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| J Biol Chem, 1997 Jun 6, 272(23), 14643 - 9 A repertoire of novel antibacterial diastereomeric peptides with selective cytolytic activity; Oren Z et al.; The increase in infectious diseases and bacterial resistance to antibiotics has resulted in intensive studies focusing on the use of linear, alpha-helical, cytolytic peptides from insects and mammals as potential drugs for new target sites in bacteria . Recent studies with diastereomers of the highly potent cytolytic peptides, pardaxin and melittin, indicate that alpha-helical structure is required for mammalian cells lysis but is not necessary for antibacterial activity . Thus, hydrophobicity and net positive charge of the polypeptide might confer selective antibacterial lytic activity . To test this hypothesis, a series of diastereomeric model peptides (12 amino acids long) composed of varying ratios of leucine and lysine were synthesized, and their structure and biological function were investigated . Peptide length and the position of D-amino acids were such that short peptides with stretches of only 1-3 consecutive L-amino acids that cannot form an alpha-helical structure were constructed . Circular dichroism spectroscopy showed that the peptides do not retain any detectable secondary structure in a hydrophobic environment . This enabled examination of the sole effect of hydrophobicity and positive charge on activity . The data reveal that modulating hydrophobicity and positive charge is sufficient to confer antibacterial activity and cell selectivity . A highly hydrophobic diastereomer that permeated both zwitterionic and negatively charged phospholipid vesicles, lysed eukaryotic and prokaryotic cells . In contrast, a highly positively charged diastereomer that only permeated slightly negatively charged phospholipid vesicles had low antibacterial activity and could not lyse eukaryotic cells . In the boundary between high hydrophobicity and high positive charge, the diastereomers acquired selective and potent antibacterial activity . Furthermore, they were completely resistant to human serum inactivation, which dramatically reduces the activity of native antibacterial peptides . In addition, a strong synergistic effect was observed at nonlethal concentrations of the peptides with the antibiotic tetracycline on resistant bacteria . The results are discussed in terms of proposed mechanisms of antibacterial activity, as well as a new strategy for the design of a repertoire of short, simple, and easily manipulated antibacterial peptides as potential drugs in the treatment of infectious diseases. Nature, 1997 Jun 5, 387(6633), 627 - 30 Three-dimensional organization of a human water channel; Cheng A et al.; Aquaporins (AQP) are members of the major intrinsic protein (MIP) superfamily of integral membrane proteins and facilitate water transport in various eukaryotes and prokaryotes . The archetypal aquaporin AQP1 is a partly glycosylated water-selective channel that is widely expressed in the plasma membranes of several water-permeable epithelial and endothelial cells . Here we report the three-dimensional structure of deglycosylated, human erythrocyte AQP1, determined at 7 A resolution in the membrane plane by electron crystallography of frozen-hydrated two-dimensional crystals . The structure has an inplane, intramolecular 2-fold axis of symmetry located in the hydrophobic core of the bilayer . The AQP1 monomer is composed of six membrane-spanning, tilted alpha-helices . These helices form a barrel that encloses a vestibular region leading to the water-selective channel, which is outlined by densities attributed to the functionally important NPA boxes and their bridges to the surrounding helices . The intramolecular symmetry within the AQP1 molecule represents a new motif for the topology and design of membrane protein channels, and is a simple and elegant solution to the problem of bidirectional transport across the bilayer. Gene, 1997 Jun 3, 191(2), 149 - 53 An improved GFP cloning cassette designed for prokaryotic transcriptional fusions; Miller WG et al.; A new gfp cloning cassette designed for prokaryotic transcriptional fusions has been constructed . This cassette consists of gfp (containing the S65T 'red-shift' {Heim et al . (1995) Nature 373, 663-664} and F64L 'protein solubility' {Cormack et al . (1996) Gene 173, 33-38} mutations) flanked by convenient restriction sites, a translational enhancer, and a consensus ribosome binding site with an optimized spacer region . gfp fusion strains containing this cassette demonstrate from 40- to 80-fold greater fluorescence intensity than wild-type gfp fusion strains . Additionally, this cassette confers sufficient fluorescence to recipient cells to be used in low copy-number plasmids, with promoters conferring low levels of transcription, and in bacterial taxa other than Escherichia, such as Pseudomonas. Protein Eng, 1997 Jun, 10(6), 673 - 6 Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method; Cserzo M et al.; A new, simple method for predicting transmembrane segments in integral membrane proteins has been developed . It is based on low-stringency dot-plots of the query sequence against a collection of non-homologous membrane proteins using a previously derived scoring matrix {Cserzo et al., 1994, J . Mol . Biol., 243, 388-396} . This so-called dense alignment surface (DAS) method is shown to perform on par with earlier methods that require extra information in the form of multiple sequence alignments or the distribution of positively charged residues outside the transmembrane segments, and thus improves prediction abilities when only single-sequence information is available or for classes of membrane proteins that do not follow the 'positive inside' rule. Biochem Mol Biol Int, 1997 Jun, 42(2), 325 - 8 Expression of soluble, active human macrophage colony stimulating factor in Escherichia coli; Jiang X et al.; Human macrophage colony stimulating factor (M-CSF) has been successfully overexpressed in Escherichia coli AD494 (DE3) with an expression level of approximate 26% of the total cellular proteins . The truncated human M-CSF gene encoding the amino-terminal 149 amino acids was subcloned into the prokaryotic expression vector pET11d under the control of the inducible T7 promoter . Nearly 40% of the recombinant protein was in the soluble fraction which showed obvious stimulating effects on mouse macrophage colony formation and had an M-CSF specific activity of approximately 1 x 10(6) units/mg soluble protein. Bioorg Med Chem, 1997 Jun, 5(6), 1097 - 105 In vitro evolution used to define a protein recognition site within a large RNA domain; Sapag A et al.; A minimum of 460 nucleotides of 16S ribosomal RNA are needed to fold the target site for E . coli ribosomal protein S4, although a much smaller region within this large domain is protected from chemical reagents by the protein . Starting with a 531-nucleotide tRNA fragment, cycles of mutagenesis, selection with S4, and amplification ('in vitro evolution') were used to obtain a pool of 30 RNA sequences selected for S4 recognition but approximately 30% different from wild type . Numerous compensatory base pair changes have largely preserved the same secondary structure among these RNAs as found in wild-type sequences . A 20-base deletion and a single nucleotide insertion are among several unusual features found in most of the selected sequences and also prevalent among other prokaryotic rRNAs . Most of the compensatory base changes and selected features are located outside of the region protected by S4 from chemical reagents . It was unexpected that S4 would select for RNA structures throughout such a large domain; the selected features are probably contributing indirectly to S4 recognition by promoting correct tertiary folding of the region actually contacted by S4 . The role of S4 may be to stabilize this domain (nearly one-third of the 16S rRNA) in its proper conformation for ribosome function. Hybridoma, 1997 Jun, 16(3), 273 - 5 Monoclonal antibody NM11 recognizes a C-terminal epitope shared by p300 and CBP; Dallas PB et al.; The epitope recognized by the monoclonal antibody NM11, previously shown to recognize both CBP and p300, has been mapped here to the C-terminal third of p300 and CBP by Western analysis of p300 and CBP prokaryotic fusion proteins . More precise epitope mapping, carried out by screening a plasmid expression library derived from small randomly generated CBP cDNA fragments localizes the NM11 epitope to a 21 amino acid stretch spanning amino acids 2071-2091 near the CBP C-terminus . CBP and p300 differ by three noncontiguous residues within this 21 amino acid region, a difference that does not detectably affect the reactivity of NM11. EMBO J, 1997 Jun, 16(12), 3655 - 65 FIS activates sequential steps during transcription initiation at a stable RNA promoter; Muskhelishvili G et al.; FIS (factor for inversion stimulation) is a small dimeric DNA-bending protein which both stimulates DNA inversion and activates transcription at stable RNA promoters in Escherichia coli . Both these processes involve the initial formation of a complex nucleoprotein assembly followed by local DNA untwisting at a specific site . We have demonstrated previously that at the tyrT promoter three FIS dimers are required to form a nucleoprotein complex with RNA polymerase . We now show that this complex is structurally dynamic and that FIS, uniquely for a prokaryotic transcriptional activator, facilitates sequential steps in the initiation process, enabling efficient polymerase recruitment, untwisting of DNA at the transcription startpoint and finally the escape of polymerase from the promoter . Activation of all these steps requires that the three FIS dimers bind in helical register . We suggest that FIS acts by stabilizing a DNA microloop whose topology is coupled to the local topological transitions generated during the initiation of transcription. Mol Microbiol, 1997 Jun, 24(6), 1109 - 17 Roles for motility in bacterial-host interactions; Ottemann KM et al.; The ability to move in a directed manner may confer distinct advantages upon host-adapted prokaryotes . Potential benefits of motility include increased efficiency of nutrient acquisition, avoidance of toxic substances, the ability to translocate to preferred hosts and access optimal colonization sites within them, and dispersal in the environment during the course of transmission . The costs of motility also may be significant . These include the metabolic burden of synthesizing flagellar components, the energetic expense of fuelling flagellar motors and the presentation of polymeric and highly antigenic targets to the immune system . It is therefore not surprising that synthesis of the motility apparatus is usually subject to strict control . Studies of a variety of bacterial-host interactions demonstrate roles for motility, and its regulation, at points throughout the infectious cycle. Exp Parasitol, 1997 Jun, 86(2), 133 - 43 Uptake and cellular localization of exogenous lipids by Giardia lamblia, a primitive eukaryote; Stevens TL et al.; Giardia lamblia trophozoites are unable to carry out de novo lipid synthesis . It is therefore likely that lipids are acquired from the small intestine of the host, in which the trophozoites are exposed to free and conjugated fatty acids, various sterols, phospholipids, bile acids, and bile-lipid mixed micelles . Here we show that G . lamblia is capable of taking up exogenous phosphatidylcholine (PC), phosphatidylinositol (PI), sphingomyelin (SM), cholesterol, ceramide (Cer), and fatty acids . Results from epifluorescence and high-resolution confocal microscopy suggest that fluorescent analogs of SM and PC were accumulated in the plasma membranes, whereas palmitic acid and Cer were localized intracellularly . Interestingly, many of these analogs were also concentrated in perinuclear regions . Similar labeling patterns were observed when the fluorescent analogs were delivered to the parasite via liposomes . To test whether G . lamblia was capable of esterifying exogenous fatty acids into membrane or cellular phospholipids, trophozoites were pulse-labeled with 3H-labeled palmitic or myristic acids and the phospholipids analyzed by thin-layer chromatography . Results document that G . lamblia was able to incorporate exogenous fatty acids into various phospholipids, i.e., PI, PC, PE, and PG . Interestingly, a major portion of radiolabeled fatty acids was incorporated into PG, a phospholipid characteristic of prokaryotic membranes. Genome, 1997 Jun, 40(3), 342 - 56 The evolution of species-type specificity in the global DNA sequence organization of mitochondrial genomes; Hill KA et al.; Prokaryote genomes and nuclear genomes of eukaryotes have a global DNA sequence organization that is species type specific, determined primarily by nearest-neighbor nucleotide associations, and independent of gene function and sequence length . The determinants of such a global structure have remained largely uncharacterized . The monophyletic and endosymbiotic origin of mitochondria permit examination of the influence of evolutionary time and host species type . Different global structures were seen among (i) protozoan and plant (ii) fungal, (iii) algal (iv) nematode, (v) echinoderm, (vi) insect, and (vii) vertebrate species followed examination of 28 complete mitochondrial genomes using chaos representation and measure of short-sequence representation . The mitochondrial genomes have biases in single-nucleotide and dinucleotide representation, specifically, an overrepresentation of A and T nucleotides and CC/GG and AG/CT dinucleotides and a deficiency of CG dinucleotides, in all but one genome . Dinucleotide representation is similar among (i) mitochondrial genomes of more closely related species; (ii) mitochondrial genomes and the Mycoplasma capricolum genome, a proposed progenitor of mitochondrial genomes; and (iii) mitochondrial genomes of diverse species, more so than between the mitochondrial and the nuclear genome of the same or a closely related species . It is hypothesized that sufficient evolutionary time has permitted host-specific constraints to affect nuclear and mitochondrial genomes and that different species type specific constraints influence nuclear and mitochondrial genome global structure. Protein Sci, 1997 Jun, 6(6), 1129 - 38 The chemistry and enzymology of the type I signal peptidases; Dalbey RE et al.; The discovery that proteins exported from the cytoplasm are typically synthesized as larger precursors with cleavable signal peptides has focused interest on the peptidases that remove the signal peptides . Here, we review the membrane-bound peptidases dedicated to the processing of protein precursors that are found in the plasma membrane of prokaryotes and the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoidal membrane of eukaryotes . These peptidases are termed type I signal (or leader) peptidases . They share the unusual feature of being resistant to the general inhibitors of the four well-characterized peptidase classes . The eukaryotic and prokaryotic signal peptidases appear to belong to a single peptidase family . This review emphasizes the evolutionary concepts, current knowledge of the catalytic mechanism, and substrate specificity requirements of the signal peptidases. Microbiol Mol Biol Rev, 1997 Jun, 61(2), 262 - 80 Energetics of syntrophic cooperation in methanogenic degradation; Schink B; Fatty acids and alcohols are key intermediates in the methanogenic degradation of organic matter, e.g., in anaerobic sewage sludge digestors or freshwater lake sediments . They are produced by classical fermenting bacteria for disposal of electrons derived in simultaneous substrate oxidations . Methanogenic bacteria can degrade primarily only one-carbon compounds . Therefore, acetate, propionate, ethanol, and their higher homologs have to be fermented further to one-carbon compounds . These fermentations are called secondary or syntrophic fermentations . They are endergonic processes under standard conditions and depend on intimate coupling with methanogenesis . The energetic situation of the prokaryotes cooperating in these processes is problematic: the free energy available in the reactions for total conversion of substrate to methane attributes to each partner amounts of energy in the range of the minimum biochemically convertible energy, i.e., 20 to 25 kJ per mol per reaction . This amount corresponds to one-third of an ATP unit and is equivalent to the energy required for a monovalent ion to cross the charged cytoplasmic membrane . Recent studies have revealed that syntrophically fermenting bacteria synthesize ATP by substrate-level phosphorylation and reinvest part of the ATP-bound energy into reversed electron transport processes, to release the electrons at a redox level accessible by the partner bacteria and to balance their energy budget . These findings allow us to understand the energy economy of these bacteria on the basis of concepts derived from the bioenergetics of other microorganisms. Endocr Rev, 1997 Jun, 18(3), 306 - 60 Steroid receptor interactions with heat shock protein and immunophilin chaperones; Pratt WB et al.; We have provided a historical perspective on a body of steroid receptor research dealing with the structure and physiological significance of the untransformed 9S receptor that has often confused both novice and expert investigators . The frequent controversies and equivocations of earlier studies were due to the fact that the native, hormone-free state of these receptors is a large multiprotein complex that resisted description for many years because of its unstable and dynamic nature . The untransformed 9S state of the steroid and dioxin receptors has provided a unique system for studying the function of the ubiquitous, abundant, and conserved heat shock protein, hsp90 . The hormonal control of receptor association with hsp90 provided a method of manipulating the receptor heterocomplex in a manner that was physiologically meaningful . For several steroid receptors, binding to hsp90 was required for the receptor to be in a native hormone-binding state, and for all of the receptors, hormone binding promoted dissociation of the receptor from hsp90 and conversion of the receptor to the DNA-binding state . Although the complexes between tyrosine kinases and hsp90 were discovered earlier, the hormonal regulation or steroid receptor association with hsp90 permitted much more rapid and facile study of hsp90 function . The observations that hsp90 binds to the receptors through their HBDs and that these domains can be fused to structurally different proteins bringing their function under hormonal control provided a powerful linkage between the hormonal regulation of receptor binding to hsp90 and the initial step in steroid hormone action . Because the 9S receptor hsp90 heterocomplexes could be physically stabilized by molybdate, their protein composition could be readily studied, and it became clear that these complexes are multiprotein structures containing a number of unique proteins, such as FKBP51, FKBP52, CyP-40, and p23, that were discovered because of their presence in these structures . Further analysis showed that hsp90 itself exists in a variety of native multiprotein heterocomplexes independent of steroid receptors and other 'substrate' proteins . Cell-free systems can now be used to study the formation of receptor heterocomplexes . As we outlined in the scheme of Fig . 1, the multicomponent receptor-hsp90 heterocomplex assembly system is being reconstituted, and the importance of individual proteins, such as hsp70, p60, and p23, in the assembly process is becoming recognized . It should be noted that our understanding of the mechanism and purpose of steroid receptor heterocomplex assembly is still at an early stage . We can now speculate on the roles of receptor-associated proteins in receptor action, both as individuals and as a group, but their actual functions are still vague or unknown . We can make realistic models about the chaperoning and trafficking of steroid receptors, but we don't yet know how these processes occur, we don't know where chaperoning occurs in the cell (e.g . Is it limited to the cytoplasm? Is it a diffuse process or does chaperoning occur in association with structural elements?), and, with the exception of the requirement for hormone binding, we don't know the extent to which the hsp90-based chaperone system impacts on steroid hormone action . It is not yet clear how far the discovery of this hsp90 heterocomplex assembly system will be extended to the development of a general understanding of protein processing in the cell . Because this assembly system is apparently present in all eukaryotic cells, it probably performs an essential function for many proteins . The bacterial homolog of hsp90 is not an essential protein, but hsp90 is essential in eukaryotes, and recent studies indicate that the development of the cell nucleus from prokaryotic progenitors was accompanied by the duplication of genes for hsp90 and hsp70 (698) . (ABSTRACT TRUNCATED) Comput Appl Biosci, 1997 Jun, 13(3), 231 - 4 'TransMem': a neural network implemented in Excel spreadsheets for predicting transmembrane domains of proteins; Aloy P et al.; MOTIVATION: Genomic sequences from different organisms, even prokaryotic, have plenty of orphan ORFs, making necessary methods for the prediction of protein structure and function . The prediction of the presence of hydrophobic transmembrane (HTM) stretches is a valuable clue for this . RESULTS: The program . TransMem, based on a neural network and running on personal computers (either Apple Macintosh or PC, using Excel worksheets), for the prediction and distribution of amino acid residues in transmembrane segments of integral membrane proteins is reported . The percentage of residue predictive accuracy obtained for the set of proteins tested is 93%, ranging from 99.9% for the best to 71.7% for the worst prediction . The segment-based accuracy is 93.6%; 63.6% of the protein set match any of the predicted and observed segment locations . AVAILABILITY: TransMem is available upon request or by anonymous up: IP address: luz.uab.es, directory/pub/ TransMem . It is also placed on the EMBL file server (ftp:/(/)ftp.ebi.ac.uk/pub/software/mac/TransMem ). Appl Environ Microbiol, 1997 Jun, 63(6), 2421 - 31 Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain; Roth BL et al.; A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria . SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells . Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent . SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser . The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms . Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry . Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli . Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests . These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility. Appl Environ Microbiol, 1997 Jun, 63(6), 2411 - 20 An immunological approach to detect phosphate stress in populations and single cells of photosynthetic picoplankton; Scanlan DJ et al.; In the marine cyanobacterium Synechococcus sp . strain WH7803, PstS is a 32-kDa cell wall-associated phosphate-binding protein specifically synthesized under conditions of restricted inorganic phosphate (P1) availability (D . J . Scanlan, N . H . Mann, and N . G . Carr, Mol . Microbiol . 10:181-191, 1993) . We have assessed its use as a potential diagnostic marker for the P status of photosynthetic picoplankton . Expression of PstS in Synechococcus sp . strain WH7803 was observed when the P1 concentration fell below 50 nM, demonstrating that the protein is induced at concentrations of P1 typical of oligotrophic conditions . PstS expression could be specifically detected by use of standard Western blotting (immunoblotting) techniques in natural mesocosm samples under conditions in which the N/P ratio was artificially manipulated to force P depletion . In addition, we have developed an immunofluorescence assay that can detect PstS expression in single Synechococcus cells both in laboratory cultures and natural samples . We show that antibodies raised against PstS cross-react with P-depleted Prochlorococcus cells, extending the use of these antibodies to both major groups of prokaryotic photosynthetic picoplankton . Furthermore, DNA sequencing of a Prochlorococcus pstS homolog demonstrated high amino acid sequence identity (77%) with the marine Synechococcus sp . strain WH7803 protein, including those residues in Escherichia coli PstS known to be directly involved in phosphate binding. J Mol Evol, 1997 Jun, 44(6), 632 - 6 Relationships between genomic G+C content, RNA secondary structures, and optimal growth temperature in prokaryotes; Galtier N et al.; G:C pairs are more stable than A:T pairs because they have an additional hydrogen bond . This has led to many studies on the correlation between the guanine+cytosine (G+C) content of nucleic acids and temperature over the last 20 years . We collected the optimal growth temperatures (Topt) and the G+C contents of genomic DNA; 23S, 16S, and 5S ribosomal RNAs; and transfer RNAs for 764 prokaryotic species . No correlation was found between genomic G+C content and Topt, but there were striking correlations between the G+C content of ribosomal and transfer RNA stems and Topt . Two explanations have been proposed-neutral evolution and selection pressure-for the approximate equalities of G and C (respectively, A and T) contents within each strand of DNA molecules . Our results do not support the notion that selection pressure induces complementary oligonucleotides in close proximity and therefore numerous secondary structures in prokaryotic DNA, as the genomic G+C content does not behave in the same way as that of folded RNA with respect to optimal growth temperature. J Mol Evol, 1997 Jun, 44(6), 625 - 31 On the informational content of overlapping genes in prokaryotic and eukaryotic viruses; Pavesi A et al.; In genetic language a peculiar arrangement of biological information is provided by overlapping genes in which the same region of DNA can code for functionally unrelated messages . In this work, the informational content of overlapping genes belonging to prokaryotic and eukaryotic viruses was analyzed . Using information theory indices, we identified in the regions of overlap a first pattern, exhibiting a more uniform base composition and more severe constraints in base ordering with respect to the nonoverlapping regions . This pattern was found to be peculiar to coliphage, avian hepatitis B virus, human lentivirus, and plant luteovirus families . A second pattern, characterized by the occurrence of similar compositional constraints in both types of coding regions, was found to be limited to plant tymoviruses . At the level of codon usage, a low degree of correlation between overlapping and nonoverlapping coding regions characterized the first pattern, whereas a close link was found in tymoviruses, indicating a fine adaptation of the overlapping frame to the original codon choice of the virus . As a result of codon usage correlation analysis, deductions concerning the origin and evolution of several overlapping frames were also proposed . Comparison of amino acid composition revealed an increased frequency of amino acid residues with a high level of degeneracy (arginine, leucine, and serine) in the proteins encoded by overlapping genes; this peculiar feature of overlapping genes can be viewed as a way with which they may expand their coding ability and gain new, specialized functions. Mol Reprod Dev, 1997 Jun, 47(2), 140 - 7 Expression of bonnet monkey (Macaca radiata) zona pellucida-3 (ZP3) in a prokaryotic system and its immunogenicity; Kaul R et al.; An internal fragment (978 bp) corresponding to the bonnet monkey (Macaca radiata) ZP3, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by PCR from a full-length cDNA clone . The amplified Bam HI and SacI restricted fragment was cloned in frame downstream of the T5 promoter under lac operator control for expression in the pQE-30 vector . Recombinant ZP3 (r-ZP3) was expressed as a poly-histidine fusion protein in E . coli strains SG13009{pREP4} and BL-21(DE3) . Immunoblot with a murine monoclonal antibody, MA-451 (raised against porcine ZP3 beta-a homologue of bonnet ZP3, and cross-reactive with bonnet zona pellucida) revealed a predominant band of 50 kDa besides degraded fragments . Optimum expression of r-ZP3 was observed at 0.5 mM IPTG . Antisera generated in monkeys against synthetic peptides from the N-(23-45 aa residues) and C-(300-322 and 324-347 aa residues) termini of the deduced bonnet monkey precursor ZP3 sequence reacted with the r-ZP3 protein in ELISA . The r-ZP3 expressed in SG13009{pREP4} was purified on Ni-NTA resin under denaturing conditions and conjugated with diphtheria toxoid (DT) . Immunization of a female rabbit and six female bonnet monkeys with the r-ZP3-DT conjugate generated antibodies reactive with r-ZP3 in ELISA . Rabbit r-ZP3 antiserum reacted with porcine ZP3 beta and bonnet r-ZP3 but failed to react with porcine ZP3 alpha in a Western blot . Moreover, antisera when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida . The availability of r-ZP3 will further help in evaluating its efficacy for fertility regulation and understanding the autoimmune oophoritis associated with ZP3 immunization in nonhuman primates. J Mol Biol, 1997 May 30, 269(1), 102 - 12 Initiation of bacteriophage phi29 DNA replication in vivo: assembly of a membrane-associated multiprotein complex; Bravo A et al.; Initiation of in vitro phage phi29 DNA replication requires the formation of a heterodimer between a free molecule of terminal protein (TP), which acts as primer, and the viral DNA polymerase . We have analyzed membrane vesicles from phi29-infected Bacillus subtilis cells by quantitative immunoblot techniques . During phage DNA synthesis, large amounts of the viral proteins p1 and free TP were recovered in membrane fractions, as well as a low percentage of the total viral DNA polymerase . Interestingly, the amount of DNA polymerase in membrane fractions increased when viral DNA replication was blocked . Both protein p1 and free TP showed affinity for membranes in the absence of viral DNA . The association of protein p1 with membranes was abolished when the C-terminal 43 amino acid residues were deleted . The above results, together with the critical role of protein p1 for in vivo phi29 DNA replication, led us to conclude that a preliminary stage in the initiation of in vivo phi29 DNA replication could be the assembly of a membrane-associated multiprotein complex containing at least protein p1, free TP and DNA polymerase . Membrane-attachment of this complex could be directly mediated by both protein p1 and free TP . The ability of free TP to bind to membranes and to prime phi29 DNA replication would enable a nascent viral DNA molecule to become membrane-associated when its synthesis begins . We postulate that a general function of the TPs covalently linked to linear DNA genomes in prokaryotes might be, in addition to act as primer, to anchor the linear DNA molecule to the bacterial membrane. Nature, 1997 May 29, 387(6632 Suppl), 84 - 7 The nucleotide sequence of Saccharomyces cerevisiae chromosome IX; Churcher C et al.; Large-scale systematic sequencing has generally depended on the availability of an ordered library of large-insert bacterial or viral genomic clones for the organism under study . The generation of these large insert libraries, and the location of each clone on a genome map, is a laborious and time-consuming process . In an effort to overcome these problems, several groups have successfully demonstrated the viability of the whole-genome random 'shotgun' method in large-scale sequencing of both viruses and prokaryotes . Here we report the sequence of Saccharomyces cerevisiae chromosome IX, determined in part by a whole-chromosome 'shotgun', and describe the particular difficulties encountered in the random 'shotgun' sequencing of an entire eukaryotic chromosome . Analysis of this sequence shows that chromosome IX contains 221 open reading frames (ORFs), of which approximately 30% have been sequenced previously . This chromosome shows features typical of a small Saccharomyces cerevisiae chromosome. Science, 1997 May 23, 276(5316), 1261 - 4 Ligand-specific opening of a gated-porin channel in the outer membrane of living bacteria; Jiang X et al.; Ligand-gated membrane channels selectively facilitate the entry of iron into prokaryotic cells . The essential role of iron in metabolism makes its acquisition a determinant of bacterial pathogenesis and a target for therapeutic strategies . In Gram-negative bacteria, TonB-dependent outer membrane proteins form energized, gated pores that bind iron chelates (siderophores) and internalize them . The time-resolved operation of the Escherichia coli ferric enterobactin receptor FepA was observed in vivo with electron spin resonance spectroscopy by monitoring the mobility of covalently bound nitroxide spin labels . A ligand-binding surface loop of FepA, which normally closes its transmembrane channel, exhibited energy-dependent structural changes during iron and toxin (colicin) transport . These changes were not merely associated with ligand binding, but occurred during ligand uptake through the outer membrane bilayer . The results demonstrate by a physical method that gated-porin channels open and close during membrane transport in vivo. J Biol Chem, 1997 May 23, 272(21), 13986 - 90 Reverse gyrase from Methanopyrus kandleri . Reconstitution of an active extremozyme from its two recombinant subunits; Krah R et al.; Reverse gyrases are ATP-dependent type I 5'-topoisomerases that positively supercoil DNA . Reverse gyrase from Methanopyrus kandleri is unique as the first heterodimeric type I 5'-topoisomerase described, consisting of a 138-kDa subunit involved in the hydrolysis of ATP (RgyB) and a 43-kDa subunit that forms the covalent complex with DNA during the topoisomerase reaction (RgyA) . Here we report the reconstitution of active reverse gyrase from the two recombinant proteins overexpressed in Escherichia coli . Both proteins have been purified by column chromatography to >90% homogeneity . RgyB has a DNA-dependent ATPase activity at high temperature (80 degrees C) and is independent of the presence of RgyA . RgyA alone has no detectable activity . The addition of RgyA to RgyB reconstitutes positive supercoiling activity, but the RgyB and RgyA subunits form a stable heterodimer only after being heated together . This is the first case in which it has been possible to reconstitute an active heterodimeric enzyme of a hyperthermophilic prokaryote from recombinant proteins. Biochim Biophys Acta, 1997 May 22, 1326(1), 1 - 6 Cloning of a cDNA encoding a putative metal-transporting P-type ATPase from Arabidopsis thaliana; Tabata K et al.; Metal-transporting P-type ATPases were recently proposed to constitute a newly emerged sub-family of cation-transporting P-type ATPases, and are known to occur widely in prokaryotes and eukaryotes . However, no instance has been reported for higher plants . A cDNA clone encoding a metal-transporting P-type ATPase was thus searched for, if present, and was identified in Arabidopsis thaliana . The amino acid sequence, predicted from the determined nucleotide sequence for the cloned cDNA, shows all the critical features common to known metal-transporting P-type ATPases . This plant P-type ATPase has a typical metal-binding motif at its N-terminal portion . The newly isolated Arabidopsis gene, named PAA1, provides us with the first instance of putative metal-transporting P-type ATPases in higher plants . Some results of genomic analyses for this gene are also presented. Gene, 1997 May 20, 191(1), 47 - 50 Cloning and nucleotide sequence of Bacillus stearothermophilus pyruvate carboxylase; Kondo H et al.; A gene for prokaryotic pyruvate carboxylase (PC) was cloned from Bacillus stearothermophilus . It has an open reading frame of 3441 base pairs which can code for a protein of 128,353 Da . Not only the molecular size and domain organization but also the deduced amino acid sequence of B . stearothermophilus PC are similar to those of eukaryotic PCs. J Mol Biol, 1997 May 16, 268(4), 724 - 38 Molecular and immunological analysis of an ABC transporter complex required for cytochrome c biogenesis; Goldman BS et al.; The helABC genes are predicted to encode an ATP-binding cassette (ABC) transporter necessary for heme export for ligation in bacterial cytochrome c biogenesis . The recent discoveries of homologs of the helB and helC genes in plant mitochondrial genomes suggest this is a highly conserved transporter in prokaryotes and some eukaryotes with the HelB and HelC proteins comprising the transmembrane components . Molecular genetic analysis in the Gram-negative bacterium Rhodobacter capsulatus was used to show that the helABC and helDX genes are part of an operon linked to the secDF genes . To facilitate analysis of this transporter, strains with non-polar deletions in each gene, epitope and reporter-tagged HelABCD proteins, and antisera specific to the HelA and HelX proteins were generated . We directly demonstrate that this transporter is present in the cytoplasmic membrane as an HelABCD complex . The HelB and HelC but not HelD proteins are necessary for the binding and stability of the HelA protein, the cytoplasmic subunit containing the ATP-binding region . In addition we show that the HelA protein co-immunoprecipitates with either the HelC or HelD proteins . Thus, the HelABCD heme export complex is distinguished by the presence of four membrane-associated subunits and represents a unique subfamily of ABC transporters. J Mol Biol, 1997 May 16, 268(4), 704 - 11 Only one nucleotide insertion to the long variable arm confers an efficient serine acceptor activity upon Saccharomyces cerevisiae tRNA(Leu) in vitro; Himeno H et al.; Several tRNA species have a long variable arm composed of over ten nucleotides, which are relevant to those specific to serine, leucine and tyrosine in prokaryotes, while there are only serine and leucine-specific tRNAs in eukaryotes . To clarify the evolutionary aspects of the identity determination mechanism of these tRNAs, the tRNA(Ser) recognition in Saccharomyces cerevisiae was studied . Unmodified tRNA(Leu) transcript had serylation ability of low efficiency, but native tRNA(Leu) did not, indicating that some modification of tRNA(Leu) serves as a negative identity determinant for seryl-tRNA synthetase . Changing the discriminator base did not seriously affect the serine accepting efficiency . The tRNA(Leu) transcript possessing the variable arm of tRNA(Ser) was efficiently aminoacylated with serine . Eventually, it was found that only one nucleotide insertion to the variable arm of tRNA(Leu) was sufficient to confer an efficient serine accepting activity . The mode of serine tRNA recognition is similar to that in Escherichia coli in that the end of the long variable arm, but not the anticodon or discriminator base, is important . However, S . cerevisiae seryl-tRNA synthetase adopts a substantially different mechanism for rejection of tRNA(Leu) from that of its E . coli counterpart. J Biol Chem, 1997 May 16, 272(20), 13365 - 71 Activated alleles of yeast SLN1 increase Mcm1-dependent reporter gene expression and diminish signaling through the Hog1 osmosensing pathway; Fassler JS et al.; Two-component signal transduction systems involving histidine autophosphorylation and phosphotransfer to an aspartate residue on a receiver molecule have only recently been discovered in eukaryotes, although they are well studied in prokaryotes . The Sln1 protein of Saccharomyces cerevisiae is a two-component regulator involved in osmotolerance . Phosphorylation of Sln1p leads to inhibition of the Hog1 mitogen-activated protein kinase osmosensing pathway . We have discovered a second function of Sln1p by identifying recessive activated alleles (designated nrp2) that regulate the essential transcription factor Mcm1 . nrp2 alleles cause a 5-fold increase in the activity of an Mcm1-dependent reporter, whereas deletion of SLN1 causes a 10-fold decrease in reporter activity and a corresponding decrease in expression of Mcm1-dependent genes . In addition to activating Mcm1p, nrp2 mutants exhibit reduced phosphorylation of Hog1p and increased osmosensitivity suggesting that nrp2 mutations shift the Sln1p equilibrium toward the phosphorylated state . Two nrp2 mutations map to conserved residues in the receiver domain (P1148S and P1196L) and correspond to residues implicated in bacterial receivers to control receiver phosphorylation state . Thus, it appears that increased Sln1p phosphorylation both stimulates Mcm1p activity and diminishes signaling through the Hog1 osmosensing pathway. EMBO J, 1997 May 15, 16(10), 2826 - 35 Recombination between DNA repeats in yeast hpr1delta cells is linked to transcription elongation; Prado F et al.; The induction of recombination by transcription activation has been documented in prokaryotes and eukaryotes . Unwinding of the DNA duplex, disruption of chromatin structure or changes in local supercoiling associated with transcription can be indirectly responsible for the stimulation of recombination . Here we provide genetic and molecular evidence for a specific mechanism of stimulation of recombination by transcription . We show that the induction of deletions between repeats in hpr1delta cells of Saccharomyces cerevisiae is linked to transcription elongation . Molecular analysis of different direct repeat constructs reveals that deletions induced by hpr1delta are specific for repeat constructs in which transcription initiating at an external promoter traverses particular regions of the DNA flanked by the repeats . Transcription becomes HPR1 dependent when elongating through such regions . Both the induction of deletions and the HPR1 dependence of transcription were abolished when a strong terminator was used to prevent transcription from proceeding through the DNA region flanked by the repeats . In contrast to previously reported cases of transcription-induced recombination, there was no correlation between high levels of transcripts and high levels of recombination . Our study provides evidence that direct repeat recombination can be induced by transcriptional elongation. EMBO J, 1997 May 15, 16(10), 2756 - 68 Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme; Duong F et al.; Escherichia coli preprotein translocase contains a membrane-embedded trimeric complex of SecY, SecE and SecG (SecYEG) and the peripheral SecA protein . SecYE is the conserved functional 'core' of the SecYEG complex . Although sufficient to provide sites for high-affinity binding and membrane insertion of SecA, and for its activation as a preprotein-dependent ATPase, SecYE has only very low capacity to support translocation . The proteins encoded by the secD operon--SecD, SecF and YajC--also form an integral membrane heterotrimeric complex (SecDFyajC) . Physical and functional studies show that these two trimeric complexes are associated to form SecYEGDFyajC, the hexameric integral membrane domain of the preprotein translocase 'holoenzyme' . Either SecG or SecDFyajC can support the translocation activity of SecYE by facilitating the ATP-driven cycle of SecA membrane insertion and de-insertion at different stages of the translocation reaction . Our findings show that each of the prokaryote-specific subunits (SecA, SecG and SecDFyajC) function together to promote preprotein movement at the SecYE core of the translocase. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5237 - 42 Characteristic enrichment of DNA repeats in different genomes; Cox R et al.; Using computer programs developed for this purpose, we searched for various repeated sequences including inverted, direct tandem, and homopurine-homopyrimidine mirror repeats in various prokaryotes, eukaryotes, and an archaebacterium . Comparison of observed frequencies with expectations revealed that in bacterial genomes and organelles the frequency of different repeats is either random or enriched for inverted and/or direct tandem repeats . By contrast, in all eukaryotic genomes studied, we observed an overrepresentation of all repeats, especially homopurine-homopyrimidine mirror repeats . Analysis of the genomic distribution of all abundant repeats showed that they are virtually excluded from coding sequences . Unexpectedly, the frequencies of abundant repeats normalized for their expectations were almost perfect exponential functions of their size, and for a given repeat this function was indistinguishable between different genomes. J Biol Chem, 1997 May 9, 272(19), 12824 - 30 Reconstitution of yeast and Arabidopsis RNA polymerase alpha-like subunit heterodimers; Larkin RM et al.; Two subunits of about 36-44 kDa and 13-19 kDa in the eukaryotic nuclear RNA polymerases share limited amino acid sequence similarity to the alpha subunit in Escherichia coli RNA polymerase . The alpha subunit in the prokaryotic enzyme has a stoichiometry of 2, but the stoichiometry of the alpha-like subunits in the eukaryotic enzymes is not entirely clear . To gain insight into the subunit stoichiometry and assembly pathway for eukaryotic RNA polymerases, in vitro reconstitution experiments have been carried out with recombinant alpha-like subunits from yeast and plant RNA polymerase II . The large and small alpha-like subunits from each species formed stable heterodimers in vitro, but neither the large or small alpha-like subunits formed stable homodimers . Furthermore, mixed heterodimers were formed between corresponding subunits of yeast and plants, but were not formed between corresponding subunits in different RNA polymerases from the same species . Our results suggest that RNA polymerase II alpha-like heterodimers may be the equivalent of alpha homodimers found in E . coli RNA polymerase. Biochim Biophys Acta, 1997 May 2, 1352(1), 102 - 12 Expression of bovine mitochondrial elongation factor Ts in Escherichia coli and characterization of the heterologous complex formed with prokaryotic elongation factor Tu; Xin H et al.; When bovine mitochondrial elongation factor Ts (EF-Ts(mt)) is expressed in Escherichia coli, it forms a tightly associated complex with E . coli EF-Tu (EF-Tu(Eco) x Ts(mt)) . This complex is active in poly(U)-directed polymerization and this activity is inhibited by kirromycin . The EF-Tu(Eco) x Ts(mt) complex does not bind guanine nucleotides detectably and is not dissociated to a significant extent by either GDP or GTP . A portion of the EF-Tu(Eco) x Ts(mt) complex can be dissociated by aa-tRNA in the presence of GTP . The heterologous complex cannot be dissociated completely in the presence of either the 8 M urea or 8 M guanidine hydrochloride, suggesting that EF-Ts(mt) has an unusually tight interaction with E . coli EF-Tu . The EF-Tu(Eco) x Ts(mt) complex can be dissociated by denaturation using 2 M guanidine thiocyanate . Free EF-Ts(mt) can then be purified and renatured . The refolded EF-Ts(mt) is active in stimulating the activity of expressed mitochondrial EF-Tu (EF-Tu(mt)) in poly(U)-directed polymerization . Almost all the EF-Ts(mt) molecules appear to refold into a conformation which can interact with EF-Tu(mt) . Protease mapping of EF-Ts(mt) indicates that the first 54 residues fold into an independent domain . Analysis of deletion derivatives of EF-Ts(mt) indicates that extensive regions of this factor are required for its tight interaction with EF-Tu. Biochim Biophys Acta, 1997 May 2, 1352(1), 91 - 101 Mechanistic studies of the translational elongation cycle in mammalian mitochondria; Woriax VL et al.; Polyclonal antibodies have been prepared against both components of the bovine liver mitochondrial translational elongation factor Tu and Ts complex (EF-Tu x Ts(mt)) . The antibodies against EF-Tu(mt) cross-react somewhat with Escherichia coli EF-Tu and wheat germ EF-1alpha . The antibodies against EF-Ts(mt) cross-react little, if at all, with E . coli EF-Ts or with EF-Ts from Euglena gracilis chloroplasts . These polyclonal antibodies have been used to investigate the relative amounts of EF-Tu(mt) and EF-Ts(mt) in bovine liver mitochondria and in cultured cells . The results of this analysis suggest that there is a 1:1 ratio of EF-Tu(mt) to EF-Ts(mt) in mammalian mitochondria . Intermediate complexes formed during the elongation cycle of protein synthesis in bovine liver mitochondria have also been investigated . The EF-Tu x Ts(mt) complex is quite resistant to dissociation by guanine nucleotides . This complex will, however, dissociate in the presence of GTP and Phe-tRNA resulting in the formation of a ternary complex comparable to that observed in prokaryotes . Kinetic data suggest that the use of the ternary complex in chain elongation increases the rate of Phe-tRNA binding to ribosomes, suggesting that it is a true intermediate in the elongation cycle . Sucrose gradient analysis indicates that the binding of EF-Tu(mt) to ribosomes can be detected in the presence of Phe-tRNA and a non-hydrolyzable analog of GTP . These results suggest that, in contrast to previous thinking, the basic features of the elongation cycle in mammalian mitochondria are quite similar to those in prokaryotes. J Biol Chem, 1997 May 2, 272(18), 12030 - 4 Calmodulin promotes dimerization of the oxygenase domain of human endothelial nitric-oxide synthase; Hellermann GR et al.; The active form of endothelial nitric-oxide synthase (eNOS) is a homodimer . The activity of the enzyme is regulated in vivo by calcium signaling involving the binding of calmodulin (CAM), which triggers the activation of eNOS . We have examined the possible role of calcium-mediated CAM binding in promoting dimerization of eNOS through the oxygenase domain of the enzyme . A recombinant form of the oxygenase domain of human eNOS was expressed in a prokaryotic expression system . This recombinant domain contains the catalytic cytochrome P-450 site for arginine oxidation by molecular oxygen as well as the binding sites for tetrahydrobiopterin and Ca2+-CAM but lacks the reductase domain and associated FAD, FMN, and NADPH binding sites . Binding of Ca2+-CAM caused an association of monomeric eNOS oxygenase domain as determined by changes in fluorescence, both intrinsic and extrinsic, and by gel filtration, chemical cross-linking, and particle-sizing . Dimerization of the domain was not dependent on the presence of the substrate, arginine, or the cofactor, tetrahydrobiopterin . A truncated form of the eNOS oxygenase domain lacking the Ca2+-CAM binding region did not undergo self-association to form dimers . These results show that the eNOS reductase domain is not required for Ca2+-CAM-induced dimerization of eNOS and suggest that this dimerization may be a primary event in the activation of eNOS by Ca2+. Res Virol, 1997 May-Jun, 148(3), 207 - 13 Inhibition of prokaryotic cell growth by HIV1 Vpr; Bodeus M et al.; We have cloned the nef, vif, vpr and vpu genes of HIV1 in the pGEX system to produce auxiliary proteins of HIV1 as N-terminal fusions with glutathione S-transferase (GST) . Some GST proteins are difficult to obtain under standard conditions . The synthesis and solubility varied considerably from one protein to another . We investigated the reasons for the poor production of GST-Vpr, GST-Vpu and GST-Vif . Interestingly, using this GST prokaryotic model, we demonstrated that Vpr, which is known to block the cell cycle of mammalian and yeast cells at the G2 phase, is also bacteriostatic for Escherichia coli . The effect on E . coli was specific to Vpr, and was not linked to the expression of the other HIV1 proteins . This suggests that Vpr interferes with components of cell replication that are conserved from prokaryotes to eukaryotes . Thus, E . coli appears to be a convenient model system for studies on the function of Vpr. Zentralbl Veterinarmed B, 1997 May, 44(3), 139 - 45 Eukaryotic and prokaryotic cell functions required for invasion of Staphylococcus aureus into bovine mammary epithelial cells; Almeida RA et al.; Eukaryotic and prokaryotic cellular functions required for invasion of Staphylococcus aureus into bovine mammary epithelial cells were investigated . Two strains of S . aureus isolated from milk of cows with clinical mastitis, a primary bovine mammary epithelial cell culture and a bovine mammary epithelial cell line were pretreated with inhibitors of nucleic acid and protein synthesis . In addition, mammary epithelial cells were pretreated with inhibitors of receptor-mediated endocytosis and oxidative phosphorylation . Protein and nucleic acid synthesis in prokaryotic and eukaryotic cells and eukaryotic oxidative phosphorylation were required for invasion of S . aureus into mammary epithelial cells . Inhibition of receptor-mediated endocytosis caused a significant reduction in the number of invading S . aureus . These results suggest that invasion of S . aureus into bovine mammary epithelial cells occurs through a receptor-mediated endocytosis process . Furthermore, eukaryotic oxidative metabolism, protein synthesis and nucleic acid synthesis as well as bacterial protein synthesis are required for bacterial invasion. Mol Microbiol, 1997 May, 24(3), 449 - 56 Polypeptide chain release factors; Buckingham RH et al.; Newly synthesized polypeptide chains are released from peptidyl-tRNA when the ribosome encounters a stop signal on mRNA . Extra-ribosomal proteins (release factors) play an essential role in this process . Although the termination process was first discovered in the late 1960s, much of the mechanism has remained obscure . However, important steps have recently been made in both prokaryotic and eukaryotic organisms in unlocking the secrets of this vital stage in protein synthesis . In this review we summarize these advances and focus attention on the remaining areas of uncertainty, particularly with respect to the models that have been proposed for the action of the GTP-hydrolysing termination factors in prokaryotes and eukaryotes, i.e . RF3 and eRF3. Anal Biochem, 1997 May 1, 247(2), 305 - 9 Capillary electrophoresis assay for ubiquitin carboxyl-terminal hydrolases with chemically synthesized ubiquitin-valine as substrate; Franklin K et al.; Ubiquitin is expressed in eukaryotic cells as precursors, fused via its carboxyl terminus either to other ubiquitin sequences in linear polyubiquitin arrays or to specific ribosomal proteins . In some of the polyubiquitin fusions a single amino acid (e.g., valine in humans) is attached to the carboxyl terminus . These gene products are rapidly (probably cotranslationally) cleaved by ubiquitin carboxyl-terminal hydrolase (UCH) enzymes; therefore, although ubiquitin precursors are suitable substrates for assays of UCH activity, they are difficult to isolate from nucleated cells . While the recombinant approach allows the production of ubiquitin precursors in prokaryotic cells (which do not contain the ubiquitin system), proteins produced in this manner require purification and may also be susceptible to modification by bacterial enzymes, e.g., adventitious proteolysis . As an alternative we have chemically synthesized human ubiquitin-valine . In the assay described here the cleavage of ubiquitin-valine to ubiquitin (77 and 76 residue proteins, respectively) by a purified recombinant Drosophila UCH was monitored by capillary electrophoresis . Mass spectrometry verified the precise cleavage of ubiquitin-valine, confirming that this synthetic protein is a UCH substrate . Synthetic ubiquitin-valine may serve as a generic substrate for UCHs allowing the purification and identification of new members of this enzyme family. Trends Biochem Sci, 1997 May, 22(5), 172 - 6 Two-component signal transducers and MAPK cascades; Wurgler-Murphy SM et al.; Two-component signal transducers, which are characterized by the histidine-to-aspartate phospho-transfer mechanism, were once thought to be restricted to prokaryotes . They have, however, now been identified in diverse eukaryotic species including plant, fungus, yeast and slime mold . In yeast, a two-component osmosensor has been found to regulate a mitogen-activated protein kinase (MAPK) cascade, a ubiquitous eukaryotic signaling module. Artif Cells Blood Substit Immobil Biotechnol, 1997 May, 25(3), 261 - 74 Perfluorochemicals and cell biotechnology; Lowe KC et al.; Perfluorochemical (PFC) liquids have properties, especially high gas solubility, which make these compounds useful in medicine and biotechnology . PFCs are being employed to facilitate respiratory gas supply to both prokaryotic and eukaryotic cells and, in some systems, to improve biomass production and yields of commercially-important cellular products . Animal (including human) and plant cells have also been cultured at the interface between PFC liquids and aqueous culture medium, while fluorocarbon polymers have been employed as gaspermeable membranes in eukaryotic cell cultures . This paper presents an overview of the applications and beneficial effects of PFCs in microbial, animal and plant culture systems . PFCs have been compared with other physical and chemical options for manipulating respiratory gas supply to cultured cells . PFC-facilitated improvements in cell culture technology will have increasingly important biotechnological implications. J Pharmacol Exp Ther, 1997 May, 281(2), 992 - 7 Expression of hygR in transgenic mice causes resistance to toxic effects of hygromycin B in vivo; Aubrecht J et al.; Aminoglycoside antibiotics are indispensable for treatment of serious bacterial infections, and despite careful attention to dosage regimens, nephrotoxicity and ototoxicity still cause concern . In the present study, we tested whether side effects of aminoglycoside therapy could be limited by expression of prokaryotic genes of antibiotic resistance in vivo . We characterized the acute and tissue-specific toxicity of hygromycin B in transgenic mice bearing the hygromycin B phosphotransferase (hygR) gene under control of a constitutive promoter . We characterized the tissue-specific expression of hygR mRNA and also investigated the acute toxicity of hygromycin B in hygR and wild-type mice . The hygR mRNA reached its highest levels in brain and reached intermediate levels in spleen, muscle, kidney, liver and testis . The lowest levels were detected in heart and lungs . The hygR expression in transgenic animals caused an 89-fold increase in the approximate lethal dose of hygromycin B compared with wild-type mice . Serum biochemical analysis of hygR and wild-type mice treated with lethal doses of hygromycin B indicated liver and kidney damage measured as ALT, AST and BUN . On the morphological level, these changes led to acute tubular nephrosis in wild-type mice and acute liver damage in hygR mice . Our results show that constitutive expression of the bacterial hygR gene in transgenic mice in vivo confers resistance to hygromycin B. J Bacteriol, 1997 May, 179(10), 3188 - 95 Molecular characterization and cellular localization of TpLRR, a processed leucine-rich repeat protein of Treponema pallidum, the syphilis spirochete; Shevchenko DV et al.; Automated Edman degradation was used to obtain N-terminal and internal amino acid sequences from a 26-kDa protein in isolated Treponema pallidum outer membranes (OMs) . The resulting sequences enabled us to PCR amplify from T . pallidum DNA a 275-bp fragment of the corresponding gene . The complete nucleotide sequence of the gene was determined from fragments amplified by long-distance PCR . Primer extension verified the assigned translational start of the open reading frame (ORF) and putative upstream promoter elements . The ORF encoded a highly basic (pI 9.6) 26-kDa protein which contained an N-terminal 25-amino-acid leader peptide terminated by a signal peptidase I cleavage site . The mature protein contained seven tandemly spaced copies (as well as an eighth incomplete copy) of a leucine-rich repeat (LRR), a motif previously identified in a number of prokaryotic and eukaryotic proteins . Accordingly, the polypeptide was designated T . pallidum leucine-rich repeat protein (TpLRR) . Although Triton X-114 phase partitioning showed that TpLRR was hydrophilic, cell localization studies showed that most of the antigen was associated with the peptidoglycan-cytoplasmic membrane complex rather than being freely soluble in the periplasmic space . Immunoblot studies showed that syphilis patients develop a weak antibody response to the antigen . Lastly, the lrr(T . pallidum) gene was mapped to a 60-kb SfiI-SpeI fragment of the T . pallidum chromosome which also contains the rrnA and flaA genes . The function(s) of TpLRR is currently unknown; however, protein-protein and/or protein-lipid interactions mediated by its LRR motifs may facilitate interactions between components of the T . pallidum cell envelope. Protein Sci, 1997 May, 6(5), 1047 - 56 Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli; Vickery LE et al.; The hscA and hscB genes of Escherichia coli encode novel chaperone and co-chaperone proteins, designated Hsc66 and Hsc20, respectively . We have overproduced and purified Hsc66 and Hsc20 in high yield in E . coli and describe their initial characterization including absorbance, fluorescence, and circular dichroism spectra . Immunoblot analyses of E . coli cultures using antisera to Hsc66 and Hsc20 raised in rabbits establish that Hsc66 and Hsc20 are constitutively expressed at levels corresponding to cell concentration approximately 20 microM and approximately 10 microM, respectively . The levels do not change appreciably following heat shock (44 degrees C), but a small increase in Hsc20 is observed following a shift to 10 degrees C . Purified Hsc66 exhibits a low intrinsic ATPase activity (approximately 0.6 min-1 at 37 degrees C), and Hsc20 was found to stimulate this activity up to 3.8-fold with half-maximal stimulation at a concentration approximately 5 microM . These findings suggest that Hsc66 and Hsc20 comprise a molecular chaperone system similar to the prokaryotic DnaK/DnaJ and eukaryotic hsp70/hsp40 systems . Sequence differences between Hsc66 and Hsc20 compared to other members of this chaperone family, however, suggest that the Hsc66/Hsc20 system will display different peptide binding specificity and that it is likely to be subject to different regulatory mechanisms . The high level of constitutive expression and the lack of a major response to temperature changes suggest that Hsc66 and Hsc20 play an important cellular role(s) under non-stress conditions. Nat Med, 1997 May, 3(5), 567 - 70 The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol; Telenti A et al.; Ethambutol (EMB), a frontline antituberculous drug, targets the mycobacterial cell wall, a unique structure among prokaryotes which consists of an outer layer of mycolic acids covalently bound to peptidoglycan via the arabinogalactan . EMB inhibits the polymerization of cell wall arabinan, and results in the accumulation of the lipid carrier decaprenol phosphoarabinose, which suggests that the drug interferes with the transfer of arabinose to the cell wall acceptor . Unfortunately, resistance to EMB has been described in up to 4% of clinical isolates of Mycobacterium tuberculosis and is prevalent among isolates from patients with multidrug-resistant tuberculosis . We used resistance to EMB as a tool to identify genes participating in the biosynthesis of the mycobacterial cell wall . This approach led to the identification of the embCAB gene cluster, recently proposed to encode for mycobacterial arabinosyl transferases . Resistance to EMB results from an accumulation of genetic events determining overexpression of the Emb protein(s), structural mutation in EmbB, or both . Further characterization of these proteins might provide information on targets for new chemotherapeutic agents and might help development of diagnostic strategies for the detection of resistant M . tuberculosis. Int Arch Allergy Immunol, 1997 May-Jul, 113(1-3), 142 - 5 In vivo analysis of the cytoplasmic domain of mIgE antibodies; Achatz G et al.; All immunoglobulin molecules exist in two forms, one integrated into the plasma membrane and displayed on the cell surface as the B cell receptor {1}, the other secreted into tissue fluids as the soluble antibodies . Any given B lymphocyte has the potential of producing both forms . The membrane-bound form and the secreted form produced by a single B cell have identical light chains and identical heavy chains except for a short segment at the C terminus . This segment consists of a 'spacer' sequence, followed by a stretch of hydrophobic amino acids (transmembrane region) and the cytoplasmic region which differs in size between the Ig classes . Little is known about the function of intracellular tails of IgG, IgA and IgE . They differ from those of IgM and IgD, which have only three intracellular amino acids (Lys Val Lys) . The intracellular parts of IgG, IgA and IgE are longer . Using a gene targeting technique by homologous recombination in ES cells {2, 3}, combined with the prokaryotic CRE-recombinase system {4}, we constructed two mice . One with the membrane exons and a mutated cytoplasmic tail in place (KVKdelta tail), and one with essentially only the sequence coding for the secreted form of IgE (delta M1M2) . Measurements of the steady-state level of IgE showed that in delta M1M2 mice IgE can only be detected at a minimal level, whereas in KVKdelta tail mice serum IgE is reduced by about 50% . These data allow us to speculate about a specific function of the cytoplasmic tail of mIgE antibodies . We think that the cytoplasmic tail of IgE is involved in signal transduction which leads to the expression of high quantities of secreted IgE. Infect Immun, 1997 May, 65(5), 1824 - 9 Characterization of the nucleotide sequence of the groE operon encoding heat shock proteins chaperone-60 and -10 of Francisella tularensis and determination of the T-cell response to the proteins in individuals vaccinated with F . tularensis; Ericsson M et al.; The groE operon of Francisella tularensis LVS, encoding the heat shock proteins chaperone-10 (Cpn10) and Cpn60, was sequenced and characterized, and the T-cell response of LVS-vaccinated individuals to the two proteins and the third major chaperone, Ft-DnaK, was assayed . The cpn10 and cpn60 genes were amplified by PCR with degenerate oligonucleotides derived from the N-terminal sequence of the two proteins . The sequence analysis revealed the expected two open reading frames, encoding proteins with estimated Mrs of 10,300 and 57,400 . The deduced amino acid sequences closely resembled Cpn10 and Cpn60 proteins of other prokaryotes . The genes constituted a bicistronic operon, the cpn10 gene preceding the cpn60 gene . Upstream of the cpn10 gene, an inverted repeat and motifs similar to -35 and -10 sequences of sigma70-dependent but not of sigma32-dependent promoters of Escherichia coli were found . The inverted repeat of the operon resembled so-called hairpin loops identified in other characterized prokaryotic groE operons lacking sigma32-dependent promoters . Primer extension analysis disclosed one and the same transcription start, irrespective of the presence or absence of heat or oxidative stress . After separation of lysates of the F . tularensis LVS organism by two-dimensional gel electrophoresis, DnaK, Cpn60, and Cpn10 were extracted and used as antigens in T-cell tests . When compared to those from nonvaccinated individuals, T cells from individuals previously vaccinated with live F . tularensis LVS showed an increased proliferative response to DnaK and Cpn60 but not to Cpn10 . The present data will facilitate further studies of the involvement of the heat shock proteins in protective immunity to tularemia. J Clin Microbiol, 1997 May, 35(5), 1071 - 6 Molecular cloning and characterization of a recombinant Histoplasma capsulatum antigen for antibody-based diagnosis of human histoplasmosis; Chandrashekar R et al.; Immunological cross-reactivity among fungi has hampered the development of specific serodiagnostic assays for histoplasmosis . We report the molecular cloning and characterization of a Histoplasma capsulatum cDNA (GH17) that encodes an antigen with immunodiagnostic potential . GH17 is an 810-bp cDNA which encodes a protein of 211 amino acid residues . The GH17 sequence has almost no significant homology with other sequences in GenBank . Southern blot analysis suggests that GH17 is confined to a single location in the genomic DNA of H . capsulatum . Immunoblots indicated that the protein product of GH17 (expressed as a 140-kDa beta-galactosidase fusion protein) was recognized by antibodies in 18 of 18 sera from histoplasmosis patients, but not by antibodies in sera from patients or animals infected with other fungi . GH17 was expressed in a prokaryotic expression vector, pPROEX-1, and recombinant protein was purified by preparative electrophoresis . Antibodies raised to this protein bound to a 60-kDa native antigen in immunoblots of H . capsulatum yeast antigen extract . These results suggest that GH17 encodes an H . capsulatum antigen that may be useful for the diagnosis of histoplasmosis in humans. Gene, 1997 Apr 29, 190(1), 113 - 8 Involvement of host factors in transcription from baculovirus very late promoters -- a review; Hasnain SE et al.; The baculovirus expression vector system has emerged as the system of choice for the expression of a number of heterologous genes of both prokaryotic and eukaryotic origin . This system utilizes the baculovirus very late, hyperactive polyhedrin and p10 promoters to drive the transcription of foreign genes . Regulation of transcription from these promoters is presently not well understood even though a number of viral gene products that may be important for transcription have been identified . Fresh insight into host-virus interactions during baculovirus pathogenesis is now offered by the identification of insect host factors that interact with transcriptionally essential motifs of these promoters as well as cis-acting enhancer-like elements upstream from the promoter. Gene, 1997 Apr 29, 190(1), 17 - 26 Polypurine/polypyrimidine sequences as cis-acting transcriptional regulators; Brahmachari SK et al.; Genome sequence information has generated increasing evidence for the claim that repetitive DNA sequences present within and around genes could play a important role in the regulation of gene expression . Polypurine/polypyrimidine sequences {poly(Pu/Py)} have been observed in the vicinity of promoters and within the transcribed regions of many genes . To understand whether such sequences influence the level of gene expression, we constructed several prokaryotic and eukaryotic expression vectors incorporating poly(Pu/Py) repeats both within and upstream of a reporter gene, lacZ (encoding beta-galactosidase), and studied its expression in vivo . We find that, in contrast to the situation in Escherichia coli, the presence of poly(Pu/Py) sequences within the gene does not significantly inhibit gene expression in mammalian cells . On the other hand, the presence of such sequences upstream of lacZ leads to a several-fold reduction of gene expression in mammalian cells . Similar down-regulation was observed when a structural cassette containing poly(Pu/Py) sequences upstream of lacZ was integrated into yeast chromosome V . Sequence analysis of the nine totally sequenced yeast chromosomes shows that a large number of such sequences occur upstream of ORFs . On the basis of our experimental results and DNA sequence analysis, we propose that these sequences can function as cis-acting transcriptional regulators. DNA Res, 1997 Apr 28, 4(2), 161 - 8 Compilation of all genes encoding two-component phosphotransfer signal transducers in the genome of Escherichia coli; Mizuno T; Bacteria have devised sophisticated His-Asp phosphorelay signaling systems for eliciting a variety of adaptive responses to their environment, which are generally referred to as the "two-component regulatory system." The widespread occurrence of the His-Asp phosphorelay signaling in both prokaryotes and eukaryotes implies that it is a powerful device for a wide variety of adaptive responses of cells to their environment . The two-component signal transducers contain one or more of three common and characteristic phosphotransfer signaling domains, named the "transmitter, receiver, and histidine-containing phosphotransfer (HPt) domains." The recently determined entire genomic sequence of Escherichia coli allowed us to compile systematically a complete list of genes encoding such two-component signal transduction proteins . The results of such an effort, made in this study, revealed that at least 62 open reading frames (ORFs) were identified as putative members of the two-component signal transducers in this single species . Among them, 32 were identified as response regulator and 23 were identified as orthodox sensory kinases . In addition, E . coli has five hybrid sensory kinases . The precise location of each ORF was mapped on a physical map of the entire E . coli genome . All of these ORFs were then compiled and annotated extensively. Mol Gen Genet, 1997 Apr 28, 254(4), 345 - 50 A novel homologue of the prokaryotic htrA gene is differentially expressed in the alga Haematococcus pluvialis following stress; Hershkovits G et al.; The alga Haematococcus pluvialis is able to respond to environmental stress by changing from the motile, vegetative green cell form to red stationary aplanospores . This differentiation program is accompanied by dramatic morphological changes that must result, at least in part, from differential gene expression . To begin to identify genes that are differentially expressed as a response to stress, we applied the differential display technique to identify and isolate differentially expressed RNA sequences . Here we report on the isolation and characterization of one such RNA sequence that codes for Haematococcus htrA, a member of a heat shock serine protease family previously described only in prokaryotes . Interestingly, database searches of mouse and human cDNA sequences showed that a previously unreported homologue is also found in a number of tissues . htrA mRNA was not detectable in vegetative cells, but was found at high levels in developing aplanospores . Evidence presented suggests that RNA transcripts encoding this protein are differentially spliced, and that the different splice products are differentially expressed during the developmental process . These experiments provide the groundwork upon which the developmental program of H . pluvialis can be investigated . In addition, they indicate that the htrA family of heat shock serine proteases may play an important role in stress response in higher organisms as well as in bacteria. J Mol Biol, 1997 Apr 25, 268(1), 15 - 20 Six molecules of SV40 large T antigen assemble in a propeller-shaped particle around a channel; San Martin MC et al.; The large T antigen of simian virus 40 (SV40) is a multifunctional regulatory protein, responsible for both the control of viral infection and the required alterations of cellular processes . T antigen is the only viral protein required for viral DNA replication . It binds specifically to the viral origin and as a helicase unwinds the SV40 DNA bidirectionally . The functional complex is a double hexameric oligomer . In the absence of DNA, but in the presence of ATP or a non-hydrolyzable analog, T antigen assembles into hexamers, which are active as a helicase when a partially single-stranded (3') entry site exists on the substrate . We have used negative staining electron microscopy, single particle image processing and three-dimensional reconstruction with a new algebraic reconstruction techniques (ART) algorithm to study the structure of these hexameric particles in the presence of different nucleotide cofactors (ATP, ADP, and the non-hydrolyzable analogs ATPgammaS and AMP-PNP) . In every case a strong 6-fold structure was found, with the six density maxima arranged in a ring-like particle around a channel, and a well-defined vorticity . Because these structural features have recently been found in other prokaryotic helicases, they seem to be strongly related to the activity of the protein, which suggests a general functional model conserved through evolution. Biochemistry, 1997 Apr 22, 36(16), 5084 - 96 Mechanism of action of base release by Escherichia coli Fpg protein: role of lysine 155 in catalysis; Rabow LE et al.; Fpg protein (formamidopyrimidine/8-oxoguanine DNA N-glycosylase) is a DNA repair enzyme that catalyzes the removal of oxidized purines, most notably the mutagenic 7-hydro-8-oxoguanine (8oxoGua) lesion, by an N-glycosylase action . Additionally, Fpg protein catalyzes beta and delta elimination reactions subsequent to removal of the base lesions, as well as the analogous chemistry at abasic sites (AP sites) . In this report, we show that of the two lysines that are conserved among the various putative prokaryotic Fpg proteins, a site specific alteration in one of them (lysine 155 changed to alanine) displays meaningful changes in substrate activities . However, lysine 155 is not required for the postulated covalent enzyme-substrate imine intermediate as demonstrated by trapping of the mutant protein-oligonucleotide complexes with cyanide or cyanoborohydride . The K155A mutant shows a decrease in activity with the 8oxoGua-substrate of approximately 50-fold under both k(cat)/Km and k(cat) conditions . This mutant also displays a similar reduction in activity with an oligonucleotide substrate possessing a single 2'-deoxy-8-oxonebularine site . In contrast, activity for a site specific 7-methylformamidopyrimidine-modified oligonucleotide is reduced approximately 3-4-fold, a much more modest decrease in activity . Interestingly, there is a concomitant increase in AP lyase activity above wild-type for the K155A mutant (1.6-fold increase in k(cat), 32-fold increase in k(cat)/Km), demonstrating retention of functional beta and delta lyase activities . Together these observations are readily accommodated by a model requiring a direct interaction of lysine 155 with the C8 oxygen of 8-oxopurines . Thus, conservation of this amino acid residue during evolution appears to be essential for specific incision of the mutagenic 8oxoGua base lesion by Fpg protein. Front Biosci, 1996 Oct 01, 1, d309 - 17 HSP90--news from the front; Jakob U; The 90 kDa heat shock protein Hsp90 is a highly conserved and very abundant protein in the cytosol of both eukaryotic and prokaryotic cells . The main focus in the recent years has been concerned Hsp90's interaction with untransformed steroid receptors and newly synthesized kinases . Within these heterocomplexes, Hsp90 acts in concert with several other heat shock and non heat shock proteins to mediate important regulatory effects . These roles of Hsp90 leave unexplained its high abundance and heat shock regulation . More recently, however, Hsp90 has been identified as an ATP independent molecular chaperone, which binds transiently to folding intermediates in vitro, prevents aggregation and supports the refolding of the intermediates to the native state . The finding that Hsp90 interacts with late, probably highly structured, folding intermediates led to the suggestion that Hsp90 might function as a general chaperone for well structured not yet native polypeptides . This explanation provides the missing link between Hsp90 on the one hand as a highly specialized binding protein and Hsp90 on the other hand as a rather promiscuous molecular chaperone. Nature, 1997 Apr 10, 386(6625), 627 - 30 Activation of prokaryotic transcription through arbitrary protein-protein contacts; Dove SL et al.; Many transcriptional activators in prokaryotes are known to bind near a promoter and contact RNA polymerase, but it is not clear whether a protein-protein contact between an activator and RNA polymerase is enough to activate gene transcription . Here we show that contact between a DNA-bound protein and a heterologous protein domain fused to RNA polymerase can elicit transcriptional activation; moreover, the strength of this engineered protein-protein interaction determines the amount of gene activation . Our results indicate that an arbitrary interaction between a DNA-bound protein and RNA polymerase can activate transcription . We also find that when the DNA-bound 'activator' makes contact with two different components of the polymerase, the effect of these two interactions on transcription is synergistic. FEBS Lett, 1997 Apr 7, 406(1-2), 69 - 74 Distribution of sequence-dependent curvature in genomic DNA sequences; Gabrielian A et al.; The distribution of inherent, sequence-dependent curvature was calculated for a number of prokaryotic (M . genitalium, H . influenzae, M . jannaschii), viral (adenovirus 2, equine herpes virus 1), phage (M13, lambda), eukaryotic (S . cerevisiae) and mitochondrial genomes as well as E . coli and human genomic fragments . The genomic averages are in the range of 6-8 degrees/helical turn and only about 20% of DNA is curved less than 3 degrees/helical turn . The prokaryotes and phages appear to have a consistently higher frequency of curved DNA in their genomes than the other genomes tested . Long, highly curved segments, similar to artificially designed curved DNA, are apparently absent from the genomes . Short, curved segments, differing in G+C content may provide environmentally modulated conformational signals for gene regulation . A WWW-server was constructed for the prediction of curved sites from DNA sequences .. J Biol Chem, 1997 Apr 4, 272(14), 9182 - 8 Characterization of the COQ5 gene from Saccharomyces cerevisiae . Evidence for a C-methyltransferase in ubiquinone biosynthesis; Barkovich RJ et al.; Ubiquinone (coenzyme Q or Q) is a lipophilic metabolite that functions in the electron transport chain in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes . Q-deficient mutants of Saccharomyces cerevisiae fall into eight complementation groups (coq1-coq8) . Yeast mutants from the coq5 complementation group lack Q and as a result are respiration-defective and fail to grow on nonfermentable carbon sources . A nuclear gene, designated COQ5 was isolated from a yeast genomic library based on its ability to restore growth of a representative coq5 mutant on media containing glycerol as the sole carbon source . The DNA segment responsible for the complementation contained an open reading frame (GenBankTM accession number Z49210Z49210) with 44% sequence identity over 262 amino acids to UbiE, which is required for a C-methyltransferase step in the Q and menaquinone biosynthetic pathways in Escherichia coli . Both the ubiE and COQ5 coding sequences contain sequence motifs common to a wide variety of S-adenosyl-L-methionine-dependent methyltransferases . A gene fusion expressing a biotinylated form of Coq5p retains function, as assayed by the complementation of the coq5 mutant . This Coq5-biotinylated fusion protein is located in mitochondria . The synthesis of two farnesylated analogs of intermediates in the ubiquinone biosynthetic pathway is reported . These reagents have been used to develop in vitro C-methylation assays with isolated yeast mitochondria . These studies show that Coq5p is required for the C-methyltransferase step that converts 2-methoxy-6-polyprenyl-1, 4-benzoquinone to 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinone. Fungal Genet Biol, 1997 Apr, 21(2), 228 - 37 Characterization of mycobionts of photomorph pairs in the peltigerineae (lichenized ascomycetes) based on internal transcribed spacer sequences of the nuclear ribosomal DNA; Goffinet B et al.; The "one fungus-two photomorphs" hypothesis suggests that certain lichenized fungi can establish a symbiotic relationship with either a eukaryotic or a prokaryotic photobiont . Such pairs of photomorphs are well know from cephalodiate Peltigerineae . Using an ascomycete-specific primer we amplified the internal transcribed spacer region of the nrDNA repeat of the mycobiont from total "lichen DNA" extracts of Peltigera malacea, photomorphs of P . aphthosa, P . britannica, and P . leucophlebia, Nephroma expallidum, and photomorphs of N . arcticum . Comparisons of 5.8S sequences suggest that the sequences obtained belong to the mycobiont and thus, that the ascomycete-specific primer is adequate for amplifying fungal DNA from total lichen-DNA extracts . The strict identity of nucleotide sequences of the internal transcribed spacer region of the nrDNA repeat between joined-photomorphs supports the one fungus-two photomorphs hypothesis . Photomorph may thus primarily reflect phenotypic plasticity of photomorphic fungi in response to changing environmental conditions . The cyanomorph recently reported for P . leucophlebia is shown to be based on a misidentified specimen of P . aphthosa . Comparisons of the ITS sequences further supports recognizing P . aphthosa, P . britannica, and P . leucophlebia at the species rather than the infraspecific level. Mol Biotechnol, 1997 Apr, 7(2), 145 - 51 The use of monoclonal antibodies with colloidal gold-labeled probes in postembedding immunoelectron microscopy; Suarez CE et al.; This article focuses on procedures for the use of monoclonal antibodies (MAb) in immunoelectron microscopy (IEM) for the subcellular localization of antigens or to relate function with structure in prokaryotic and eukaryotic cells using postembedding immunoelectron microscopy techniques . The use of MAbs greatly increases the specificity and quality of information when used in combination with gold-labeled probes . Because of its specificity, the reactivity of MAb may be very sensitive to antigenic changes resulting from the process of sample preparation when performing IEM studies . Specific protocols for each particular combination of epitope/MAb must be usually specifically devised, since it is impossible to predict an experimental system that will successfully preserve structures and antigenic determinants for every combination . In this article, we discuss critical technical aspects that usually result in improved resolution when the procedure is used to identify structure in diverse prokaryotic and eukaryotic cells. Mol Microbiol, 1997 Apr, 24(1), 19 - 28 Functional importance of RNA interactions in selection of translation initiation codons; Sprengart ML et al.; RNA base pairing between the initiation codon and anticodon loop of initiator tRNA is essential but not sufficient for the selection of the 'correct' mRNA translational start site by ribosomes . In prokaryotes, additional RNA interactions between small ribosomal subunit RNA and mRNA sequences just upstream of the start codon can efficiently direct the ribosome to the initiation site . Although there is presently no proof for a similar important ribosomal RNA interaction in eukaryotes, the 5' non-coding regions of their mRNAs and 'consensus sequences' surrounding initiation codons have been shown to be strong determinants for initiation-site selection, but the exact mechanisms are not yet understood . Intramolecular base pairing in mRNA and participation of translation initiation factors can strongly influence the formation of mRNA-small ribosomal subunit-initiator tRNA complexes and modulate translational activities in both prokaryotes and eukaryotes . Only recently has it been appreciated that alternative mechanisms may also contribute to the selection of initiation codons in all organisms . Although direct proof is currently lacking, there is accumulating evidence that additional cis-acting mRNA elements and trans-acting proteins may form specific 'bridging' interactions with ribosomes during translation initiation. J Endocrinol, 1997 Apr, 153(1), 139 - 50 Recombinant gilthead seabream (Sparus aurata) insulin-like growth factor-I: subcloning, expression in Escherichia coli, purification and characterization; Fine M et al.; Gilthead seabream (Sparus aurata) insulin-like growth factor-I (gsIGF-I) cDNA coding for the mature protein was cloned in a pGEM-3Z vector, and then transferred into prokaryotic expression vector pET-11a and expressed in Escherichia coli BL21(DE3) cells upon induction with isopropyl thiogalactoside . The expressed protein contained within the inclusion-body pellet was solubilized in 4.5 M urea, refolded for 24 h at pH 11.3 in the presence of catalytic amounts of cysteine and purified to over 98% purity, as a monomeric methionyl-gsIGF-I . Amino acid composition and N-terminal sequence confirmed the identity to be the predicted protein . Binding assays of the 125I-gsIGF-I to gilthead seabream or carp (Cyprinus carpio) sera resulted in high specific binding, indicating the existence of one or more IGF-binding proteins . In binding experiments to crude gilthead seabream brain homogenate, using human (h) IGF-I as a ligand, the respective IC50 value of hIGF-I was about fourfold lower than that of gsIGF-I . Recombinant gsIGF-I exhibited mitogenic activity in a mouse mammary gland-derived MME-L1 cell line which was approximately 200-fold lower than that of hIGF-1 . Binding experiments to intact MME-L1 cells suggests that this difference most likely results from a correspondingly lower affinity for IGF-I receptor in these cells . In contrast, the activities of gsIGF-I and hIGF-I measured by 35S uptake by gill arches from the goldfish (Carassius auratus) were identical, indicating that the recombinant gsIGF-I is biologically active. Gene, 1997 Apr 1, 188(2), 285 - 90 Cloning and expression of cDNA encoding heart-type isoform of AMP deaminase; Wang X et al.; Mouse cDNA for the heart-type (H) isoform of AMP deaminase (EC 3.5.4.6., AMPD) has been isolated and characterized . The cDNA for the Ampd gene expressed in heart is predicted to encode a peptide of 766 amino acids with a molecular mass of 88 kDa . The coding region of this cDNA was quite homologous to the human AMPD3 gene which encodes the erythrocyte-type (E) isoform of AMPD, although the H-isoform of rodent AMPD was reported to be immunologically distinct from the E-isoform of human AMPD . The non-coding region of the isolated cDNA is not homologous to human AMPD3, while rodent Ampd1 has similarity to human AMPD1 in the non-coding region as well as in the coding region . The transcripts for the cloned cDNA were expressed in heart, slow-twitch skeletal muscle and non-muscle tissues . Prokaryotic expression showed that this cDNA encodes a catalytically active enzyme which reacts to the specific antibody raised to the H-isoform of AMPD. J Mass Spectrom, 1997 Apr, 32(4), 370 - 8 Conserved motifs as the basis for recognition of homologous proteins across species boundaries using peptide-mass fingerprinting; Cordwell SJ et al.; Two-dimensional gel electrophoresis of any biological system presently resolves a plethora of highly purified proteins for which no function or identity has been determined . Theoretical and experimental data were used to demonstrate that peptide-mass fingerprinting (PMF) could aid in the recognition of conserved motifs across species boundaries, and thereby assist in attributing putative function to some of these molecules . Amino acids residue substitutions produced by biological diversity and phylogenetic distance combine to highlight regions of functional significance within proteins . Using 10 prokaryotic and two eukaryotic elongation factors (EF), up to 25 peptide fragments (> 800 Da) per molecule were compared across species boundaries within a 12 x 12 contingency table (66 cross-species comparisons), based upon the degree of molecular mass and amino acid sequence identity . Total amino acid sequence identity ranged from 29.4-80.9% for these molecules . Peptide fragments with homologous sequence across three or more EF were defined as containing, or being near to, conserved functional motifs . Twelve such fragments (> 800 Da) were found in this group of proteins . In addition, an 808.9 Da peptide of unknown functional significance was seen to occur in three of the 12 molecules studied and in another three EF-Tu molecules . At the 83% (five of six residues) identity level, this fragment was found in a further 35 EF-Tu molecules and in 14 unrelated proteins . Further investigation should reveal a role for this fragment (motif) in structural integrity or protein function . A FASTA search conducted on a peptide fragment containing a conserved GTP-binding motif (GHVDHGK) of EF-Tu from Euglena gracilis was used as an example to putatively attribute partial function to three hypothetical proteins derived from DNA sequencing initiatives. Protein Sci, 1997 Apr, 6(4), 892 - 902 Metal- and DNA-binding properties and mutational analysis of the transcription activating factor, B, of coliphage 186: a prokaryotic C4 zinc-finger protein; Pountney DL et al.; Coliphage 186 B is a 72-amino acid protein belonging to the Ogr family of analogous transcription factors present in P2-like phage, which contain a Cys-X2-Cys-X22-Cys-X4-Cys presumptive zinc-finger motif . The molecular characterization of these proteins has been hampered by their insolubility, a difficulty overcome in the present study by obtaining B as a soluble cadmium-containing derivative (CdB) . Atomic absorption spectroscopy showed the presence of one atom of cadmium per molecule of purified CdB . The UV absorption spectrum revealed a shoulder at 250 nm, characteristic of CysS-Cd(II) ligand-to-metal charge-transfer transitions, and the difference absorption coefficient after acidification (delta epsilon 248, 24 mM-1 cm-1) indicated the presence of a Cd(Cys-S)4 center . Gel mobility shift analysis of CdB with a 186 late promoter demonstrated specific DNA-binding (KD, app 3-4 microM) and the protein was shown to activate transcription in vitro from a promoter-reporter plasmid construct . The B DNA-binding site was mapped by gel shift and DNAase I cleavage protection experiments to an area between-70 and -43 relative to the transcription start site, coincident with the consensus sequence, GTTGT-N8-TNANCCA, from -66 to -47 of the 186 and P2 late promoters . Inactive B point mutants were obtained in the putative DNA-binding loop of the N-terminal zinc-finger motif and in a central region thought to interact with the Escherichia coli RNA polymerase alpha-subunit . A truncated B mutant comprising the first 53 amino acids (B1-53) exhibited close to wild-type activity, showed a DNA-binding affinity similar to that of the full-length protein, and could be reconstituted with either Cd or Zn . Gel permeation analysis revealed that B1-53 was a majority dimeric species whereas wild-type B showed larger oligomers . 186 B therefore exhibits a potentially linear organization of functional regions comprising an N-terminal C4 zinc-finger DNA-binding region, a dispensable C-terminal region involved in protein self-association, and a central region that interacts with RNA polymerase. Hum Mol Genet, 1997 Apr, 6(4), 519 - 25 Inhibition of fibrillin 1 expression using U1 snRNA as a vehicle for the presentation of antisense targeting sequence; Montgomery RA et al.; This study examines whether the mimicking of selected properties of naturally occurring antisense RNAs in prokaryotes allows efficient inhibition of gene expression by in situ-expressed recombinant molecules in mammalian cells . Prokaryotic regulatory transcripts are expressed at high levels and have hairpin structures at their termini, features reminiscent of small nuclear RNAs (snRNAs) which are abundant and stable in the nucleus of all mammalian cells . A sequence complementary to fibrillin-1 (FBN1) mRNA, interrupted in its center by a hammerhead ribozyme, was substituted for the Sm protein binding site between the stem-loop structures of U1 snRNA . Expression of the chimeric antisense RNA resulted in dramatic inhibition of expression of fibrillin-1 message and protein in stably transfected cultured cells . The inhibitory effect was localized to the nucleus . The biological properties of U1 snRNA may provide a widely applicable vehicle for the in vivo delivery of antisense targeting sequences. Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 3465 - 70 Multi-functional acetyl-CoA carboxylase from Brassica napus is encoded by a multi-gene family: indication for plastidic localization of at least one isoform; Schulte W et al.; Three genes coding for different multifunctional acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) isoenzymes from Brassica napus were isolated and divided into two major classes according to structural features in their 5' regions: class I comprises two genes with an additional coding exon of approximately 300 bp at the 5' end, and class II is represented by one gene carrying an intron of 586 bp in its 5' untranslated region . Fusion of the peptide sequence encoded by the additional first exon of a class I ACCase gene to the jellyfish Aequorea victoria green fluorescent protein (GFP) and transient expression in tobacco protoplasts targeted GFP to the chloroplasts . In contrast to the deduced primary structure of the biotin carboxylase domain encoded by the class I gene, the corresponding amino acid sequence of the class II ACCase shows higher identity with that of the Arabidopsis ACCase, both lacking a transit peptide . The Arabidopsis ACCase has been proposed to be a cytosolic isoenzyme . These observations indicate that the two classes of ACCase genes encode plastidic and cytosolic isoforms of multi-functional, eukaryotic type, respectively, and that B . napus contains at least one multi-functional ACCase besides the multi-subunit, prokaryotic type located in plastids . Southern blot analysis of genomic DNA from B . napus, Brassica rapa, and Brassica oleracea, the ancestors of amphidiploid rapeseed, using a fragment of a multi-functional ACCase gene as a probe revealed that ACCase is encoded by a multi-gene family of at least five members. J Bacteriol, 1997 Apr, 179(7), 2435 - 9 Unfolding of the bacterial nucleoid both in vivo and in vitro as a result of exposure to camphor; Harrington EW et al.; Both prokaryotic and eukaryotic cells are sensitive to killing by camphor; however, the mechanism by which camphor kills has not been elucidated . We report here that camphor unfolds the nucleoid of Escherichia coli and that unfolding does not require DNA replication, translation, or cell division . We show that exposure of isolated nucleoids to camphor results in unfolding of the chromosome. J Biol Chem, 1997 Mar 28, 272(13), 8482 - 9 Identification of pirin, a novel highly conserved nuclear protein; Wendler WM et al.; In this article we describe the molecular cloning of Pirin, a novel highly conserved 32-kDa protein consisting of 290 amino acids . Pirin was isolated by a yeast two-hybrid screen as an interactor of nuclear factor I/CCAAT box transcription factor (NFI/CTF1), which is known to stimulate adenovirus DNA replication and RNA polymerase II-driven transcription . Pirin mRNA is expressed weakly in all human tissues tested . About 15% of all Pirin cDNAs contain a short 34-base pair insertion in their 5'-untranslated regions, indicative of alternative splicing processes . Multiple Pirin transcripts are expressed in skeletal muscle and heart . Western blots and immunoprecipitations employing monoclonal anti-Pirin antibodies reveal that Pirin is a nuclear protein . Moreover, confocal immunofluorescence experiments demonstrate a predominant localization of Pirin within dot-like subnuclear structures . Homology searches using the BLAST algorithm indicate the existence of Pirin homologues in mouse and rat . The N-terminal half of Pirin is significantly conserved between mammals, plants, fungi, and even prokaryotic organisms . Genomic Southern and Western blots demonstrate the presence of Pirin genes and their expression, respectively, in all mammalian cell lines tested . The expression pattern, the concentrated localization in subnuclear structures, and its interaction with NFI/CTF1 in the two-hybrid system classify Pirin as a putative NFI/CTF1 cofactor, which might help to gain new insights in NFI/CTF1 functions. J Theor Biol, 1997 Mar 21, 185(2), 241 - 53 An evolutionary model of a complementary circular code; Arques DG et al.; The subset X0 = {sequence: see text} of 20 trinucleotides has a preferential occurrence in frame 0 (a reading frame established by the ATG start trinucleotide) of protein (coding) genes of both prokaryotes and eukaryotes . This subset X0++ has the rarity property (6 x 10(-8)) to be a complementary maximal circular code with two permutated maximal circular codes X1 and X2 in frames 1 and 2 respectively (frame 0 shifted by one and two nucleotides respectively in the 5'-3' direction) . X0 is called a C3 code . A quantitative study of these three subsets X0, X1 and X2 in the three frames 0, 1 and 2 of eukaryotic protein genes shows that their occurrence frequencies are constant functions of the trinucleotide positions in the sequences . The frequencies of X0, X1 and X2 in frame 0 of the eukaryotic protein genes are 48.5%, 29% and 22.5% respectively . These properties are not observed in the 5' and 3' regions of eukaryotes where X0, X1 and X2 occur with variable frequencies around the random value (1/3) . Several frequency asymmetries unexpectedly observed, e.g . the frequency difference between X1 and X2 in the frame 0, are related to a new property of the C3 code X0 involving substitutions . An evolutionary model at three parameters (p, q, k) based on an independent mixing of the 20 codons (trinucleotides in frame 0) of X0 with equiprobability (1/20) followed by k approximately 5 substitutions per codon in the three codon sites in proportions p approximately 0.1, q approximately 0.1 and r = 1-p-q approximately 0.8 respectively, retrieves the frequencies of X0, X1 and X2 observed in the three frames of protein genes and explains these asymmetries. J Mol Biol, 1997 Mar 21, 267(1), 60 - 74 Region 1 of sigma70 is required for efficient isomerization and initiation of transcription by Escherichia coli RNA polymerase; Wilson C et al.; The sigma (sigma) subunit of prokaryotic RNA polymerase is essential for promoter recognition . sigma Factor directs the RNA polymerase core subunits (alpha2beta beta') to the promoter consensus elements and thereby confers selectivity for transcription initiation . The N-terminal domain (region 1.1) of Escherichia coli sigma70 has been shown to inhibit DNA binding by the C-terminal DNA recognition domains . Since DNA recognition is the first step of transcription initiation, we predicted that the N-terminal domain of sigma70 may also influence the initiation of transcription by holoenzyme (alpha2beta beta'sigma) . N-terminally truncated sigma70 derivatives were used to reconstitute holoenzyme, and transcription by the mutant holoenzymes was analyzed in vitro . Deletion of the first 75 to 100 amino acids of sigma70 (region 1.1) resulted in both a slow rate of transition from a closed promoter complex to a DNA- strand-separated open complex, as well as a reduced efficiency of transition from the open complex to a ternary initiated complex . These effects were partially reversed by the addition of a polypeptide containing region 1.1 in trans . Therefore, region 1.1 not only modulates DNA binding but is important for efficient transcription initiation, once a closed complex has formed . A deletion of the first 133 amino acids, which removes regions 1.1 and 1.2, resulted in arrest of initiation at the earliest closed complex, suggesting that region 1.2 is required for open complex formation. Gene, 1997 Mar 18, 187(2), 273 - 9 New ultrarare restriction site-carrying transposons for bacterial genomics; Mahillon J et al.; Electrophoretic separation of macrorestriction fragments containing a particular genomic interval has until recently depended on fortuitously placed native rare restriction sites . We present new IS10-based transposons carrying the yeast intron-encoded I-SceI restriction site which is absent from most prokaryotic and eukaryotic genomes . Construction of the plasmid vectors containing them is described . Analysis by conventional or Pulsed Field gel electrophoresis of the DNA fragments generated by the I-SceI digestion reveals the physical distance between genomic insertions of these transposons: use of the same approach to subdivide the chromosome of Escherichia coli K-12 into equivalently sized contiguous/nonoverlapping I-SceI fragments is demonstrated . Because coordinates for the loci delimited by their insertions can be readily determined in different isolates by either physical or genetic manipulations, these transposons allow sufficient flexibility for species-wide bacterial genomics. Biochem J, 1997 Mar 15, 322 ( Pt 3), 729 - 35 cDNA cloning and expression of the flavoprotein D-aspartate oxidase from bovine kidney cortex; Simonic T et al.; The isolation and sequencing of the complete cDNA coding for a d-aspartate oxidase, as well as the overexpression of the recombinant active enzyme, are reported for the first time . This 2022 bp cDNA, beside the coding portion, comprises a 5' untranslated tract and the whole 3' region including the polyadenylation signal and the poly(A) tail . The encoded protein comprises 341 amino acids, with the last three residues (-Ser-Lys-Leu) representing a peroxisomal targeting signal 1 (PTS1), hitherto unknown for this protein . The overexpression of recombinant d-aspartate oxidase was achieved in a prokaryotic system, and a soluble and active enzyme was obtained which accounted for about 10% of total bacterial protein . Comparisons with the known cDNAs for mammalian d-amino acid oxidase, another peroxisomal enzyme, are also made . The close structural and functional similarities shared by these enzymes at the protein level are not reflected at the nucleic acid level. Nucleic Acids Res, 1997 Mar 15, 25(6), 1170 - 6 Significance of the conserved amino acid sequence for human MTH1 protein with antimutator activity; Cai JP et al.; 8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is produced during normal cellular metabolism, and incorporation into DNA causes transversion mutation . Organisms possess an enzyme, 8-oxo-dGTPase, which catalyzes the hydrolysis of 8-oxo-dGTP to the corresponding nucleoside monophosphate, thereby preventing the occurrence of mutation . There are highly conserved amino acid sequences in prokaryotic and eukaryotic proteins containing this and related enzyme activities . To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site- directed mutagenesis of the cloned cDNA for human 8-oxo-dGTPase, and the activity and stability of mutant forms of the enzyme were examined . When lysine-38 was replaced by other amino acids, all of the mutants isolated carried the 8-oxo-dGTPase-negative phenotype . 8-Oxo-dGTPase-positive revertants, isolated from one of the negative mutants, carried the codon for lysine . Using the same procedure, the analysis was extended to other residues within the conserved sequence . At the glutamic acid-43, arginine-51 and glutamic acid-52 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild type protein . We propose that Lys-38, Glu-43, Arg-51 and Glu-52 residues in the conserved region are essential to exert 8-oxo-dGTPase activity. Structure, 1997 Mar 15, 5(3), 349 - 58 Crystal structure of the DNA-binding domain of Mbp1, a transcription factor important in cell-cycle control of DNA synthesis; Xu RM et al.; BACKGROUND: During the cell cycle, cells progress through four distinct phases, G1, S, G2 and M; transcriptional controls play an important role at the transition between these phases . MCB-binding factor (MBF), a transcription factor from budding yeast, binds to the so-called MCB (MluI cell-cycle box) elements found in the promoters of many DNA synthesis genes, and activates the transcription of those at the G1-->S phase transition . MBF is comprised of two proteins, Mbp1 and Swi6 . RESULTS: The three-dimensional structure of the N-terminal DNA-binding domain of Mbp1 has been determined by multiwavelength anomalous diffraction from crystals of the selenomethionyl variant of the protein . The structure is composed of a six-stranded beta sheet interspersed with two pairs of alpha helices . The most conserved core region among Mbp1-related transcription factors folds into a central helix-turn-helix motif with a short N-terminal beta strand and a C-terminal beta hairpin . CONCLUSIONS: Despite little sequence similarity, the structure within the core region of the Mbp1 N-terminal domain exhibits a similar fold to that of the DNA-binding domains of other proteins, such as hepatocyte nuclear factor-3gamma and histone H5 from eukaryotes, and the prokaryotic catabolite gene activator . However, the structure outside the core region defines Mbp1 as a larger entity with substructures that stabilize and display the helix-turn-helix motif. J Membr Biol, 1997 Mar 15, 156(2), 99 - 103 A novel family of ubiquitous heavy metal ion transport proteins; Paulsen IT et al.; We describe a novel diverse family of metal ion transporter (CDF) proteins (the cation diffusion facilitator (CDF) family) with members occurring in both prokaryotes and eukaryotes . Thirteen sequenced protein members of the CDF family have been identified, several of which have been shown to transport cobalt, cadmium and/or zinc . All members of the CDF family possess six putative transmembrane spanners with strongest conservation in the four N-terminal spanners, and on the basis of the analyses, we present a unified structural model . Members of the family are shown to exhibit an unusual degree of size variation, sequence divergence, and differences in cell localization and polarity . The phylogenetic tree for the CDF family reveals that prokaryotic and eukaryotic proteins cluster separately . It allows functional predictions for some uncharacterized members of this family . A signature sequence specific for the CDF family is derived. Science, 1997 Mar 14, 275(5306), 1655 - 7 RNA polymerase beta' subunit: a target of DNA binding-independent activation; Miller A et al.; The bacteriophage N4 single-stranded DNA binding protein (N4SSB) activates transcription by the Escherichia coli final sigma70-RNA polymerase at N4 late promoters . Here it is shown that the single-stranded DNA binding activity of N4SSB is not required for transcriptional activation . N4SSB interacts with the carboxyl terminus of the RNA polymerase beta' subunit in a region that is highly conserved in the largest subunits of prokaryotic and eukaryotic RNA polymerases. FEBS Lett, 1997 Mar 10, 404(2-3), 272 - 4 Aeration-dependent changes in composition of the quinone pool in Escherichia coli . Evidence of post-transcriptional regulation of the quinone biosynthesis; Shestopalov AI et al.; The aeration-dependent changes in content of various quinones in Escherichia coli were found to be unaffected by a prokaryotic translation inhibitor chloramphenicol . In addition, this process was shown to be completely intact in cells with mutated fnr, arc and appY loci . It is assumed that E . coli possesses a special system of oxygen-dependent post-transcriptional regulation of the quinone biosynthetic pathways. Cell, 1997 Mar 7, 88(5), 717 - 23 Insights into multistep phosphorelay from the crystal structure of the C-terminal HPt domain of ArcB; Kato M et al.; The histidine-containing phosphotransfer (HPt) domain is a novel protein module with an active histidine residue that mediates phosphotransfer reactions in the two-component signaling systems . A multistep phosphorelay involving the HPt domain has been suggested for these signaling pathways . The crystal structure of the HPt domain of the anaerobic sensor kinase ArcB has been determined at 2.06 A resolution . The domain consists of six alpha helices containing a four-helix bundle-folding . The pattern of sequence similarity of the HPt domains of ArcB and components in other signaling systems can be interpreted in light of the three-dimensional structure and supports the conclusion that the HPt domains have a common structural motif both in prokaryotes and eukaryotes. J Biol Chem, 1997 Mar 7, 272(10), 6174 - 8 Roles of disulfide bonds in bacterial alkaline phosphatase; Sone M et al.; Alkaline phosphatase of Escherichia coli (a homodimeric protein found in the periplasmic space) contains two intramolecular disulfide bonds (Cys-168-Cys-178 and Cys-286-Cys-336) that are formed after export to the periplasmic space . The location-specific folding character of this enzyme allowed its wide usage as a reporter of protein localization in prokaryotic cells . To study the roles of disulfide bonds in alkaline phosphatase, we eliminated each of them by Cys to Ser mutations . Intracellular stability of alkaline phosphatase decreased in the absence of either one or both of the disulfide bonds . The mutant proteins were stabilized in a DegP protease-deficient strain, allowing accumulation at significant levels and subsequent characterization . A mutant protein that lacked the N-terminally located disulfide bond (Cys-168-Cys-178) was found to have Cys-286 and Cys-336 residues disulfide-bonded, to have a dimeric structure, and to have almost full enzymatic activity . Nevertheless, the mutant protein lost the trypsin-resistant conformation that is characteristically observed for the wild-type enzyme . In contrast, mutants lacking Cys-286 and Cys-336 were monomeric and inactive . These results indicate that the Cys-286-Cys-336 disulfide bond is required and is sufficient for correctly positioning the active site region of this enzyme, but such an active conformation is still insufficient for the conformational stability of the enzyme . Thus, a fully active state of this enzyme can be formed without full protein stability, and the two disulfide bonds differentially contribute to these properties. J Biol Chem, 1997 Mar 7, 272(10), 6119 - 27 Topological rules for membrane protein assembly in eukaryotic cells; Gafvelin G et al.; Insertion into the endoplasmic reticulum membrane of model proteins with one, two, and four transmembrane segments and different distributions of positively charged residues in the N-terminal tail and the polar loops has been studied both in vitro and in vivo . Membrane insertion of these same constructs has previously been analyzed in Escherichia coli, thus making possible a detailed comparison between the topological rules for membrane protein assembly in prokaryotic and eukaryotic cells . In general, we find that positively charged residues have similar effects on the membrane topology in both systems when they are placed in the N-terminal tail but that the effects of charged residues in internal loops clearly differ . Our results rule out a sequential start-stop transfer model where successive hydrophobic segments insert with alternating orientations starting from the most N-terminal one as the only mechanism for membrane protein insertion in eukaryotic cells. Arch Microbiol, 1997 Mar 7, 167(2/3), 67 - 77 Ribosomally synthesized antimicrobial peptides: their function, structure, biogenesis, and mechanism of action Nissen-Meyer J, Nes IF. Ribosomally synthesized peptides with antimicrobial activity are produced by prokaryotes, plants, and a wide variety of animals, both vertebrates and invertebrates . These peptides represent an important defense against micro-organisms . Although the peptides differ greatly in primary structures, they are nearly all cationic and very often amphiphilic, which reflects the fact that many of these peptides kill their target cells by permeabilizing the cell membrane . Moreover, many of these peptides may roughly be placed into one of three groups: (1) those that have a high content of one (or two) amino acid(s), often proline, (2) those that contain intramolecular disulfide bonds, often stabilizing a predominantly beta-sheet structure, and (3) those with amphiphilic regions if they assume an alpha-helical structure . Most known ribosomally synthesized antimicrobial peptides have been identified and characterized during the past 15 years . As a result of these studies, insight has been gained into fundamental aspects of biology and biochemistry such as innate immunity, membrane-protein interactions, and protein modification and secretion . Moreover, it has become evident that these peptides may be developed into useful antimicrobial additives and drugs . This review presents a broad overview of the main types of ribosomally synthesized antimicrobial peptides produced by eukaryotes and prokaryotes. Biochem Biophys Res Commun, 1997 Mar 6, 232(1), 69 - 73 A novel mutation which enhances the fluorescence of green fluorescent protein at high temperatures; Kimata Y et al.; Green fluorescent protein (GFP) from Aequorea victoria is widely used as a marker of gene expression and protein localization in living cells from prokaryotes to eukaryotes . However, the total fluorescent signal from wild-type GFP is very weak when expressed in cells cultured at 37 degrees C compared to 30 degrees C or below . This characteristic makes GFP poorly suited to use as a marker in mammalian cells . Here we describe a new variant of GFP which carries a substitution of Ser147 to Pro (S147P GFP) and which emits a stronger fluorescent signal than the wild-type GFP at high temperature . When S147P is combined with the Ser65 to Thr mutation (S65T GFP), the resulting double mutant emits fluorescence which is several-fold stronger than GFP with a single S65T modification in both bacterial or mammalian cells . This S147P mutation should be useful for constructing new GFP variants which stably emit strong fluorescence at a wide range of culturing temperatures. Genetika, 1997 Mar, 33(3), 405 - 9 {Determination of the genotoxicity of fullerene C60 and fullerol using the method of somatic mosaics on cells of Drosophila melanogaster wing and SOS-chromotest}; Zakharenko LP et al.; Genotoxicity of fullerene C60 was been determined in a prokaryotic in vitro test and in an eukaryotic in vivo system . The SOS chromotest of fullerene C60 in the Escherichia coli strain PQ37 revealed no genotoxicity either with or without activation of the rat liver homogenate . To perform the somatic mutation and recombination genotoxicity test (SMART) on somatic wing cells, Drosophila melanogaster larvae were grown on a standard medium with or without fullerene dope . No statistically significant differences were observed at the same fullerene concentrations in the SOS chromotest (0.45 micrograms/ml) . Only at the highest possible fullerene concentration of 2.24 micrograms per 1 ml medium, a slight genotoxic effect was observed in wing cells . Fullerol demonstrates no mutagenic effect at a concentration of 2.46 mg/ml. Genes Cells, 1997 Mar, 2(3), 167 - 84 Signal transduction via the histidyl-aspartyl phosphorelay; Egger LA et al.; The histidyl-aspartyl phosphorelay, formerly described as the two-component system, is the predominant mode of signal transduction in bacteria . Adaptation to environmental changes occurs through a sensor histidine protein kinase and a response regulator . The histidine protein kinase is usually a transmembrane receptor and the response regulator is a cytoplasmic protein . Together the histidyl-aspartyl phosphorelay proteins mediate reversible phosphorylation events that control downstream effectors . Following autophosphorylation at a conserved histidine residue, the histidine kinase serves as a phospho-donor for the response regulator . Once phosphorylated, the response regulator mediates changes in gene expression or cellular locomotion . The EnvZ-OmpR phosphorelay system in Escherichia coli, which monitors external osmolarity and responds by differentially modulating the expression of the OmpF and OmpC major outer membrane porins, will be described as a model system . While histidine kinases were thought to be present only in prokaryotes, they have recently been identified in eukaryotic systems . Here, we review the unique and conserved features of this growing family of signal transducers. J Pineal Res, 1997 Mar, 22(2), 102 - 6 Melatonin production in an aerobic photosynthetic bacterium: an evolutionarily early association with darkness; Tilden AR et al.; Melatonin was measured in a species of aerobic photosynthetic bacteria, Erythrobacter longus, grown in either constant light or constant dark . A radioimmunoassay was used to quantify melatonin levels and thin-layer chromatography to confirm the identity of melatonin immunoactivity . Melatonin levels were significantly higher (nearly 2.3-fold) in the dark-grown than in the light-grown samples . Also, the homogenates of the dark-grown bacteria retained melatonin-producing enzymatic activity, whereas the light-grown homogenates did not; melatonin levels extracted from the dark-grown homogenates increased with increasing extraction time, reaching as high as 29.2 ng.mg-1 protein at 120 min . Removal of membrane fragments from homogenates did not influence melatonin levels in light-grown homogenate, but this procedure increased melatonin levels in dark-grown homogenate, indicating that at least some of the enzymes in the pathway of melatonin production are not membrane-bound . This study is the second to demonstrate the presence of melatonin at the prokaryotic level, supporting the evidence that melatonin appeared very early in evolution . Its function in prokaryotes has not been determined, but may relate to its antioxidative actions. Biofizika, 1997 Mar-Apr, 42(2), 354 - 62 {Fourier analysis of nucleotide sequences . Periodicity in E . coli promoter sequences}; Kutuzova GI et al.; Fourier spectra of E . coli promoter DNA sequences have been obtained . The periodical structure of individual promoter sequences is characterized . E . coli promoter sequences are classified according to their Fourier spectra using three feature sets: 1--the number of peaks in Fourier spectra; 2--values of power spectra for promoter primary structures and their similarity with physical periodicities in the backbone of polynucleotide; 3--the presence of blocks made of equal nucleotides . The comparison of Fourier spectra of promoter sequences and corresponding genes is provided . The conclusion that different ways of stabilization of promoter secondary structure in the case of different primary structure periodicities is drawn . The intermittence of AT- and GC-blocks, and variety of Fourier spectra mean DNA hydrate shell in DNA promoters is non-contiguous and non-stable at junction points . Characteristic features of prokaryotic promoters Fourier spectra differ from human promoters. Antonie Van Leeuwenhoek, 1997 Mar, 71(3), 257 - 63 Bacterial evolution and metabolism; Trevors JT; This article examines the relationship between (or dependence of) bacterial evolution in prokaryotes and metabolism, and the changing physical-chemical conditions present during early evolution. Plant Mol Biol, 1997 Mar, 33(5), 911 - 22 Brassica napus cDNAs encoding fatty acyl-CoA synthetase; Fulda M et al.; From a cDNA library of developing siliques of rapeseed (Brassica napus L.) we have isolated five full-length clones encoding polypeptides of the AMP-binding protein family . Two cDNAs encode fatty acyl-CoA synthetase activity (EC 6.2.1.3) . The deduced polypeptides share about 52% identical amino acids . After expression in Escherichia coli the predicted enzymatic activity was confirmed by in vitro assays and product analysis . The enzymatic activity for one of the clones was characterized in detail by determination of the K(m) for oleic acid ( 10.4 microm) and the pH optimum (between 7 and 8) . For the three additional clones no enzymatic activities could be demonstrated after expression in E . coli, although two of them exhibit similarity to either eukaryotic or prokaryotic acyl-CoA synthetases . The sequences are compared to a number of related expressed sequence tags from Brassica and Arabidopsis . Potential subcellular locations and functions of the deduced polypeptides within plant cells are discussed. Mol Microbiol, 1997 Mar, 23(6), 1099 - 106 RNase E: still a wonderfully mysterious enzyme; Cohen SN et al.; Ribonuclease E (RNase E), which is encoded by an essential Escherichia coli gene known variously as rne, ams, and hmp, was discovered initially as an rRNA-processing enzyme but it is now known to have a general role in RNA decay . Multiple functions, including the ability to cleave RNA endonucleolytically in AU-rich single-strand regions, RNA-binding capabilities, and the ability to interact with polynucleotide phosphorylase and other proteins implicated in the processing and degradation of RNA, are encoded by its 1,061 amino acid residues . The presence of homologues and functional analogues of the rne gene in a variety of prokaryotic and eukaryotic species suggests that its functions have been highly conserved during evolution . While much has been learned in recent years about the structure and functions of RNase E, there is continuing mystery about possible additional activities and molecular interactions of this enzyme. Microbiologia, 1997 Mar, 13(1), 45 - 56 Structure and composition of freshwater microbial mats from a sulfur spring ("Font Pudosa", NE Spain); Martinez A et al.; Different types of microbial mats developing on the wall on a non-thermal sulfur freshwater spring have been studied . Both, light and electron microscopy as well as HPLC analysis of photosynthetic pigments revealed their structure and composition . Prokaryotic chlorophylls and carotenoids helped in the taxonomical assignment of the main photosynthetic groups . "Inverted position" mats (Mat-I) were dominated by Chromatiaceae; they were located closed to the water outlets (0.3 mM sulfide) . "Normal position", that is, cyanobacterial-covered mats (Mat-II and Mat-IV), developed elsewhere on the stone walls at lower sulfide concentrations . A third type of mat (Mat-III), covered by chemolithotrophic bacteria, was distinguishable at the water-air interface, strongly attached to the walls of the spring . Up to six physiological types of microorganisms have been recognized: cyanobacteria, Chromatiaceae, purple nonsulfur bacteria . Chlorobiaceae, Chloroflexaceae, and chemolithotrophic bacteria . Cyanobacteria Lyngbya-like, Oscillatoria-like and Pseudanabaena sp . were found . The diversity of Chromatiaceae (six morpho-/pigment types of the genus Chromatium, plus two non identified Chromatiaceae, named PB1 and PB2 were observed) was noticeable . Chemolithotrophic bacteria were represented by the genera Beggiatoa and Thiothrix . Finally, small numbers of Chloroflexus-like bacteria and Chlorobium limicola were found in all the studied mats. Microbiology, 1997 Mar, 143 ( Pt 3), 971 - 8 Characterization of the ftsH gene of Bacillus subtilis; Lysenko E et al.; Members of the AAA-protein family are found in both prokaryotes and eukaryotes . These ATPases are involved in a number of diverse activities ranging from protein secretion to cell cycle control . This paper reports the functional analysis of the Bacillus subtilis ftsH gene, which encodes a member of this protein family . In cells containing reduced levels of a truncated FtsH protein cell growth was impaired under certain nutritional conditions . In a hypersaline environment FtsH was required in increased amounts for the cells' recovery from osmotic stress . In the absence of FtsH the abundance of several of the major penicillin-binding proteins (PBP2A and 2B) in the cytoplasmic membrane was affected . Lastly, it has been established that FtsH is required for entry into the developmental life cycle. Bioessays, 1997 Mar, 19(3), 233 - 40 The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair; Lieber MR; Unlike the most well-characterized prokaryotic polymerase, E . coli DNA pol l, none of the eukaryotic polymerases have their own 5' to 3' exonuclease domain for nick translation and Okazaki fragment processing . In eukaryotes, FEN-1 is an endo- and exonuclease that carries out this function independently of the polymerase molecules . Only seven nucleases have been cloned from multicellular eukaryotic cells . Among these, FEN-1 is intriguing because it has complex structural preferences; specifically, it cleaves at branched DNA structures . The cloning of FEN-1 permitted establishment of the first eukaryotic nuclease family, predicting that S . cerevisiae RAD2 (S . pombe Rad13) and its mammalian homolog, XPG, would have similar structural specificity . The FEN-1 nuclease family includes several similar enzymes encoded by bacteriophages . The crystal structures of two enzymes in the FEN-1 nuclease family have been solved and they provide a structural basis for the interesting steric requirements of FEN-1 substrates . Because of their unique structural specificities, FEN-1 and its family members have important roles in DNA replication, repair and, potentially, recombination . Recently, FEN-1 was found to specifically associate with PCNA, explaining some aspects of FEN-1 function during DNA replication and potentially in DNA repair. Bioessays, 1997 Mar, 19(3), 209 - 14 The genetics of phototransduction and circadian rhythms in Arabidopsis; Millar AJ et al.; A wide range of biological processes, in all eukaryotes and in some prokaryotes, are controlled by rhythms with a period close to 24 hours . The circadian oscillator, which is responsible for generating these rhythms, is controlled by light signals that maintain its synchrony with the environmental day/night cycle . Higher plants exhibit many circadian rhythms, including rhythms in the transcription of specific genes . Molecular tools derived from such clock-controlled genes have led to the identification of several circadian rhythm mutants in the genetic model, Arabidopsis thaliana . The extensive understanding of photoperception in this species will make it a powerful system with which to investigate the light regulation of circadian rhythms . We compare Arabidopsis rhythms to the results from other systems, and discuss these data with respect to the current phototransduction models. Trends Microbiol, 1997 Mar, 5(3), 102 - 9 DNA gyrase as a drug target; Maxwell A; DNA gyrase is a remarkable enzyme, catalysing the seemingly complex reaction of DNA supercoiling . As gyrase is essential in prokaryotes, it is a good target for antibacterial agents . These agents have diverse chemical structures and interact with gyrase in a variety of ways. Mol Microbiol, 1997 Mar, 23(5), 945 - 54 The Bordetella pertussis sigma subunit of RNA polymerase confers enhanced expression of fha in Escherichia coli; Steffen P et al.; We have cloned the rpoD gene coding for the major sigma factor of Bordetella pertussis . The deduced amino acid sequence reveals a protein of 733 residues which has extensive amino acid homology with the principal sigma factors of a number of divergent prokaryotes . It is larger than most sigma factors identified to date, having a molecular mass of 81.3 kDa . We have designated this factor sigma 80 . In a heterologous complementation assay, B . pertussis rpoD was able to complement the Escherichia coli rpoD temperature-sensitive mutant UQ285 . Furthermore, B . pertussis rpoD conferred better specificity to the E . coli RNA polymerase, allowing increased expression of the B . pertussis virulence-associated tha promoter, but could not activate the ptx and cya promoters in the E . coli UQ285 strains carrying the B . pertussis bvg locus . We discuss the implications of these results on the mechanisms involved in the activation of virulence-associated promoters. Methods, 1997 Mar, 11(3), 267 - 78 Study of redox-regulated transcription factors in prokaryotes; Demple B; Several prokaryotic regulatory proteins that respond to changes in oxygen tension or the presence of oxidative agents have now been identified . The Fnr protein governs the expression of numerous genes during anaerobic growth, both as a transcriptional activator and as a repressor . OxyR protein responds to cellular exposure to H2O2 to stimulate transcription of several defense proteins . SoxR protein is triggered by superoxide or nitric oxide to activate a multigene regulon for antioxidant defense and antibiotic resistance . Each of these proteins has been purified and characterized for DNA binding and transcriptional activity in vitro . Fnr, OxyR, and SoxR all seem to respond directly to redox signals generated in the cell, and their in vitro properties support this view: Fnr has an oxygen-sensitive {4Fe-4S} center essential for DNA binding; OxyR may be activated via oxidation of a key cysteine residue; and SoxR activation depends on redox-sensitive {2Fe-2S} centers . Basic methods for genetic and biochemical analysis in these systems are presented, with emphasis on detailed methods for SoxR that illustrate general approaches for all the systems. Protein Sci, 1997 Mar, 6(3), 543 - 55 A proposed architecture for the central domain of the bacterial enhancer-binding proteins based on secondary structure prediction and fold recognition; Osuna J et al.; The expression of genes transcribed by the RNA polymerase with the alternative sigma factor sigma 54 (E sigma 54) is absolutely dependent on activator proteins that bind to enhancer-like sites, located far upstream from the promoter . These unique prokaryotic proteins, known as enhancer-binding proteins (EBP), mediate open promoter complex formation in a reaction dependent on NTP hydrolysis . The best characterized proteins of this family of regulators are NtrC and NifA, which activate genes required for ammonia assimilation and nitrogen fixation, respectively . In a recent IRBM course (@ontiers of protein structure prediction," IRBM, Pomezia, Italy, 1995; see web site one of us (J.O.) participated in the elaboration of the proposal that the Central domain of the EBPs might adopt the classical mononucleotide-binding fold . This suggestion was based on the results of a new protein fold recognition algorithm (Map) and in the mapping of correlated mutations calculated for the sequence family on the same mononucleotide-binding fold topology . In this work, we present new data that support the previous conclusion . The results from a number of different secondary structure prediction programs suggest that the Central domain could adopt an alpha/beta topology . The fold recognition programs ProFIT 0.9, 3D PROFILE combined with secondary structure prediction, and 123D suggest a mononucleotide-binding fold topology for the Central domain amino acid sequence . Finally, and most importantly, three of five reported residue alterations that impair the Central domain . ATPase activity of the E sigma 54 activators are mapped to polypeptide regions that might be playing equivalent roles as those involved in nucleotide-binding in the mononucleotide-binding proteins . Furthermore, the known residue substitution that alter the function of the E sigma 54 activators, leaving intact the Central domain ATPase activity, are mapped on region proposed to play an equivalent role as the effector region of the GTPase superfamily. Mol Biol Evol, 1997 Mar, 14(3), 287 - 98 Evolutionary distances between nucleotide sequences based on the distribution of substitution rates among sites as estimated by parsimony; Tourasse NJ et al.; The rate of evolution of macromolecules such as ribosomal RNAs and proteins varies along the molecule because structural and functional constraints differ between sites . Many studies have shown that ignoring this variation in computing evolutionary distances leads to severe underestimation of sequence divergences, and thus can lead to misleading evolutionary tree inferences . We propose here a new parsimony-based method for computing evolutionary distances between pairs of sequences that takes into account this variation and estimates it from the data . This method applies to the number of substitutions per site in ribosomal RNA genes as well as to the number of nonsynonymous substitutions per codon for protein-coding genes and is especially suitable when large data sets (> or = 100 sequences) are analyzed . First, starting from a phylogeny constructed with usual distances, the maximum-parsimony method is used to infer the distribution of the number of substitutions that have occurred at each site (or codon) along this tree . This distribution is then fitted to an "invariant + truncated negative binomial" distribution that allows for invariant sites . Maximum-likelihood fitting of this distribution to different data sets showed that it agreed very well with real data . Noticeably, allowing for invariant sites seemed to be very important . Finally, two distance estimates were developed by introducing the distribution of site variability into the substitution models of Jukes and Cantor and of Kimura . The use of different numbers of aligned sequences (up to 1,000 rRNA sequences) showed that the parameters of the model are very sensitive to the number of sequences used to estimate them . However, if at least 100 sequences are considered, the two new distance estimates are quite stable with respect to the number of sequences used to fit the distribution . This stability is true for low as well as for high evolutionary distances . These new distances appeared to be much better estimates of the number of substitutions per site than the classical distances of Jukes and Cantor and of Kimura, which both greatly underestimate this number, so that they can serve as indexes to detect saturation . We conclude that the new distances are particularly suitable for phylogenetic analysis when very distantly related species and relatively large data sets are considered . Trees reconstructed using these distances are generally different from those constructed by means of the classical estimates . Using this new method, we showed that the mean evolutionary distance between Prokaryotes and Eukaryotes is substantially higher for the small-subunit than for the large-subunit rRNAs . This suggests than the former might have experienced a drastic change during the early evolution of Eukaryotes. Trends Genet, 1997 Mar, 13(3), 98 - 104 Genome organization, natural genetic engineering and adaptive mutation; Shapiro JA; Bacterial evolution is considered in the light of molecular discoveries about genome organization, biochemical mechanisms of genetic change, and cellular control networks . Prokaryotic genetic determinants are organized as modular composites of coding sequences and protein-factor binding sites joined together during evolution . Studies of genetic change have revealed the existence of biochemical functions capable of restructuring the bacterial genome at various levels and joining together different sequence elements . These natural genetic engineering systems can be subject to regulation by signal transduction networks conveying information about the extracellular and intracellular environments . Mu-mediated araB-lacZ coding sequence fusions provide one example of adaptive mutation (increased formation of useful mutations under selection) and illustrate how physiological regulation can modulate the activity of a natural genetic engineering system under specific conditions. Anal Biochem, 1997 Mar 1, 246(1), 111 - 7 Synthesis and use of a substrate for the detection of isopeptidase activity; Loewy AG et al.; We have developed a substrate to assay for an isopeptidase, an enzyme capable of cleaving the Nepsilon-(gamma-glutamic) lysine bond which crosslinks polypeptide chains . This substrate consists of modified lysine (N-alpha-{3H}acetyl-l-lysine-N-methylamide or ALMA), linked by its epsilon-amino group to a gamma-carboxyl amide group of casein with guinea pig liver transglutaminase or Factor XIIIa . We used this substrate to demonstrate the release of {3H}ALMA from {3H}ALMA-casein in a culture medium of Bacillus cereus and in brain homogenates of 12- to 14-day-old embryonic chicks . The prokaryotic and the eukaryotic enzymes resemble each other in that both are activated by Ca2+ or Mg2+ and by alkaline phosphatase and both are inhibited by ATP . The {3H}ALMA-casein is a sensitive substrate able to measure reliably specific activities as low as 10(-8) micromol of {3H}ALMA/min/microg protein . The special advantage of this substrate is that the initial rate of ALMA-casein cleavage is not affected significantly by the levels of protease contaminants we have encountered . We were able to rule out alternative mechanisms such as gamma-glutamyl transpeptidase, gamma-glutamyl cyclotransferase, and the reversal of transglutaminase . We conclude that an isopeptidase mechanism most plausibly accounts for the ALMA release. J Gen Virol, 1997 Mar, 78 ( Pt 3), 641 - 7 The inhibitory effects of antisense RNA on hepatitis B virus surface antigen synthesis; Wu J et al.; Antisense RNA-mediated inhibition of gene expression has the potential for gene therapy of virus infections . We studied the inhibitory effect of antisense RNA directed against the hepatitis B virus (HBV) genome on the expression of the HBV surface antigen (HBsAg) . Three prokaryotic antisense RNA expression constructs were produced which expressed antisense RNA complementary to the entire coding region (1.4 kb) and to 1.0 kb and 582 bp of the 5' region of HBsAg mRNA, respectively . In an in vitro translation system, all three antisense RNAs showed concentration-dependent inhibitory effects on translation of HBsAg mRNA . In a coupled in vitro transcription and translation system, concentration-dependent inhibition of HBsAg synthesis was observed for all above mentioned antisense RNAs . Three mammalian antisense RNA expression vectors were then constructed, expressing the same antisense RNAs as used above . Transfection of the vectors into Hep3B cells (an HBsAg secreting cell line) resulted in almost complete blockage of HBsAg production, whereas control vector transfected cells secreted high levels of HBsAg . The inhibitory effect lasted for more than 10 months post-transfection . To examine the possible mechanism of the antisense RNA effect in the cell line, we measured HBV mRNA levels in the transfected cells and found that the mRNA levels in the antisense RNA expressing cells were much lower than those in the control cells . Therefore, in Hep3B cells, the antisense RNAs inhibited HBsAg synthesis, at least partially, through the reduction of HBV mRNA levels. J Bacteriol, 1997 Mar, 179(5), 1721 - 6 Characterization of the reverse gyrase from the hyperthermophilic archaeon Pyrococcus furiosus; Borges KM et al.; The reverse gyrase gene rgy from the hyperthermophilic archaeon Pyrococcus furiosus was cloned and sequenced . The gene is 3,642 bp (1,214 amino acids) in length . The deduced amino acid sequence has relatively high similarity to the sequences of the Methanococcus jannaschii reverse gyrase (48% overall identity), the Sulfolobus acidocaldarius reverse gyrase (41% identity), and the Methanopynrus kandleri reverse gyrase (37% identity) . The P . furiosus reverse gyrase is a monomeric protein, containing a helicase-like module and a type I topoisomerase module, which resembles the enzyme from S . acidocaldarius more than that from M . kandleri, a heterodimeric protein encoded by two separate genes . The control region of the P . furiosus rgy gene contains a typical archaeal putative box A promoter element which is located at position -26 from the transcription start identified by primer extension experiments . The initiating ATG codon is preceded by a possible prokaryote-type ribosome-binding site . Purified P . furiosus reverse gyrase has a sedimentation coefficient of 6S, suggesting a monomeric structure for the native protein . The enzyme is a single polypeptide with an apparent molecular mass of 120 kDa, in agreement with the gene structure . The sequence of the N terminus of the protein corresponded to the deduced amino acid sequence . Phylogenetic analysis indicates that all known reverse gyrase topoisomerase modules form a subgroup inside subfamily IA of type I DNA topoisomerases (sensu Wang {J . C . Wang, Annu . Rev . Biochem . 65:635-692, 1996}) . Our results suggest that the fusion between the topoisomerase and helicase modules of reverse gyrase occurred before the divergence of the two archaeal phyla, Crenoarchaeota and Euryarchaeota. Nucleic Acids Res, 1997 Mar 1, 25(5), 1009 - 14 Mammalian cDNA and prokaryotic reporter sequences silence adjacent transgenes in transgenic mice; Clark AJ et al.; The ovine beta-lactoglobulin gene is expressed efficiently and at high levels in the mammary gland of transgenic mice . In contrast, when this gene is linked to a second gene construct comprising a mammalian cDNA or a CAT reporter sequence it fails to be expressed in the majority of transgenic lines generated . We suggest that mammalian cDNAs and prokaryotic reporter sequences can serve as active foci for gene silencing in the mammalian genome. Mol Gen Genet, 1997 Feb 27, 253(6), 761 - 5 Nucleotide sequence analysis of the junctions of the terminal inverted repeats of the Streptomyces lividans linear chromosome; Volff JN et al.; The junctions of the Streptomyces lividans chromosomal terminal inverted repeats (TIRs) were isolated from cosmid clones as 6.2 kb PstI and 2.9 kb BamHI fragments, respectively . The fragments were completely sequenced . In each of the fragments just one open reading frame could be identified . One putative gene product showed significant similarities to a sensor and the other to a transcriptional regulator protein of prokaryotic two-component signalling systems . Next to one TIR numerous long direct repeats were found within a region of about 400 bp. Mol Gen Genet, 1997 Feb 27, 253(6), 666 - 73 Identification, sequence analysis, and expression of the lepB gene for a leader peptidase in Rhodobacter capsulatus; Klug G et al.; The leader peptidase (signal peptidase I) gene, lepB, of Rhodobacter capsulatus has been cloned and sequenced . The amino acid sequence of the predicted protein exhibits similarity to other known bacterial leader peptidases . R . capsulatus belongs to the alpha-subdivision of purple bacteria and thus is a relative of mitochondria in eukaryotes . Like the yeast mitochondrial inner membrane proteases IMP1 and IMP2, the leader peptidase from Rhodobacter has only one membrane-spanning segment . Sequence comparison of the Rhodobacter Lep protein with IMP1 and IMP2 did not reveal a higher overall similarity than between other prokaryotic signal peptidases and the mitochondrial enzymes . Expression studies using lacZ fusions in combination with primer extension analysis provide evidence for a weak promoter located a short distance from the transcription start of the lepB gene . Failure to establish a Rhodobacter strain with a disrupted lepB gene indicates that this gene is essential. Biochemistry, 1997 Feb 25, 36(8), 2041 - 50 Characterization of human recombinant annexin II tetramer purified from bacteria: role of N-terminal acetylation; Kang HM et al.; Annexin II tetramer (AIIt) is a Ca2+-dependent, phosphatidylserine-binding, and F-actin-bundling phosphoprotein which is localized to both the extracellular and cytoplasmic surfaces of the plasma membrane . The tetramer is composed of two p36 heavy chains and two p11 light chains . We have produced prokaryotic cDNA expression constructs for both p36 and p11 . Both proteins were expressed in large amounts in Escherichia coli upon induction with IPTG . Electrospray ionization mass spectrometry and amino acid sequence analysis of purified recombinant p36 (rp36) and recombinant p11 (rp11) suggested that the recombinant proteins were identical to their native counterparts except for the lack of N-terminal acetylation of rp36 . Furthermore, the non-acetylated rp36 bound rp11 and formed AIIt . The circular dichroism spectra and urea denaturation profiles of acetylated AIIt and non-acetylated rAIIt were identical . In addition, both the acetylated AIIt and non-acetylated rAIIt were similar in their Ca2+ dependence and concentration dependence of phospholipid liposome aggregation, chromaffin granule aggregation, and F-actin bundling . These results suggest that N-terminal acetylation of p36 is not in fact necessary for binding of the protein to p11 and that N-terminal acetylation does not affect the conformational stability of AIIt or the in vitro activities of AIIt . The availability of large amounts of rAIIt will facilitate further characterization of the structure-function relationships of the protein. J Mol Biol, 1997 Feb 21, 266(2), 246 - 68 Mapping to nucleotide resolution of pseudouridine residues in large subunit ribosomal RNAs from representative eukaryotes, prokaryotes, archaebacteria, mitochondria and chloroplasts; Ofengand J et al.; The pseudouridine (psi) residues present in the high molecular mass RNA from the large ribosomal subunit (LSU) have been sequenced from representative species of the eukaryotes, prokaryotes and archaebacteria, and from mitochondrial and chloroplast organelles . Ribosomes from Bacillus subtilis, Halobacter halobium, Drosphilia melanogaster, Mus musculus, Homo sapiens, mitochondria of M . musculus, H . sapiens and Trypanosoma brucei, and Zea mays chloroplasts were examined, resulting in the exact localization of 190 psi residues . The number of psi residues per RNA varied from one in the mitochondrial RNAs to 57 in the cytoplasmic LSU RNA of D . melanogaster and M . musculus . Despite this, all of the psi residues were found in three domains, II, IV and V . All three are at or have been linked to the peptidyl transferase center according to the literature . Comparison of the sites for psi among the species examined revealed four conserved or semi-conserved segments . One is the region 1911 to 1917, which contains three psi or modified psi in almost all species examined . This site is also juxtaposed to the decoding site of the 30 S subunit in the 70 S ribosome and has been implicated in the fidelity of codon recognition . Three additional sites were at the peptidyl transferase center itself . The juxtaposition of the conserved sites for psi with the two important functions of the ribosome, codon recognition and peptide bond formation, implies an important role for psi in ribosome function . We report some new putative modified nucleosides in LSU RNAs as detected by reverse transcription, correct a segment of the sequence of Z . mays chloroplasts and D . melanogaster LSU RNA, correlate the secondary structural context for all known psi residues in ribosomal RNA, and compare the sites for psi with those known for methylated nucleosides in H . sapiens. J Biol Chem, 1997 Feb 21, 272(8), 5192 - 8 Conversion from archaeal geranylgeranyl diphosphate synthase to farnesyl diphosphate synthase . Two amino acids before the first aspartate-rich motif solely determine eukaryotic farnesyl diphosphate synthase activity; Ohnuma S et al.; Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors for a variety of important natural products, such as sterols, carotenoids, and prenyl quinones . Although FPP synthase and GGPP synthase catalyze similar consecutive condensations of isopentenyl diphosphate with allylic diphosphates and have several homologous regions in their amino acid sequences, nothing is known about how these enzymes form the specific products . To locate the region that causes the difference of final products between GGPP synthase and FPP synthase, we constructed six mutated archaeal GGPP synthases whose regions around the first aspartate-rich motif were replaced with the corresponding regions of FPP synthases from human, rat, Arabidopsis thaliana, Saccharomyces cerevisiae, Escherichia coli, Bacillus stearothermophilus, and from some other related mutated enzymes . From the analysis of these mutated enzymes, we revealed that the region around the first aspartate-rich motif is essential for the product specificity of all FPP synthases and that the mechanism of the chain termination in eukaryotic FPP synthases (type I) is different from those of prokaryotic FPP synthases (type II) . In FPP synthases of type I, two amino acids situated at the fourth and the fifth positions before the motif solely determine their product chain length, while the product specificity of the type II enzymes is determined by one aromatic amino acid at the fifth position before the motif, two amino acids inserted in the motif, and other modifications . These data indicate that FPP synthases have evolved from the progenitor corresponding to the archaeal GGPP synthase in two ways. J Biol Chem, 1997 Feb 21, 272(8), 4896 - 903 Mutational analysis of a fatty acyl-coenzyme A synthetase signature motif identifies seven amino acid residues that modulate fatty acid substrate specificity; Black PN et al.; Fatty acyl-CoA synthetase (fatty acid:CoA ligase, AMP-forming; EC 6.2.1.3) catalyzes the formation of fatty acyl-CoA by a two-step process that proceeds through the hydrolysis of pyrophosphate . In Escherichia coli this enzyme plays a pivotal role in the uptake of long chain fatty acids (C12-C18) and in the regulation of the global transcriptional regulator FadR . The E . coli fatty acyl-CoA synthetase has remarkable amino acid similarities and identities to the family of both prokaryotic and eukaryotic fatty acyl-CoA synthetases, indicating a common ancestry . Most notable in this regard is a 25-amino acid consensus sequence, DGWLHTGDIGXWXPXGXLKIIDRKK, common to all fatty acyl-CoA synthetases for which sequence information is available . Within this consensus are 8 invariant and 13 highly conserved amino acid residues in the 12 fatty acyl-CoA synthetases compared . We propose that this sequence represents the fatty acyl-CoA synthetase signature motif (FACS signature motif) . This region of fatty acyl-CoA synthetase from E . coli, 431NGWLHTGDIAVMDEEGFLRIVDRKK455, contains 17 amino acid residues that are either identical or highly conserved to the FACS signature motif . Eighteen site-directed mutations within the fatty acyl-CoA synthetase structural gene (fadD) corresponding to this motif were constructed to evaluate the contribution of this region of the enzyme to catalytic activity . Three distinct classes of mutations were identified on the basis of growth characteristics on fatty acids, enzymatic activities using cell extracts, and studies using purified wild-type and mutant forms of the enzyme: 1) those that resulted in either wild-type or nearly wild-type fatty acyl-CoA synthetase activity profiles; 2) those that had little or no enzyme activity; and 3) those that resulted in lowering and altering fatty acid chain length specificity . Among the 18 mutants characterized, 7 fall in the third class . We propose that the FACS signature motif is essential for catalytic activity and functions in part to promote fatty acid chain length specificity and thus may compose part of the fatty acid binding site within the enzyme. Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1293 - 7 Mutations in the mariner transposase: the D,D(35)E consensus sequence is nonfunctional; Lohe AR et al.; Genetic analysis of eukaryote transposases and comparison with their prokaryote counterparts have been greatly hindered by difficulty in isolating mutations . We describe a simple eye-color screen that facilitates isolation and analysis of mutations in the mariner transposase in Drosophila melanogaster . Use of ethyl methanesulfonate and site-directed mutagenesis has identified 18 residues that are critical for in vivo excision of a target mariner element . When the mutations were examined in heterozygous mutant/nonmutant genotypes, more than half of the mutant transposase proteins were found to reduce the activity of the wild-type transposase, as assayed by the frequency of germline excision of a target element . Remarkably, transposase function is obliterated when the D,D(34)D acidic, ion-binding domain is replaced with the consensus sequence D,D(34)E found in the nematode Tc1 transposase and in many other transposases in the superfamily . A number of mutations strongly complement wild-type transposase in a dominant-negative manner, suggestive of subunit interactions in the excision reaction; these mutations are located in a small region that includes part of the D,D(34)D motif . Transposase function also is eliminated by a mutation in the inferred initiation codon and by a mutation in a putative nuclear localization signal. FEMS Microbiol Lett, 1997 Feb 15, 147(2), 181 - 8 Transposon induced mutation in Gluconobacter oxydans with special reference to its direct-glucose oxidation metabolism; Gupta A et al.; Transposons are important genetic tools for mutation studies and for location of genes in prokaryotes . However, very little published work is available on transposon mutagenesis in Gluconobacter oxydans . We report here Tn5-induced mutation in a keto acid-producing strain of G . oxydans ATCC 9937 with special reference to the direct-glucose oxidation pathway operative in this organism . In this study, a mutant deficient in glucose dehydrogenase (GDH) activity has been developed by Tn5 mutagenesis . The data of plasmid profiles in the wild-type and the GDH- mutant are indicative of transposition in the first instance on one of the three plasmids (pVJ1) harboured by the organism, resulting in rearrangement of the plasmid and finally stable transposition of Tn5 on the main genome . The final location of Tn5 on the genome has been established by DNA hybridisation studies. Nucleic Acids Res, 1997 Feb 15, 25(4), 814 - -21 Cloning and characterization of HUPF1, a human homolog of the Saccharomyces cerevisiae nonsense mRNA-reducing UPF1 protein; Applequist SE et al.; Levels of most nonsense mRNAs are normally reduced in prokaryotes and eukaryotes when compared with that of corresponding functional mRNAs . Genes encoding polypeptides that selectively reduce levels of nonsense mRNA have so far only been identified in simple eukaryotes . We have now cloned a human cDNA whose deduced amino acid sequence shows the highest degree of homology to that of UPF1, a bona fide Saccharomyces cerevisiae group I RNA helicase required for accelerated degradation of nonsense mRNA . Based on the total sequence of the shorter yeast UPF1 protein, the overall identity between the human protein and UPF1 is 51% . Besides NTPase and other RNA helicase consensus motifs, UPF1 and its human homolog also share similar putative zinc finger motifs that are absent in other group I RNA helicases . Northern blot analysis with the human cDNA probe revealed two transcripts in several human cell lines . Further, antibodies raised against a synthetic peptide of the human polypeptide detected a single 130 kDa polypeptide on Western blots from human and mouse cells . Finally, immunofluorescence and Western blot analyses revealed that the human and mouse polypeptides, like yeast UPF1, are expressed in the cytoplasm, but not in the nucleus . We have thus identified the first mammalian homolog of yeast UPF1, a protein that regulates levels of nonsense mRNA, and we tentatively name this protein human HUPF1 (for human homolog of UPF1). Eur J Biochem, 1997 Feb 15, 244(1), 258 - 64 Purification and characterization of a new type of glutamine synthetase from cyanobacteria; Garcia-Dominguez M et al.; The cyanobacterium Synechocystis sp . PCC 6803 contains two genes encoding two different types of glutamine synthetases (GS), glnA and glnN . The first codes for a typical prokaryotic GS type I and the second one codes for a GS type III, different in amino acid sequence to the prokaryotic GSI and the eukaryotic GSII . The glnN gene has been expressed in Escherichia coli and the corresponding protein purified almost to homogeneity (92%) . The native enzyme (500 kDa) was composed of six identical subunits with an apparent molecular mass of 80 kDa . The protein was strongly stabilized in the presence of Mn2+ but not with other divalent cations . Biosynthetic activity of GSIII required the same substrates and cofactors as GSI and GSII enzymes . Apparent Km values for ATP, glutamate and ammonium were 0.43 mM, 0.9 mM and 0.19 mM, respectively . The enzyme was weakly inhibited by several amino acids and strongly inhibited by ADP . Synechocystis GSIII was also inhibited by L-methionine sulfoximine and DL-phosphinotricin, two transition-state analogs of the GS reaction mechanism . GSIII has also been purified from nitrogen-starved Synechocystis 6803 glnA mutant cells, demonstrating that the GS activity, strongly induced under nitrogen starvation in these cells, corresponds to the glnN gene product . In addition, a Synechocystis 6803 glnN mutant lacks the corresponding 80-kDa protein (GSIII) . Polyclonal antibodies specific for GSIII cross-react with GSIII from other cyanobacteria . In all the strains analysed, levels of GSIII protein increased under nitrogen deficiency . These data suggest that GSIII is specifically required under conditions of nitrogen starvation. Nucleic Acids Res, 1997 Feb 15, 25(4), 873 - 6 Atomic force microscopic demonstration of DNA looping by GalR and HU; Lyubchenko YL et al.; Regulation of gene transcription in both prokaryotes and eukaryotes involves formation of various DNA-multiprotein complexes of higher order structure through communication between distant regions of DNA . The communication between distant DNA sites occurs by interaction between proteins bound to the sites by looping out the intervening DNA segments . The repression of transcription of two overlapping promoters of the gal operon in Escherichia coli requires Gal repressor (GalR) and the histone-like protein HU . Both in vivo and in vitro data support a proposed HU containing complex responsive to induction in which GalR molecules bound to two distant operator sites interact by looping out DNA . We successfully applied atomic force microscope (AFM) imaging to visualize galDNA complexes with proteins . We report GalR mediated DNA looping in which HU plays an obligatory role by helping GalR tetramerization . Supercoiling of DNA, which is also critical for GalR action, may stabilize the DNA loops by providing an energetically favorable geometry of the DNA. Biochemistry, 1997 Feb 11, 36(6), 1525 - 31 Differential scanning microcalorimetry indicates that human defensin, HNP-2, interacts specifically with biomembrane mimetic systems; Lohner K et al.; alpha-Defensins are antimicrobial peptides with 29-35 amino acid residues and cysteine-stabilized amphiphilic, triple-stranded beta-sheet structures . We used high-precision differential scanning microcalorimetry to investigate the effects of a human neutrophil alpha-defensin, HNP-2, on the phase behavior of model membranes mimicking bacterial and erythrocyte cell membranes . In the presence of this positively charged peptide, the phase behavior of liposomes containing negatively charged phosphatidylglycerol was markedly altered even at a high lipid-to-peptide molar ratio of 500:1 . Addition of HNP-2 to liposomes mimicking bacterial membranes (mixtures of dipalmitoylphosphatidylglycerol and -ethanolamine) resulted in phase separation owing to some domains being peptide-poor and others peptide-rich . The latter are characterized by an increase of the main transition temperature, most likely arising from electric shielding of the phospholipid headgroups by the peptide . On the other hand, HNP-2 did not affect the phase behavior of membranes mimicking erythrocyte membranes (equimolar mixtures of dipalmitoylphosphatidylcholine and sphingomyelin) as well as the pure single components . This is in contrast to melittin, which significantly affected the phase behavior of choline phospholipids in accordance with its unspecific lytic activity . These results support the hypothesis of preferential interaction of defensins with negatively charged membrane cell surfaces, a common feature of bacterial cell membranes, and demonstrate that HNP-2 discriminates between model membrane systems mimicking prokaryotic and eukaryotic cell membranes. Biochim Biophys Acta, 1997 Feb 7, 1350(2), 109 - 14 cDNA cloning and prokaryotic expression of maize calcium-dependent protein kinases; Saijo Y et al.; Using degenerate oligonucleotide primers corresponding to conserved regions of the calcium-dependent protein kinase (CDPK) family, we carried out a polymerase chain reaction and obtained four distinct partial-length cDNAs from a maize leaf library . We then used these clones as probes for conventional screening and isolated 19 longer clones from another cDNA library of maize seedlings . These clones were classified into four groups based on their DNA cross-hybridization . Two full-length cDNAs, designated as ZmCDPK9 and ZmCDPK7, were sequenced and characterized . The predicted protein of each clone was a typical CDPK with eleven canonical subdomains of protein kinases, and four EF-hand calcium-binding motifs in its N-terminal and C-terminal halves, respectively . The catalytic and regulatory domains were linked by a well-conserved junction domain . The N-terminus of the protein also contained a consensus sequence for an N-myristoylation signal . Northern blot analysis showed that the transcription level of each gene was higher in roots and etiolated leaves than in green leaves . To confirm the calcium dependency of the maize enzymes, the entire coding region of ZmCDPK9 was subcloned into an expression vector so that it was in frame with the vector-encoded peptide tags . A cell-free extract of Escherichia coli transformed with the recombinant plasmid exhibited calcium-dependent phosphorylation activity, using casein as a substrate. Proc Natl Acad Sci U S A, 1997 Feb 4, 94(3), 814 - 9 Stochastic mechanisms in gene expression; McAdams HH et al.; In cellular regulatory networks, genetic activity is controlled by molecular signals that determine when and how often a given gene is transcribed . In genetically controlled pathways, the protein product encoded by one gene often regulates expression of other genes . The time delay, after activation of the first promoter, to reach an effective level to control the next promoter depends on the rate of protein accumulation . We have analyzed the chemical reactions controlling transcript initiation and translation termination in a single such "genetically coupled" link as a precursor to modeling networks constructed from many such links . Simulation of the processes of gene expression shows that proteins are produced from an activated promoter in short bursts of variable numbers of proteins that occur at random time intervals . As a result, there can be large differences in the time between successive events in regulatory cascades across a cell population . In addition, the random pattern of expression of competitive effectors can produce probabilistic outcomes in switching mechanisms that select between alternative regulatory paths . The result can be a partitioning of the cell population into different phenotypes as the cells follow different paths . There are numerous unexplained examples of phenotypic variations in isogenic populations of both prokaryotic and eukaryotic cells that may be the result of these stochastic gene expression mechanisms. J Biomol Struct Dyn, 1997 Feb, 14(4), 449 - 57 Segmented structure of separate and transposable DNA and RNA elements as suggested by their size distributions; Trifonov EN; A collection of about 1000 different eukaryotic and prokaryotic DNA mobile and separate elements is compiled from literature-transposons, plasmids, extrachromosomal circular DNA, insertion sequences, as well as viral genomes and separate genome segments . Only small elements are collected, upto 2000 base pairs . Analysis of the sequence length distributions of the elements reveals that certain sizes are clearly preferred, namely those which correspond to multiples of about 345 bp in eukaryotes and multiples of about 210 bp in prokaryotes . This provides additional evidence in support of the theory (1) that segmented structure is characteristic of not only protein-coding sequences (2) but rather of genomes in general . In particular, it confirms the prediction (1) that mobile and separate elements would also be segmented. Mol Microbiol, 1997 Feb, 23(4), 813 - 23 Repetitive sequences found in the chromosome of the myxobacterium Nannocystis exedens are similar to msDNA: a possible retrotransposition event in bacteria; Lampson BC et al.; The first reverse transcriptase (RT) to be found in a prokaryotic cell is encoded by an element called a retron which resides in the chromosome of many different bacteria . In addition, all retrons code for a functionally obscure RNA-DNA satellite molecule called msDNA . msDNA is synthesized from an RNA template by the retron-encoded RT . An unusual retron element is described here from the myxobacterium Nannocystis exedens . This retron does not appear to have a typical RT gene in close proximity (1 kb) to the gene msd (which encodes the DNA strand of msDNA) . The gene msr (which encodes the RNA strand of msDNA) appears to be duplicated and flanks both sides of the msd gene . Also discovered throughout the chromosome of this bacterium is a set of repeated sequences related to msDNA . These repeat sequences match only part of the sequences of msDNA and may have become incorporated into the chromosome of this bacterium by reverse transcription. Arch Microbiol, 1997 Feb-Mar, 167(2-3), 67 - 77 Ribosomally synthesized antimicrobial peptides: their function, structure, biogenesis, and mechanism of action; Nissen-Meyer J et al.; Ribosomally synthesized peptides with antimicrobial activity are produced by prokaryotes, plants, and a wide variety of animals, both vertebrates and invertebrates . These peptides represent an important defense against micro-organisms . Although the peptides differ greatly in primary structures, they are nearly all cationic and very often amphiphilic, which reflects the fact that many of these peptides kill their target cells by permeabilizing the cell membrane . Moreover, many of these peptides may roughly be placed into one of three groups: (1) those that have a high content of one (or two) amino acid(s), often proline, (2) those that contain intramolecular disulfide bonds, often stabilizing a predominantly beta-sheet structure, and (3) those with amphiphilic regions if they assume an alpha-helical structure . Most known ribosomally synthesized antimicrobial peptides have been identified and characterized during the past 15 years . As a result of these studies, insight has been gained into fundamental aspects of biology and biochemistry such as innate immunity, membrane-protein interactions, and protein modification and secretion . Moreover, it has become evident that these peptides may be developed into useful antimicrobial additives and drugs . This review presents a broad overview of the main types of ribosomally synthesized antimicrobial peptides produced by eukaryotes and prokaryotes. Genomics, 1997 Feb 1, 39(3), 370 - 6 Two novel human members of an emerging mammalian gene family related to mono-ADP-ribosylating bacterial toxins; Koch-Nolte F et al.; Mono-ADP-ribosylation is one of the posttranslational protein modifications regulating cellular metabolism, e.g., nitrogen fixation, in prokaryotes . Several bacterial toxins mono-ADP-ribosylate and inactivate specific proteins in their animal hosts . Recently, two mammalian GPI-anchored cell surface enzymes with similar activities were cloned (designated ART1 and ART2) . We have now identified six related expressed sequence tags (ESTs) in the public database and cloned the two novel human genes from which these are derived (designated ART3 and ART4) . The deduced amino acid sequences of the predicted gene products show 28% sequence identity to one another and 32-41% identity vs the muscle and T cell enzymes . They contain signal peptide sequences characteristic of GPI anchorage . Southern Zoo blot analyses suggest the presence of related genes in other mammalian species . By PCR screening of somatic cell hybrids and by in situ hybridization, we have mapped the two genes to human chromosomes 4p14-p15.1 and 12q13.2-q13.3 . Northern blot analyses show that these genes are specifically expressed in testis and spleen, respectively . Comparison of genomic and cDNA sequences reveals a conserved exon/intron structure, with an unusually large exon encoding the predicted mature membrane proteins . Secondary structure prediction analyses indicate conserved motifs and amino acid residues consistent with a common ancestry of this emerging mammalian enzyme family and bacterial mono(ADP-ribosyl)transferases . It is possible that the four human gene family members identified so far represent the "tip of an iceberg," i.e., a larger family of enzymes that influences the function of target proteins via mono-ADP-ribosylation. Plant J, 1997 Feb, 11(2), 327 - 37 Identification and functional significance of a new class of non-consensus-type plastid promoters; Kapoor S et al.; The promoter regions of most plastid transcription units have been reported to consist of prokaryotic -10 and -35-like consensus sequences . However, a few promoters with no homology to the consensus elements have also been characterized . A novel class of non-consensus plastid promoters--designated as non consensus type II (NC-II) promoters--that effect low-level constitutive expression of respective genes in photosynthetic as well as non-photosynthetic plastids is described in this paper . The abundance of NC-II promoter-derived transcripts remains unaltered even when light-grown seedlings are shifted to the dark . In contrast, transcripts from -10 and -35-like elements containing consensus type (CT) promoters accumulate to high levels in chloroplasts as compared with non-photosynthetic plastids of roots and cultured cells . Moreover, accumulation of these transcripts is greatly affected by light . The inhibition of plastid protein synthesis has no apparent effect on the abundance of the NC-II transcripts whereas levels of CT transcripts are greatly reduced . In vivo tagetitoxin (a plastid transcription inhibitor) treatment also reduces the levels of CT transcripts with no apparent inhibitory effect on the accumulation of NC-II transcripts . The accumulation of transcripts from both classes of promoters, however, is reduced when cytoplasmic protein synthesis is inhibited by in vivo treatment with cycloheximide . The results are suggestive of the possible existence of at least two distinctive systems for the synthesis and/or maintenance of plastid transcripts which differentiate between two classes of transcripts in a promoter-type specific manner. J Endocrinol, 1997 Feb, 152(2), 245 - 55 Distinct expression patterns and biological activities of two isoforms of the mouse orphan receptor TR2; Lee CH et al.; An alternatively spliced variant of a testis-specific nuclear orphan receptor TR2-11 was identified and designated as TR2-11-t . As a result of retaining intron 5 of this gene, TR2-11-t mRNA encoded a truncated receptor with the complete ligand-binding domain deleted . Protein expression of both isoforms was confirmed using a prokaryotic expression system . In the mouse, the expression of the two TR2 isoforms was elevated in the testis with distinct profiles beginning at puberty . TR2-11 expression increased at postnatal day 18, peaked between day 20 and day 24 and remained at high levels throughout adulthood, whereas TR2-11-t expression was elevated transiently at postnatal day 24 . Among separated primary germ cells and established testicular cell lines, TR2-11 was expressed highly in meiotic and postmeiotic germ cells and weakly in a Leydig cell line and a germ cell line, but not expressed in a Sertoli cell line . In contrast, TR2-11-t was expressed at a much lower level in all the testicular cell types examined . In adult testes blocked at germ cell development by vitamin A depletion or hypophysectomy, TR2-11 expression was dramatically reduced whereas TR2-11-t was highly elevated . Based upon the RNA expression patterns of these isoforms, it was suggested that TR2-11 was specific to meiotic and postmeiotic germ cells whereas TR2-11-t was enriched in early germ cell populations such as premeiotic cells . The biological activities of TR2-11 and TR2-11-t on a direct repeat 5-type retinoic acid (RA) response element (RARE)-containing reporter gene was examined in Cos cells . TR2-11 repressed RA induction of this reporter whereas TR2-11-t enhanced RA induction of the same reporter, and the opposite biological effects of these isoforms were dose-dependent . Gel-shift experiments provided evidence for a direct interaction of TR2-11, but not TR2-11-t, with DNA fragments containing this RARE . Opposite roles of TR2-11 and TR2-11-t on RA induction of promoters containing this particular RARE are suggested. Toxicol Appl Pharmacol, 1997 Feb, 142(2), 360 - 6 Cloning, sequencing, and recombinant expression of NAT1, NAT2, and NAT3 derived from the C3H/HeJ (rapid) and A/HeJ (slow) acetylator inbred mouse: functional characterization of the activation and deactivation of aromatic amine carcinogens; Fretland AJ et al.; An acetylator polymorphism has been described in the mouse and the inbred strains C3H/HeJ and A/HeJ constitute rapid and slow acetylators, respectively . The NAT1, NAT2, and NAT3 genes from C3H/HeJ and A/HeJ acetylator inbred mouse strains were amplified using the polymerase chain reaction, cloned into the plasmid vector pUC19, and sequenced . They were then subcloned into the prokaryotic expression vector pKK223-3 and expressed in Escherichia coli strain JM105 . The 870-bp nucleotide coding region of NAT1 and NAT3 did not differ between the rapid and slow acetylator mouse strains, or from that of previously published mouse NAT1 and NAT3 sequences . However, NAT2 did differ between the rapid and slow acetylator strains with an A296 T transition which causes a (Asn99-->Ile) substitution in the deduced amino acid sequence . Recombinant NAT1, NAT2, and NAT3 proteins catalyzed N-, O-, and N,O-acetyltransferase activities . NAT3 catalyzed aromatic amine N-acetyltransferase activities at very low rates, which confirms a previous study . Apparent K(m) and Vmax kinetic constants for N-acetylation were 5- to 10-fold lower for recombinant mouse NAT1 than NAT2 . Intrinsic clearances for recombinant mouse NAT1- and NAT2-catalyzed N-acetylation of aromatic amine carcinogens were comparable . Both recombinant mouse NAT1 and NAT2 catalyzed the metabolic activation of N-hydroxyarylamine (O-acetylation) and N-hydroxyarylamide (N,O-acetylation) carcinogens . Recombinant mouse NAT3 catalyzed N,O-acetylation at very low rates, while O-acetylation was undetectable . No difference was observed between rapid and slow acetylator recombinant NAT2 proteins to activate aromatic amines by O- or N,O-acetylation, in substrate specificity, expression of immunoreactive protein, electrophoretic mobility, or N-acetyltransferase Michaelis-Menten kinetic constants . However, the slow acetylator recombinant NAT2 protein was over 10-fold less stable than rapid acetylator recombinant NAT2 . These studies demonstrate metabolic activation and deactivation by recombinant mouse NAT1, NAT2, and NAT3 proteins and confirm and extend previous studies on the molecular basis for the acetylation polymorphism in the mouse. Plant Cell, 1997 Feb, 9(2), 199 - 207 An enzyme similar to animal type II photolyases mediates photoreactivation in Arabidopsis; Ahmad M et al.; The important issue of photoreactivation DNA repair in plants has become even more interesting in recent years because a family of genes that are highly homologous to photoreactivating DNA repair enzymes but that function as blue light photoreceptors has been isolated . Here, we report the isolation of a novel photolyase-like sequence from Arabidopsis designated PHR1 (for photoreactivating enzyme) . It shares little sequence similarity with either type I photolyases or the cryptochrome family of blue light photoreceptors . Instead, the PHR1 gene encodes an amino acid sequence with significant homology to the recently characterized type II photolyases identified in a number of prokaryotic and animal systems . PHR1 is a single-copy gene and is not expressed in dark-grown etiolated seedlings: the message is light inducible, which is similar to the expression profile for photoreactivation activity in plants . The PHR1 protein complements a photolyase-deficient mutant of Escherichia coli and thus confers photoreactivation activity . In addition, an Arabidopsis mutant that is entirely lacking in photolyase activity has been found to contain a lesion within this Arabidopsis type II photolyase sequence . We conclude that PHR1 represents a genuine plant photolyase gene and that the plant genes with homology to type I photolyases (the cryptochrome family of blue light photoreceptors) do not contribute to photoreactivation repair, at least in the case of Arabidopsis. Microbiology, 1997 Feb, 143 ( Pt 2), 513 - 8 2-phenylethylamine catabolism by Escherichia coli K-12: gene organization and expression; Hanlon SP et al.; A gene encoding phenylacetaldehyde dehydrogenase (PAD), the enzyme involved together with a copper-topaquinone-containing amine oxidase in the initial steps of 2-phenylethylamine catabolism, was located at 31.1 min on the Escherichia coli K-12 genetic map . It was immediately adjacent to the gene encoding the amine oxidase but transcribed in the opposite direction . The purified PAD acted almost equally well on phenylacetaldehyde, 4-hydroxyphenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde . It had a subunit size of 54 kDa and its deduced amino acid sequence was approximately 40% identical to various eukaryotic and prokaryotic aldehyde dehydrogenases . A third gene encoding a positive regulatory protein required for expression of the amine oxidase and PAD genes was located next to the PAD gene . A gene previously located in this position was reported to encode a second amine oxidase but this was not confirmed . The nucleotide sequence from 1447 to 1450 kb on the E . coli K-12 physical map has been determined. Nat Struct Biol, 1997 Feb, 4(2), 153 - 7 Crystal structure of human mitochondrial single-stranded DNA binding protein at 2.4 A resolution; Yang C et al.; We solved the crystal structure of the homotetrameric single-stranded DNA binding (SSB) protein from human mitochondria at a resolution of 2.4 A . The tetramer is formed by two dimers interacting head-to-head and shows D2 symmetry . Sequence-related tetrameric SSB proteins occur in prokaryotes and eukaryotic mitochondria; this is the first report of an atomic resolution structure of this type of protein . Using biochemical data and analysis of sequence homologies, we were able to correlate the functional properties with structure . We propose that ssDNA wraps around the tetrameric HsmtSSB protein through electropositive channels guided by flexible loops. Curr Opin Struct Biol, 1997 Feb, 7(1), 86 - 93 Making DNA do a U-turn: IHF and related proteins; Rice PA; IHF and HU belong to a family of proteins that introduce sharp bends into DNA and act as accessory factors in a variety of cellular processes in prokaryotes . In addition to the crystal structure of IHF bound to DNA, the past year has seen a number of advances in the understanding of the interactions of these proteins with DNA in solution. Curr Opin Struct Biol, 1997 Feb, 7(1), 110 - 6 The ternary complex of EF-Tu and its role in protein biosynthesis; Clark BF et al.; The past year has seen a breakthrough in our structural understanding of how aminoacyl-tRNAs are selected and transported to the ribosomal A-site in order to decode genetic information contained in messenger RNA . All aminoacyl-tRNAs are recognized by the elongation factor EF-Tu in prokaryotes or EF-1alpha in eukaryotes . The recent determination of the structure of the ternary complex of aminoacyl-tRNA, EF-Tu and a GTP analogue shows how the CCA end of all aminoacyl-tRNA structures can be accommodated in a specific binding site on EF-Tu-GTP, and how part of the T-helix can be recognized by EF-Tu in a non-sequence-specific way . Furthermore, the structure of the ternary complex shows striking structural similarity to the structure of another prokaryotic elongation factor, EF-G, the tRNA translocase, in its GDP or empty form . This observation has led to the proposal of a general macromolecular mimicry of RNA and protein, which predicts elements of RNA-like structures will occur in other translation factors, such as initiation factors and release factors, that interact with similar sites on the ribosome. Genes Dev, 1997 Feb 1, 11(3), 299 - 308 The phantom ligand effect: allosteric control of transcription by the retinoid X receptor; Schulman IG et al.; Regulation of gene expression via allosteric control of transcription is one of the fundamental concepts of molecular biology . Studies in prokaryotes have illustrated that binding of small molecules or ligands to sequence-specific transcription factors can produce conformational changes at a distance from the binding site . These ligand-induced changes can dramatically alter the DNA binding and/or trans-activation abilities of the target transcription factors . In this work, analysis of trans-activation by members of the steroid and thyroid hormone receptor superfamily identifies a unique form of allosteric control, the phantom ligand effect . Binding of a novel ligand (LG100754) to one subunit (RXR) of a heterodimeric transcription factor results in a linked conformational change in the second noncovalently bound subunit of the heterodimer (RAR) . This conformational change results in both the dissociation of corepressors and association of coactivators in a fashion mediated by the activation function of the non-liganded subunit . Without occupying the RAR hormone binding pocket, binding of LG100754 to RXR mimics exactly the effects observed when hormone is bound to RAR . Thus, LG100754 behaves as a phantom ligand. Biophys J, 1997 Feb, 72(2 Pt 1), 866 - 75 Quantification of DNA patchiness using long-range correlation measures; Viswanathan GM et al.; We introduce and develop new techniques to quantify DNA patchiness, and to quantify characteristics of its mosaic structure . These techniques, which involve calculating two functions, alpha(l) and beta(l), measure correlations at length scale l and detect distinct characteristic patch sizes embedded in scale-invariant patch size distributions . Using these new methods, we address a number of issues relating to the mosaic structure of genomic DNA . We find several distinct characteristic patch sizes in certain genomic sequences, and compare, contrast, and quantify the correlation properties of different sequences, including a number of yeast, human, and prokaryotic sequences . We exclude the possibility that the correlation properties and the known mosaic structure of DNA can be explained either by simple Markov processes or by tandem repeats of dinucleotides . We find that the distinct patch sizes in all 16 yeast chromosomes are similar . Furthermore, we test the hypothesis that, for yeast, patchiness is caused by the alternation of coding and noncoding regions, and the hypothesis that in human sequences patchiness is related to repetitive sequences . We find that, by themselves, neither the alternation of coding and noncoding regions, nor repetitive sequences, can fully explain the long-range correlation properties of DNA. Endocrinology, 1997 Feb, 138(2), 588 - 93 Characterization of soluble, disulfide bond-stabilized, prokaryotically expressed human thyrotropin receptor ectodomain; Bobovnikova Y et al.; To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein; it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges . However, the reducing environment of the Escherichia coli (E . coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products . To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21-415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E . coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm . After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21-35 . This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E . coli (strain alpha F'), where the majority of the recombinant receptor was insoluble . The soluble recombinant receptor was affinity purified and characterized . Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein . This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes . Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant. J Virol, 1997 Feb, 71(2), 1576 - 82 Interaction of an adenovirus 14.7-kilodalton protein inhibitor of tumor necrosis factor alpha cytolysis with a new member of the GTPase superfamily of signal transducers; Li Y et al.; The adenovirus (Ad) 14.7-kDa E3 protein (E3-14.7K), which can inhibit tumor necrosis factor alpha (TNF-alpha) cytolysis, was used to screen HeLa cell cDNA libraries for interacting proteins in the yeast two-hybrid system . A new member of the low-molecular-weight (LMW) GTP-binding protein family with Ras and ADP-ribosylation factor homology was discovered by this selection and has been named FIP-1 (14.7K-interacting protein) . FIP-1 colocalized with Ad E3-14.7K in the cytoplasm especially near the nuclear membrane and in discrete foci on or near the plasma membrane . Its interaction with E3-14.7K was dependent on the FIP-1 GTP-binding domain . The stable expression of FIP-1 antisense message partially protected the cells from TNF-alpha cytolysis . FIP-1 was associated transiently with several unknown phosphorylated cellular proteins within 15 min after treatment with TNF-alpha . FIP-1 mRNA was expressed ubiquitously but at higher levels in human skeletal muscle, heart, and brain . In addition to homology to other LMW GTP-binding proteins, FIP-1 has regions of homology to two prokaryotic metalloproteases . However, there was no homology between FIP-1 and any of the recently isolated death proteins in the TNF-alpha or Fas/APO1 cytolytic pathway and no interaction with several members of the Bcl-2 family of inhibitors of apoptosis . These data suggest that FIP-1, as a cellular target for Ad E3-14.7K, is either a new intermediate on a previously described pathway or part of a novel TNF-alpha-induced cell death pathway . FIP-1 has two consensus sequences for myristoylation which would be expected to facilitate membrane association and also has sequences for Ser/Thr as well as Tyr phosphorylation that could affect its function. J Biol Chem, 1997 Jan 31, 272(5), 2969 - 76 Selective reporter expression in mast cells using a chymase promoter; Liao Y et al.; Primate alpha-chymases are mast cell neutral proteases that are involved in regulating several regulatory peptides including angiotensin II . Because of significant substrate specificity differences among the chymase group of enzymes, animal models that overexpress primate chymases are crucial for delineating the in vivo function of these enzymes . Activation of alpha-prochymase requires processing enzymes and proteoglycans found in mast cell secretory granules . Thus, the development of models overexpressing active primate chymase requires a mast cell-specific promoter . We show that the 571-base pair (bp) 5'-upstream sequence of the baboon chymase gene, which encodes an alpha-chymase, coupled to the prokaryotic lacZ gene allows the targeting of beta-galactosidase to mast cells in transgenic mice . Tissue expression of the transgene is similar to the expression of the endogenous mouse alpha-chymase mouse mast cell protease-5 . A mouse mast cell line that endogenously expresses mouse mast cell protease-5 (JKras mast cells) also selectively supports the expression of this transgene . In vitro transcription studies in JKras mast cells shows the critical role of a GATA cis-regulatory motif in baboon chymase promoter, located approximately 430-bp upstream of the transcription start site . These results suggest that the 571-bp domain of the baboon chymase promoter contains most, if not all, of the mast cell-specific region of the promoter . We describe here for the first time a promoter that directs expression of transgenes specifically to mouse mast cells . This promoter should be generally applicable for dominant expression of mast cell regulatory proteins. Biochemistry, 1997 Jan 28, 36(4), 768 - 79 Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region; Wang Y et al.; Aminoglycoside antibiotics can bind to many different types of RNA molecules . It was of interest to determine the nature of the selectivity of binding of aminoglycosides to important, biologically relevant RNA targets . Fluorescence anisotropy methods were developed to quantitatively measure aminoglycoside affinities to constructs of the HIV-1 RRE transcriptional activation region and the prokaryotic rRNA decoding region which is the natural antibacterial target of the aminoglycosides . A fluorescent analog of Rev34-50 (Fl-Rev34-50) was prepared and shown by fluorescence anisotropy measurements to bind to the HIV-1 RRE region with a stoichiometry of 1 and a dissociation constant of 7.6 nM . RRE RNA is a target for the arginine rich Rev protein, and the binding is known to be mimicked by Rev34-50 . The binding is driven by a strongly negative enthalpic term . Aminoglycosides compete with Fl-Rev34-50 binding and competition experiments with semisynthetic aminoglycosides and neomycin B and tobramycin show binding affinities in the 1-2 microM range . The binding of aminoglycosides to this construct is thus not highly selective . A prokaryotic rRNA construct was also prepared and shown to bind a fluorescent dye labeled derivative of the antibiotic paromomycin (CRP) stoichiometrically with a dissociation constant of 0.16 microM . Competition experiments with other aminoglycosides showed binding in the micromolar range, with limited specificity for aminoglycoside type, suggesting that much of the aminoglycoside molecule is not involved in binding . The relatively modest specificity in the binding of aminoglycoside described above is to be contrasted to the subnanomolar affinities and specificity of aminoglycoside binding found using in vitro selected RNA molecules (Wang et al., 1996). Mol Gen Genet, 1997 Jan 27, 253(4), 484 - 91 The Listeria monocytogenes gene ctpA encodes a putative P-type ATPase involved in copper transport; Francis MS et al.; A Tn917 transposon derivative was used to construct a lacZ transcriptional fusion mutant in Listeria monocytogenes DRDC8 that displayed increased beta-galactosidase activity in response to cation stress . A 4.3 kb fragment of L . monocytogenes chromosomal DNA flanking the lacZ fusion was cloned and sequenced . A 1962 bp open reading frame was identified, and designated ctpA . Analysis of the deduced 653 amino acid sequence revealed significant similarity to the family of ATP-dependent enzymes involved in copper transport in prokaryotes and eukaryotes . CtpA is distinctive by virtue of an N-terminal truncation in the domain responsible for cation binding . Growth of ctpA insertion mutants was restricted by the copper-chelating agent 8-hydroxyquinoline . DNA/RNA hybridisation studies revealed that levels of ctpA mRNA were increased following growth in media containing low and high copper concentrations . These results suggest the isolation of a region of DNA that encodes a novel copper-transporting system in L . monocytogenes. J Biol Chem, 1997 Jan 24, 272(4), 2053 - 5 FtsY, the prokaryotic signal recognition particle receptor homologue, is essential for biogenesis of membrane proteins; Seluanov A et al.; In mammalian cells, many secretory proteins are targeted to the endoplasmic reticulum co-translationally, by the signal recognition particle (SRP) and its receptor . In Escherichia coli, the targeting of secretory proteins to the inner membrane can be accomplished post-translationally . Unexpectedly, despite this variance, E . coli contains essential genes encoding Ffh and FtsY with a significant similarity to proteins of the eukaryotic SRP machinery . In this study, we investigated the possibility that the prokaryotic SRP-like machinery is involved in biogenesis of membrane proteins in E . coli . The data presented here demonstrate that the SRP-receptor homologue, FtsY, is indeed essential for expression of integral membrane proteins in E . coli, indicating that, in the case of this group of proteins, FtsY and the mammalian SRP receptor have similar functions. Biochem Biophys Res Commun, 1997 Jan 23, 230(3), 602 - 6 Detection of membrane associated thioredoxin on human cell lines; Wollman EE et al.; Thioredoxin (TRX) is a ubiquitous dithiol-oxidoreduction enzyme broadly expressed in cells from prokaryote to eukaryote organisms . Human thioredoxin (human TRX) gene, previously cloned in our laboratory, codes for a 12-kDa protein found in the culture supernatant of several hemopoietic human cell lines . This protein is secreted by a nonclassical pathway . The role of the secreted enzyme as a signalling soluble mediator was demonstrated, but nothing is known about a membrane associated form of thioredoxin which could be involved in cell/cell contacts and accessory signal function . Thus, we performed experiments to determine if human TRX is also expressed at the cell surface . We report here positive results based upon indirect immunofluorescence flow cytometric analysis of different human cell lines (HeLa, U 937, Jurkat, 3B6, Daudi and Raji) using a cross reactive sheep anti E . coli TRX polyclonal antibody demonstrating a significant expression of human TRX at the surface of human cells. Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 475 - 8 Ribonucleotide reductase in the archaeon Pyrococcus furiosus: a critical enzyme in the evolution of DNA genomes? Riera J, Robb FT, Weiss R, Fontecave M. Ribonucleotide reductase (RNR), the enzyme responsible for deoxyribonucleotide synthesis, has been isolated from Pyrococcus furiosus, a deeply branching hyperthermophilic, strictly anaerobic archaeon . Its gene has been cloned, sequenced, and shown to harbor two insertions encoding inteins . The purified enzyme absolutely requires adenosylcobalamin for activity, a trait that defines it as a member of class II (adenosyl-cobalamin-dependent) prokaryotic RNRs . On the other hand, the archaeal RNR has significant amino acid sequence homology with class I (aerobic non-heme iron-dependent) and class III (anaerobic iron-sulfur-dependent) RNRs present in eukaryotes and bacteria, respectively . It is proposed that this enzyme may be the closest possible relative of the original RNR, which allowed the key "RNA world" to "DNA world" transition, and that the different classes of present-day RNRs are the products of divergent evolution. Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 418 - 21 Fetal mouse selenophosphate synthetase 2 (SPS2): characterization of the cysteine mutant form overproduced in a baculovirus-insect cell system; Kim IY et al.; A novel gene detected in mouse embryonic sites of hematopoiesis was cloned and shown to be a eukaryotic analog of the Escherichia coli selenophosphate synthetase gene . Unlike the E . coli enzyme, which is not a selenoprotein, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the gene in a position corresponding to the essential cysteine of the E . coli enzyme . An ionized selenol group in place of a cysteine sulfhydryl group could render this mammalian selenocysteine-containing enzyme a more active catalyst . The native cDNA clone and also a mutant form containing a TGC (cysteine) codon in place of TGA were expressed in a baculovirus-insect cell system . Based on recovery of purified proteins, expression of the mutant enzyme was about 40 times higher than wild-type enzyme . The cysteine mutant enzyme exhibited selenophosphate synthetase activity in the assay that measures selenide-dependent AMP formation from ATP . Although expression of wild-type enzyme has not been optimized, the mutant form of the fetal mouse enzyme can be produced in amounts sufficient for isolation in homogeneous form and precise physicochemical and mechanistic studies allowing direct comparison with the analogous cysteine-containing prokaryotic enzyme. Biochemistry, 1997 Jan 21, 36(3), 586 - 603 Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: crystallographic evidence; Sawaya MR et al.; The reaction catalyzed by Escherichia coli dihydrofolate reductase (ecDHFR) cycles through five detectable kinetic intermediates: holoenzyme, Michaelis complex, ternary product complex, tetrahydrofolate (THF) binary complex, and THF.NADPH complex . Isomorphous crystal structures analogous to these five intermediates and to the transition state (as represented by the methotrexate-NADPH complex) have been used to assemble a 2.1 A resolution movie depicting loop and subdomain movements during the catalytic cycle (see Supporting Information) . The structures suggest that the M20 loop is predominantly closed over the reactants in the holoenzyme, Michaelis, and transition state complexes . But, during the remainder of the cycle, when nicotinamide is not bound, the loop occludes (protrudes into) the nicotinamide-ribose binding pocket . Upon changing from the closed to the occluded conformation, the central portion of the loop rearranges from beta-sheet to 3(10) helix . The change may occur by way of an irregularly structured open loop conformation, which could transiently admit a water molecule into position to protonate N5 of dihydrofolate . From the Michaelis to the transition state analogue complex, rotation between two halves of ecDHFR, the adenosine binding subdomain and loop subdomain, closes the (p-aminobenzoyl)glutamate (pABG) binding crevice by approximately 0.5 A . Resulting enhancement of contacts with the pABG moiety may stabilize puckering at C6 of the pteridine ring in the transition state . The subdomain rotation is further adjusted by cofactor-induced movements (approximately 0.5 A) of helices B and C, producing a larger pABG cleft in the THF.NADPH analogue complex than in the THF analogue complex . Such movements may explain how THF release is assisted by NADPH binding . Subdomain rotation is not observed in vertebrate DHFR structures, but an analogous loop movement (residues 59-70) appears to similarly adjust the pABG cleft width, suggesting that these movements are important for catalysis . Loop movement, also unobserved in vertebrate DHFR structures, may preferentially weaken NADP+ vs NADPH binding in ecDHFR, an evolutionary adaptation to reduce product inhibition in the NADP+ rich environment of prokaryotes. J Biol Chem, 1997 Jan 17, 272(3), 1885 - 90 Human transcription-repair coupling factor CSB/ERCC6 is a DNA-stimulated ATPase but is not a helicase and does not disrupt the ternary transcription complex of stalled RNA polymerase II; Selby CP et al.; Transcription is coupled to repair in Escherichia coli and in humans . Proteins encoded by the mfd gene in E . coli and by the ERCC6/CSB gene in humans, both of which possess the so-called helicase motifs, are required for the coupling reaction . It has been shown that the Mfd protein is an ATPase but not a helicase and accomplishes coupling, in part, by disrupting the ternary complex of E . coli RNA polymerase stalled at the site of DNA damage . In this study we overproduced the human CSB protein using the baculovirus vector and purified and characterized the recombinant protein . CSB has an ATPase activity that is stimulated strongly by DNA; however, it neither acts as a helicase nor does it dissociate stalled RNA polymerase II, suggesting a coupling mechanism in humans different from that in prokaryotes . CSB is a DNA-binding protein, and it also binds to XPA, TFIIH, and the p34 subunit of TFIIE . These interactions are likely to play a role in recruiting repair proteins to ternary complexes formed at damage sites. Eur J Biochem, 1997 Jan 15, 243(1-2), 442 - 51 Cloning, sequencing and developmental expression of phosphofructokinase from Dictyostelium discoideum; Estevez AM et al.; Phosphofructokinase (PFK) from Dictyostelium discoideum is a non-allosteric enzyme that lacks any of the characteristic regulatory mechanisms of PFK from other cells . We have determined the DNA sequence and analyzed the amino acid sequence of D . discoideum PFK, as an initial step toward understanding the peculiar properties of this enzyme . Three overlapping fragments, two of cDNA and one of genomic DNA, were isolated, which together could encode the complete sequence of D . discoideum PFK . The constructed full-length cDNA coded for a protein of 834 amino acids, with a calculated molecular mass of 92.4 kDa, which was similar to other eukaryotic and prokaryotic PFK . Alignments of the amino acid sequence with other isozymes revealed that many of the amino acid residues assigned to binding sites of substrates and allosteric effectors are conserved in this enzyme, but changes were also found that may contribute to the absence of allosteric mechanisms . A phylogenetic tree for the eukaryotic PFK family was constructed and showed that the N-terminal domain clustered with those of yeast subunits, whereas the C-terminal domain was more related to PFK from metazoa . Southern blotting indicated that D . discoideum PFK is encoded by a single gene . The enzyme is present throughout the life cycle of D . discoideum, with a gradual decrease of its expression during development. Genomics, 1997 Jan 15, 39(2), 231 - 4 The cloning of a human ABC gene (ABC3) mapping to chromosome 16p13.3; Connors TD et al.; The ATP binding cassette (ABC) transporters, or traffic ATPases, constitute a large family of proteins responsible for the transport of a wide variety of substrates across cell membranes in both prokaryotic and eukaryotic cells . We describe a human ABC protein with regions of strong homology to the recently described murine ABC1 and ABC2 transporters . The gene for this novel protein, human ABC3, maps near the polycystic kidney disease type 1 (PKD1) gene on chromosome 16p13.3 . The ABC3 gene is expressed at highest levels in lung compared to other tissues. Anal Biochem, 1997 Jan 15, 244(2), 328 - 39 Domain-directed polymerase chain reaction capable of distinguishing bacterial from host DNA at the single-cell level: characterization of a systematic method to investigate putative bacterial infection in idiopathic disease; Steinman CR et al.; The use of a broad-range PCR to target conserved sequences in the bacterial ribosome has long been recognized as an approach to investigating idiopathic human diseases for the presence of bacteria . An important example is rheumatoid arthritis where the hypothesis of a bacterial etiology remains viable despite failure of previous methods to identify a causative organism . Practical implementation of this strategy, however, was impeded by requirements unique to the study of these diseases . Hence, an adequately characterized method for achieving this has not appeared . We now describe such a method based on the use of a broadly reactive pair of deoxyinosine-containing primers . Detailed characterization addressing these requirements provided evidence that DNA from at least 96% of potential prokaryotic pathogens would be detected . The sensitivity was shown to approximate that of a single organism per reaction . Importantly, this sensitivity was shown to be maintained among multiple targets and to be unimpaired by a large excess of human DNA . Similar results were obtained with extracts of inflamed human synovial fluid to which as little as 0.01 pg of bacterial DNA or, alternately, a single organism per reaction was added . This method was also shown correctly to detect the causative bacteria in clinically infected synovial fluids, further documenting its applicability to such specimens . Finally, it was converted to an in situ method by which bacterial sequences were histochemically localized to Michaelis-Guttman bodies in tissue sections from a patient with malakoplakia, a poorly understood infectious disease . The broad reactivity and high sensitivity achieved by this approach translate into a high likelihood of detecting an unknown bacterium if present in a clinical specimen . Conversely, if properly controlled, the failure of this method to detect such organisms can provide evidence supporting rejection of the bacterial etiology hypothesis, an important aspect unique to this approach . For those bacterial sequences that are detected, the ability to localize them in situ would help define their histologic context and hence facilitate their pathogenetic interpretation . The present method should therefore be appropriate for use in systematically studying rheumatoid arthritis as well as a number of other important idiopathic disorders where a bacterial etiology is suspect. Nature, 1997 Jan 9, 385(6612), 176 - 81 Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA; Bochkarev A et al.; The single-stranded-DNA-binding proteins (SSBs) are essential for DNA function in prokaryotic and eukaryotic cells, mitochondria, phages and viruses . The structures of four SSBs have been solved, but the molecular details of the interaction of SSBs with DNA remain speculative . We report here the crystal structure at 2.4 A resolution of the single-stranded-DNA-binding domain of human replication protein A (RPA) bound to DNA . Replication protein A is a heterotrimeric SSB that is highly conserved in eukaryotes . The largest subunit, RPA70, binds to single-stranded (ss)DNA and mediates interactions with many cellular and viral proteins . The DNA-binding domain, which lies in the middle of RPA70, comprises two structurally homologous subdomains oriented in tandem . The ssDNA lies in a channel that extends from one subdomain to the other . The structure of each RPA70 subdomain is similar to those of the bacteriophage SSBs, indicating that the mechanism of ssDNA-binding is conserved. Biochemistry, 1997 Jan 7, 36(1), 34 - 40 Active site directed mutagenesis of 3 beta/17 beta-hydroxysteroid dehydrogenase establishes differential effects on short-chain dehydrogenase/reductase reactions; Oppermann UC et al.; Mutagenetic replacements of conserved residues within the active site of the short-chain dehydrogenase/reductase (SDR) superfamily were studied using prokaryotic 3 beta/17 beta-hydroxysteroid dehydrogenase (3 beta/17 beta-HSD) from Comamonas testosteroni as a model system . The results provide novel data to establish Ser 138 as a member of a catalytically important "triad" of residues also involving Tyr151 and Lys155 . A Ser-->Ala exchange at position 138 results in an almost complete (> 99.9%) loss of enzymatic activity, which is not observed with a Ser-->Thr replacement . This indicates that an essential factor for catalysis is the ability of side chain 138 to form hydrogen bond interactions . Mutations in the NAD(H) binding region, in strands beta A, beta D, and adjacent turns, reveal two additional residues, Thr12 and Asn87, which are important for correct binding of the coenzyme and with a differential effect on the reactions catalyzed . Thus, mutation of Thr12 to Ala results in a complete loss of the 3 beta-dehydrogenase activity, whereas the 3-oxoreductase activity remains unchanged . On the other hand, a T12S substitution yields a protein with unaltered catalytic constants for both reactions, revealing that a specific hydrogen bond is critical for the dehydrogenase activity . Our interpretation of the available crystal structure of 3 alpha/20 beta-HSD from Streptomyces hydrogenans suggests a hydrogen bond in that enzyme between the Thr12 side chain and the backbone NH of Asn87 rather than the coenzyme, indicating that this hydrogen bond to the beta D strand might determine a crucial difference between the reductive and the oxidative reaction types . Similarly, mutation of Asn87 to Ala results in an 80% reduction of kcat/Km in the dehydrogenase direction but also unchanged 3-oxoreductase properties . It appears that the binding of NAD+ to the protein is influenced by local structural changes involving strand beta D and turn beta A to alpha B. Proc Natl Acad Sci U S A, 1997 Jan 7, 94(1), 277 - 82 Molecular phylogeny of Archaea from soil; Bintrim SB et al.; Cultivation methods have contributed to our present knowledge about the presence and diversity of microbes in naturally occurring communities . However, it is well established that only a small fraction of prokaryotes have been cultivated by standard methods and, therefore, the prokaryotes that are cultivated may not reflect the composition and diversity within those communities . Of the two prokaryotic phylogenetic domains, Bacteria and Archaea, members of the former have been shown to be ubiquitous in nature, with ample evidence of vast assemblages of uncultured organisms . There is also now increasingly compelling evidence that the Archaea, which were once thought to occupy a limited number of environments, are also globally widespread . Here we report the use of molecular phylogenetic techniques, which are independent of microbial cultivation, to conduct an assessment of Archaea in a soil microbial community . Small subunit ribosomal RNA genes of Archaea were amplified from soil and cloned . Phylogenetic and nucleotide signature analyses of these cloned small subunit ribosomal RNA gene sequences revealed a cluster of Archaea from a soil microbial community that diverge deeply from the crenarchaeotal line of descent and has the closest affiliation to the lineage of planktonic Archaea . The identification and phylogenetic classification of this archaeal lineage from soil contributes to our understanding of the ecological significance of Archaea as a component of microbial communities in non-extreme environments. Biochim Biophys Acta, 1997 Jan 4, 1337(1), 113 - 22 A 3-hydroxy-3-methylglutaryl-CoA lyase gene in the photosynthetic bacterium Rhodospirillum rubrum; Baltscheffsky M et al.; A 1.2 kb long DNA segment from Rhodospirillum rubrum has been sequenced (EMBL/GenBank accession number: U41280) . This DNA segment includes the first sequenced gene for a putative 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, termed hmgL, from a photosynthetic organism . The sequenced segment also contains a ribosome-binding site and two clusters of possible-35 and -10 promotor sequences preceding the hmgL gene . Translation of the gene would yield a 303 amino-acid-long protein with a calculated molecular weight of 31.1 kDa . This protein shows 55-60% identity and approx . 75% similarity, including conservative substitutions, with the three eukaryotic and the single prokaryotic HMG-CoA lyases which previously have been sequenced . The R . rubrum enzyme showed stronger homology to the chicken HMG-CoA lyase than to the other bacterial protein . Significant sequence similarity was also found with homocitrate synthases from nitrogen-fixing prokaryotes . In contrast to the other sequenced prokaryotic HMG-CoA lyase (from Pseudomonas mevalonii), the R . rubrum hmgL does not seem to appear in an operon together with a HMG-CoA reductase . The hmgL gene was transcribed in photosynthetically grown cells as judged by amplification of cDNAs synthesised from DNA-free total RNA . In addition, HMG-CoA lyase activity was found in R . rubrum cells grown anaerobically in the light with leucine as the carbon source. Gene, 1997 Jan 3, 184(1), 55 - 63 DNA sequence and secondary structure of the mitochondrial small subunit ribosomal RNA coding region including a group-IC2 intron from the cultivated basidiomycete Agrocybe aegerita; Gonzalez P et al.; Due to their structural complexity and their evolutionary dimension, rRNAs are the most investigated nucleic acids in prokaryotes, eukaryotes and organelles . However, no complete sequence of a mitochondrial small subunit (SSU) rRNA was available in the basidiomycotina subdivision . The mitochondrial gene encoding the SSU rRNA of the cultivated basidiomycete Agrocybe aegerita was cloned and its complete nucleotide sequence achieved; the 5'- and 3'-ends were localized by nuclease SI mapping, leading to a size of 3277 nt . The secondary structure of the SSU rRNA (1906 nt in size) possessed all the helices and loops of the prokaryotic model; a unique modification was found in a conserved nucleotide predicted by the model: the nt 487 was A instead of C . The same modification, has been found in all the partial basidiomycete mitochondrial sequences available in databases . The Agrocybe aegerita SSU rRNA was characterized by large and unusual extensions leading to additional helices in the variable domains V4, V6 and V9, which were the longest of the known prokaryotic or mitochondrial SSU rRNAs . Nucleotide sequence analysis indicated a 1371-bp intron, belonging to subgroup-IC2, located in a conserved loop in the 3'-part of the SSU rRNA . This intron, which is the second example reported in a fungal mitochondrial SSU rDNA, encoded a putative protein (407 aa) sharing homologies with endonucleases involved in group-I intron mobility . This report constitutes the first complete mitochondrial SSU rRNA sequence and secondary structure of any member of the basidiomycotina subdivision. FEBS Lett, 1997 Jan 3, 400(2), 188 - 92 Biochemical characterization of Pkn2, a protein Ser/Thr kinase from Myxococcus xanthus, a Gram-negative developmental bacterium; Udo H et al.; Pkn2, a protein Ser/Thr kinase, from the developmental bacterium Myxococcus xanthus was expressed under a T7 promoter in Escherichia coli and purified . Purified Pkn2 retained the autophosphorylation activity with the Km value of 177 microM for ATP and 73 nmol/min/mg for Vmax . The optimum pH and temperature were determined to be 7.5 and 35 degrees C, respectively . The autophosphorylation activity was inhibited by staurosporine with the IC50 value of 400 nM while H-7 and genistein had little effect on this kinase . Pkn2 appears to be unique for its higher manganese dependence . This is the first biochemical characterization of the prokaryotic protein Ser/Thr kinase. J Biol Chem, 1997 Jan 3, 272(1), 572 - 9 A point mutation in Escherichia coli DNA helicase II renders the enzyme nonfunctional in two DNA repair pathways . Evidence for initiation of unwinding from a nick in vivo; Brosh RM Jr et al.; Biosynthetic errors and DNA damage introduce mismatches and lesions in DNA that can lead to mutations . These abnormalities are susceptible to correction by a number of DNA repair mechanisms, each of which requires a distinct set of proteins . Escherichia coli DNA helicase II has been demonstrated to function in two DNA repair pathways, methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair . To define further the role of UvrD in DNA repair a site-specific mutant was characterized . The mutation, uvrDQ251E, resides within helicase motif III, a conserved segment of amino acid homology found in a superfamily of prokaryotic and eukaryotic DNA helicases . The UvrD-Q251E protein failed to complement the mutator and ultraviolet light-sensitive phenotypes of a uvrD deletion strain indicating that the mutant protein is inactive in both mismatch repair and excision repair . Biochemical characterization revealed a significant defect in the ability of the mutant enzyme to initiate unwinding at a nick . The elongation phase of the unwinding reaction was nearly normal . Together, the biochemical and genetic data provide evidence that UvrD-Q251E is dysfunctional because the mutant protein fails to initiate unwinding at the nick(s) used to initiate excision and subsequent repair synthesis . These results provide direct evidence to support the notion that helicase II initiates unwinding from a nick in vivo in mismatch repair and excision repair. Chin J Biotechnol, 1997, 13(1), 37 - 42 Construction and application of a prokaryotic vector which expresses the protein that can be quickly purified by IMAC; Li F et al.; A vector was constructed by inserting a pair of complementary oligo nucleotides encoding 6 histidine residues into the polylinker's upstream of the prokaryotic high expression vector pBV220 . The resultant vector is named pBV222 . Proteins expressed by this vector will have a 6-histidine tail as an affinity handle fused to their N-terminus and can be quickly purified by one-step immobilized metal affinity chromatography (IMAC) . This plasmid was verified by restriction mapping and DNA sequencing . When GM-CSF and IL-2 cDNA were closed into pBV222, expressed proteins in the inclusion body showed the predicted molecular weight and biological activity . The expressed bacteria were dissolved in 6 mol/L guanidine.HCl and the supernatant was loaded directly to IMAC . IL-2 and GM-CSF fusion proteins were eluted by the pH gradient, and over 90% purity was achieved. Cancer Chemother Pharmacol, 1997, 40 Suppl, S3 - 8 Multidrug resistance: molecular mechanisms and clinical relevance; Ling V; Multidrug resistance (MDR) describes the phenomenon of simultaneous resistance to unrelated drugs . It has been a decade since the P-glycoprotein (Pgp) gene, which is associated with a form of MDR caused by reduced drug accumulation, was cloned . Thus, this would seem to be an appropriate time to evaluate our understanding of this form of MDR . The two MDR genes identified in humans to date (the MDR-associated protein {MRP} and Pgp genes) are structurally similar and both are members of the ATP-binding cassette (ABC) transporter family . Although the physiological role of MRP is not yet understood, one Pgp gene (mdr1) plays an important role in the blood-tissue barrier and the other (mdr2/3) is involved in phospholipid transport in the liver . A variety of compounds (chemosensitizing agents) can interfere with Pgp and MRP function; such agents may improve the efficacy of conventional therapy when used in combination with such regimens . Determining the roles cellular MDR mechanisms play in patients' response to chemotherapy is a major challenge . Using Pgp and MRP as molecular markers to detect MDR tumor cells is technically demanding, and solid tumors in particular contain heterogeneous cell populations . Since MDR requires Pgp or MRP gene expression, clinically relevant gene expression thresholds need to be established; sequential samples from individual patients are valuable for correlating MDR gene expression with the clinical course of disease . Studies in leukemias, myelomas, and some childhood cancers show that Pgp expression correlates with poor response to chemotherapy . However, in some cases, inclusion of a reversing or chemosensitizing agent such as verapamil or cyclosporin A has improved clinical efficacy . Such agents may inactivate Pgp in tumor cells or affect Pgp function in normal cells, resulting in altered pharmacokinetics . It would be interesting to determine whether patients who fail treatment in the presence of chemosensitizing agents acquire other MDR mechanisms . The ABC transporter superfamily in prokaryotes and eukaryotes is involved in the transport of substrates ranging from ions to large proteins . Of the 15 or more ABC transporter genes characterized in human cells, two (Pgp and MRP) cause MDR . Therefore, it would be relevant to determine the number of such genes present in the human genome; however, extrapolating from the number of ABC transporter genes in bacteria, the human gene probably contains a minimum of 200 ABC transporter superfamily members . Thus, tumor cells can potentially use many ABC transporters to mount resistance to known and future therapeutic agents . The challenge will be to determine which ABC transporters are clinically relevant . Despite the potential of tumor cells to protect themselves, a variety of malignancies can be successfully treated with chemotherapy . This may provide unique insights. DNA Seq, 1997, 7(5), 301 - 6 Localization of an open reading frame with homology to human aspartoacylase upstream from psbA in the prokaryote Prochlorococcus marinus CCMP 1375; Hess WR; An open reading frame encoding a polypeptide with significant homology (about 28% identity and 16% similarity) to human aspartoacylase (ASPA) was identified in the genome of Prochlorococcus marinus CCMP 1375, an oxyphototrophic bacterium . Sequence alignments show that, in particular, the regions previously suggested to form the catalytic core of human ASPA are evolutionarily conserved and nearly identical to that found in the Prochlorococcus putative ASPA . A glutamate at position 273 is located central to the strongly conserved INEAAY motif and corresponds to a glutamate-285 in human ASPA . A point mutation at this site, resulting in a Glu285Ala missense mutation, was identified previously as the molecular basis for Canavan disease, a form of leukodystrophy . The data provide new insights about conserved and hence functionally important domains of ASPA . The detection of this gene in an unicellular prokaryote demonstrates that this enzymatic activity is more widespread than expected previously . Furthermore, bacteria may be used as simple model organisms to study different forms of ASPA. DNA Seq, 1997, 7(5), 261 - 5 Nucleotide sequence of the Treponema pallidum eno gene; Stamm LV et al.; We determined the nucleotide sequence of the enolase (eno) gene of Treponema pallidum, the noncultivable agent of syphilis . The deduced amino acid sequence of T . pallidum enolase (Eno) is 432 amino acids long with a predicted molecular mass of 46.7 kDa . The Eno amino acid sequence has a high degree of homology to the amino acid sequences of prokaryotic and eukaryotic Eno . This is the first eno sequence reported for a bacterium in the order Spirochaetales. DNA Seq, 1997, 7(3-4), 141 - 51 Genomic structure and sequence analysis of the valyl-tRNA synthetase gene of the Japanese pufferfish, Fugu rubripes; Lim EH et al.; The genomic sequence and exon-intron organisation of the valyl-tRNA synthetase gene in the Japanese pufferfish, Fugu rubripes, have been determined . This single-copy Fugu gene spans 8.5 kb, about 2.5 times smaller than that in man (21 kb) . It contains 29 exons, with the largest intron being 1008 bp . The predicted polypeptide consists of 1217 amino acids, with a molecular weight of 138 kD and an isoelectric point of 7.27 . It shares 40% identity in the overlapping region with its homolog in bacteria, 47% with yeast, and 67% with man . The Fugu gene has an additional N-terminal sequence which shows strong similarity to elongation factory-1gamma, a feature it shares only with the human sequence, but not with any other lower eukaryote or prokaryote studied so far . This N-terminal segment is encoded in the first six exons, suggesting their capture by a translocation through introns . Indeed, the acquisition of extra domains to perform related functions in RNA splicing and translation of polypeptides has already been observed in other aminoacyl-tRNA synthetases . Two cDNA sequences of human valyl-tRNA synthetase have been published, with discrepancies between them . Aided by comparisons with the Fugu gene, three of these discrepancies have been resolved, involving the elucidation of the sequence and positions of two introns . This compact vertebrate genome has demonstrated its value as a tool for the analysis of genes at the genomic level. Annu Rev Biochem, 1997, 66, 173 - 97 Polyadenylation of mRNA in prokaryotes; Sarkar N; The 3'-ends of both prokaryotic and eukaryotic mRNA are polyadenylated, but the poly(A) tracts of prokaryotic mRNA are generally shorter, ranging from 15 to 60 adenylate residues and associated with only 2-60% of the molecules of a given mRNA species . The sites of polyadenylation of bacterial mRNA are diverse and include the 3'-ends of primary transcripts, the sites of endonucleolytic processing in the 3' untranslated and intercistronic regions, and sites within the coding regions of mRNA degradation products . The diversity of polyadenylation sites suggests that mRNA polyadenylation in prokaryotes is a relatively indiscriminate process that can occur at all mRNA's 3'-ends and does not require specific consensus sequences as in eukaryotes . Two poly(A) polymerases have been identified in Escherichia coli . They are encoded by unlinked genes, neither of which is essential for growth, suggesting significant functional overlap . Polyadenylation promotes the degradation of a regulatory RNA that inhibits the replication of bacterial plasmids and may play a similar role in the degradation of mRNA . However, under certain conditions, poly(A) tracts may lead to mRNA stabilization . Their ability to bind S1 ribosomal protein suggests that poly(A) tracts may also play a role in mRNA translation. Annu Rev Biochem, 1997, 66, 117 - 72 Basic mechanisms of transcript elongation and its regulation; Uptain SM et al.; Ternary complexes of DNA-dependent RNA polymerase with its DNA template and nascent transcript are central intermediates in transcription . In recent years, several unusual biochemical reactions have been discovered that affect the progression of RNA polymerase in ternary complexes through various transcription units . These reactions can be signaled intrinsically, by nucleic acid sequences and the RNA polymerase, or extrinsically, by protein or other regulatory factors . These factors can affect any of these processes, including promoter proximal and promoter distal pausing in both prokaryotes and eukaryotes, and therefore play a central role in regulation of gene expression . In eukaryotic systems, at least two of these factors appear to be related to cellular transformation and human cancers . New models for the structure of ternary complexes, and for the mechanism by which they move along DNA, provide plausible explanations for novel biochemical reactions that have been observed . These models predict that RNA polymerase moves along DNA without the constant possibility of dissociation and consequent termination . A further prediction of these models is that the polymerase can move in a discontinuous or inchworm-like manner . Many direct predictions of these models have been confirmed . However, one feature of RNA chain elongation not predicted by the model is that the DNA sequence can determine whether the enzyme moves discontinuously or monotonically . In at least two cases, the encounter between the RNA polymerase and a DNA block to elongation appears to specifically induce a discontinuous mode of synthesis . These findings provide important new insights into the RNA chain elongation process and offer the prospect of understanding many significant biological regulatory systems at the molecular level. Annu Rev Biochem, 1997, 66, 93 - 116 Bacterial cell division and the Z ring; Lutkenhaus J et al.; Bacterial cell division occurs through the formation of an FtsZ ring (Z ring) at the site of division . The ring is composed of the tubulin-like FtsZ protein that has GTPase activity and the ability to polymerize in vitro . The Z ring is thought to function in vivo as a cytoskeletal element that is analogous to the contractile ring in many eukaryotic cells . Evidence suggests that the Z ring is utilized by all prokaryotic organisms for division and may also be used by some eukaryotic organelles . This review summarizes our present knowledge about the formation, function, and evolution of the Z ring in prokaryotic cell division. Acta Biochim Pol, 1997, 44(1), 79 - 82 Autonomous replication of a wheat DNA sequence in isolated wheat nuclei; Szurmak B et al.; A fragment of wheat nuclear DNA was shown to be able to replicate autonomously in wheat nuclei . The fragment was 637-bp long and contained telomeric repeats at both termini . Its replication was manifested by the appearance of a radioactive reaction product of the same approximate size . Further analyses showed that the linear, double-stranded reaction product was labelled along its entire length and contained the same number of similarly located HaeIII sites as did the fragment tested . Prokaryotic DNA remained unlabelled under the same assay conditions. Virus Genes, 1997, 14(2), 163 - 5 A member of the immunoglobulin superfamily in bacteriophage T4; Bateman A et al.; We report a prediction that the highly immunogenic outer capsid (Hoc) protein of the prokaryotic phage T4 contains three tandem immunoglobulin-like domains . Immunoglobulin-like folds have previously been identified in prokaryotic proteins but these share no recognizable sequence similarity with eukaryotic immunoglobulin superfamily (IgSF) folds, and may represent products of convergent evolution . In contrast, the Hoc immunoglobulin-like folds are proposed, based on immunoglobulin-like sequence consensus matches detected by hidden Markov modeling . We propose that the Hoc immunoglobulin-like domains and eukaryotic immunoglobulin-like domains are likely to be related by divergence from a common ancestor. Biochimie, 1997, 79(1), 7 - 11 Molecular characterization of the prokaryotic efp gene product involved in a peptidyltransferase reaction; Aoki H et al.; The translation factor EF-P is required for efficient prokaryotic peptide bond synthesis on 70S ribosomes from fMet-tRNAfMet . This protein has been purified from Escherichia coli cells and the gene, efp, encoding it has been cloned and sequenced . We have isolated recombinant clones which overexpress a protein that co-migrates with purified EF-P upon SDS-PAGE analysis . Using these clones, we report the purification, crystallization and initial characterization of the efp gene product . The mechanism by which EF-P stimulates peptide-bond synthesis was studied using several antibiotics that inhibit translocation, peptide-bond synthesis and decoding . The stimulation of peptidyltransferase by EF-P was not inhibited by antibiotics that affect translocation and occupation of the A site (in the elongation state), ie thiostrepton, viomycin, neomycin and fusidic acid but was inhibited by streptomycin as well as by inhibitors of peptidyltransferase, chloramphenicol and lincomycin . This observation and the requirement for L16 but not for the L7/L12 nor L6 or L11 r-proteins suggest that the binding site for EF-P may overlap the peptidyltransferase center of the ribosome. Adv Exp Med Biol, 1997, 419, 99 - 107 Sequence and structural links between distant ADP-ribosyltransferase families; Bazan JF et al.; The low resolution structure of the Pseudomonas aeroginosa exotoxin A (ETA) presented in 1986 provided the first tantalizing three-dimensional view of an ADP-ribosyl-transferase (ADPRT) catalytic domain . The major features of this protein fold have recurred in the more recently solved crystal structures of the cholera toxin-related heat-labile enterotoxin (LT), diphtheria toxin (DT) and pertussis toxin (PT) . A core set of alpha + beta elements define a minimal, conserved scaffold with remarkably plastic sequence requirements-only a single glutamic acid residue critical to catalytic activity is invariant . Other interchangeable residues in locations important for catalysis and binding are suggested by the cocrystal structures of DT with the inhibitor ApUp, ETA with bound AMP and nicotinamide, and DT with substrate NAD-in close accord with labeling and mutagenic data . Faint sequence resemblances that were earlier noticed among prokaryotic ADPRTs have now been securely extended by the structural concordance between toxin folds; more recently, eukaryotic ADPRTs have surfaced and their sequences can be reliably threaded into the conserved core fold . We will briefly summarize efforts in Palo Alto and Hamburg to explore these latter relationships, and to mount a rigorous search for new ADPRT families in the growing sequence databases. Adv Exp Med Biol, 1997, 419, 25 - 33 ADP-ribosylarginine hydrolases and ADP-ribosyltransferases . Partners in ADP-ribosylation cycles; Moss J et al.; Mono-ADP-ribosylation is a reversible modification of arginine residues in proteins, with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases constituting opposing arms of a putative ADP-ribosylation cycle . The enzymatic components of an ADP-ribosylation cycle have been identified in both prokaryotic and eukaryotic systems . The regulatory significance of the cycle has been best documented in prokaryotes . As shown by Ludden and coworkers, ADP-ribosylation controls the activity of dinitrogenase reductase in the phototropic bacterium Rhodospirillum rubrum . ADP-ribosylation of other amino acids, such as cysteine, has also been demonstrated, lending credence to the hypothesis that this modification is heterogeneous . In eukaryotes, the functional relationship between ADP-ribosyltransferases and ADP-ribosylarginine hydrolases is less well documented . The transferase-catalyzed reaction results in sterospecific formation of alpha-ADP-ribosylarginine from beta-NAD; ADP-ribosylarginine hydrolases specifically cleave the alpha-anomer, leading to release of ADP-ribose and regeneration of the free guanidino group of arginine . The two reactions can thus be coupled in vitro . Coupling in vivo is dependent on cellular localization . The deduced amino acid sequences of ADP-ribosyltransferases from avian and mammalian tissues have common consensus sequences involved in catalytic activity but, in some instances, enzyme-specific cellular localization signals . The presence of amino- and carboxy-terminal signal sequences is consistent with the glycosylphosphatidylinositol(GPI)-anchoring to the cell surface . The muscle and lymphocyte transferases ADP-ribosylate integrins . Some transferases lack the carboxy- terminal signal sequence needed for GPI-anchoring . Most ADP-ribosylarginine hydrolase activity is cytosolic, although perhaps some is located at the cell surface . Deduced amino acid sequences of hydrolases from a number of mammalian species are consistent with their cytoplasmic localization . Katada and coworkers have determined, however, that auto-ADP-ribosylated RT6, a GPI-linked protein, is metabolized by a hydrolase-like activity, consistent with the existence of an ADP-ribosylation cycle . ADP-ribosyl RT6 may be internalized, thereby coming in contact with the cytosolic hydrolase; alternatively, a novel form of the hydrolase may be located at the surface . The mechanism of coupling of ADP-ribosyltransferases and hydrolases in eukaryotic ADP-ribosylation cycles has yet to be clarified. Fold Des, 1997, 2(2), 101 - 8 Ribosomes and ribosomal RNA as chaperones for folding of proteins; Kudlicki W et al.; BACKGROUND: Provocative recent reports indicate that the large subunits of either prokaryotic or eukaryotic ribosomes have the capacity to promote refolding of denatured enzymes . RESULTS: Salt-washed Escherichia coli ribosomes are shown to promote refolding of denatured rhodanese . The ability of the ribosomes to carry out renaturation is a property of the 50S ribosomal subunit, specifically the 23S rRNA . Refolding and release of enzymatically active rhodanese leaves the ribosomes in an inactive state or conformation for subsequent rounds refolding . Inactive ribosomes can be activated by elongation factor G (EF-G) plus GTP or by cleavage of their 23S rRNA by alpha-sarcin . Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex . CONCLUSIONS: Large subunits of E . coli ribosomes, specifically 23S rRNA, have the capacity to mediate refolding of denatured rhodanese . Refolding activity is related to the state or conformation of ribosomes that is promoted by EF-G . Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex. Immunogenetics, 1997, 45(6), 353 - 64 Physical mapping and structural analysis of new gene families RT1.S and Rps2r in the grc region of the rat major histocompatibility complex; Salgar SK et al.; Five new genes were identified in the growth and reproduction complex (grc) region: RT1.S1, RT1.S2, Rps2r1, Rps2r2, and Rps2r3 . The class Ib RT1.S1 and RT1.S2 genes have five distinct exons (1, 4, 5, 6, 7) similar to other class I major histocompatibility complex genes but the conventional exons 2 and 3 are absent . The genes are 97% similar, have CAAT and TATA boxes much upstream of the conventional position, obey the GT/AG rule in their exon-intron boundaries, and are transcribed at a low level in thymus and testis but not in the liver and spleen . The region between exon 1 and exon 4 was analyzed by obtaining transcripts by reverse transcription-polymerase chain reaction (RT-PCR) amplification which revealed the presence of four alternatively spliced mRNA transcripts of RT1.S1: 1) S1-1 (clones 14 and 16) has no exon between exons 1 and 4; 2) S1-2 (clones 7 and 8) has an exon of 45 nucleotides that can translate into 15 amino acids; 3) S1-3 (clone 5) has an exon of 42 nucleotides with a stop codon; and 4) S1-4 (clone 10) has two exons of 42 and 38 nucleotides, respectively, with stop codons . Only one RT1.S2 mRNA transcript was obtained, and it has an exon of 45 nucleotides between exon 1 and exon 4 which can form a peptide identical to the S1-2 isoform for that region . The 45 nucleotide exon between exon 1 and exon 4 was unique for RT1.S1 and RT1.S2 and only matched a sequence in the RT1.O intron region (nucleotides 2905 - 2949) . The three ribosomal-protein-S2-related (Rps2r) genes are 94% - 98% similar; they are related to the genes encoding ribosomal protein S2 of the black rat and the LLRep3 genes of the mouse and the human and to the genes encoding Saccharomyces cerevisiae S4, Escherichia coli S5, and other members of prokaryote S5 family . The Rps2r1 gene is located just outside of the grc region . The Rps2r2 and Rps2r3 genes are in the grc and have multiple stop codons in their genomic sequences . The Rps2r1 mRNA transcript was identified by RT-PCR in the thymus and testis but not in the liver and spleen. Growth Factors, 1997, 14(1), 39 - 57 Amphibian FGF-1 is structurally and functionally similar to but antigenically distinguishable from its mammalian counterpart; Patrie K et al.; Recent studies have shown that fibroblast growth factors (FGF) play an important role in the diverse cellular mechanisms involved with vertebrate development . One system which has received a great deal of attention is the developing limb in part because of the extensive epithelial-mesenchymal interactions that take place during this process . Because it closely parallels the developmental process of the limb and is a model for wound repair, the phenomenon of amphibian limb regeneration has been used to investigate the role of FGF in these processes . We have recently reported on the cloning and functional characterization of an FGF receptor (FGFR) isolated from amphibian regenerative tissue . In this report, we describe the isolation and characterization of an FGF-1 molecule from the newt, Notophthalmus viridescens . Amino acid sequence comparisons indicate that the newt FGF-1 exhibits between 79 to 83% identity with FGF-1 from mammalian and avian species . The full length cDNA of the newt FGF-1 was cloned into a prokaryotic expression vector and purified from E . coli . Although the newt FGF-1 shares a high degree of primary amino acid sequence similarity with other FGF-1 molecules, the recombinant protein was not detected in a Western blot analysis using a polyclonal antibody directed against mammalian FGF-1 . Despite the antigenic divergence, the newt FGF-1 was capable of binding to NIH/3T3 and Chinese hamster ovary cells overexpressing mammalian and amphibian FGFRs with dissociation constants comparable to those reported for mammalian FGF-1 . Newt FGF-1 could also be cross-linked to receptors on the surface of NIH/3T3 cells . In addition, it elicits a mitogenic response in NIH/3T3 cells indistinguishable from human recombinant FGF-1. J Gastroenterol Hepatol, 1997 Jan, 12(1), 54 - 7 Effect of quinolone antibiotics on hepatic growth and protein synthesis following partial hepatectomy in rats; Minuk GY et al.; Quinolone antibiotics inhibit eukaryotic as well as prokaryotic cell growth and protein synthesis . To determine whether these properties adversely affect hepatic growth and recovery following surgical resection, five groups of healthy, adult male rats (n = 7-8/group) were treated for 10 days with equal volumes of either ofloxacin (50 mg/kg), fleroxacin (25 mg/kg), ciprofloxacin (25 mg/kg), norfloxacin (15 mg/kg) or sterile saline (controls) prior to 70% partial hepatectomy (PH) and daily thereafter until death . Restituted liver mass, DNA and protein synthesis rates were determined at 24, 48 and 72 h PH . The results of the study revealed that all parameters of hepatic regeneration were similar in the five study groups at each time interval . To ensure that an effect on hepatic regeneration was not dose-dependent, additional experiments were performed where 1, 10 and 100 mg/kg ciprofloxacin was administered and DNA synthesis was measured 24 h post-PH . Once again, the results were similar to sterile saline-treated controls . These findings suggest that the quinolone antibiotics are unlikely to have an adverse effect on hepatic recovery following surgical resection of the liver and are safe to use in that setting. Int Rev Cytol, 1997, 171, 1 - 78 Biology of plant cells in microgravity and under clinostating; Kordyum EL; Experimental data on plant cell reproduction, growth, and differentiation in spaceflight and under clinostating that partially reproduce the biological effects of microgravity are elucidated . The rearrangements of organelle structural and functional organization in unicellular plant organisms as well as in meristematic, differentiating, and differentiated cells of multicellular organisms in these conditions are considered . The focus is on the changes in the interrelations of prokaryotic and eukaryotic organisms under altered gravity . Ideas on the acceleration of differentiation and aging of cells in microgravity and clinostating and the organism's adaptive possibilities for carrying out its own functions are discussed. Annu Rev Neurosci, 1997, 20, 567 - 94 The molecules of mechanosensation; Garcia-Anoveros J et al.; Mechanosensation, the transduction of mechanical forces into a cellular electrochemical signal, enables living organisms to detect touch; vibrations, such as sound; accelerations, including gravity; body movements; and changes in cellular volume and shape . Ion channels directly activated by mechanical tension are thought to mediate mechanosensation in many systems . Only one channel has been cloned that is unequivocably mechanically gated: the MscL channel in bacteria . Genetic screens for touch-insensitive nematodes or flies promise to identify the proteins that constitute a mechanosensory apparatus in eukaryotes . In Caenorhabditis elegans, the mec genes thus identified encode molecules for a candidate structure, which includes a "degenerin" channel tethered to specialized extracellular and intracellular structural proteins . In hair cells of the inner ear, evidence suggests that an extracellular tip link pulls on a channel, which attached intracellularly to actin via a tension-regulating myosin 1beta . The channel and the tip link have not been cloned . Because degenerins and MscL homologs have not been found outside of nematodes and prokaryotes, respectively, and because intracellular and extracellular accessory structures apparently differ among organs and species, it may be that mechanosensory channel complexes evolved multiple times. Annu Rev Neurosci, 1997, 20, 91 - 123 Cloned potassium channels from eukaryotes and prokaryotes; Jan LY et al.; Potassium channels contribute to the excitability of neurons and signaling in the nervous system . They arise from multiple gene families including one for voltage-gated potassium channels and one for inwardly rectifying potassium channels . Features of potassium permeation, channel gating and regulation, and subunit interaction have been analyzed . Potassium channels of similar design have been found in animals ranging from jellyfish to humans, as well as in plants, yeast, and bacteria . Structural similarities are evident for the pore-forming alpha subunits and for the beta subunits, which could potentially regulate channel activity according to the level of energy and/or reducing power of the cell. Protein Eng, 1997 Jan, 10(1), 1 - 6 Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites; Nielsen H et al.; We have developed a new method for the identification of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequence . The method performs significantly better than previous prediction schemes and can easily be applied on genome-wide data sets . Discrimination between cleaved signal peptides and uncleaved N-terminal signal-anchor sequences is also possible, though with lower precision . Predictions can be made on a publicly available WWW server. Steroids, 1997 Jan, 62(1), 117 - 23 P450BM-3; a tale of two domains--or is it three? Peterson JA, Sevrioukova I, Truan G, Graham-Lorence SE. Over 400 P450s have been identified to date in prokaryotes and eukaryotes, plants and animals, mitochondria and endoplasmic reticulum . These enzymes function in areas such as metabolism and steroidogenesis . The eukaryotic members of this gene superfamily of proteins have proved difficult to study because of the hydrophobic nature of their substrates, their various redox partners, and membrane association . To better understand the structure/function relationship of P450s-what determines substrate specificity and selectivity, what determines redox-partner binding, and which regions are involved in membrane binding-we have compared the three crystallized, soluble bacterial P450s (two class I and one class II) and a model of a steroidogenic, eukaryotic P450 (P450arom), to define which structural elements form a conserved structural fold for P450s, what determines specificity of substrate binding and redox-partner binding, and which regions are potentially involved in membrane association . We believe that there is a conserved structural fold for all P450s that can be used to model those P450s that prove intransigent to structural determination . However, although there appears to be a conserved structural core among P450s, there is sufficient sequence variability that no two P450s are structurally identical . NADPH-P450 reductase transfers electrons from NADPH to P450 during the P450 catalytic cycle . This enzyme has usually been thought of as a simple globular protein; however, sequence analysis has shown that NADPH-P450 reductase is related to two separate flavoprotein families, ferredoxin nucleotide reductase (FNR) and flavodoxin . Recent studies by Wolff and his colleagues have shown that the FAD-binding FNR domain and FMN-binding flavodoxin domain of human NADPH-P450 reductase can be independently expressed in Escherichia coli . The subdomains can be used to reconstitute, however poorly, the monooxygenase activity of the P450 system . We have been utilizing the reductase domain of P450BM-3 to study the mechanism of electron transfer from NADPH to P450 in this complex multidomain protein . We have overexpressed both the FNR subdomain and the flavodoxin subdomain in E . coli and fully reconstituted the cytochrome c reductase activity of this enzyme . Our studies have shown that electron transfer from NADPH through the reductase domain to the P450 requires shuttling of the FMN subdomain between the reductase subdomain and the P450 . Studies of the factors that control the molecular recognition and interaction among these three proteins are complicated by the weakness of the association and changes in the strength of the interaction depending on the redox state of each of the components . How these structural and mechanistic studies of a soluble bacterial P450 can be extended to gain a better understanding of the control of membrane-bound eukaryotic P450-dependent redox systems is discussed. Anal Biochem, 1997 Jan 1, 244(1), 40 - 4 A rapid fluorescence bioassay for the determination of selenium on agar plates; Liu Z et al.; The essential trace element selenium (Se) is involved in the form of selenocysteine at the active site of several prokaryotic and eukaryotic proteins called selenoproteins . These proteins have recently attracted attention particularly in relation to their application to human health and new characteristics of the genetic code . We have recently described a selenium bioassay based on a recombinant DNA construct in which the expression of the lac' Z gene in Escherichia coli is proportionally and specifically driven by UGA-directed selenocysteine incorporation . Here we have further developed this bioassay for more rapid and sensitive detection and measurement of selenium that permits screening of the selenium status on agar plates . Again, the inclusion of selenium into the lac'Z-fusion product is reflected by the level of beta-galactosidase activity, which in turn is reflected by the intensity of fluorescence on agar plates . This fluorescing agent is a 4-methylumbelliferyl moiety which is released through the cleavage by the enzyme of 4-methylumbelliferyl-beta-D-galactoside . The intensity of the fluorescence is easily detected by uv irradiation and photographed by polaroid or video cameras. Nucleic Acids Res, 1997 Jan 1, 25(1), 98 - 102 Small RNA database; Gu J et al.; The small RNA database is a compilation of all the small size RNA sequences available to date, including nuclear, nucleolar, cytoplasmic and mitochondrial small RNAs from eukaryotic organisms and small RNAs from prokaryotic cells as well as viruses . Currently, about 600 small RNA sequences are in our database . It also gives the sources of individual RNAs and their GenBank accession numbers . The small RNA database can be accessed through WWW(World Wide Web) . Our WWW URL address is: html . The new small RNA sequences published since our last compilation are listed in this paper. J Gen Virol, 1997 Jan, 78 ( Pt 1), 13 - 21 The cleavage activities of aphthovirus and cardiovirus 2A proteins; Donnelly ML et al.; The primary 2A/2B polyprotein cleavage of aphtho-and cardioviruses is mediated by their 2A proteins cleaving C-terminally . Whilst the aphthovirus 2A region is only 16 aa (possibly 18 aa) long, the cardiovirus 2A protein is some 150 aa . We have previously shown that foot-and-mouth disease virus (FMDV) 2A is able to mediate cleavage in an artificial (chloramphenicol acetyltransferase/FMDV 2A/beta-glucuronidase {CAT-2A-GUS}) polyprotein system devoid of any other FMDV sequences with high (approximately 85%), although not complete, cleavage . In this paper we show that insertion of upstream FMDV capsid protein 1 D sequences increases the activity . In addition, we have demonstrated that the cardiovirus Theiler's murine encephalomyelitis virus(TME) 2A protein, when linked to GUS in a single ORF, is able to cleave at its own C terminus with high efficiency--if not completely . The C-terminal 19 aa of TME 2A, together with the N-terminal proline residue of protein 2B, were inserted into the CAT/GUS artificial polyprotein system (in a single ORF) . This recombinant {CAT-deltaTME2A-GUS} polyprotein was able to mediate cleavage with high (approximately 85%) efficiency--directly comparable to the activity observed when FMDV 2A was inserted . A similar insertion into {CAT-GUS} of the C-terminal 19 aa of the cardiovirus encephalomyocarditis virus (EMC) 2A, together with the N-terminal proline residue of protein 2B, produced a {CAT-delta EMC2A-GUS} polyprotein which also mediated cleavage at approximately 85% . Analysis of the products of expression of these artificial polyproteins in a prokaryotic translation system did not, apparently, reveal any GUS cleavage product. Clin Diagn Lab Immunol, 1997 Jan, 4(1), 49 - 56 Human humoral responses to antigens of Mycobacterium tuberculosis: immunodominance of high-molecular-mass antigens; Laal S et al.; The selection of antigens of Mycobacterium tuberculosis for most studies of humoral responses in tuberculosis patients has been restricted to molecules that were either immunodominant in immunized animals or amenable to biochemical purification rather than those that were reactive with the human immune system . Delineation of antigens that elicit humoral responses during the natural course of disease progression in humans has been hindered by the presence of cross-reactive antibodies to conserved regions on ubiquitous prokaryotic antigens in sera from healthy individuals and tuberculosis patients . The levels of cross-reactive antibodies in the sera were reduced by preadsorption with Escherichia coli lysates, prior to studying their reactivity against a large panel of M . tuberculosis antigens to which the human immune system may be exposed during natural infection and disease . Thus, reactivity against pools of secreted, cellular, and cell wall-associated antigens of M . tuberculosis was assessed by an enzyme-linked immunosorbent assay (ELISA) . Initial results suggested that the secreted protein preparation contained antigens most frequently recognized by the humoral responses of pulmonary tuberculosis patients . The culture filtrate proteins were subsequently size fractionated by preparative polyacrylamide gel electrophoresis, characterized by reaction with murine monoclonal antibodies to known antigens of M . tuberculosis by an ELISA, and assessed for reactivity with tuberculous and nontuberculous sera . Results show that a secreted antigen of 88 kDa elicits a strong antibody response in a high percentage of patients with pulmonary tuberculosis . This and other antigens identified on the basis of their reactivity with patient sera may prove useful for developing serodiagnosis for tuberculosis. Mol Microbiol, 1997 Jan, 23(1), 1 - 10 Plasmid replication and partition in Escherichia coli: is the cell membrane the key? Firshein W, Kim P. The DNA-membrane complex has been the subject of intensive investigation for over 35 years as the possible site for DNA replication in the prokaryotic cell and the site through which newly synthesized chromosomes are segregated into daughter cells . However, the molecular mechanisms which control these phenomena are, for the most part, poorly understood despite genetic, biochemical, and morphologic evidence in favour of their existence . This is probably due to the transient nature and non-covalent interactions that occur between DNA and the membrane . In addition, there is a paucity of knowledge concerning the nature of the membrane receptors for DNA and whether the membrane plays simply a structural or metabolic role in the two processes . Plasmids can provide important insights into the role of the membrane in replication and partitioning because the plasmid life cycle is relatively simple, with replication occurring during the cell cycle and partitioning during cell division . The replicon model of Jacob et al . (1963, Cold Spring Harbor Symp Quant Biol 28: 329-348) still represents a good conceptual framework (with modifications) to explain how plasmid replication and partitioning are linked by the membrane . In its simplest form, the model focuses on specific membrane binding sites (possibly along the equator of the cell) for plasmid (or bacterial) replication, with the membrane acting as a motive force to separate the newly synthesized replicons and their attached sites into daughter cells . Indeed, proteins involved in both plasmid replication and partitioning have been found in membrane fractions and some plasmids require membrane binding for initiation and an active partitioning . We propose that several factors are critical for both plasmid DNA replication and partitioning . One factor is the extent of negative supercoiling (brought about by an interplay of various topoisomerases, but most importantly by DNA gyrase) . Supercoiling is known to be critical for initiation of DNA replication but may also be important for the formation of a partition complex in contact with the cell membrane . Another factor is the presence of specific subdomains of the membrane which can interact specifically with origin DNA and possibly other regions involved in partitioning . Such domains may be induced transiently or be present at all times during the cell cycle. Appl Environ Microbiol, 1997 Jan, 63(1), 91 - 8 Phylogenetic diversity of bacterial symbionts of Solemya hosts based on comparative sequence analysis of 16S rRNA genes; Krueger DM et al.; The bacterial endosymbionts of two species of the bivalve genus Solemya from the Pacific Ocean, Solemya terraeregina and Solemya pusilla, were characterized . Prokaryotic cells resembling gram-negative bacteria were observed in the gills of both host species by transmission electron microscopy . The ultrastructure of the symbiosis in both host species is remarkably similar to that of all previously described Solemya spp . By using sequence data from 16S rRNA, the identity and evolutionary origins of the S . terraeregina and S . pusilla symbionts were also determined . Direct sequencing of PCR-amplified products from host gill DNA with primers specific for Bacteria 16S rRNA genes gave a single, unambiguous sequence for each of the two symbiont species . In situ hybridization with symbiont-specific oligonucleotide probes confirmed that these gene sequences belong to the bacteria residing in the hosts gills . Phylogenetic analyses of the 16S rRNA gene sequences by both distance and parsimony methods identify the S . terraeregina and S . pusilla symbionts as members of the gamma subdivision of the Proteobacteria . In contrast to symbionts of other bivalve families, which appear to be monophyletic, the S . terraeregina and S . pusilla symbionts share a more recent common ancestry with bacteria associating endosymbiotically with bivalves of the superfamily Lucinacea than with other Solemya symbionts (host species S . velum, S . occidentalis, and S . reidi) . Overall, the 16S rRNA gene sequence data suggest that the symbionts of Solemya hosts represent at least two distinct bacterial lineages within the gamma-Proteobacteria . While it is increasingly clear that all extant species of Solemya live in symbiosis with specific bacteria, the associations appear to have multiple evolutionary origins. Appl Environ Microbiol, 1997 Jan, 63(1), 50 - 6 Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel; Massana R et al.; Newly described phylogenetic lineages within the domain Archaea have recently been found to be significant components of marine picoplankton assemblages . To better understand the ecology of these microorganisms, we investigated the relative abundance, distribution, and phylogenetic composition of Archaea in the Santa Barbara Channel . Significant amounts of archaeal rRNA and rDNA (genes coding for rRNA) were detected in all samples analyzed . The relative abundance of archaeal rRNA as measured by quantitative oligonucleotide hybridization experiments was low in surface waters but reached higher values (20 to 30% of prokaryotic rRNA) at depths below 100 m . Probes were developed for the two major groups of marine Archaea detected . rRNA originating from the euryarchaeal group (group II) was most abundant in surface waters, whereas rRNA from the crenarchaeal group (group I) dominated at depth . Clone libraries of PCR-amplified archaeal rRNA genes were constructed with samples from 0 and 200 m deep . Screening of libraries by hybridization with specific oligonucleotide probes, as well as subsequent sequencing of the cloned genes, indicated that virtually all archaeal rDNA clones recovered belonged to one of the two groups . The recovery of cloned rDNA sequence types in depth profiles exhibited the same trends as were observed in quantitative rRNA hybridization experiments . One representative of each of 18 distinct restriction fragment length polymorphism types was partially sequenced . Recovered sequences spanned most of the previously reported phylogenetic diversity detected in planktonic crenarchaeal and euryarchaeal groups . Several rDNA sequences appeared to be harbored in archaeal types which are widely distributed in marine coastal waters . In total, data suggest that marine planktonic crenarchaea and euryarchaea of temperate coastal habitats thrive in different zones of the water column . The relative rRNA abundance of the crenarchaeal group suggests that its members constitute a significant fraction of the prokaryotic biomass in subsurface coastal waters. Mol Cell Biol, 1997 Jan, 17(1), 46 - 53 An intact Raf zinc finger is required for optimal binding to processed Ras and for ras-dependent Raf activation in situ; Luo Z et al.; The function of the c-Raf-1 zinc finger domain in the activation of the Raf kinase was examined by the creation of variant zinc finger structures . Mutation of Raf Cys 165 and Cys 168 to Ser strongly inhibits the Ras-dependent activation of c-Raf-1 by epidermal growth factor (EGF) . Deletion of the Raf zinc finger and replacement with a homologous zinc finger from protein kinase C gamma (PKC gamma) (to give gamma/Raf) also abrogates EGF-induced activation but enables a vigorous phorbol myristate acetate (PMA)-induced activation . PMA activation of gamma/Raf does not require endogenous Ras or PKCs and probably occurs through a PMA-induced recruitment of gamma/Raf to the plasma membrane . The impaired ability of EGF to activate the Raf zinc finger variants in situ is attributable, at least in part, to a major decrement in their binding to Ras-GTP; both Raf zinc finger variants exhibit decreased association with Ras (V12) in situ upon coexpression in COS cells, as well as diminished binding in vitro to immobilized, processed COS recombinant Ras(V12)-GTP . In contrast, Raf binding to unprocessed COS or prokaryotic recombinant Ras-GTP is unaffected by Raf zinc finger mutation . Thus, the Raf zinc finger contributes an important component to the overall binding to Ras-GTP in situ, through an interaction between the zinc finger and an epitope on Ras, distinct from the effector loop, that is present only on prenylated Ras. DNA Res, 1996 Dec 31, 3(6), 407 - 14 Compilation of all genes encoding bacterial two-component signal transducers in the genome of the cyanobacterium, Synechocystis sp . strain PCC 6803; Mizuno T et al.; Bacteria have devised sophisticated signaling systems for eliciting a variety of adaptive responses to their environment, which are generally referred to as the "two-component regulatory system." The widespread occurrence of the two-component systems in both prokaryotes and eukaryotes implies that it is a powerful device for a wide variety of adaptive responses of cells to their environment . The two-component signal transducers contain one or more of three conserved and characteristic phosphotransfer signaling domains, named the "transmitter, receiver, and alternative transmitter." The recently determined entire genomic sequence of Synechocystis sp . strain PCC 6803 allowed us to compile systematically a complete list of genes encoding such two-component signal transduction proteins . The results of such an effort, made in this study, revealed that at least 80 ORFs were identified as members of the two-component signal transducers in this single species of cyanobacteria. Neurosci Lett, 1996 Dec 27, 221(1), 29 - 32 Parameters influencing ectopic gene expression in Aplysia neurons; Kaang BK; DNA microinjection for expressing exogenous genes in Aplysia neurons has been very useful for analyzing not only functions of encoded proteins, but also regulations of gene expression in the nervous system . Some of the factors that affect expression of foreign genes microinjected into Aplysia neurons are described . The effect of the DNA form (supercoiled or linear) and promoter modification in the expression vectors are analyzed as well as buffer composition and DNA concentration in the microinjection solution . The time course of reporter gene expression was also monitored . Reporter gene expression was first detected as early as 1 h and maintained at a high level even until 7 days after microinjection . The presence of AP-1 enhancer in the promoter region of the expression vectors was essential in driving a high-level constitutive expression of reporter genes . Particularly, a pNEX derivative containing eight copies of AP-1 enhancer drove constitutive overexpression more effectively than ones harboring either four or 12 copies of AP-1 enhancer . We also found that a prokaryotic promoter/operator from E . coli lacZ gene placed upstream from an eukaryotic enhancer/promoter repressed the expression of the downstream reporter gene in Aplysia neurons. Cell, 1996 Dec 27, 87(7), 1295 - 306 Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn; Rice PA et al.; Integration host factor (IHF) is a small heterodimeric protein that specifically binds to DNA and functions as an architectural factor in many cellular processes in prokaryotes . Here, we report the crystal structure of IHF complexed with 35 bp of DNA . The DNA is wrapped around the protein and bent by >160 degrees, thus reversing the direction of the helix axis within a very short distance . Much of the bending occurs at two large kinks where the base stacking is interrupted by intercalation of a proline residue . IHF contacts the DNA exclusively via the phosphodiester backbone and the minor groove and relies heavily on indirect readout to recognize its binding sequence . One such readout involves a six-base A tract, providing evidence for the importance of a narrow minor groove. J Biol Chem, 1996 Dec 27, 271(52), 33242 - 55 Multiple specific CytR binding sites at the Escherichia coli deoP2 promoter mediate both cooperative and competitive interactions between CytR and cAMP receptor protein; Perini LT et al.; Binding of cAMP receptor protein (CRP) and CytR mediates both positive and negative control of transcription from Escherichia coli deoP2 . Transcription is activated by CRP and repressed by a multi-protein CRP.CytR.CRP complex . The latter is stabilized by cooperative interactions between CRP and CytR . Similar interactions at the other transcriptional units of the CytR regulon coordinate expression of the transport proteins and enzymes required for nucleoside catabolism . A fundamental question in both prokaryotic and eukaryotic gene regulation is how combinatorial mechanisms of this sort regulate differential expression . To understand the combinatorial control mechanism at deoP2, we have used quantitative footprint and gel shift analysis of CRP and CytR binding to evaluate the distribution of ligation states . By comparison to distributions for other CytR-regulated promoters, we hope to understand the roles of individual states in differential gene expression . The results indicate that CytR binds specifically to multiple sites at deoP2, including both the well recognized CytR site flanked by CRP1 and CRP2 and also sites coincident with CRP1 and CRP2 . Binding to these multiple sites yields both cooperative and competitive interactions between CytR and CRP . Based on these findings we propose that CytR functions as a differential modulator of CRP1 versus CRP2-mediated activation . Additional high affinity specific sites are located at deoP1 and near the middle of the 600-base pair sequence separating P1 and P2 . Evaluation of the DNA sequence requirement for specific CytR binding suggests that a limited array of contiguous and overlapping CytR sites exists at deoP2 . Similar extended arrays, but with different arrangements of overlapping CytR and CRP sites, are found at the other CytR-regulated promoters . We propose that competition and cooperativity in CytR and CRP binding are important to differential regulation of these promoters. Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15244 - 8 Independent evolution of the prochlorophyte and green plant chlorophyll a/b light-harvesting proteins; La Roche J et al.; The prochlorophytes are oxygenic prokaryotes differing from other cyanobacteria by the presence of a light-harvesting system containing both chlorophylls (Chls) a and b and by the absence of phycobilins . We demonstrate here that the Chl a/b binding proteins from all three known prochlorophyte genera are closely related to IsiA, a cyanobacterial Chl a-binding protein induced by iron starvation, and to CP43, a constitutively expressed Chl a antenna protein of photosystem II . The prochlorophyte Chl a/b protein (pcb) genes do not belong to the extended gene family encoding eukaryotic Chl a/b and Chl a/c light-harvesting proteins . Although higher plants and prochlorophytes share common pigment complements, their light-harvesting systems have evolved independently. J Mol Biol, 1996 Dec 20, 264(5), 1002 - 12 Structural role of a buried salt bridge in the 434 repressor DNA-binding domain; Pervushin K et al.; The independently folding 63-residue N-terminal DNA-binding domain of the 434 repressor, 434(1-63), contains a buried Arg10-Glu35 salt bridge . A corresponding salt bridge is found in a variety of prokaryotic and eukaryotic DNA-binding proteins with helix-turn-helix motifs . Here, the NMR solution structures of 434(1-63) and the mutant protein 434{R10M}(1-63) were determined to investigate the structural role of this salt bridge . Both proteins contain the same type of global fold, with five alpha-helices and a helix-turn-helix motif formed by the helices II and III . The primary structural difference caused by the Arg10 --> Met mutation is a translation of helix I along its axis relative to the helix II-turn-helix III motif . This limited conformational change is paralleled by a 9 kJ M(-1) decrease of the stability of the folded mutant protein in aqueous solution at pH 4.8 . It affects the pKa value of Glu19 as well as the population of a hydrogen bond between the backbone amide proton of Asn16 and the side-chain carboxylate group of Glu19 . Using the crystal structure of the 434 repressor dimer complexed with the operator DNA as a basis, model building of the DNA complex with the NMR structure of 434{R10M}(1-63) shows that Asn16, which is located on the protein surface, makes direct contact with the DNA and indicates that the point mutation Arg10 --> Met should also lead to modifications of the protein-protein contacts in the complex. J Mol Biol, 1996 Dec 20, 264(5), 968 - 80 A network of heterogeneous hydrogen bonds in GNRA tetraloops; Jucker FM et al.; RNA hairpin loops containing a GNRA consensus sequence are the most frequently occurring hairpins in a variety of prokaryotic and eukaryotic RNAs . These tetraloops play important functional roles in RNA folding, in RNA-RNA tertiary interactions and as protein binding sites . Homo and heteronuclear NMR spectroscopy have been used to determine the structures of the most abundant members of the GNRA tetraloop family: the GAGA, GCAA and GAAA loops closed by a C-G base pair . Analysis of the structures of these three hairpin loops reveals a network of heterogeneous hydrogen bonds . The loops contain a G-A base pair, a G base-phosphate hydrogen bond and several 2' OH-base hydrogen bonds . These intramolecular interactions and the extensive base stacking in the loop help explain the high thermodynamic stability and give insight into the diverse biological roles of the GNRA RNA hairpins. J Biol Chem, 1996 Dec 20, 271(51), 32580 - 5 H1 binding unwinds DNA . Evidence from topological assays; Ivanchenko M et al.; The preference of the linker histones to bind to superhelical DNA in comparison with linear or relaxed molecules suggests that these proteins might, in turn, change the twist and/or writhe of DNA molecules upon binding . In order to explore such a possibility, we looked for changes in the linking number of plasmid pBR322 caused by H1 binding, using assays that involve nicking and resealing of DNA strands . Two types of enzymes were used, eukaryotic topoisomerase I and prokaryotic DNA ligase . The results revealed that H1 binding causes unwinding of the DNA, with the unwinding angle being approximately 10 degrees . The globular domain of histone H1 is also capable of unwinding DNA, but to a lesser degree. Biochemistry, 1996 Dec 17, 35(50), 16319 - 27 Casein kinase I alpha and alpha L: alternative splicing-generated kinases exhibit different catalytic properties; Zhang J et al.; Casein kinase I (CKI) is a family of serine/threonine protein kinases found in all eukaryotes examined to date . Here, the rat CKI isoforms alpha and alpha L were cloned and expressed in both eukaryotic and prokaryotic systems . Characterization of the genomic DNA flanking the exon unique to CKI alpha L demonstrated that CKI alpha and CKI alpha L arise by the alternative splicing of a common pre-mRNA molecule . To the best of our knowledge, the alpha L isoform is the only known active serine/threonine kinase to contain an insert within its catalytic domain . Tissue distribution of each splicing isoform was examined by RT-PCR, immunoprecipitation, and Western blotting . Both isoforms were expressed in all tissues tested but at different levels . Bacterially expressed CKI alpha isoforms were active and therefore biochemically characterized . CKI alpha and CKI alpha L proteins were demonstrated to have casein kinase I catalytic properties . More importantly, the recombinant isoform proteins exhibited differences in binding and activity toward common CKI substrates . These observations demonstrate that the alpha L insert within the kinase domain modulates substrate kinetics . These kinetic differences suggest that CKI alpha and CKI alpha L may perform different biological roles. Eur J Biochem, 1996 Dec 15, 242(3), 779 - 87 Purification and spectroscopic characterization of a recombinant chloroplastic hemoglobin from the green unicellular alga Chlamydomonas eugametos; Couture M et al.; Hemoglobins (Hb), which have the important task of delivering molecular oxygen by facilitating its reversible binding to the heme, are now thought to have evolved in all groups of organisms including prokaryotes, fungi, plants and animals . Our recent finding of a light-inducible chloroplastic Hb in the green unicellular alga Chlamydomonas eugametos has further extend this idea, while raising questions about the function that an Hb could play in a high oxygen environment such as in the chloroplast . In order to understand the role played by this new Hb, we have undertaken its biochemical characterization . To facilitate the characterization of Chlamydomonas Hb, which represents less than 0.01% of the soluble protein in the green alga, the protein has been expressed in Escherichia coli and purified to apparent homogeneity . The purified recombinant protein possesses a non-covalently bound iron-protoporphyrin IX heme . The oxy form of the recombinant Hb . purified directly from bacterial cells, is very stable, with a measured half-life of 7 days at pH 8 and has an ultraviolet/visible spectrum similar to those of the related cytoplasmic Hbs of the ciliated protozoa Paramecium and Tetrahymena and of the cyanobacterium Nostoc commune . In contrast to what has been reported for oxymyoglobins and oxyhemoglobins, the dioxygen molecule bound to the L1637 Hb can be reduced by the electron-transfer mediator phenazine methosulfate in the presence of NADPH, indicating that the heme pocket of Chlamydomonas Hb may be more accessible to small molecules . With regard to this we found that when the small reducing agent sodium dithionite is used to reduce the met form, it must be removed anaerobically from the Hb prior to oxygenation of the protein to stably produce the oxy form . Otherwise, the oxy form is obtained readily from the met form under an oxygenic atmosphere when ferredoxin and ferredoxin NADP+ reductase are used to enzymically reduce the Hb . Finally, the spectra of the deoxy and met forms were unusual, the heme being partly low-spin at physiological pH . These results confirm the existence of a reversible oxygen-binding protein in the chloroplast of C . eugametos . The unusual spectral and biochemical properties of the protein may reflect a specialized function for this Hb. Mol Gen Genet, 1996 Dec 13, 253(3), 315 - 23 scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis; Brondijk TH et al.; A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counter-parts . An open reading frame of 1311 bp was found . The deduced 437 amino acid sequence showed a high degree of identity to the beta-succinyl-CoA synthetase of Escherichia coli (46%), the mitochondrial beta-succinyl-CoA synthetase from pig (48%) and the hydrogenosomal beta-succinyl-CoA synthetase from Trichomonas vaginalis (49%) . The G + C content of the succinyl-CoA synthetase coding sequence (43.8%) was considerably higher than that of the 5' (14.8%) and 3' (13.3%) non-translated flanking sequences, as has been observed for other genes from N . frontalis . The codon usage pattern was biased, with only 34 codons used and a strong preference for a pyrimidine (T) in the third positions of the codons . The coding sequence of the beta-succinyl-CoA synthetase cDNA was cloned in an E . coli expression vector encoding a 6(His) tag . The recombinant protein was purified by affinity binding and used to produce polyclonal antibodies . The anti-succinyl-CoA synthetase serum recognized a 45 kDa protein from a N . frontalis fraction enriched for hydrogenosomes and similar polypeptides in two related anaerobic fungi, Piromyces rhizinflata (45 kDa) and Caecomyces communis (47 kDa) . Immunocytochemical experiments suggest that succinyl-CoA synthetase is located in the hydrogenosomal matrix . Staining for SCS activity in native electrophoretic gels revealed a band with an apparent molecular weight of approximately 330 kDa . The C-terminus of the succinyl-CoA synthetase sequence was devoid of the typical targeting signals identified so far in microbody proteins, indicating that N . frontalis uses a different signal for sorting SCSB into hydrogenosomes . Based on comparisons with other proteins we propose a putative N-terminal targeting signal for succinyl-CoA synthetase of N . frontalis that shows some of the features of mitochondrial targeting sequences. Gene, 1996 Dec 12, 183(1-2), 53 - 60 Genomic targeting with an MBP-Cre fusion protein; Kolb AF et al.; The Cre recombinase of bacteriophage P1 catalyses site-specific recombination between lox-recombination target sites both in prokaryotic and eukaryotic cells and has thus become a popular tool in genetic research . Stable, Cre-mediated integration of DNA sequences at pre-existing lox sites in the eukaryotic genome is facilitated when a Cre recombinase protein rather than a cre-expression plasmid is used to direct site-specific recombination (Baubonis and Sauer (1993) Nucleic Acids Res., 21, 2025-2029) . We bacterially produced a Cre recombinase containing a nuclear localisation signal as a fusion protein with the E . coli maltose binding protein (MBP) and purified the protein by one step affinity chromatography . Subsequent cleavage with the protease factor Xa releases the Cre recombinase including the nuclear localisation signal from the maltose binding protein . Surprisingly, we found that the recombination activity of the uncleaved MBP-Cre fusion protein is virtually identical to that of the native Cre recombinase . This suggests that the MBP portion of the fusion protein behaves as a separate protein domain which does not interfere with Cre activity and can thus be used as an independent molecular tag . Additionally, the fusion protein is very resistant to proteolytic degradation and active over a wide range of temperatures . It efficiently catalyses excision and integration reactions in vitro and in eukaryotic cells . Finally, we could show that, by using MBP-Cre, it is possible to concomitantly excise a lox-flanked DNA sequence from a plasmid and integrate it into a pre-existing lox site in the genome in one transfection experiment . Vector backbone sequences which might have undesirable effects can thereby be excluded . The MBP-Cre fusion protein described here will be a useful tool not only for the catalysis of Cre-mediated recombination reactions in vitro and in vivo but also for the analysis of the mechanism of site-specific recombination. Mol Biochem Parasitol, 1996 Dec 2, 83(1), 81 - 91 Stable coexpression of a drug-resistance gene and a heterologous gene in an ancient parasitic protozoan Giardia lamblia; Yu DC et al.; Manipulation of gene expression in Giardia lamblia, one of the most ancient eukaryotes, may provide insights into the evolutionary transition from prokaryotes to eukaryotes . Two recent successes in transient expression of the firefly luciferase (luc) gene in G . lamblia were mediated by a 5'-untranslated region (UTR) of the Giardia glutamate dehydrogenase (gdh) gene and a giardiavirus (GLV) genomic transcript, respectively . We now report a stable coexpression of luc gene with a neomycin phosphotransferase (neo(r)) gene in G . lamblia . An in vitro transcript of the construct pC670-Neo; containing the neo(r) encoding region flanked with the 5'670 nucleotides (nt) and the 3'2022 nt portion of GLV positive strand RNA, was electroporated into G . lamblia trophozoites that were infected with GLV . G418-resistant Giardia trophozoites were cloned, and the neo(r) mRNA in these clones was found to increase with increasing G418 pressure . This drug resistance remained stable upon continuous in vitro cultivation in the absence of G418 for over 15 days . Another plasmid pNeo/GDH/Luc, was constructed by inserting luc gene downstream from the neo(r) gene and the 193 nt 5' portion of gdh gene in pC670-Neo, and its bicistronic in vitro transcript was introduced into GLV-infected G . lamblia by electroporation . The transfectants demonstrated G418-resistance and persistent luciferase activity at levels parallel to the amount of G418 used for selection, peaking at a level of several thousand-fold above the background . Taken together, these data indicate that the neo(r) gene provides an effective selection marker for transformation of Giardia trophozoites, and the bicistronic RNA transfection vector may open the way for functional analysis of other genes in Giardia. Rev Sci Tech, 1996 Dec, 15(4), 1233 - 40 Animal mycoplasmoses: a general introduction; Nicolet J; The nature of the smallest prokaryotes with autonomous replication, i.e . the mycoplasmas, which constitute the class Mollicutes, is presented in brief . These micro-organisms are extracellular parasites of the mucous membranes in animals . Mycoplasmas may cause infections, known as mycoplasmoses . The author describes current knowledge of the pathogenicity of mycoplasmas, especially in regard to the diverse effects on the immune system and the variability of the superficial antigens . Relatively few species of pathogenic mycoplasmas cause mycoplasmoses (both sporadic and endemic diseases) which have significant socio-economic consequences . The control and prevention of endemic mycoplasmoses can only be achieved with effective diagnostic tools and an efficient structure of epidemiological surveillance . The author recommends the encouragement of biological and genetic research on the mycoplasmas and the co-ordination of the development of diagnostic tests, including evaluation and validation in various epidemiological field situations. Biokhimiia, 1996 Dec, 61(12), 2173 - 80 {The major cytoplasmic mRNP protein, p50, is required for efficient mRNA translation in vitro}; Kovrigina EA et al.; The major cytoplasmic mRNP protein of somatic cells, p50, is the member of the Y-box-binding transcription factor family and can control gene expression at two levels including mRNA transcription and translation . It has been demonstrated that p50 is responsible for the inactive state of mRNA within free mRNPs . In this work, we show that the Y-box-containing DNA (Y-DNA) predominantly binds to p50 in rabbit reticulocyte lysates and causes translation inhibition of the endogenous and exogenous globin mRNA and prokaryotic beta-galactosidase mRNA . Preincubation of Y-DNA with purified p50 prevents the inhibition . Inhibition of protein biosynthesis by the Y-DNA is not due to the degradation or functional inactivation of mRNA . The inhibition is associated with the decay of polyribosomes and dissociation of a newly synthesized protein from the ribosomes . The data indicate that Y-DNA inhibits protein biosynthesis predominantly at the initiation stage and that p50 is an essential component of the translation initiation apparatus. Bioorg Khim, 1996 Dec, 22(12), 883 - 90 {Low molecular weight GTPase of the rac family from the bovine retina . Structure of cDNA and its expression in a prokaryotic system}; Petrov VM et al.; cDNA with a high degree of homology to the rac1 gene of the Ras-like protein from the HL-60 cell line was isolated from a bovine retina cDNA library . The homologies of the nucleotide and deduced amino acid sequences were 90 and 98%, respectively . The product of cDNA expression in E . coli cells purified by chromatography displayed GTP-binding and GTPase activities . The data suggest that the Rac1 protein is one of the low-molecular GTP-binding proteins in photoreceptor membranes of vertebrates. Immunol Lett, 1996 Dec, 54(2-3), 215 - 9 The multidrug transporters--proteins of an ancient immune system; Sarkadi B et al.; The multidrug resistance proteins, discovered as membrane transporters producing chemotherapy-resistance in cancer, are functioning as complex cellular defence systems through recognition and energy-dependent removal of a large variety of toxic agents . The multidrug transporters belong to the ATP-binding cassette (ABC) transporters, present both in prokaryotes and eukaryotes and built from a combination of characteristic membrane-spanning helices and cytoplasmic ATP-binding domains . In mammals the MDR1 (P-glycoprotein) extrudes large hydrophobic compounds and provides the basis of the blood-brain and the blood-testis barrier for such molecules . The multidrug resistance-associated protein (MRP) and its homologues have a major role in the cellular export of large organic anions, including e.g . conjugated bile salts and glutathione-conjugates . The substrate recognition, that is the self and non-self discrimination and the ATP-dependent foreign agent extrusion are directly coupled within the structure of these large plasma membrane proteins . Here we suggest that the multidrug transporters are essential parts of our immune-defence system, working as 'cellular antitoxic' mechanisms. Fungal Genet Biol, 1996 Dec, 20(4), 289 - 98 Polarized Growth of Fungal Hyphae Is Defined by an Alkaline pH Gradient Robson GD, Prebble E, Rickers A, Hosking S, Denning DW, Trinci APJ, Robertson W. Robson, G . D., Prebble, E., Rickers, A., Hosking, S., Denning, D . W., Trinci, A . P . J., and Robertson, W . 1996 . Polarized growth of fungal hyphae is defined by an alkaline pH gradient . Fungal Genetics and Biology 20, 289-298 . Polarized cell growth is exhibited by a diverse range of eukaryotic and prokaryotic cells . The events which are responsible for this growth are poorly understood . However, the existence of ion gradients may play an important role in establishing and driving cell polarity . Using a pH-sensitive, ratiometric fluorescent dye to monitor intracellular pH in growing fungal hyphae, we report a gradient at the extending hyphal tip that is up to 1.4 pH units more alkaline than more distal regions . Both the magnitude and the length of the pH gradient were strongly correlated with the rate of hyphal extension and eradication of the gradient-arrested growth . These results suggest that alkaline pH gradients may be integral to hyphal extension in fungi. Insect Biochem Mol Biol, 1996 Dec, 26(10), 997 - 1009 Developmental analysis of a female-specific 16S rRNA gene from mycetome-associated endosymbionts of a mealybug, Planococcus lilacinus; Kantheti P et al.; A clone showing female-specific expression was identified from an embryonic cDNA library of a mealybug, Planococcus lilacinus . In Southern blots this clone (P7) showed hybridization to genomic DNA of females, but not to that of males . However, P7 showed no hybridization to nuclei of either sex, raising the possibility that it was extrachromosomal in origin . In sectioned adult females P7 hybridized to an abdominal organ called the mycetome . The mycetome is formed by mycetocytes, which are polyploid cells originating from the polar bodies and cleavage nuclei that harbour maternally transmitted, intracellular symbionts . Electron microscopy confirmed the presence of symbionts within the mycetocytes . Sequence analysis showed that P7 is a 16S rRNA gene, confirming its prokaryotic origin . P7 transcripts are localized to one pole in young embryos but are found in the pole as well as in the germ band during later stages of development . P7 expression is detectable in young embryos of both sexes but the absence of P7 in third instar and adult males suggests that this gene, and hence the endosymbionts, are subject to sex-specific elimination. Glycobiology, 1996 Dec, 6(8), 811 - 6 Dolichyl-phosphomannose synthase from the archae Thermoplasma acidophilum; Zhu BC et al.; Archae (formerly Archaebacteria) comprise an entire kingdom of organisms placed halfway between prokaryotes and eucaryotes in evolution . This class of organisms lacks murein cell wall and is devoid of organelles, yet Archae synthesize and export N-linked and O-linked glycoproteins utilizing only the plasma membrane . Study of glycosylation systems in Archae is extremely interesting because the plasma membrane must perform many functions normally carried out by the endoplasmic reticulum and Golgi in eucaryotes . This report represents the first glycosyl transferase system enzyme demonstrated from archae showing a functional relationship with homologous eucaryotic enzymes . Archae dolichyl-phosphoryl-mannose synthase was purified 1070-fold from Thermoplasma acidophilum by column chromatography on Sephacryl S-200, Cibacron blue 3GA-agarose, Octyl-Sepharose, and hydroxylapatite in the presence of 0.2% polioxyethylene 9 lauryl ether . The enzyme activity was stimulated by MgCl2 (20 mM optimum) and exhibited a pH optimum at 6.0 . Although the native polyisoprenoid has not been isolated or characterized, the enzyme prefers dolichyl phosphate (dol-P) to C55-polyisoprenol as an acceptor, and the Km value for dol-P was calculated to be 2.6 microM . Amphomycin, an inhibitor of dol-P-Man synthase, blocked mannosyl transfer to the endogenous lipids, proteins, and to dol-P; 100 micrograms/ml amphomycin inhibited 97% of mannosyl transfer to dol-P, and 50% to endogenous acceptors, indicating direct transfer from GDP-mannose to some intermediates or final structures . The size range of {3H}Man-oligosaccharides from acid-labile manno-lipid product was from dp 1 to 4 . dol-P-Man synthase activity could be correlated directly with a 42 kDa band on SDS/polyacrylamide gel electrophoresis. Vet Microbiol, 1996 Dec, 53(3-4), 261 - 9 Characterization of enzootic nasal tumor virus capsid antigen; Rosati S et al.; The RT-PCR was carried out on tumor tissue from sheep with enzootic nasal tumor (ENT), using primers designed from conserved amino acid regions of related type D retroviruses . A 591 bp PCR fragment, corresponding to 90% of the capsid antigen was cloned, sequenced and expressed in E . coli . Alignment with ovine pulmonary carcinoma (OPC) virus showed 93% nucleotide and 96% amino acid homology . No amplification occurred when DNA from ovine fetal cell line was used as template . The recombinant protein, highly expressed in prokaryotic system, reacted in immunoblot with mouse antiserum to Mason Pfizer monkey virus (MPMV) p27, as well as sera from OPC and ENT diseased animals . Preliminary application of this antigen in ELISA suggested its potential use to detect seropositive animals in infected flocks. Microbiology, 1996 Dec, 142 ( Pt 12), 3515 - 24 Characterization of the gene for an immunodominant 72 kDa lipoprotein of Mycoplasma mycoides subsp . mycoides small colony type; Cheng X et al.; With the aim of characterizing specific immunogenic proteins of Mycoplasma mycoides subsp . mycoides small colony (SC) type, the aetiological agent of contagious bovine pleuropneumonia, a gene encoding a major immunogenic protein of 72 kDa named P72 was cloned and expressed in Escherichia coli . The expressed protein was of the same apparent molecular mass as that produced by the parent strain . The predicted molecular mass of P72, based on the DNA-deduced amino acid sequence, was 61.118 kDa, significantly lower than the apparent molecular mass of endogenous or recombinant P72 on SDS-PAGE . Analysis of the amino acid sequence revealed a typical prokaryotic signal peptidase II-membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein . P72 was shown to be a lipoprotein and its surface location was confirmed by trypsin treatment of whole cells . An unassigned gene encoding a peptide with some similarity to P72 was found on the genome sequence of M . capricolum subsp . capricolum but not on that of Mycoplasma genitalium . The P72 gene was detected in 11/11 M . mycoides subsp . mycoides SC strains . Antiserum against recombinant P72 reacted strongly with 12/12 strains of M . mycoides subsp . mycoides SC, weakly with Mycoplasma bovine group 7 strain PG50, but not with other members of the 'mycoides cluster' or closely related mycoplasmas . Cows experimentally contact-infected with M . mycoides subsp . mycoides SC developed a humoral response against P72 within 35 d . P72 is a specific antigenic membrane lipoprotein of M . mycoides subsp . mycoides SC with potential for use in development of diagnostic reagents . It seems to belong to a family of lipoproteins of the "mycoides cluster'. Curr Opin Genet Dev, 1996 Dec, 6(6), 686 - 91 Biodiversity, genomes, and DNA sequence databases; Leipe DD; There are approximately 1.4 million organisms on this planet that have been described morphologically but there is no comparable coverage of biodiversity at the molecular level . Little more than 1% of the known species have been subject to any molecular scrutiny and eukaryotic genome projects have focused on a group of closely related model organisms . The past year, however, has seen an approximately 80% increase in the number of species represented in sequence databases and the completion of the sequencing of three prokaryotic genomes . Large-scale sequencing projects seem set to begin coverage of a wider range of the eukaryotic diversity, including green plants, microsporidians and diplomonads. Microbiol Rev, 1996 Dec, 60(4), 575 - 608 Proton-dependent multidrug efflux systems; Paulsen IT et al.; Multidrug efflux systems display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents . This review examines multidrug efflux systems which use the proton motive force to drive drug transport . These proteins are likely to operate as multidrug/proton antiporters and have been identified in both prokaryotes and eukaryotes . Such proton-dependent multidrug efflux proteins belong to three distinct families or superfamilies of transport proteins: the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, and the resistance/ nodulation/cell division (RND) family . The MFS consists of symporters, antiporters, and uniporters with either 12 or 14 transmembrane-spanning segments (TMS), and we show that within the MFS, three separate families include various multidrug/proton antiport proteins . The SMR family consists of proteins with four TMS, and the multidrug efflux proteins within this family are the smallest known secondary transporters . The RND family consists of 12-TMS transport proteins and includes a number of multidrug efflux proteins with particularly broad substrate specificity . In gram-negative bacteria, some multidrug efflux systems require two auxiliary constituents, which might enable drug transport to occur across both membranes of the cell envelope . These auxiliary constituents belong to the membrane fusion protein and the outer membrane factor families, respectively . This review examines in detail each of the characterized proton-linked multidrug efflux systems . The molecular basis of the broad substrate specificity of these transporters is discussed . The surprisingly wide distribution of multidrug efflux systems and their multiplicity in single organisms, with Escherichia coli, for instance, possessing at least nine proton-dependent multidrug efflux systems with overlapping specificities, is examined . We also discuss whether the normal physiological role of the multidrug efflux systems is to protect the cell from toxic compounds or whether they fulfil primary functions unrelated to drug resistance and only efflux multiple drugs fortuitously or opportunistically. Genetics, 1996 Dec, 144(4), 1455 - 62 A dominant negative effect of eth-1r, a mutant allele of the Neurospora crassa S-adenosylmethionine synthetase-encoding gene conferring resistance to the methionine toxic analogue ethionine; Barra JL et al.; eth-1r, a thermosensitive allele of the Neurospora crassa S-adenosylmethionine (AdoMet) synthetase gene that confers ethionine resistance, has been cloned and sequenced . Replacement of an aspartic amino acid residue (D48-->N48), perfectly conserved in prokaryotic, fungal and higher eukaryotic AdoMet synthetases, was found responsible for both thermosensitivity and ethionine resistance conferred by eth-1r . Gene fusion constructs, designed to overexpress eth-1r in vivo, render transformant cells resistant to ethionine . Dominance of ethionine resistance was further demonstrated in eth-1+/eth-1r partial diploids carrying identical gene doses of both alleles . Heterozygous eth-1+/eth-1r cells have, at the same time, both the thermotolerance conferred by eth-1+ and the ethionine-resistant phenotype conferred by eth-1r . AdoMet levels and AdoMet synthetase activities were dramatically decreased in heterozygous eth-1+/ eth-1r cells . We propose that this negative effect exerted by eth-1r results from the in vivo formation of heteromeric eth-1+/eth-1r AdoMet synthetase molecules. Eur J Immunol, 1996 Dec, 26(12), 2831 - 40 Protective immunization with plasmid DNA containing the outer surface lipoprotein A gene of Borrelia burgdorferi is independent of an eukaryotic promoter; Simon MM et al.; Plasmid DNA encoding the outer surface lipoprotein A (OspA) of Borrelia burgdorferi under the control of either strong eukaryotic/viral or its own bacterial promoter was injected intramuscularly (m . tibialis anterior) or intradermally into BALB/c and AKR/N mice . OspA-specific antibodies and OspA-reactive T helper 1 cells (Th1) were induced only with those plasmids containing the ospA structural gene including its own regulatory control region immediately upstream . In the absence of the ospA promoter, no or only marginal immune responses to OspA were obtained, even when strong eukaryotic promoter/enhancer elements were present . Together with the finding that the ospA promoter is active in a mouse B-lymphoma line, the data suggest that spirochetes are able to express at least part of their genes in the mammalian environment . Mice previously vaccinated with the relevant ospA plasmid DNA were protected against subsequent experimental challenge with a virulent strain of B . burgdorferi, as measured by the appearance of antibodies to a prominent protective epitope (LA-2) and the failure to re-isolate spirochetes from ear biopsies . In addition, C.B-17 severe-combined immunodeficient mice could be protected against infection by passive transfer of immune sera from ospA plasmid DNA-inoculated normal mice . Protective LA-2-related antibody titers obtained after repeated immunization persisted for 200 days and longer . This simple procedure of immunization using plasmid DNA consisting of a prokaryotic gene under the control of its own promoter holds great promise for the development of alternative subunit vaccines against bacterial infections, including Lyme disease . In addition, the availability of this novel prokaryotic promoter element now allows the study of the basis for the differential expression of bacterial genes in prokaryotic and eukaryotic environments. Protein Sci, 1996 Dec, 5(12), 2429 - 37 Crystal structure of dUTP pyrophosphatase from feline immunodeficiency virus; Prasad GS et al.; We have determined the crystal structure of dUTP pyrophosphatase (dUTPase) from feline immunodeficiency virus (FIV) at 1.9 A resolution . The structure has been solved by the multiple isomorphous replacement (MIR) method using a P6(3) crystal form . The results show that the enzyme is a trimer of 14.3 kDa subunits with marked structural similarity to E . coli dUTPase . In both enzymes the C-terminal strand of an anti-parallel beta-barrel participates in the beta-sheet of an adjacent subunit to form an interdigitated, biologically functional trimer . In the P6(3) crystal form one trimer packs on the 6(3) screw-axis and another on the threefold axis so that there are two independent monomers per asymmetric unit . A Mg2+ ion is coordinated by three asparate residues on the threefold axis of each trimer . Alignment of 17 viral, prokaryotic, and eukaryotic dUTPase sequences reveals five conserved motifs . Four of these map onto the interface between pairs of subunits, defining a putative active site region; the fifth resides in the C-terminal 16 residues, which is disordered in the crystals . Conserved motifs from all three subunits are required to create a given active site . With respect to viral protein expression, it is particularly interesting that the gene for dUTPase (DU) resides in the middle of the Pol gene, the enzyme cassette of the retroviral genome . Other enzymes encoded in the Pol polyprotein, including protease (PR), reverse transcriptase (RT), and most likely integrase (IN), are dimeric enzymes, which implies that the stoichiometry of expression of active trimeric dUTPase is distinct from the other Pol-encoded enzymes . Additionally, due to structural constraints, it is unlikely that dUTPase can attain an active form prior to cleavage from the polyprotein. Plant Physiol, 1996 Dec, 112(4), 1641 - 7 Identification of a chloroplast coenzyme A-binding protein related to the peroxisomal thiolases; Yang LM et al.; A 30-kD coenzyme A (CoA)-binding protein was isolated from spinach (Spinacea oleracea) chloroplast soluble extracts using affinity chromatography under conditions in which 95% of the total protein was excluded . The 30-kD protein contains an eight-amino-acid sequence, DVRLYYGA, that is identical to a region in a 36-kD protein of unknown function that is encoded by a kiwifruit (Actinidia deliciosa) cDNA . Southern blotting also detected a spinach gene that is related to the kiwifruit cDNA . The kiwifruit 36-kD protein that was synthesized in Escherichia coli was imported into chloroplasts and cleaved to a 30-kD form; it was processed to the same size in an organelle-free assay . Furthermore, the kiwifruit protein specifically bound to CoA . The kiwifruit protein contains a single cysteine within a domain that is related to the peroxisomal beta-ketoacyl-CoA thiolases, which catalyze the CoA-dependent degradative step of fatty acid beta-oxidation . Within 50 amino acids surrounding the cysteine, considered to be part of the thiolase active site, the kiwifruit protein shows approximately 26% sequence identity with the mango, cucumber, and rat peroxisomal thiolases . N-terminal alignment with these enzymes, relative to the cysteine, indicates that the 36-kD protein is cleaved after serine-58 during import, agreeing with the estimated size (approximately 6 kD) of a transit peptide . The 30-kD protein is also related to the E . coli and mitochondrial thiolases, as well as to the acetoacetyl-CoA thiolases of prokaryotes . Features distinguish it from members of the thiolase family, suggesting that it carries out a related but novel function . The protein is more distantly related to chloroplast beta-ketoacyl-acyl carrier protein synthase III, the initial condensing enzyme of fatty acid synthetase that utilizes acetyl-CoA. J Virol, 1996 Dec, 70(12), 8944 - 60 Recombinant adenoviruses with large deletions generated by Cre-mediated excision exhibit different biological properties compared with first-generation vectors in vitro and in vivo; Lieber A et al.; In vivo gene transfer of recombinant E1-deficient adenoviruses results in early and late viral gene expression that elicits a host immune response, limiting the duration of transgene expression and the use of adenoviruses for gene therapy . The prokaryotic Cre-lox P recombination system was adapted to generate recombinant adenoviruses with extended deletions in the viral genome (referred to here as deleted viruses) in order to minimize expression of immunogenic and/or cytotoxic viral proteins . As an example, an adenovirus with a 25-kb deletion that lacked E1, E2, E3, and late gene expression with viral titers similar to those achieved with first-generation vectors and less than 0.5% contamination with E1-deficient virus was produced . Gene transfer was similar in HeLa cells, mouse hepatoma cells, and primary mouse hepatocytes in vitro and in vivo as determined by measuring reporter gene expression and DNA transfer . However, transgene expression and deleted viral DNA concentrations were not stable and declined to undetectable levels much more rapidly than those found for first-generation vectors . Intravenous administration of deleted vectors in mice resulted in no hepatocellular injury relative to that seen with first-generation vectors . The mechanism for stability of first-generation adenovirus vectors (E1a deleted) appeared to be linked in part to their ability to replicate in transduced cells in vivo and in vitro . Furthermore, the deleted vectors were stabilized in the presence of undeleted first-generation adenovirus vectors . These results have important consequences for the development of these and other nonintegrating vectors for gene therapy. Genomics, 1996 Dec 1, 38(2), 192 - 8 A human homolog of bacterial acetolactate synthase genes maps within the CADASIL critical region; Joutel A et al.; CADASIL, a recently identified autosomal dominant condition characterized by the recurrence of subcortical infarcts leading to dementia, was previously mapped to chromosome 19p13.1 within a 2-cM interval, D19S226-D19S199 . No recombination event was observed with D19S841, a highly polymorphic microsatellite marker isolated from a cosmid mapped to this region . We recently identified within this cosmid a conserved sequence that we used to screen a fetal brain cDNA library and isolated an ubiquitous and abundantly transcribed gene . We did not detect any mutation of this gene in CADASIL patients, suggesting that it is not implicated in this disorder . Interestingly, this gene encodes a putative protein homologous to several thiamine pyrophosphate-binding proteins previously identified in bacteria, yeast, and plants . The proteins with the highest degree of similarity were the acetolactate synthase enzymes which, in prokaryotes, are involved in the branched chain amino acid biosynthetic pathway, raising fascinating questions on the yet unknown function of this gene in mammals. J Clin Microbiol, 1996 Dec, 34(12), 3120 - 8 Prokaryotic DNA sequences in patients with chronic idiopathic prostatitis; Krieger JN et al.; Half of all men experience symptoms of prostatitis at some time in their lives, but the etiology is unknown for more than 90% of patients . Optimal clinical and culture methods were used to select 135 men with chronic prostatitis refractory to multiple previous courses of antimicrobial therapy . The subjects had no evidence of structural or functional lower genitourinary tract abnormalities of bacteriuria or bacterial prostatitis by traditional clinical tests, or of urethritis or urethral pathogens by culture . Specific PCR assays detected Mycoplasma genitalium, Chlamydia trachomatis, or Trichomonas vaginalis in 10 patients (8%) . Broad-spectrum PCR tests detected tetracycline resistance-encoding genes, tetM-tetO-tetS, in 25% of patients and 16S rRNA in 77% of subjects . The tetM-tetO-tetS-positive cases constituted a subset of the 16S rRNA-positive cases . Patients with 16S rRNA were more likely to have > or = 1,000 leukocytes per mm3 in their expressed prostatic secretion than men whose prostate biopsy specimens were negative for 16S rRNA (P < 0.001) . Based on direct sequencing and repetitive cloning, multiple sources of 16S rRNA were observed in individual patients . Sequences of 29 cloned PCR products revealed 16S rRNAs distinct from those of common skin and gut flora . These findings suggest that the prostate can harbor microorganisms that are not detectable by traditional approaches . These organisms may be associated with inflammation in the expressed prostatic secretions . Molecular methods hold great promise for identifying culture-resistant microorganisms in patients with chronic prostatitis. J Cell Biochem, 1996 Dec 1, 63(3), 358 - 65 Regulation of c-fos is affected by electromagnetic fields; Rao S et al.; The goal of the present study was to determine if regulatory regions of the c-fos gene were responsive to electromagnetic field exposure . The research design used transfected cells to increase the sensitivity of assays designed to identify changes following exposure . HeLa cells were transiently transfected with plasmids containing upstream regulating regions of c-fos up to -700 base pairs, coupled with the prokaryotic reporter gene CAT . Cells were exposed to an environmentally relevant EMF of 60 Hz at 60 mGrms . CAT expression above control levels in transfected cells (region +42 to -700 bp) was observed following 5 min exposure to the electromagnetic field, with a peak at 20 min . The expression was at basal levels following 40 min exposure . Deletion analysis of upstream DNA narrowed the responsive region to 138 base pairs from -363 to -225, which contains the SRE/AP-1 sites. Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 14030 - 5 Biochemical characterization and intracellular localization of the Menkes disease protein; Yamaguchi Y et al.; Menkes disease is a fatal neurodegenerative disorder of childhood due to the absence or dysfunction of a putative copper-transporting P-type ATPase encoded on the X chromosome . To elucidate the biosynthesis and subcellular localization of this protein, polyclonal antisera were generated against a bacterial fusion protein encoding the 4th to 6th copper-binding domains in the amino terminus of the human Menkes protein . RNA blot analysis revealed abundant Menkes gene expression in several cell lines, and immunoblotting studies utilizing this antiserum readily detected a 178-kDa protein in lysates from these cells . Pulse-chase studies indicate that this protein is synthesized as a single-chain polypeptide which is modified by N-linked glycosylation to a mature endoglycosidase H-resistant form . Sucrose gradient fractionation of HeLa cell lysates followed by immunoblotting of individual fractions with antibodies to proteins of known intracellular location identified the Menkes ATPase in fractions similar to those containing the cation-independent mannose-6-phosphate receptor . Consistent with this observation, confocal immunofluorescence studies of these same cells localized this protein to the trans-Golgi network and a vesicular compartment with no expression in the nucleus or on the plasma membrane . Taken together, these data provide a unique model of copper transport into the secretory pathway of mammalian cells which is compatible with clinical observations in affected patients and with recent data on homologous proteins identified in prokaryotes and yeast. Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13617 - 22 Chemical nature of the light emitter of the Aequorea green fluorescent protein; Niwa H et al.; The jellyfish Aequorea victoria possesses in the margin of its umbrella a green fluorescent protein (GFP, 27 kDa) that serves as the ultimate light emitter in the bioluminescence reaction of the animal . The protein is made up of 238 amino acid residues in a single polypeptide chain and produces a greenish fluorescence (lambda max = 508 nm) when irradiated with long ultraviolet light . The fluorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser65-Tyr66-Gly67- . GFP has been used extensively as a reporter protein for monitoring gene expression in eukaryotic and prokaryotic cells, but relatively little is known about the chemical mechanism by which fluorescence is produced . To obtain a better understanding of this problem, we studied a peptide fragment of GFP bearing the chromophore and a synthetic model compound of the chromophore . The results indicate that the GFP chromophore consists of an imidazolone ring structure and that the light emitter is the singlet excited state of the phenolate anion of the chromophore . Further, the light emission is highly dependent on the microenvironment around the chromophore and that inhibition of isomerization of the exo-methylene double bond of the chromophore accounts for its efficient light emission. Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13600 - 4 Sucrose biosynthesis in a prokaryotic organism: Presence of two sucrose-phosphate synthases in Anabaena with remarkable differences compared with the plant enzymes; Porchia AC et al.; Biosynthesis of sucrose-6-P catalyzed by sucrose-phosphate synthase (SPS), and the presence of sucrose-phosphate phosphatase (SPP) leading to the formation of sucrose, have both been ascertained in a prokaryotic organism: Anabaena 7119, a filamentous heterocystic cyanobacterium . Two SPS activities (SPS-I and SPS-II) were isolated by ion-exchange chromatography and partially purified . Four remarkable differences between SPSs from Anabaena and those from higher plants were shown: substrate specificity, effect of divalent cations, native molecular mass, and oligomeric composition . Both SPS-I and SPS-II accept Fru-6-P (K(m) for SPS-I = 0.8 +/- 0.1 mM; K(m) for SPS-II = 0.7 +/- 0.1 mM) and UDP-Glc as substrates (K(m) for SPS-I = 1.3 +/- 0.4 mM; K(m) for SPS-II = 4.6 +/- 0.4 mM), but unlike higher plant enzymes, they are not specific for UDP-Glc . GDP-Glc and TDP-Glc are also SPS-I substrates (K(m) for GDP-Glc = 1.2 +/- 0.2 mM and K(m) for TDP-Glc = 4.0 +/- 0.4 mM), and ADP-Glc is used by SPS-II (K(m) for ADP-Glc = 5.7 +/- 0.7 mM) . SPS-I has an absolute dependence toward divalent metal ions (Mg2+ or Mn2+) for catalytic activity, not found in plants . A strikingly smaller native molecular mass (between 45 and 47 kDa) was determined by gel filtration for both SPSs, which, when submitted to SDS/PAGE, showed a monomeric composition . Cyanobacteria are, as far as the authors know, the most primitive organisms that are able to biosynthesize sucrose as higher plants do. Biochemistry, 1996 Nov 26, 35(47), 14999 - 5008 Template strand gap bypass is a general property of prokaryotic RNA polymerases: implications for elongation mechanisms; Liu J et al.; It has previously been shown that T7 RNA polymerase is capable of bypassing gaps on the template strand ranging in size from 1 to 24 nucleotides . This as well as other observations suggested a role for the nontemplate strand during elongation . To establish the generality of this gap bypassing event, we have extended these studies to SP6 and Escherichia coli RNA polymerases . SP6 RNA polymerase bypasses template gaps from 1 to 19 nucleotides in size with various degrees of efficiency and produces runoff transcripts of decreasing length corresponding to increasing gap size . RNA sequence analysis of the resulting runoff transcripts revealed that SP6 RNA polymerase faithfully transcribed both parts of the template strand flanking the gapped region . Similar experiments were carried out with E . coli RNA polymerase (a multiple subunit enzyme) and indicate that it is also capable of gap bypass albeit with reduced efficiency compared to T7 and SP6 RNA polymerases . It appears that the ability to bypass gaps present on the DNA template strand is a general property of prokaryotic RNA polymerases . These results have implications with respect to the mechanism of elongation and the role of the nontemplate strand in transcription. J Biol Chem, 1996 Nov 22, 271(47), 30126 - 35 Purification of storage granule protein-23 . A novel protein identified by phage display technology and interaction with type I plasminogen activator inhibitor; Lang IM et al.; Type 1 plasminogen activator inhibitor (PAI-1) is a key regulator of the fibrinolytic cascade that is stored in a rapidly releasable form within platelet alpha-granules . To identify proteins that may participate in the targeting or storage of this potent inhibitor, this report investigates the applicability of utilizing filamentous bacteriophages to display proteins expressed by cells containing a regulated secretory pathway and their enrichment based upon an interaction with PAI-1 . For this purpose, RNA was extracted from AtT-20 cells (i.e . a classical model cell system for intracellular protein sorting), reverse transcribed, amplified using polymerase chain reaction primers containing internal restriction sites, and cloned into the phagemid pCOMB3H for expression as fusion constructs with the bacteriophage gene III protein . Escherichia coli was transformed with the phagemids and infected with VCSM13 helper phage, and the resulting AtT-20 cDNA-bacteriophage library was enriched by panning against solid- and solution-phase PAI-1 . The enriched cDNA library was subcloned into a prokaryotic expression vector system that replaces the gene III protein with a decapeptide tag for immunologic quantitation . One novel cDNA clone (i.e . A-61), which preferentially recognized solution-phase PAI-1 and reacted positively with antibodies derived from a rabbit immunized with alpha-granules, was subcloned into the prokaryotic expression vector pTrcHis to create a construct containing an N-terminal six-histidine purification tag . This construct was expressed in E . coli, purified by nickel-chelate chromatography followed by preparative SDS-polyacrylamide gel electrophoresis, and utilized for the generation of polyclonal antibodies . Immunoblotting analysis employing antibodies against the purified A-61 construct revealed a 23-kDa protein present in the regulated secretory pathway of AtT-20 cells . The 23-kDa molecule was purified from media conditioned by AtT-20 cells by ion exchange chromatography on DEAE-Sephacel, molecular sieve chromatography on Sephacryl S-100, chromatofocusing on Polybuffer exchanger 94, and affinity chromatography on PAI-1-Sepharose . N-terminal amino acid sequencing of a 16-kDa Lys-C proteolytic fragment of the 23-kDa storage granule protein was employed to confirm its identity with the cDNA sequence of clone A-61 . These data indicate that phage display of cDNA libraries fused to the C-terminal region of the gene III protein and their enrichment via an interaction with a target molecule can be utilized to define other proteins present within a particular cellular pathway. Science, 1996 Nov 22, 274(5291), 1367 - 71 Structure of the A site of Escherichia coli 16S ribosomal RNA complexed with an aminoglycoside antibiotic; Fourmy D et al.; Aminoglycoside antibiotics that bind to 30S ribosomal A-site RNA cause misreading of the genetic code and inhibit translocation . The aminoglycoside antibiotic paromomycin binds specifically to an RNA oligonucleotide that contains the 30S subunit A site, and the solution structure of the RNA-paromomycin complex was determined by nuclear magnetic resonance spectroscopy . The antibiotic binds in the major groove of the model A-site RNA within a pocket created by an A-A base pair and a single bulged adenine . Specific interactions occur between aminoglycoside chemical groups important for antibiotic activity and conserved nucleotides in the RNA . The structure explains binding of diverse aminoglycosides to the ribosome, their specific activity against prokaryotic organisms, and various resistance mechanisms, and provides insight into ribosome function. Biochem J, 1996 Nov 15, 320 ( Pt 1), 129 - 35 Production of the R2 subunit of ribonucleotide reductase from herpes simplex virus with prokaryotic and eukaryotic expression systems: higher activity of R2 produced by eukaryotic cells related to higher iron-binding capacity; Lamarche N et al.; The R2 subunit of ribonucleotide reductase from herpes simplex virus type 2 was overproduced with prokaryotic and eukaryotic expression systems . The recombinant R2 purified by a two-step procedure exhibited a 3-fold higher activity when produced in eukaryotic cells . Precise quantification of the R2 concentration at each step of the purification indicated that the activity was not altered during the purification procedure . Moreover, we have observed that the level of R2 expression, in eukaryotic cells as well as in prokaryotic cells, did not influence R2 activity . Extensive characterization of the recombinant R2 purified from eukaryotic and prokaryotic expression systems has shown that both types of pure R2 preparations were similar in their 76 kDa dimer contents (more than 95%) and in their ability to bind the R1 subunit . However, we have found that the higher activity of R2 produced in eukaryotic cells is more probably related to a higher capability of binding the iron cofactor as well as a 3-fold greater ability to generate the tyrosyl free radical. J Biol Chem, 1996 Nov 15, 271(46), 29407 - 14 DnaK heat shock protein of Escherichia coli maintains the negative supercoiling of DNA against thermal stress; Ogata Y et al.; Plasmid DNA in exponentially growing Escherichia coli immediately relaxes after heat shock, and the relaxed state of DNA rapidly reverts to the original state with exposure to conditions of heat shock . We have now obtained genetic and biochemical evidence indicating that DnaK heat shock protein of E . coli, a prokaryotic homologue of hsp70, is involved in this re-supercoiling of DNA . As re-supercoiling of DNA did not occur in an rpoH amber mutant, it seems likely that heat shock proteins are required for this reaction . Plasmid DNA in a dnaK deletion mutant relaxed excessively after temperature shift-up, and the re-supercoiling of DNA was not observed . DNAs incubated with a crude cell extract prepared from the dnaK mutant were more relaxed than seen with the extract from its isogenic wild-type strain, and the addition of purified DnaK protein to the mutant extract led to an increase in the negative supercoiling of DNA . Moreover, reaction products of purified DNA gyrase more negatively supercoiled in the presence of DnaK protein . Based on these results, we propose that DnaK protein plays a role in maintaining the negative supercoiling of DNA against thermal stress. J Biol Chem, 1996 Nov 8, 271(45), 28703 - 9 Amino acid sequence homology between N- and C-terminal halves of a carbonic anhydrase in Porphyridium purpureum, as deduced from the cloned cDNA; Mitsuhashi S et al.; Carbonic anhydrase (CA) from Porphyridium purpureum, a unicellular red alga, was purified >209-fold to a specific activity of 1,147 units/mg protein . cDNA clones for this CA were isolated . The longest clone, comprising 1,960 base pairs, contained an open reading frame which encoded a 571-amino acid polypeptide with a calculated molecular mass of 62,094 Da . The N- and C-terminal halves of the putative mature Porphyridium CA have amino acid sequence homology to each other (>70%) and to other prokaryotic-type CAs . Both regions contain, at equivalent positions, one set of three possible zinc-liganding amino acid residues conserved among prokaryotic-type CAs . CA purified from Porphyridium contained two atoms of zinc per molecule . We propose that the Porphyridium CA has evolved by duplication of an ancestral CA gene followed by the fusion of the duplicated CA gene . The CA truncated into the putative mature form was overexpressed in Escherichia coli, and the expressed protein was active . Clones expressing separately the N- and C-terminal halves of the CA were constructed . CA activity was present in extracts of E . coli cells expressing the N-terminal half, while no detectable activity was found in cells expressing the C-terminal half. J Theor Biol, 1996 Nov 7, 183(1), 1 - 7 A chromatin switch; Bodnar JW et al.; Cellular and molecular biological approaches that study eukaryotic gene regulation have led to separate models which describe structure and mechanism with differing precision . Using principles of combinatorial and cooperative interactions inherent in both models, we have extended concepts derived from a "genetic switch" for prokaryotes to a chromatin switch for eukaryotes composed of DNA, transactivators, nucleosomes and the nuclear matrix . We present a consensus model for gene regulation that uses a simple Monte Carlo method for simulating condensation and extension of chromatin . Such a chromatin switch can be modulated by known biochemical and molecular modifications, and the transactivator binding sites or enhancers within DNA domains can be organized into a hierarchy to control cell cycling and differentiation. Vet Immunol Immunopathol, 1996 Nov, 54(1-4), 355 - 63 Novel viral vaccines for livestock; Babiuk LA et al.; Recent advances in our understanding of virulence factors of viruses and the proteins or glycoproteins involved in inducing neutralizing antibodies or cell mediated immunity are forming the foundation for the development of a new generation of viral vaccines . Using bovine herpesvirus as an example, we have identified glycoproteins gB, gC, and gD as important targets for inducing neutralizing antibody responses, with gD being able to induce the highest neutralizing and cellular responses . For subunit vaccine development, the glycoproteins were produced in both prokaryotic and eukaryotic expression systems . Glycoproteins produced in eukaryotic systems were very effective in stimulating a broad range of immune responses in cattle . These glycoproteins were then formulated into effective vaccines that prevented both virus shedding and clinical disease . Herpesviruses also served as an excellent model for the identification and deletion of specific genes which lead to attenuation . In herpesviruses, two major classes of genes can be deleted . Class I includes glycoprotein genes that are nonessential for virus replication in vitro, and Class II includes genes involved in nucleic acid metabolism . these gene deleted regions can then be replaced with genes coding for protective antigens of other pathogens to develop multivalent vaccines in a single vector . Similar approaches are being used for other viruses including vaccinia virus and adenovirus . Finally, we introduced plasmids coding for protective antigens, gB, gC, and gD, into animals and developed immunity to these antigens . This approach has the potential to revolutionize vaccination regimes of the future. Matrix Biol, 1996 Nov, 15(5), 301 - 10; discussion 311-2 Exon shuffling and other ways of module exchange; Patthy L; Thanks to recent improvements in techniques used for the detection of homologies, it is now clear that module exchange played a major role in protein evolution . Analysis of the genes of various modular proteins has identified a large number of cases where gene assembly was facilitated by intronic recombination--i.e., the proteins were formed by exon shuffling . Studies of the principles and mechanistic details of exon shuffling, however, revealed that this powerful evolutionary mechanism could become significant only after the appearance of spliceosomal introns typical of higher eukaryotes . Although exon shuffling is the most efficient way of constructing modular proteins, recent studies on the evolution of multidomain proteins of prokaryotes emphasize that intronic recombination is not an absolute prerequisite of module exchange. Plant Mol Biol, 1996 Nov, 32(4), 611 - 20 Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase; Brinch-Pedersen H et al.; In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS) . In order to modify the flux through the aspartate family pathway in barley and enhance the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS . As a result, leaves of primary transformants (T0) exhibited a 14-fold increase of free lysine and an 8-fold increase in free methionine . In mature seeds of the DHPS transgenics, there was a 2-fold increase in free lysine, arginine and asparagine and a 50% reduction in free proline, while no changes were observed in the seeds of the two AK transgenic lines analysed . When compared to that of control seeds, no differences were observed in the composition of total amino acids . The introduced genes were inherited in the T1 generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0-9.5-fold increase for DHPS . T1 seeds of DHPS transformants showed the same changes in free amino acids as observed in T0 seeds . It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine. Microbiology, 1996 Nov, 142 ( Pt 11), 3147 - 61 Gene arrangement and organization in a approximately 76 kb fragment encompassing the oriC region of the chromosome of Mycobacterium leprae; Fsihi H et al.; A continuous 75627 bp segment of the Mycobacterium leprae chromosome spanning the oriC region was sequenced . The gene order at this locus was similar to that found in the replication origin region of many other prokaryotes, particularly Mycobacterium tuberculosis and Streptomyces coelicolor . As in the case of several Gram-positive bacteria, essential genes involved in basic cellular functions, such as DNA or RNA metabolism (dnaA, dnaB, dnaN, gyrB, gyrA, pcnB, recF, rnpA, ssb), cell wall synthesis (ponA, pbpA) and probably cell division (gidB, rodA) were found . Strikingly, the gidA gene was absent from this part of the genome and there was no rRNA operon near oriC . The gyrA gene harbours an intein coding sequence indicating that protein splicing is required to produce the mature A subunit of DNA gyrase . Among the many other noteworthy features were ORFs encoding putative serine/threonine protein kinases and a protein phosphatase, three tRNA genes, one M . leprae-specific repetitive element and a glnQ pseudogene. Acta Biotheor, 1996 Nov, 44(3-4), 209 - 18 Elements of a unifying theory of biology; Norris V et al.; To discover a unifying theory of biology, it is necessary first to believe in its existence and second to seek its elements . Such a theory would explain the regulation of the cell cycle, differentiation and the origin of life . Some elements of the theory may be obtained by considering both eukaryotic and prokaryotic cell cycles . These elements include cytoskeletal proteins, calcium, cyclins, protein kinase C, phosphorylation, transcriptional sensing, autocatalytic gene expression and the physical properties of lipids . Other more exotic candidate elements include the dynamic enzoskeleton, ATP generation, mechanotransduction, the piezoelectric effect and resonance . Bringing these disparate elements together--and discovering others--will require extensive collaborations between specialists from different sciences . This can only be achieved within the context of an integrated approach to biology. Mol Microbiol, 1996 Nov, 22(4), 729 - 37 Increased distal gene transcription by the elongation factor RfaH, a specialized homologue of NusG; Bailey MJ et al.; The Escherichia coli RfaH protein is required for the expression of operons directing synthesis and export of the toxin haemolysin, the lipopolysaccharide core, and the F-factor sex pilus . Mutation of rfaH increases transcriptional polarity along all three operons . By demonstrating strong RfaH-dependent suppression of transcription polarity in vitro, we have established RfaH as a novel transcriptional activator, and we reveal that RfaH is a homologue of the essential protein NusG that modulates general transcriptional pausing and termination in prokaryotes . Full transcription of the distal genes from an upstream promoter required RfaH and the 5' cls-acting ops element, both in vivo and in vitro . In vivo the requirement for the ops element was suppressed by overexpressing RfaH, and in vitro the presence of ops lowered the concentration of RfaH required to stimulate transcript elongation . We suggest that RfaH directs transcript elongation in an analogous way to NusG, but does so in a subset of bacterial operons primarily engaged in the production of extracellular components required for virulence and fertility. Mol Microbiol, 1996 Nov, 22(4), 643 - 53 Specific binding of the Listeria monocytogenes transcriptional regulator PrfA to target sequences requires additional factor(s) and is influenced by iron; Bockmann R et al.; The PrfA protein, which is a member of the Crp/Fnr family of prokaryotic transcription activators, regulates the virulence genes of Listeria monocytogenes . In this work, specific binding of PrfA to its target DNA was determined by electrophoretic mobility-shift assays (EMSAs) using cell-free extracts from the two L . monocytogenes strains EGD and NCTC 7973 . PrfA-specific binding differs between the two strains, even when the concentration of PrfA was adjusted to similar levels . Both strains exhibited increased PrfA-specific binding after a shift into minimal essential medium (MEM) without showing a significant change in the amount of PrfA protein, relative to extracts from bacteria grown in brain-heart infusion (BHI) . The purified PrfA protein from strain EGD produced in Escherichia coli did not exhibit specific binding to the target DNA but did so upon addition of PrfA-free extracts from various Listeria species and Bacillus subtilis . The observed activation of PrfA seems to be caused by a PrfA-activating factor (Paf), which is probably a protein since elevated temperature, but not RNase treatment, destroyed the activation potential of such PrfA-free extracts . Moreover, fractionation of these extracts by sucrose gradient centrifugation yielded the Paf activity in a fraction sedimenting at 3.2 S . Specific binding of PrfA-containing extracts from strain EGD to the hly and actA promoter sequences was strongly inhibited by iron, whereas that of extracts from strain NCTC 7973 was only slightly reduced . The iron effect seems to be mediated by Paf rather than by PrfA itself. Mol Microbiol, 1996 Nov, 22(4), 631 - 42 Chlamydia trachomatis CTP synthetase: molecular characterization and developmental regulation of expression; Wylie JL et al.; Chlamydia trachomatis is a nucleotide parasite, being entirely dependent on its host eukaryotic cell for a supply of ATP, GTP, and UTP . Chlamydiae are not, however, auxotrophic for CTP, as they are able both to transport CTP from the host and synthesize CTP de novo via a chlamydial CTP synthetase . This study addresses the developmental regulation of CTP synthetase over the course of the C . trachomatis life cycle . Given the distinct life stages of C . trachomatis, analysis of temporal changes in gene expression and regulation of protein activity is the key to unravelling the mechanism of pathogenesis of this bacterium . The results of immunodetection analysis indicate that CTP synthetase is present in C . trachomatis elementary bodies and reticulate bodies and that it is widespread in other chlamydial strains . Reverse transcriptase-polymerase chain reaction (RT-PCR) and metabolic labelling experiments show that CTP synthetase is transcribed and translated primarily during the mid- and late stages of the chlamydial growth cycle . In addition, C . trachomatis CTP synthetase was transcribed with the CTP utilizing enzyme CMP-2-keto-3-deoxy-octanoic acid synthetase (CMP-KDO synthetase) as part of a polycistronic mRNA . The co-expression of these two enzymes suggests a role for CTP synthetase in lipopolysaccharide biosynthesis, potentially channelling CTP directly to CMP-KDO synthetase . The ability of the intact operon to complement CTP synthetase and CMP-KDO deficiencies in mutant Escherichia coli strains indicates that both enzymes are efficiently translated from a single messenger RNA . Kinetic analysis revealed that the C . trachomatis CTP synthetase possessed co-operativity patterns typical of both prokaryotic and eukaryotic CTP synthetases . However, the K(m) of the enzyme for UTP was lower than that of E . coli CTP synthetase, presumably in response to the low intracellular concentration of this nucleotide in C . trachomatis. Yeast, 1996 Nov, 12(14), 1459 - 69 Cloning and sequencing of the LYS1 gene encoding homocitrate synthase in the yeast Yarrowia lipolytica; Perez-Campo FM et al.; The alpha-aminoadipate pathway for the biosynthesis of lysine is present only in fungi and euglena . The first step in the pathway is the condensation of acetyl-CoA and alpha-ketoglutarate into homocitrate, and this step is carried out by the enzyme homocitrate synthase (EC 4.1.3.21) . In spite of extensive genetic analysis, no mutation affecting this step has been isolated until now in model organisms such as Saccharomyces cerevisiae or Neurospora crassa, although identification of mutations affecting the structural gene (LYS1) for homocitrate synthase was reported in the yeast Yarrowia lipolytica several years ago . Here we used these mutants for the cloning and sequencing of the Yarrowia LYS1 gene . The LYS1 gene encodes a predicted 446 amino acid polypeptide, with a molecular mass of 48442 Da . The Lys1p sequence displays two regions, one near the N-terminal section and the other in the central region, that contain conserved signatures found in prokaryotic homocitrate synthases (nifV genes of Azotobacter vinelandii and Klebsiella pneumoniae), as well as in all alpha-isopropyl malate synthases so far described . A putative mitochondrial targeting signal of 41-45 amino acids is predicted at the N-terminus . The Lys1p sequence shows 84% identity at the amino acid level with the putative product of open reading frame D1298 of S . cerevisiae . Northern blot hybridizations revealed a LYS1 transcript of approximately 1.7 kb in Y . lipolytica . Deletion of the LYS1 gene resulted in a Lys- phenotype . Our results indicate that we cloned the structural gene for homocitrate synthase in Y . lipolytica, and that the enzyme is encoded by a single gene in this yeast. Eur J Biochem, 1996 Nov 1, 241(3), 865 - 71 Bacterial triterpenoids of the hopane series from the prochlorophyte Prochlorothrix hollandica and their intracellular localization; Simonin P et al.; 35-O-beta-Galacturonopyranosyl-, 35-O-beta-3,5-anhydro-galacturonopyranosyl- and 35-O-alpha-altruronopyranosylbacteriohopanetetrol accompanied by their 2 beta-methyl homologues have been isolated from Prochlorothrix hollandica . We report here C35 triterpenoids of the hopane series in a prochlorophyte, a group of prokaryotic oxigenic phototrophs of the cyanobacterial lineage . Like many cyanobacteria, P . hollandica contains a mixture of non-methylated as well as 2 beta-methylhopanoids . After side-chain cleavage by periodic acid oxidation followed by sodium borohydride reduction, these hopanoids could be localized in cell walls and thylakoids, in accordance with their role as membrane stabilizers. Plant Physiol, 1996 Nov, 112(3), 1127 - 34 Cloning of an enzyme that synthesizes a key nucleotide-sugar precursor of hemicellulose biosynthesis from soybean: UDP-glucose dehydrogenase; Tenhaken R et al.; Hemicellulose is a major component of primary plant cell walls . Many of the glycosyl residues found in hemicellulose are derived from the sugar precursor UDP-glucuronic acid, which can be converted into UDP-arabinose, UDP-apiose, UDP-galacturonic acid, and UDP-xylose . The enzyme controlling the biosynthesis of UDP-glucuronic acid, UDP-glucose dehydrogenase (EC 1.1.1.22), was cloned from soybean (Glycine max {L.} Merr.) by an antibody screening procedure . This enzyme is surprisingly homologous to the bovine sequence, which is the only other eukaryotic UDP-glucose dehydrogenase sequence known . The characteristic motifs of the enzyme, the catalytic center, a NAD-binding site, and two proline residues for main chain bends, are conserved within the prokaryotic and eukaryotic sequences . The soybean full-length cDNA clone encodes a protein of 480 amino acids with a predicted size of 52.9 kD . The enzyme is highly expressed in young roots, but lower expression levels were observed in expanding tissues of the epicotyl and in young leaves . The expression pattern of the enzyme in different developmental stages strengthens the argument that UDP-glucose dehydrogenase is a key regulator for the availability of hemicellulose precursors. Nucleic Acids Res, 1996 Nov 1, 24(21), 4227 - 33 Characterization of a protein recognizing minor groove binders-damaged DNA; Colella G et al.; By using electromobility shift assay (EMSA), we have identified a protein able to recognize the DNA only if it was previously reacted with minor groove binders . This protein binds with very high affinity AT containing DNA treated with minor groove binders such as distamycin A, Hoechst 33258 and 33342, CC-1065 and ethidium bromide minor groove intercalator, but not with major groove binders such as quinacrine mustard, cisplatin or melphalan, or with topoisomerase I inhibitor camptothecin or topoisomerase II inhibitor doxorubicin . This protein was found to be present in different extracts of human, murine and hamster cells, with the human protein which appears to have a molecular weight slightly lower than that of the other species . This protein was found to be expressed both in cancer and normal tissues . By using molecular ultrafiltration techniques as well as southwestern analysis it was estimated that the apparent molecular weight is close to 100 kDa . We can exclude an identity between this protein and other proteins, with a similar molecular weight previously reported to be involved in DNA damage recognition/repair, such as topoisomerase I, mismatch repair activities such as the prokaryotic MutS protein and its human homologue hMSH2 or proteins of the nucleotide excision repair system such as ERCC1, -2, -3 and -4. J Bacteriol, 1996 Nov, 178(22), 6635 - 9 The nucleotide sequence of a linear plasmid of Borrelia burgdorferi reveals similarities to those of circular plasmids of other prokaryotes; Barbour AG et al.; A linear plasmid of Borrelia burgdorferi had 16,927 bp, a G+C content of 23.1%, a relative deficiency of CpG dinucleotides, and open reading frames A to O . The OrfC and OrfE proteins were similar to hypothetical proteins encoded by circular plasmids of B . burgdorferi . The OrfM and OrfN proteins were similar to replication proteins of circular plasmids of other bacteria. J Bacteriol, 1996 Nov, 178(22), 6618 - 22 Eukaryotic interference with homoserine lactone-mediated prokaryotic signalling; Givskov M et al.; Acylated homoserine lactones (AHLs) play a widespread role in intercellular communication among bacteria . The Australian macroalga Delisea pulchra produces secondary metabolites which have structural similarities to AHL molecules . We report here that these metabolites inhibited AHL-controlled processes in prokaryotes . Our results suggest that the interaction between higher organisms and their surface-associated bacteria may be mediated by interference with bacterial regulatory systems. Curr Genet, 1996 Nov, 30(5), 367 - 80 The role of protein degradation in mitochondrial function and biogenesis; Rep M et al.; It has been known for a long time that mitochondria contain their own protein-degradation systems . Only recently, however, have genes for mitochondrial proteases been identified and the powerful techniques of molecular biology been applied to gain insight into the role of protein degradation in mitochondrial biogenesis . It is now clear that the mitochondrial proteases that are involved in the initial stages of degradation are similar to prokaryotic ATP-dependent proteases, and that a division of labour exists between soluble and membrane-bound systems . These systems are essential for the biogenesis of fully functional mitochondria . Their natural targets are currently being identified, and their co-operation with chaperones and possible dual functions as chaperones/proteases are being investigated. Biol Rev Camb Philos Soc, 1996 Nov, 71(4), 637 - 702 Protein targeting and translocation; a comparative survey; Baker A et al.; The last few years has seen enormous progress in understanding of protein targeting and translocation across biological membranes . Many of the key molecules involved have been identified, isolated, and the corresponding genes cloned, opening up the way for detailed analysis of the structure and function of these molecular machines . It has become clear that the protein translocation machinery of the endoplasmic reticulum is very closely related to that of bacteria, and probably represents an ancient solution to the problem of how to get a protein across a membrane . One of the thylakoid translocation systems looks as if it will also be very similar, and probably represents a pathway inherited from the ancestral endosymbiont . It is interesting that, so far, there is a perfect correlation between thylakoid proteins which are present in photosynthetic prokaryotes and those which use the sec pathway in chloroplasts; conversely, OE16 and 23 which use the delta pH pathway are not found in cyanobacteria . To date, no Sec-related proteins have been found in mitochondria, although these organelles also arose as a result of endosymbiotic events . However, virtually nothing is known about the insertion of mitochondrially encoded proteins into the inner membrane . Is the inner membrane machinery which translocates cytoplasmically synthesized proteins capable of operating in reverse to export proteins from the matrix, or is there a separate system? Alternatively, do membrane proteins encoded by mitochondrial DNA insert independently of accessory proteins? Unlike nuclear-encoded proteins, proteins encoded by mtDNA are not faced with a choice of membrane and, in principle, could simply partition into the inner membrane . The ancestors of mitochondria almost certainly had a Sec system; has this been lost along with many of the proteins once encoded in the endosymbiont genome, or is there still such a system waiting to be discovered? The answer to this question may also shed light on the controversy concerning the sorting of the inter-membrane space proteins cytochrome c1 and cytochrome b2, as the conservative-sorting hypothesis would predict re-export of matrix intermediates via an ancestral (possibly Sec-type) pathway . Whereas the ER and bacterial systems clearly share homologous proteins, the protein import machineries of mitochondria and chloroplasts appear to be analogous rather than homologous . In both cases, import occurs through contact sites and there are separate translocation complexes in each membrane, however, with the exception of some of the chaperone molecules, the individual protein components do not appear to be related . Their similarities may be a case of convergent rather than divergent evolution, and may reflect what appear to be common requirements for translocation, namely unfolding, a receptor, a pore complex and refolding . There are also important differences . Translocation across the mitochondrial inner membrane is absolutely dependent upon delta psi, but no GTP requirement has been identified . In chloroplasts the reverse is the case . The roles of delta psi and GTP, respectively, remain uncertain, but it is tempting to speculate that they may play a role in regulating the import process, perhaps by controlling the assembly of a functional translocation complex . In the case of peroxisomes, much still remains to be learned . Many genes involved in peroxisome biogenesis have been identified but, in most cases, the biochemical function remains to be elucidated . In this respect, understanding of peroxisome biogenesis is at a similar stage to that of the ER 10 years ago . The coming together of genetic and biochemical approaches, as with the other organelles, should provide many of the answers. Biochem J, 1996 Nov 1, 319 ( Pt 3), 919 - 27 cDNA and genomic cloning and expression of the P48 monocytic differentiation/activation factor, a Mycoplasma fermentans gene product; Hall RE et al.; P48 is a 48 kDa monocytic differentiation/activation factor previously purified from the conditioned medium of the Reh human pre-B cell leukaemia cell line . It induces growth arrest and differentiation of HL-60 human promyelocytic leukaemia cells along the monocytic pathway and the production of the cytokines interleukin 1, tumour necrosis factor-alpha and interleukin 6 in human monocytes and monocytic cell lines . The cDNA for P48 was cloned from Reh cellular RNA using 3' reverse amplification of cDNA ends . Southern blot probing with P48 cDNA revealed hybridization with DNA from Reh and Molt-4 cells, but not with DNA from human peripheral blood mononuclear cells . Subsequent studies using PCR and Southern analysis revealed P48 sequences in DNA isolated from Mycoplasma fermentans but not M . hominis, M.iowae, M.synoviae or M.lypophilum . Although initial studies using Mycoplasma culture and hybridization techniques had failed to reveal Mycoplasma infection in our Reh and Molt-4 cell lines, subsequent PCR studies using Mycoplasma genus-specific rRNA primrs revealed Mycoplasma sequences in these cell lines . Using the P48 cDNA probe, we isolated a genomic clone from M . fermentans DNA which was found to be 98.5% identical with the P48 cDNA clone, and the deduced amino acid sequence agreed with N-terminal microsequencing data for P48 protein purified from the Reh cell line conditioned medium . The 5' end of the gene has a number of consensus sequences characteristic of prokaryotic genes, and the deduced amino acid sequence has a number of features suggesting that P48 is a lipoprotein . The P48 cDNA was expressed in pMAL in Escherichia coli, and the 60 kDa expressed fusion protein was found to react with anti-P48 antibodies on Western blots . This is consistent with a pMAL fusion protein representing the sum of the 42 kDa maltose-binding protein and 18 kDa of P48 recombinant protein, suggesting that native P48 has significant post-translational modification . Consistent with this, Northern blot studies revealed a single 1 kb transcript . The recombinant fusion protein was found to possess anti-proliferative activity against HL-60 cells, and antibodies against recombinant P48 were found to block the biological activity of native P48 isolated from conditioned medium . These studies demonstrate that P48, a molecule with immunomodulatory and haematopoietic differentiation activities, is derived from M . fermentans or a closely related species . P48 may be important in the pathophysiology of Mycoplasma infections and may be useful in dissecting the mechanisms involved in mammalian haematopoietic cell differentiation, immune function and cytokine biosynthesis. Mol Biol Evol, 1996 Nov, 13(9), 1233 - 41 Evolution of the frequency (frq) clock locus in Ascomycete fungi; Lewis MT et al.; The frequency (frq) locus of Neurospora crassa plays a key role in the organization of circadian rhythms . Similar timing systems have been found in nearly all eukaryotes as well as some prokaryotes; thus, frq may be an excellent gene with which to conduct evolutionary studies . To investigate, we used the cloned frq locus from ascomycete fungi representing two classical taxonomic classes and three orders to examine two open questions in ascomycete evolution . Class Pyrenomycetidae is represented by several species of Neurospora, Sordaria fimicola, and Chromocrea spinulosa; class Loculoascomycetidae is represented by the marine fungus Leptosphaeria australiensis . Generation of detailed restriction maps of homologs from the Neurospora species allows analysis of evolutionary relationships among these closely related species . A maximum-parsimony tree based on these restriction data suggests that Neurospora tetrasperma groups more closely with Neurospora sitophila than with Neurospora crassa using the homothallic species Neurospora galapagosensis as an outgroup . A maximum-parsimony tree derived using amino acid sequences from Neurospora crassa, Sordaria fimicola, Chromocrea spinulosa, and Leptosphaeria australiensis surprisingly suggests that Leptosphaeria austral should be classified within Pyrenomycetes rather than in a separate class . This suggestion is based on the observations that Leptosphaeria groups with Chromocrea on an evolutionary tree, is more closely related to Neurospora and Sordaria than is Chromocrea, and shares a conserved intron with Chromocrea . Together, these data show that frq is a useful gene with which to conduct evolutionary studies. J Virol, 1996 Nov, 70(11), 7706 - 12 Human recombinant antibody fragments neutralizing human immunodeficiency virus type 1 reverse transcriptase provide an experimental basis for the structural classification of the DNA polymerase family; Gargano N et al.; We describe in this paper the binding and biochemical properties of two human antibody fragments directed against the human immunodeficiency virus type 1 reverse transcriptase (RT) . These fragments were isolated from a synthetic combinatorial library of human Fab antibody fragments displayed on the surface of filamentous phage . The antibody fragments were selected by using recombinant heterodimeric human immunodeficiency virus type 1 RT purified from insect cells as a solid-phase selector . This procedure led to the isolation of two antibody fragments that completely neutralize the RNA-dependent DNA polymerase activity of RT at nanomolar concentrations . Both antibody fragments bind only to the enzymatically active form of the RT . The inhibitory activity of the anti-RT antibody fragments is competitive with respect to the template primer . The antibody fragments also neutralize the activities of RTs from avian and murine retroviruses and of DNA polymerases of prokaryotic origin as well as human DNA polymerase alpha . Thus, the antibody fragments selected and characterized in this study appear to recognize a structural fold that is common to the different DNA polymerases and necessary for their activity . The results provide an immunological experimental basis for a purely structural and evolutionary classification of the polymerase family. J Bacteriol, 1996 Nov, 178(21), 6238 - 49 Transcriptional control mediated by the ArcA two-component response regulator protein of Escherichia coli: characterization of DNA binding at target promoters; Lynch AS et al.; ArcA protein bearing an amino-terminal, oligohistidine extension has been purified, and its DNA binding activity has been characterized with or without prior incubation with carbamoyl phosphate . Electrophoretic mobility shift assays and DNase I protection assays indicate that where the phosphorylated form of the ArcA protein (ArcA-P) is expected to act as a transcriptional repressor (e.g., of lctPRD and gltA-sdhCDAB), the effect is likely to be mediated by sequestration of cis-controlling transcriptional regulatory elements . In contrast, in the case of cydAB, for which ArcA-P is expected to function as a transcriptional activator, two discrete binding sites have been identified upstream of a known promoter, and activation from these sites is likely to be mediated by a mechanism typical of the type I class of prokaryotic transcriptional activators . An additional ArcA-P binding site has also been located downstream of the known promoter, and a distinct role for this site in the regulation of the cydAB operon during anoxic growth transitions is suggested . These results are discussed within the framework of an overall model of signaling by the Arc two-component signal transduction system in response to changes in aerobiosis. J Bacteriol, 1996 Nov, 178(21), 6200 - 8 Characterization of the region encoding the CO-induced hydrogenase of Rhodospirillum rubrum; Fox JD et al.; In the photosynthetic bacterium Rhodospirillum rubrum, the presence of carbon monoxide (CO) induces expression of several proteins . These include carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase . Together these enzymes catalyze the following conversion: CO + H2O --> CO2 + H2 . This system enables R . rubrum to grow in the dark on CO as the sole energy source . Expression of this system has been shown previously to be regulated at the transcriptional level by CO . We have now identified the remainder of the CO-regulated genes encoded in a contiguous region of the R . rubrum genome . These genes, cooMKLXU, apparently encode proteins related to the function of the CO-induced hydrogenase . As seen before with the gene for the large subunit of the CO-induced hydrogenase (cooH), most of the proteins predicted by these additional genes show significant sequence similarity to subunits of Escherichia coli hydrogenase 3 . In addition, all of the newly identified coo gene products show similarity to subunits of NADH-quinone oxidoreductase (energy-conserving NADH dehydrogenase I) from various eukaryotic and prokaryotic organisms . We have found that dicyclohexylcarbodiimide, an inhibitor of mitochondrial NADH dehydrogenase I (also called complex I), inhibits the CO-induced hydrogenase as well . We also show that expression of the cooMKLXUH operon is regulated by CO and the transcriptional activator CooA in a manner similar to that of the cooFSCTJ operon that encodes the subunits of CODH and related proteins. J Neurochem, 1996 Nov, 67(5), 1882 - 96 Increased activity-regulating and neuroprotective efficacy of alpha-secretase-derived secreted amyloid precursor protein conferred by a C-terminal heparin-binding domain; Furukawa K et al.; Proteolytic cleavage of beta-amyloid precursor protein (beta APP) by alpha-secretase results in release of one secreted form (sAPP) of APP (sAPP alpha), whereas cleavage by beta-secretase releases a C-terminally truncated sAPP (sAPP beta) plus amyloid beta-peptide (A beta) . beta APP mutations linked to some inherited forms of Alzheimer's disease may alter its processing such that levels of sAPP alpha are reduced and levels of sAPP beta increased . sAPP alpha s may play important roles in neuronal plasticity and survival, whereas A beta can be neurotoxic . sAPP alpha was approximately 100-fold more potent than sAPP beta in protecting hippocampal neurons against excitotoxicity, A beta toxicity, and glucose deprivation . Whole-cell patch clamp and calcium imaging analyses showed that sAPP beta was less effective than sAPP alpha in suppressing synaptic activity, activating K+ channels, and attenuating calcium responses to glutamate . Using various truncated sAPP alpha and sAPP beta APP695 products generated by eukaryotic and prokaryotic expression systems, and synthetic sAPP peptides, the activity of sAPP alpha was localized to amino acids 591-612 at the C-terminus . Heparinases greatly reduced the actions of sAPP alpha s, indicating a role for a heparin-binding domain at the C-terminus of sAPP alpha in receptor activation . These findings indicate that alternative processing of beta APP has profound effects on the bioactivity of the resultant sAPP products and suggest that reduced levels of sAPP alpha could contribute to neuronal degeneration in Alzheimer's disease. Gene, 1996 Oct 31, 178(1-2), 63 - 9 Cloning, expression, purification and characterization of DNA topoisomerase I of Mycobacterium tuberculosis; Yang F et al.; The complete gene encoding Topoisomerase 1 (Topo I) from Mycobacterium tuberculosis (MTb), Erdman strain, has been isolated and sequenced . The coding region of this gene is 2700 nt encoding a polypeptide of 900 amino acids with a calculated molecular mass of 99353 Da . The amino-acid sequence identity compared to E . coli and Synechococcus Topo I is 22 and 30%, respectively . The gene was expressed in E . coli BL21(DE3) and purified to near homogeneity . Recombinant MTb Topo I is enzymatically active, relaxing negatively supercoiled DNA in a magnesium-dependent, ATP-independent reaction . Spermidine, a typical inhibitor of prokaryotic type I DNA topoisomerase, inhibits the activity . Unlike the more well-characterized E . coli Topo I, MTb Topo I does not contain a zinc-finger DNA-binding motif in the C-terminal domain of the protein. Mol Gen Genet, 1996 Oct 28, 252(6), 676 - 83 The 3' untranslated regions of chloroplast genes in Chlamydomonas reinhardtii do not serve as efficient transcriptional terminators; Rott R et al.; A general characteristic of the 3' untranslated regions of plastid mRNAs is an inverted repeat sequence that can fold into a stem-loop structure . These stem-loops are superficially similar to structures involved in prokaryotic transcription termination, but were found instead to serve as RNA 3' end processing signals in spinach chloroplasts, and in the atpB mRNA of Chlamydomonas reinhardtii chloroplasts . In order to carry out a broad study of the efficiency of the untranslated sequences at the 3' ends of chloroplast genes in Chlamydomonas to function as transcription terminators, we performed in vivo run-on transcription experiments using Chlamydomonas chloroplast transformants in which different 3' ends were inserted into the chloroplast genome between a petD promoter and a reporter gene . The results showed that none of the 3' ends that were tested, in either sense or antisense orientation, prevented readthrough transcription, and thus were not highly efficient transcription terminators . Therefore, we suggest that most or all of the 3' ends of mature mRNAs in Chlamydomonas chloroplasts are formed by 3' end processing of longer precursors.
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