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.; I