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 w