassette (ABC) transporter in the human peroxisome membrane . It is defective in X chromosome-linked adrenoleukodystrophy (ALD), a neurodegenerative disorder with impaired peroxisomal oxidation of very long chain fatty acids . We report cloning and characterization of PXA1, a yeast gene encoding a protein (Pxa1p) exhibiting high similarity to ALDp . Disruption of PXA1 results in impaired growth on oleic acid and reduced ability to oxidize oleate . Pxa1p is peroxisome associated; however, in the PXA1 mutant yeast, as in ALD cells, peroxisomes are morphologically intact . Disruption of a second yeast gene, YKL741, which encodes a more distantly related ALDp homolog (Yk174p), in either wild-type or PXA1 mutant yeast, results in a growth phenotype identical to that of the PXA1 mutant . This result suggests that Yk1741p and Pxa1p may be subunits of the same transporter . Sequence analysis of Pxa1p, ALDp, and related ABC transporters reveals a possible fatty acid binding domain and a 14-amino acid EAA-like motif, previously described only in prokaryotes . Because of the similarities in sequence and function, we propose that Pxa1p is the Saccharomyces cerevisiae ortholog of ALDp. J Biol Chem, 1995 Jun 16, 270(24), 14292 - 6 Identification of the region within the neuroendocrine polypeptide 7B2 responsible for the inhibition of prohormone convertase PC2; van Horssen AM et al.; The highly conserved polypeptide 7B2 and the subtilisin-related prohormone convertases PC1/PC3 and PC2 are broadly distributed in neurons and endocrine cells and are localized to secretory granules . We recently showed that recombinant 7B2 is in vitro a potent inhibitor of PC2 activity, but not of PC1/PC3, and that newly synthesized 7B2 is transiently associated with proPC2 in vivo . In the present study, in vitro mutagenesis was used to identify the region within the 7B2 sequence responsible for the inhibition of PC2 . Mutant proteins were produced in a prokaryotic expression system and their effects on PC1/PC3 and PC2 activities were studied by two different in vitro enzyme assays . None of the 7B2 mutant proteins inhibited PC1/PC3 activity . Truncation studies revealed that a short segment within the COOH-terminal portion of 7B2 is critical for its inhibitory effect on PC2 . This segment contains a pair of basic amino acid residues which may represent a recognition motif for PC2 . Single amino acid substitutions within this Lys171-Lys172 site strongly diminished and a double mutation abolished the inhibitory potency of 7B2 . Our results indicate that, although amino acid residues directly surrounding this dibasic pair also contribute to PC2 inhibition, the Lys171-Lys172 site is particularly important for the ability of 7B2 to inhibit PC2. Genes Dev, 1995 Jun 15, 9(12), 1469 - 78 Circadian orchestration of gene expression in cyanobacteria; Liu Y et al.; We wanted to identify genes that are controlled by the circadian clock in the prokaryotic cyanobacterium Synechococcus sp . strain PCC 7942 . To use luciferase as a reporter to monitor gene expression, bacterial luciferase genes (luxAB) were inserted randomly into the Synechococcus genome by conjugation with Escherichia coli and subsequent homologous recombination . The resulting transformed clones were then screened for bioluminescence using a new developed cooled-CCD camera system . We screened approximately 30,000 transformed Synechococcus colonies and recovered approximately 800 clones whose bioluminescence was bright enough to be easily monitored by the screening apparatus . Unexpectedly, the bioluminescence expression patterns of almost all of these 800 colonies clearly manifested circadian rhythmicity . These rhythms exhibited a range of waveforms and amplitudes, and they also showed a variety of phase relationships . We also found bioluminescence rhythms expressed by cyanobacterial colonies in which the luciferase gene set was coupled to the promoters of several known genes . Together, these results indicate that control of gene expression by circadian clocks may be more widespread than expected thus far . Moreover, our results show that screening organisms in which promoterless luciferase genes have been inserted randomly throughout the genome by homologous recombination provides an extremely sensitive method to explore differential gene expression. Nucleic Acids Res, 1995 Jun 11, 23(11), 2014 - 8 Stimulation of mouse DNA primase-catalyzed oligoribonucleotide synthesis by mouse DNA helicase B; Saitoh A et al.; Many prokaryotic and viral DNA helicases involved in DNA replication stimulate their cognate DNA primase activity . To assess the stimulation of DNA primase activity by mammalian DNA helicases, we analyzed the synthesis of oligoribonucleotides by mouse DNA polymerase alpha-primase complex on single-stranded circular M13 DNA in the presence of mouse DNA helicase B . DNA helicase B was purified by sequential chromatography through eight columns . When the purified DNA helicase B was applied to a Mono Q column, the stimulatory activity for DNA primase-catalyzed oligoribonucleotide synthesis and DNA helicase and DNA-dependent ATPase activities of DNA helicase B were co-eluted from the column . The synthesis of oligoribonucleotides 5-10 nt in length was markedly stimulated by DNA helicase B . The synthesis of longer species of oligoribonucleotides, which were synthesized at a low level in the absence of DNA helicase B, was inhibited by DNA helicase B . The stimulatory effect of DNA helicase B was marked at low template concentrations and little or no effect was observed at high concentrations . The mouse single-stranded DNA binding protein, replication protein A (RP-A), inhibited the primase activity of the DNA polymerase alpha-primase complex and DNA helicase B partially reversed the inhibition caused by RP-A. Nucleic Acids Res, 1995 Jun 11, 23(11), 1977 - 83 Occurrence of potential cruciform and H-DNA forming sequences in genomic DNA; Schroth GP et al.; We have used computer-assisted methods to search large amounts of the human, yeast and Escherichia coli genomes for inverted repeat (IR) and mirror repeat (MR) DNA sequence patterns . In highly supercoiled DNA some IRs can form cruciforms, while some MRs can form intramolecular triplexes, or H-DNA . We find that total IR and MR sequences are highly enriched in both eukaryotic genomes . In E . coli, however, only total IRs are enriched, while total MRs only occur as frequently as in random sequence DNA . We then used a set of experimentally derived criteria to predict which of the total IRs and MRs are most likely to form cruciforms or H-DNA in supercoiled DNA . We show that strong cruciform forming sequences occur at a relatively high frequency in yeast (1/19 700 bp) and humans (1/41 800 bp), but that H-DNA forming sequences are abundant only in humans (1/49 400 bp) . Strong cruciform and H-DNA forming sequences are not abundant in the E.coli genome . These results suggest that cruciforms and H-DNA may have a functional role in eukaryotes, but probably not prokaryotes. Gene, 1995 Jun 9, 158(2), 203 - 7 The Drosophila melanogaster genome contains a member of the Rh/T2/S-glycoprotein family of ribonuclease-encoding genes; Hime G et al.; Members of the Rh/T2/S-glycoprotein family of ribonuclease(RNase)-encoding genes have been found predominantly in fungi, plants and bacteria, where they have been implicated in functions as diverse as the phosphate-starvation response and self-incompatibility . We report the isolation and sequence of DmRNase-66B, the first member of this family to be found in an insect genome . This gene was identified by the analysis of a cDNA clone derived from cytological region 66B1-2 of the genome of Drosophila melanogaster . In a search of sequence databases for homologs of this gene, two animal viral proteins, gp53 of the bovine viral diarrhea virus (BVDV) and gp44/48 of the hog cholera virus (HCV), were also found to exhibit the characteristic features of this class of RNases . In all cases, the proteins contain two conserved pentameric amino-acid regions that have been shown to lie in the active site of these RNases . A series of Cys residues are also conserved in all members of this gene family . The discovery of members of this family of genes in an insect genome indicates that these RNases are widely conserved and play important roles in the animal, as well as the plant and prokaryotic kingdoms. J Mol Biol, 1995 Jun 9, 249(3), 595 - 603 Crystal structure of an RNA dodecamer containing the Escherichia coli Shine-Dalgarno sequence; Schindelin H et al.; The synthetic dodecameric RNA fragment rUAAGGAGGUGAU resembles a region upstream of the initiation site in prokaryotic mRNAs whereas the pyrimidine-rich complementary strand is identical to the last 12 nucleotides of Escherichia coli 16 S rRNA . The complex thus serves as a model for the Shine-Dalgarno interaction which is required for proper initiation of translation . The crystal structure of rUAAGGAGGUGUA.rAUCACCUCCUUA has been determined at 2.6 A resolution and refined against 2957 1 sigma(F) structure amplitudes to an R-value of 0.195 . The unit cell of the triclinic crystals contains two double-stranded RNA molecules . The conformation of the two duplexes is similar, with a root-mean-square deviation of 0.683 A between equivalent atoms, and resembles calf thymus A-DNA as determined by X-ray fiber diffraction methods . Both molecules from continuous helices that penetrate the entire crystal, but the dinucleotide step in between dodecameric duplexes has an unusual geometry with a negative twist angle . The long helices cross over each other in a characteristic manner by inserting the backbone of one molecule into the minor groove of another . These contacts are stabilized by several direct intermolecular hydrogen bonds most of which are mediated by 2'-hydroxyl groups of the ribose sugars suggesting a general mode for the interaction between RNA molecules which is different from DNA-DNA interactions. Proc Natl Acad Sci U S A, 1995 Jun 6, 92(12), 5620 - 4 Evidence for five divergent thioredoxin h sequences in Arabidopsis thaliana; Rivera-Madrid R et al.; Five different clones encoding thioredoxin homologues were isolated from Arabidopsis thaliana cDNA libraries . On the basis of the sequences they encode divergent proteins, but all belong to the cytoplasmic thioredoxins h previously described in higher plants . The five proteins obtained by overexpressing the coding sequences in Escherichia coli present typical thioredoxin activities (NADP(+)-malate dehydrogenase activation and reduction by Arabidopsis thioredoxin reductase) despite the presence of a variant active site, Trp-Cys-Pro-Pro-Cys, in three proteins in place of the canonical Trp-Cys-Gly-Pro-Cys sequence described for thioredoxins in prokaryotes and eukaryotes . Southern blots show that each cDNA is encoded by a single gene but suggest the presence of additional related sequences in the Arabidopsis genome . This very complex diversity of thioredoxins h is probably common to all higher plants, since the Arabidopsis sequences appear to have diverged very early, at the beginning of plant speciation . This diversity allows the transduction of a redox signal into multiple pathways. J Nutr, 1995 Jun, 125(6 Suppl), 1758S - 1761S Nutritional regulation of the protein kinases responsible for the phosphorylation of the alpha-ketoacid dehydrogenase complexes; Harris RA et al.; The branched-chain alpha-ketoacid dehydrogenase (BCKDH) and pyruvate dehydrogenase (PDH) complexes are regulated by phosphorylation cycles catalyzed by complex-specific protein kinases and phosphoprotein phosphatases . Molecular cloning of these mitochondrial protein kinases has established a new family of protein kinases in eukaryotes that appears related by primary sequence to the histidine protein kinase family of prokaryotes . Changes in the activities of both kinases that are stable, i.e., not caused directly by allosteric effectors, correlate inversely with the changes in the activity states of the complexes that occur in different nutritional states . For example, BCKDH kinase activity is increased and the BCKDH complex activity state is decreased in rats fed diets deficient in protein . The increase in BCKDH kinase activity is due to an increase in the amount of BCKDH kinase protein bound to the BCKDH complex . The message level for BCKDH kinase also increases in the liver of rats starved for protein, suggesting a pretranslational mechanism exists for the long-term regulation of BCKDH kinase . Starvation and high-fat feeding cause a stable increase in PDH kinase activity and a corresponding decrease in activity state of the PDH complex . The mechanism responsible has not been defined. J Nutr, 1995 Jun, 125(6 Suppl), 1677S - 1682S Role of dietary sphingolipids and inhibitors of sphingolipid metabolism in cancer and other diseases; Merrill AH Jr et al.; Sphingolipids are found in all eukaryotic and some prokaryotic organisms and participate in the regulation of cell growth, differentiation, and diverse cell functions including cell-cell communication, cell-substratum interactions and intracellular signal transduction . Nonetheless, the field of nutrition has given scant attention to these compounds so that little is known about the following fundamental questions: What is the fate of sphingolipids that are consumed in food? Does consumption of dietary sphingolipids affect the behavior of cells in the gastrointestinal tract or other organs? How do other factors in the diet affect sphingolipid metabolism? Several recent findings underscore the importance of these questions, for examples: 1) Sphingolipids are digested throughout the GI tract to ceramide and sphingosine, which are highly bioactive compounds that affect cellular regulatory pathways; 2) addition of sphingomyelin to a standard AIN diet (which is essentially devoid of sphingolipids) reduces the appearance of aberrant colonic crypts, and perhaps the number of tumors, in mice treated with a colon carcinogen; and 3) an enzyme of sphingolipid metabolism has been discovered to be the target of a class of toxic and carcinogenic mycotoxins called fumonisins . Given these recent findings, it is possible that some of the confusion that has arisen regarding the relationships between dietary fat and disease might be due to the lack of consideration of the sphingolipids that are also present. EMBO J, 1995 Jun 1, 14(11), 2661 - 9 McrB: a prokaryotic protein specifically recognizing DNA containing modified cytosine residues; Kruger T et al.; Restriction of DNA by the Escherichia coli K-12 McrBC restriction endonuclease, which consists of the two subunits McrB and McrC, depends on the presence of modified cytosine residues in a special constellation . From previous work by others it was known that restriction of 5-methylcytosine-containing DNA requires two methylated 5'-PuC sites separated by approximately 40-80 non-defined base pairs . Here we show that binding of the McrBC nuclease is mediated exclusively by the McrB subunit . McrB has a low affinity for non-methylated DNA, with which it forms low molecular weight complexes . The affinity for DNA is significantly increased, with variations depending on the sequence context, by hemi- or fully methylated 5'-PuC sites . Binding to such substrates yields high molecular weight complexes, presumably involving several McrB molecules . Methylation at unique 5'-PuC sites can be sufficient to stimulate DNA binding by McrB . As such substrates are not cleaved by the nuclease, restriction apparently requires the coordinated interaction of molecules bound to neighbouring 5'-PumC sites . The binding properties of McrB exhibit some similarities to recently identified eukaryotic proteins interacting in a non-sequence-specific manner with DNA containing methylated 5'-CpG sequences and might point to a common molecular origin of these proteins . In addition to DNA, McrB also binds GTP, an essential cofactor in DNA restriction by McrBC . McrC neither binds to DNA nor modulates the DNA binding potential of McrB . As McrC is essential for restriction it appears to predominantly function in catalysis. EMBO J, 1995 Jun 1, 14(11), 2561 - 9 Fate of linear and supercoiled multinucleosomic templates during transcription; ten Heggeler-Bordier B et al.; Electron microscopy was used to monitor the fate of reconstituted nucleosome cores during in vitro transcription of long linear and supercoiled multinucleosomic templates by the prokaryotic T7 RNA polymerase and the eukaryotic RNA polymerase II . Transcription by T7 RNA polymerase disrupted the nucleosomal configuration in the transcribed region, while nucleosomes were preserved upstream of the transcription initiation site and in front of the polymerase . Nucleosome disruption was independent of the topology of the template, linear or supercoiled, and of the presence or absence of nucleosome positioning sequences in the transcribed region . In contrast, the nucleosomal configuration was preserved during transcription from the vitellogenin B1 promoter with RNA polymerase II in a rat liver total nuclear extract . However, the persistence of nucleosomes on the template was not RNA polymerase II-specific, but was dependent on another activity present in the nuclear extract . This was demonstrated by addition of the extract to the T7 RNA polymerase transcription reaction, which resulted in retention of the nucleosomal configuration . This nuclear activity, also found in HeLa cell nuclei, is heat sensitive and could not be substituted by nucleoplasmin, chromatin assembly factor (CAF-I) or a combination thereof . Altogether, these results identify a novel nuclear activity, called herein transcription-dependent chromatin stabilizing activity I or TCSA-I, which may be involved in a nucleosome transfer mechanism during transcription. J Bacteriol, 1995 Jun, 177(12), 3504 - 11 A second branched-chain alpha-keto acid dehydrogenase gene cluster (bkdFGH) from Streptomyces avermitilis: its relationship to avermectin biosynthesis and the construction of a bkdF mutant suitable for the production of novel antiparasitic avermectins; Denoya CD et al.; A second cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH), bkdFGH, has been cloned and characterized from Streptomyces avermitilis, the soil microorganism which produces anthelmintic avermectins . Open reading frame 1 (ORF1) (bkdF, encoding E1 alpha), would encode a polypeptide of 44,394 Da (406 amino acids) . The putative start codon of the incompletely sequenced ORF2 (bkdG, encoding E1 beta) is located 83 bp downstream from the end of ORF1 . The deduced amino acid sequence of bkdF resembled the corresponding E1 alpha subunit of several prokaryotic and eukaryotic BCDH complexes . An S . avermitilis bkd mutant constructed by deletion of a genomic region comprising the 5' end of bkdF is also described . The mutant exhibited a typical Bkd- phenotype: it lacked E1 BCDH activity and had lost the ability to grow on solid minimal medium containing isoleucine, leucine, and valine as sole carbon sources . Since BCDH provides an alpha-branched-chain fatty acid starter unit, either S(+)-alpha-methylbutyryl coenzyme A or isobutyryl coenzyme A, which is essential to initiate the synthesis of the avermectin polyketide backbone in S . avermitilis, the disrupted mutant cannot make the natural avermectins in a medium lacking both S(+)-alpha-methylbutyrate and isobutyrate . Supplementation with either one of these compounds restores production of the corresponding natural avermectins, while supplementation of the medium with alternative fatty acids results in the formation of novel avermectins . These results verify that the BCDH-catalyzed reaction of branched-chain amino acid catabolism constitutes a crucial step to provide fatty acid precursors for antibiotic biosynthesis in S . avermitilis. Mol Cell Biol, 1995 Jun, 15(6), 2942 - 54 Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure; Gilbert DM et al.; Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus . DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules . In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites . Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus . Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains . Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes . When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta) . However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated . Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta . We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs. Cancer Res, 1995 Jun 1, 55(11), 2338 - 45 Mechanism of action of bisimidazoacridones, new drugs with potent, selective activity against colon cancer; Hernandez L et al.; Antitumor bisimidazoacridones are bifunctional DNA binders which have recently been shown to selectively target human colon carcinoma cells in vitro and in vivo and appear to be excellent candidates for clinical development . We have studied the mechanism of action of one bisimidazoacridone, WMC26, which is 1,000-10,000 times more toxic to human colon carcinoma cells (HCT116) than to melanoma cells (SKMEL2) in vitro . Plasmid DNA exposed to WMC26 showed enhanced digestion by DNase I at A-T-rich sites, suggesting alterations in DNA conformation upon drug binding . These results led us to investigate whether WMC26 was selectively toxic due to a specific recognition of DNA bends by repair excinucleases, as has been demonstrated with the DNA bisintercalator, ditercalinium . Both prokaryotic and eukaryotic cells with intact repair capacity were shown to be selectively sensitive to WMC26, strongly indicating that excision repair plays a role in its toxicity . Confocal microscopy studies utilizing fluorescence of the WMC26 chromophore showed compound localization in the perinuclear cytoplasmic area, as had been previously noted for ditercalinium, indicating that cytoplasmic DNA could be the target . This irreversible accumulation of compound was gradually followed by vacuolization of the cytoplasm and cell death . Cell cycle analysis of both lines treated with WMC26 or with ditercalinium showed that, while the latter induced HCT116 growth arrest at G1-G0, WMC26 also blocked the cell cycle at G2-M; SKMEL2 cells did not undergo any changes in cell cycle as a result of either treatment . Our data show that WMC26 is 10-100 times more cytotoxic than ditercalinium in vitro . Like ditercalinium, WMC26 appears to exert its toxicity via cytoplasmic elements, through a mechanism involving excision repair processes . However, its highly selective cytotoxicity may stem from additional undefined targets in sensitive colon cancer cells. J Cell Biochem, 1995 Jun, 58(2), 208 - 20 Collagen binding activity of recombinant and N-terminally modified annexin V (anchorin CII); Turnay J et al.; We have cloned the full coding cDNA sequence of chicken annexin V and of a mutant lacking 8 amino acid residues of the N-terminal tail for prokaryotic expression . Both proteins were synthesized in Escherichia coli upon induction with isopropyl thio-beta-D-galactoside, and were purified following two different protocols: one based on the ability of these proteins to interact reversibly with liposomes in the presence of calcium, and the other based on two sequential ion-exchange chromatographic steps . Spectroscopical analysis of recombinant annexin V revealed that binding of calcium did not change the circular dichroism spectra indicating no significant changes on the secondary structure; however, a conformational change affecting the exposition to the solvent of the tryptophan residue 187 was detected by analysis of fluorescence emission spectra . Recombinant annexin V binds with high affinity to collagen types II and X, and with lower affinity to collagen type I in a calcium-independent manner . Heat denaturing of collagen decreases this interaction while pepsin-treatment of collagen almost completely abolishes annexin V binding . Mutated annexin V interacts with collagen in a similar way as the nonmutated recombinant protein, indicating that the N-terminal tail of annexin V is not essential for collagen binding. Biochem Mol Biol Int, 1995 Jun, 36(2), 339 - 45 Regulation of the translation and processing of rat dopamine beta-hydroxylase by metal ions in a cell free system; Feng Z et al.; Metal ions play an important role in the metabolism of prokaryotic and eukaryotic cells . In this study we examine the effect of various metal ions on the translation, glycosylation and co-translational processing of dopamine beta-hydroxylase (DBH) in vitro . The translation of wild type DBH mRNA and constructs with site directed mutations near the putative signal cleavage site was carried out with the addition of different ions (Mg2+, Cu2+, Mn2+, Ni2+, Co2+, Zn2+, Pb2+, Fe2+, Fe3+, Ca2+) in a cell-free system in the present of microsomal membranes . Most of the metal ions inhibited translation at concentrations above 1.5 mM . The translation was more sensitive to Fe2+ than Fe3+ . Ni2+ and Cu2+ preferentially inhibited formation of the glycosylated products . Only magnesium affected the ratio of the two different processed forms in a concentration dependent manner. Curr Opin Cell Biol, 1995 Jun, 7(3), 362 - 70 Higher-order nucleoprotein complexes in transcription: analogies with site-specific recombination; Grosschedl R; Regulation of transcription involves the assembly of multiprotein complexes at enhancers and promoters . Interactions between adjacent and non-adjacent DNA-binding proteins can augment the specificity and stability of multi-component nucleoprotein complexes . Recently, several proteins have been identified that can function as 'architectural' elements in the assembly of higher-order nucleoprotein structures reminiscent of those involved in site-specific recombination in prokaryotes. J Mol Evol, 1995 Jun, 40(6), 671 - 7 Partial sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase and the phylogeny of Prochloron and Prochlorococcus (Prochlorales); Shimada A et al.; The prochlorophytes, oxygenic photosynthetic prokaryotes having no phycobiliprotein but possessing chlorophylls a and b, have been proposed to have a common ancestry with green chloroplasts, yet this is still controversal . We report here that partial sequence comparisons of the large subunit of ribulose-1,5'-bisphosphate carboxylase/oxygenase, including sequence data from two prochlorophytes, Prochlorococcus and Prochloron, indicate that Prochlorococcus is more closely related to a photosynthetic bacterium, Chromatium vinosum (gamma-purple bacteria), than to cyanobacteria, while Prochloron is closely related to the prochlorophyte Prochlorothrix and to cyanobacteria . The molecular phylogenetic tree indicates that a common ancestor of Prochlorococcus and gamma-purple bacteria branched off from the land plant lineage earlier than Prochloron, Prochlorothrix, and cyanobacteria. Immunology, 1995 Jun, 85(2), 262 - 9 Anti-dsDNA antibodies cross-react with ribosomal P proteins expressed on the surface of glomerular mesangial cells to exert a cytostatic effect; Sun KH et al.; Affinity-purified human polyclonal anti-double-stranded DNA antibodies (anti-dsDNA) exerted a cytostatic effect towards human and rat glomerular mesangial cells (MC) . In order to identify the cognate antigens for anti-dsDNA on the surface of MC, we used these autoantibodies to probe a human renal lambda gt11 cDNA expression library . Two cDNA clones encoding the cognate proteins for the autoantibodies were isolated . Sequencing analysis of the two cDNA showed that they had 98.6% homology with the gene of the P0 and 99.2% homology with the gene of the P1 human acidic ribosomal phosphoproteins (P protein) . Two galactosidase fusion proteins (125,000 and 150,000 MW) derived from the two cDNA inserts expressed in lysogenic Escherichia coli Y1089 could react with the original screening antibodies in an immunoblotting analysis . After transformation and expression of the full-length P1 clone in prokaryotic cells, the purified P1 protein was able to react with anti-dsDNA . In a cross-inhibition experiment, the dsDNA binding activity of anti-dsDNA was inhibited by a synthetic polypeptide corresponding to the carboxyl-terminal 20 amino acids of P protein and purified P1 protein in a dose-dependent manner, but this was less potent than the inhibition caused by calf thymus dsDNA . By use of well-defined systemic lupus erythematosus (SLE) sera, we found only sera containing a high titre of anti-dsDNA activity (> 300 IU/ml) reacted with P1 of rat MC lysate . Furthermore, the 38,000 and 19,000 MW macromolecules were proved to be the cognate antigens for anti-dsDNA expression on the surface of the MC, by Western blot of the MC plasma membrane lysates . These results suggest that anti-dsDNA may cross-react with ribosomal P proteins expressed on the surface of the MC and exert cytostasis towards these cells. Plant Mol Biol, 1995 Jun, 28(3), 487 - 503 Chlamydomonas reinhardtii thioredoxins: structure of the genes coding for the chloroplastic m and cytosolic h isoforms; expression in Escherichia coli of the recombinant proteins, purification and biochemical properties; Stein M et al.; Based on known amino acid sequences, probes have been generated by PCR and used for the subsequent isolation of cDNAs and genes coding for two thioredoxins (m and h) of Chlamydomonas reinhardtii . Thioredoxin m, a chloroplastic protein, is encoded as a preprotein of 140 amino acids (15,101 Da) containing a transit peptide of 34 amino acids with a very high content of Ala and Arg residues . The sequence for thioredoxin h codes for a 113 amino acid protein with a molecular mass of 11,817 Da and no signal sequence . The thioredoxin m gene contains a single intron and seems to be more archaic in structure than the thioredoxin h gene, which is split into 4 exons . The cDNA sequences encoding C . reinhardtii thioredoxins m and h have been integrated into the pET-3d expression vector, which permits efficient production of proteins in Escherichia coli cells . A high expression level of recombinant thioredoxins was obtained (up to 50 mg/l culture) . This has allowed us to study the biochemical/biophysical properties of the two recombinant proteins . Interestingly, while the m-type thioredoxin was found to have characteristics very close to the ones of prokaryotic thioredoxins, the h-type thioredoxin was quite different with respect to its kinetic behaviour and, most strikingly, its heat denaturation properties. Curr Opin Struct Biol, 1995 Jun, 5(3), 360 - 71 Statistical significance of sequence patterns in proteins; Karlin S; I discuss three recent developments in sequence analysis by the statistical method of scores . First is the identification of segments of high aggregate score in a single protein sequence . Charge clusters and hyper-charge runs are prime examples . Proteins containing hyper-charge runs are principally associated with DNA and RNA processing, chromatin structure, ion storage and exchange, and protein complex assembly . Second is the protein sequence comparisons identifying common segments having high total similarity scores . These are illustrated by comparisons within the family of prokaryotic heat shock 70 kDa proteins . Third is the scoring protocols applied to the inverse folding problem. Bioessays, 1995 Jun, 17(6), 569 - 76 Cell shape and chromosome partition in prokaryotes or, why E . coli is rod-shaped and haploid; Donachie WD et al.; In the rod-shaped cells of E . coli, chromosome segregation takes place immediately after replication has been completed . A septum then forms between the two sister chromosomes . In the absence of certain membrane proteins, cells grow instead as large, multichromosomal spheres that divide successively in planes that are at right angles to one another . Although multichromosomal, the spherical cells cannot be maintained as heterozygotes . These observations imply that, in these mutants, each individual chromosome gives rise to a separate clone of descendant cells . This suggests a model in which sites for cell division form between pairs of sister chromosomes at the time of segregation, but are not used in spherical cells until further rounds of replication have taken place, thus ensuring clonal ('hierarchical') segregation of chromosomes into progeny cells . The role of the morphogenetic membrane proteins is to convert the basically spherical cell into a cylinder that is able to divide as soon as replication and segregation have been completed, and thus to maximise the number of viable cells per genome. Curr Biol, 1995 Jun 1, 5(6), 635 - 42 Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells; Rizzuto R et al.; BACKGROUND: It has recently been demonstrated that the green fluorescent protein (GFP) of the jellyfish Aequorea victoria retains its fluorescent properties when recombinantly expressed in both prokaryotic (Escherichia coli) and eukaryotic (Caenorhabditis elegans and Drosophila melanogaster) living cells; it can therefore be used as a powerful marker of gene expression in vivo . The specific targeting of recombinant GFP within cells would allow it to be used for even more applications, but no information is yet available on the possibility of targeting GFP to intracellular organelles . RESULTS: In this study, we show that the GFP cDNA can be expressed at high levels in cultured mammalian cells; the recombinant polypeptide is highly fluorescent and is exclusively localized in the cytosol . Furthermore, we have modified the GFP cDNA to include a mitochondrial targeting sequence (and a strong immunological epitope at the amino terminus of the encoded polypeptide) . When transiently transfected into mammalian cells, this construct drives the expression of a strongly fluorescent GFP chimera which selectively localizes to the mitochondria . We also describe two of the many possible applications of this recombinant GFP in physiological studies . The targeted chimera allows the visualization of mitochondrial movement in living cells . Also, unlike dyes such as rhodamine, it reveals morphological changes induced in mitochondria by drugs that collapse the organelle membrane potential . Moreover, when GFP is cotransfected with a membrane receptor, such as the alpha 1-adrenergic receptor, the fluorescence of the GFP in intact cells can be used in recognizing the transfected cells . Thus, specific changes in intracellular Ca2+ concentration that occur in cells expressing the recombinant receptor can be identified using a classical fluorescent Ca2+ indicator . CONCLUSION: GFP is an invaluable new tool for studies of molecular biology and cell physiology . As a marker of transfection in vivo, it provides a simple means of identifying genetically modified cells to be used in physiological studies . More importantly, chimeric GFP, which in principle can be targeted to any subcellular location, can be used to monitor complex phenomena in intact living cells, such as changes in shape and distribution of organelles, and it has the potential to be used as a probe of physiological parameters. Curr Opin Genet Dev, 1995 Jun, 5(3), 349 - 53 An update and lessons from whole-genome sequencing projects; Jones SJ; A number of prokaryotic and eukaryotic genomes are currently being sequenced . Already, the nucleotide sequences of four yeast chromosomes and of 2.2 Mb from Caenorhabditis elegans have been reported . Human genomic sequences have also been used in comparative studies with both mouse and Fugu rubripes. J Vet Med Sci, 1995 Jun, 57(3), 557 - 8 A novel repetitive sequence from Mycoplasma hyopneumoniae; Harasawa R et al.; We have isolated a novel repetitive DNA element from Mycoplasma hyopneumoniae strain VPP11, the entire structure of which is distinct from those of prokaryotic transposons, insertion sequences or eukaryotic retroposons reported . Southern blot hybridization experiments indicate that at least eight copies of this element locate on the M . hyopneumoniae genome . The size of this repetitive sequence is 4,193 bp which includes 270- and 272-bp direct long terminal repeats at each terminus . The internal domain of this element defines three open reading frames. Mol Microbiol, 1995 Jun, 16(5), 921 - 9 Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae; Wieles B et al.; The thioredoxin system comprising thioredoxin (Trx), thioredoxin reductase (TR) and NADPH operates via redox-active disulphides and provides electrons for a wide variety of different metabolic processes in prokaryotic and eukaryotic cells . Thioredoxin is also a general protein disulphide reductase involved in redox regulation . In bacteria, the Trx and TR proteins previously identified were encoded by separate genes (trxA and trxB) . In this study, we report a novel genomic organization of TR and Trx in mycobacteria and show that at least three modes of organization of TR and Trx genes can exist within a single bacterial genus: (i) in the majority of mycobacterial strains the genes coding for TR and Trx are located on separate sites of the genome; (ii) interestingly, in all pathogenic Mycobacterium tuberculosis complex mycobacteria both genes are found on the same locus, overlapping in one nucleotide; (iii) in the pathogen Mycobacterium leprae, TR and Trx are encoded by a single gene . Sequence analysis of the M . leprae gene demonstrated that the N-terminal part of the protein corresponds to TR and the C-terminal part to Trx . A corresponding single protein product of approximately 49 kDa was detected in cell extracts of M . leprae . These findings demonstrate the very unusual phenomenon of a single gene coding for both the substrate (thioredoxin) and the enzyme (thioredoxin reductase), which seems to be unique to M . leprae.
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