Escherichia coli

Plant Mol Biol, 1993 Oct, 23(2), 387 - 96
Functional expression and molecular characterization of AtUBC2-1, a novel ubiquitin-conjugating enzyme (E2) from Arabidopsis thaliana; Bartling D et al.; The first member of a novel subfamily of ubiquitin-conjugating E2-proteins was cloned from a cDNA library of Arabidopsis thaliana . Genomic blots indicate that this gene family (AtUBC2) consists of two members and is distinct from AtUBC1, the only other E2 enzyme known from this species to date (M.L . Sullivan and R.D . Vierstra, Proc . Natl . Acad . Sci . USA 86 (1989) 9861-9865) . The cDNA sequence of AtUBC2-1 extends over 794 bp which would encode a protein of 161 amino acids and a calculated molecular mass of 18.25 kDa . The protein encoded by AtUBC2-1 is shown to accept 125I-ubiquitin from wheat E1 enzymes, when expressed from Escherichia coli hosts as fusion protein carrying N-terminal extensions . It is deubiquitinated in the presence of lysine and, by these criteria, is considered a functional E2 enzyme.

Plant Mol Biol, 1993 Oct, 23(2), 287 - 95
The plant mitochondrial open reading frame orf221 encodes a membrane-bound protein; Prioli LM et al.; We have shown that the open reading frame orf221 is an active mitochondrial gene which encodes a novel mitochondrial polypeptide . The orf221 sequence is common to higher plants but absent in animal and fungal mitochondria . A mitochondrial polypeptide with an apparent molecular weight of 21,000 was detected with a polyclonal antibody raised against an ORF221 fusion protein . In organello translation followed by immunoprecipitation with the anti-ORF221 antibody demonstrated that this polypeptide is encoded by the orf221 gene in plant mitochondria . The ORF221 was found to be a mitochondrial membrane protein in normal (N), cms-T, and cms-C cytoplasms of several inbred lines of maize (Zea mays L.) and in other plant species.

Am Rev Respir Dis, 1993 Oct, 148(4 Pt 1), 878 - 81
Changes of pulmonary glucocorticoid receptor and phospholipase A2 in sheep with acute lung injury after high dose endotoxin infusion; Liu LY et al.; In a sheep model of acute lung injury induced by an Escherichia coli endotoxin (5 micrograms/kg) with chronic lung lymph fistula (n = 15), we measured the changes in glucocorticoid receptor (GCR) binding capacity in lung tissue by means of radioligand binding assay . The content of cortisol and the activity of phospholipase A2 (PLA2) were also measured . The results showed that the maximal binding capacity (Bmax) of GCR in lung cytoplasma decreased continuously 2 h (113 +/- 3 versus 66 +/- 2 fmol/mg protein, p < 0.01), 4 h (105 +/- 6 versus 52 +/- 3 fmol/mg protein, p < 0.01), and 6 h (105 +/- 5 versus 37 +/- 2 fmol/mg protein, p < 0.01) after endotoxin infusion . Its affinity decreased markedly (p < 0.05) at 6 h after the infusion . The contents of cortisol in plasma elevated at 0.5 h and remained at a high level until 4 h after the infusion . PLA2 activity rose from 97 +/- 25 to 188 +/- 12 U (p < 0.05), 99 +/- 13 to 285 +/- 25 U (p < 0.01), and 106 +/- 14 to 354 +/- 32 U (p < 0.01) at 2, 4, and 6 h after endotoxin infusion, respectively . There was a negative correlation between the Bmax of GCR and PLA2 activity (r = -0.87, p < 0.01) . The findings indicate that there was a secondary GCR abnormality and a higher PLA2 activity during endotoxin-induced lung injury . The glucocorticoid hypofunction caused by reduced GCR binding capacity may accelerate the pathologic response of acute lung injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Mar Biol Biotechnol, 1993 Oct, 2(5), 280 - 90
Kinetic and physical properties of a recombinant RuBisCO from a chemoautotrophic endosymbiont; Stein JL et al.; Ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO), which catalyzes the key step in autotrophic CO2 fixation, is present at high activity in the symbiont-containing tissues of many hydrothermal vent invertebrates . The genes encoding RuBisCO from a gill endosymbiont of the hydrothermal vent gastropod Alviniconcha hessleri have been cloned, sequenced, and functionally expressed in Escherichia coli under control of the lac promoter in the vector pBlueScript . The purified protein is a hexadecamer (L8S8) with an apparent molecular weight of 554 kDa and a specific carboxylase activity of 2.9 mumol/min/mg protein . Unlike previously characterized RuBisCOs, which display sharp temperature optima, the symbiont RuBisCO maintains a high level of activity over a broad temperature span, although it is not thermally stable after extended exposure to temperatures above 50 degrees C . This funding suggests an adaptation to the thermal transients measured at vent openings where the host snail resides . The enzyme also maintains 90% of its 1 atm activity at 370 atm pressure, whereas spinach RuBisCO retains 28% activity under similar conditions . At 1 atm pressure and 20 degrees C, the Km(CO2) and the relative substrate specificity (Srel) of the symbiont enzyme were 80 and 32.5 mumol/L, respectively, which are similar to values reported for RuBisCOs from cyanobacteria and the purple photosynthetic bacteria . The relatively low specificity of the enzyme for substrate CO2 indicates that the intracellular environment of the endosymbionts may be microaerophilic for RuBisCO to maintain net carboxylation.

Wei Sheng Wu Xue Bao, 1993 Oct, 33(5), 331 - 8
{Oligonucleotide directed in vitro mutagenesis}; Su G; In this study, a method for oligonucleotide directed in vitro mutagenesis was described . It includes the following steps: clone of the gene to be mutated into the phagemid pGCI; preparation of single strand template; design and synthesis of mutated oligonucleotides; synthesis of double strand DNA . Using this method, a new BamHI site was originated at the N terminal of Stx-B and BglII site at the C terminal of Stx-B . It is ready to fuse the Stx-B gene to LamB . This method is a very useful tool for genetic engineering.

Int Surg, 1993 Oct-Dec, 78(4), 354 - 6
Neutrophil dynamics in abdominal cavity of peritonitic rats treated with antiseptics; Celdran A et al.; Peritonitis was induced in Wistar rats by intraperitoneal inoculation of pure Escherichia coli . Rats in which 2 ml of 1% povidone iodine had been injected intraperitoneally 5 min after the bacterial challenge, showed a decrease in the neutrophil percentage of the peritoneal cell population during the first hours of peritonitis compared to the control group . When the same experiment was performed with 2 ml of 0.05% chlorhexidine the percentage of neutrophils was superior to the control group in the first hours after bacterial challenge . These results concur with a previous study in which the deleterious effect of povidone iodine and the beneficial effect of chlorhexidine were demonstrated.

Indian J Biochem Biophys, 1993 Oct, 30(5), 252 - 6
Denatured supercoiled DNA--structural and biological activity; Santra CR et al.; Supercoiled DNA on treatment with NaOH followed by neutralization produces a condensed structure (form Id) . This structure does not split into topoisomers when run on long gel in presence of intercalating agents and the migration of this form does not change appreciably in presence or absence of ethidium bromide . Relaxation of form Id by topoisomerase I from pea chloroplast is facilitated more than form I . Single-stranded binding (SSB) protein binds more to form Id as evidenced from gel retardation study . Hydroxyl radical nicking is facilitated in this form . Compared to form I, this form produces half the number of transformants, but adsorption and penetration remain almost same in both the forms . Post-transformational growth using 32P labelled form I and form Id showed greater amount of degradation in form Id.

J Protein Chem, 1993 Oct, 12(5), 571 - 7
Reactivity of cysteinyl, arginyl, and lysyl residues of Escherichia coli phosphoenolpyruvate carboxykinase against group-specific chemical reagents; Bazaes S et al.; Calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli is not inactivated by a number of sulfhydryl-directed reagents {5,5'-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N'-(5-sulfo-1-naphthylethylenediamine)}, unlike phosphoenolpyruvate carboxykinase from other organisms . On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal . The substrates, ADP plus PEP in the presence of Mn2+, protect the enzyme against inactivation by the diones . Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme . Chemical modification by pyridoxal 5'-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine . The inactivation by this reagent is also prevented by the substrates . Binding stoichiometries of 1.25 and 0.30 mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively . The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.

Avian Dis, 1993 Oct-Dec, 37(4), 1092 - 6
Association of K-1 capsule, smooth lipopolysaccharides, traT gene, and Colicin V production with complement resistance and virulence of avian Escherichia coli; Wooley RE et al.; A group of complement-resistant, virulent avian Escherichia coli isolates were compared with a group of complement-sensitive, avirulent avian isolates for the presence of K-1 capsule, smooth lipopolysaccharides (LPS), the traT gene, and Colicin V (ColV) production . These parameters were selected because of their reported association with complement resistance and virulence in E . coli . Lethality in chicken embryos has also been shown to be correlated with virulence of avian E . coli for chickens . The complement-resistant, virulent E . coli isolates did not possess a K-1 capsule . Production of ColV and the presence of smooth LPS were significantly correlated with embryo lethality . There was no correlation between the presence of traT and embryo lethality . These results suggest that complement resistance and virulence in avian E . coli are associated with ColV production and smooth LPS but not with K-1 antigen or traT.

J Bioenerg Biomembr, 1993 Oct, 25(5), 483 - 92
Phosphate transport in mitochondria: past accomplishments, present problems, and future challenges; Ferreira GC et al.; The requirement of inorganic phosphate (Pi) for oxidative phosphorylation in eukaryotic cells is fulfilled through specific Pi transport systems . The mitochondrial proton/phosphate symporter (Pic) is a membrane-embedded protein which translocates Pi from the cytosol into the mitochondrial matrix . Pic is responsible for the very rapid transport of most of the Pi used in ATP synthesis . During the past five years there have been advances on several fronts . Genomic and cDNA clones for yeast, bovine, rat, and human Pic have been isolated and sequenced . Functional expression of yeast Pic in yeast strains deficient in Pi transport and expression in Escherichia coli of a chimera protein involving Pic and ATP synthase alpha subunit have been accomplished . Pic, in contrast to other members of the family of transporters involved in energy metabolism, was demonstrated to have a presequence, which optimizes the import of the precursor protein into mitochondria . Six transmembrane segments appear to be a structural feature shared between Pic and other mitochondrial anion carriers, and recent-site directed mutagenesis studies implicate structure-functional relationships to bacteriorhodopsin . These recent advances on Pic will be assessed in light of a more global interpretation of transport mechanism across the inner mitochondrial membrane.

J Bioenerg Biomembr, 1993 Oct, 25(5), 435 - 46
The mitochondrial transport protein superfamily; Walker JE et al.; The ADP/ATP, phosphate, and oxoglutarate/malate carrier proteins found in the inner membranes of mitochondria, and the uncoupling protein from mitochondria in mammalian brown adipose tissue, belong to the same protein superfamily . Established members of this superfamily have polypeptide chains approximately 300 amino acids long that consist of three tandem related sequences of about 100 amino acids . The tandem repeats from the different proteins are interrelated, and probably have similar secondary structures . The common features of this superfamily are also present in nine proteins of unknown functions characterized by DNA sequencing in various species, most notably in Caenorhabditis elegans and Saccharomyces cerevisiae . The high level expression in Escherichia coli of the bovine oxoglutarate/malate carrier, and the reconstitution of active carrier from the expressed protein, offers encouragement that the identity of superfamily members of known sequence but unknown function may be uncovered by a similar route.

Thromb Res, 1993 Oct 1, 72(1), 71 - 82
Fibrin-specific lysis of microthrombosis in endotoxemic rats by saruplase; Schneider J; The dissolution by the fibrinolytic agent saruplase of microthrombi due to disseminated intravascular coagulation (DIC) has been studied in anesthetized rats . The intravenous infusion of E . coli lipopolysaccharide (endotoxin) for 4 hours (total dose: 25 mg/kg) induced marked thrombocytopenia and hypofibrinogenemia . DIC-related microthrombosis, detected as increased deposition of 125I-labelled human fibrin, was found in the liver and the kidneys, but not in the lungs, the heart, the mesenterium, the spleen and the M . rectus abdominis of endotoxemic rats . Treatment with 1-20 micrograms/kg.min saruplase, that was infused concomitantly with endotoxin, dose-dependently and significantly reduced endotoxin-induced microthrombosis in the liver and the kidneys by 85 resp . 88% . When saruplase (20 micrograms/kg.min) was administered only during the last two hours of endotoxin infusion, liver microthrombosis was still significantly dissolved by 69%, whereas renal microthrombosis was insignificantly reduced by 34% . The inhibition of endotoxin-induced microthrombosis took place in the same dosage range as the shortening of the euglobulin clot lysis time in normal rats by saruplase as a measure of its fibrinolytic activity . Saruplase did not modify thrombocytopenia and hypofibrinogenemia in endotoxemic rats . Saruplase per se did not affect plasma fibrinogen levels . Thus, in a fibrin-selective dose range saruplase is able to dissolve microthrombosis associated with DIC in endotoxemic rats.

Mol Biol Rep, 1993 Oct, 18(3), 209 - 15
Comparison of the homologous carboxy-terminal domain and tail of alpha-crystallin and small heat shock protein; Merck KB et al.; The C-terminal domain and tail, which is the most conserved region of the alpha-crystallin/small heat shock protein (HSP) family, was obtained from rat alpha A-crystallin, bovine alpha B-crystallin and mouse HSP25 . All three domains have primarily beta-sheet conformation and less than 10% of alpha-helix, like the proteins from which they are derived . Whereas the C-terminal part of alpha A-crystallin forms dimers or tetramers, the corresponding regions of alpha B-crystallin and HSP25 form larger aggregates . The heat-protective activity, recently described for the alpha-crystallin/small HSP family, is not retained in the C-terminal domain and tail . In the course of this study some differences with the previously published sequence of HSP25 were observed, and a revision is proposed.

Mol Biol Rep, 1993 Oct, 18(3), 183 - 7
Polypyrimidine/polypurine sequence in plasmid DNA enhances formation of dimer molecules in Escherichia coli . Dimerization of plasmid DNA in Escherichia coli; Kato M; Formation of dimer molecules of a recombinant plasmid, pTIR10, which carries a pyrimidine/purine-biased stretch occurs about 6-fold more efficiently than for the control plasmid pUC19 in Escherichia coli strain JM107 . Since pyrimidine/purine-biased sequences have a potential to form unusual DNA structures, this observation suggests that the inserted sequence affects the replication process of plasmid DNA, probably by forming a triple helix under physiological conditions.

Proc Natl Acad Sci U S A, 1993 Oct 1, 90(19), 9120 - 4
Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties; Le Roux I et al.; In previous reports we have demonstrated that the 60-aa peptide corresponding to the homeodomain of the Drosophila protein Antennapedia (pAntp) translocates through the membrane of neurons in culture, accumulates in neuronal nuclei, and promotes neurite growth . To analyze the importance of specific pAntp DNA-binding properties in this phenomenon we have constructed three mutant versions of pAntp that differ in their ability to translocate through the membrane and to bind specifically the cognate sequence for homeodomains present in the promoter of HoxA5 . We demonstrate that removing two hydrophobic residues of the third helix inhibits pAntp internalization and suppresses its neurotrophic activity . We also show that pAntp neurotrophic activity is lost when mutations are introduced in positions preserving its penetration and nuclear accumulation but abolishing its capacity to bind specifically the cognate DNA-binding motif for homeoproteins . Our results strongly suggest that pAntp neurotrophicity requires both its internalization and its specific binding to homeobox cognate sequences . We propose that homeoproteins might regulate important events in the morphological differentiation of the postmitotic neuron.

Poult Sci, 1993 Oct, 72(10), 1823 - 31
Major histocompatibility complex class IV restriction fragment length polymorphism markers in replicated meat-type chicken lines divergently selected for high or low early immune response; Uni Z et al.; Information on MHC may improve the efficiency of selection for immunological traits via the application of marker assisted selection or by selecting directly for a specific restriction fragment length polymorphism (RFLP) band or MHC haplotype . An experimental procedure is presented here for identifying MHC genes that are related to early immune response . A Class IV cDNA clone was used to probe Southern blots of erythrocyte genomic DNA from chickens . Chickens were taken from the second (S2) and third (S3) generations of replicated lines divergently selected for high antibody response (HC1, HC2) or low antibody response (LC1, LC2) to Escherichia coli vaccination at 10 days of age . These selection criteria have been found to be associated with other immunological parameters . The hypothesis that these selected lines differ in their MHC loci was evaluated by comparing the frequencies of MHC RFLP markers (single RFLP bands) and haplotypes (patterns of RFLP bands) . The significant differences between LC and HC in the frequency of many MHC RFLP bands and of five MHC haplotypes indicate that early antibody production is influenced by MHC genes . The reliability of the association between the selection and frequency differences was tested and proven in most cases by analysis of the replicated lines . These differences in RFLP markers represent a change in allelic frequencies in MHC genes, probably due to selection . The results imply a connection between the Class IV genes and early antibody production, and they show the potential of prospective breeding not only by immunological phenotype but also by genotype (i.e., using RFLP markers of the MHC).

J Bacteriol, 1993 Oct, 175(19), 6186 - 93
Environmental regulation of the fim switch controlling type 1 fimbrial phase variation in Escherichia coli K-12: effects of temperature and media; Gally DL et al.; Expression of type 1 fimbriae in Escherichia coli K-12 is phase variable and associated with the inversion of a short DNA element (switch) . The fim switch requires either fimB (on-to-off or off-to-on switching) or fimE (on-to-off switching only) and is affected by the global regulators leucine-responsive regulatory protein (Lrp), integration host factor (IHF), and H-NS . Here it is shown that switching frequencies are regulated by both temperature and media and that these effects appear to be independent . fimE-promoted on-to-off switching occurs far more rapidly than previously estimated (0.3 per cell per generation in defined rich medium at 37 degrees C) and faster at lower than at higher temperatures . In direct contrast, fimB-promoted switching increases with temperature, with optima between 37 and 41 degrees C . Switching promoted by both fimB and fimE is stimulated by aliphatic amino acids (alanine, isoleucine, leucine, and valine), and this stimulation requires lrp . Furthermore, lrp appears to differentially regulate fimB- and fimE-promoted switching in different media.

Infect Immun, 1993 Oct, 61(10), 4518 - 22
Multivalent binding of K99 fimbriae to the N-glycolyl-GM3 ganglioside receptor; Willemsen PT et al.; The ganglioside N-glycolyl-GM3 binds laterally at numerous positions to K99 fimbriae, as shown by electron microscopic detection and erythrocyte-binding activity . The data demonstrate the multivalent nature of K99 fimbriae with respect to their receptor-binding sites.

Infect Immun, 1993 Oct, 61(10), 4480 - 4
Sialyloligosaccharide chains of laminin as an extracellular matrix target for S fimbriae of Escherichia coli; Virkola R et al.; S fimbriae purified from recombinant Escherichia coli HB101(pANN801-13) bound strongly to extracellular matrices of cultured endothelial and epithelial cells; only poor binding was seen with the fimbriae purified from the sfaS mutant strain HB101(pANN801-1321) . E . coli HB101(pANN801-13) adhered strongly to laminin immobilized on glass; no adhesion was seen to type I, III, IV, or V collagen . Strain HB101(pANN801-1321) failed to adhere to any of the target proteins . Adhesion to laminin of strain HB101(pANN801-13) was inhibited by sialyl-alpha-2,3-lactose as well as by periodate oxidation and neuraminidase treatment of laminin . In Western blotting, the purified S fimbriae recognized more strongly the A chain than the B chains of laminin.

J Infect Dis, 1993 Oct, 168(4), 1037 - 41
Distribution of the bundle-forming pilus structural gene (bfpA) among enteropathogenic Escherichia coli; Giron JA et al.; Enteropathogenic Escherichia coli (EPEC) express an inducible bundle-forming pilus (BFP) associated with the presence of the EPEC adherence factor (EAF) plasmid and localized adherence (LA) on HEp-2 cells . The cloned structural gene (bfpA) encoding BFP was found to be specific for EPEC, as homologous sequences were found only in EPEC and not in other enteropathogens . The bfpA probe was slightly more sensitive than the EAF probe; among EPEC strains with LA, the bfpA and EAF probes hybridized with 99% and 96% of the strains, respectively . Immunoblotting of whole cell lysates of BFP-positive, EAF-positive or -negative, and LA-positive or -negative EPEC strains revealed variations in the size (18,500-21,000) of the expressed structural subunit of BFP, suggesting differences in processing that may account for discrepancies between the bfpA, EAF, and LA . Because the bfpA probe consists primarily of sequences encoding an important EPEC virulence factor, in contrast to the unknown function of sequences contained in the EAF probe, the bfpA probe may be useful in epidemiologic studies to detect EPEC.

Rev Latinoam Microbiol, 1993 Oct-Dec, 35(4), 433 - 41
Mutagenic assessment of 1,N6-ethenodeoxyadenosine in DNA; Maldonado-Rodriguez R et al.; The mutagenic role of 1-N6-Ethenodeoxydenosine (epsilon A) was assessed by a genetic assay of mutations in the 5' coding region of the lacl gene of Escherichia coli . 1-N6-Etheno-2-deoxydadenosine 5'-triphosphate (epsilon dATP) was substituted for dATP during in vitro DNA synthesis on M13 recombinant uracil single stranded DNA bearing the lacl gene, catalyzed by the large fragment of E . coli DNA polymerase I . DNA products were transfected into a strain of E . coli lacking a chromosomal copy of the lacl gene, and i- phenotypic mutants were seen as blue plaques in the absence of inducer . Mutant progeny were characterized by dideoxy sequencing in the N-terminal region of the lacl gene where epsilon A had originally replaced A, and were found to have T-->C transitions . The frequency of base substitution mutation was different in each of three target sites tested . Taking into account the sequence changes and the coding properties at target sites, we conclude that in general, epsilon A increases the mutation frequency when compared with the control (transfection with unsubstituted DNA) . This increase was similar to that produced by in vitro primer elongation in absence of dATP . The combined results of the electrophoretic assay of primer elongation, measurements of mutation frequency, and sequence analysis of mutant phage progeny support the proposal that epsilon A in template DNA can be mutagenic through epsilon AC mispairing during in vivo replication.

Mol Microbiol, 1993 Oct, 10(1), 181 - 91
The response of the picoplanktonic marine cyanobacterium Synechococcus species WH7803 to phosphate starvation involves a protein homologous to the periplasmic phosphate-binding protein of Escherichia coli; Scanlan DJ et al.; During phosphate-limited growth the marine phycoerythrin-containing picoplanktonic cyanobacterium Synechococcus sp . WH7803 synthesizes novel polypeptides, including two abundant species of 100 kDa and 32 kDa . The 32 kDa polypeptide was localized to the cell wall, although in a related strain, Synechococcus sp . WH8103, it could be detected in both the cell wall fraction and the periplasm . The gene (designated pstS) encoding this polypeptide was cloned and shown to be present in a single copy . The deduced amino acid sequence indicated a polypeptide consisting of 326 amino acids with a calculated M(r) of 33,763 . Comparison of this sequence with that obtained by microsequencing the N-terminus of the 32 kDa polypeptide showed that the mature protein was synthesized as a precursor, the first 24 amino acid residues being cleaved between two alanine residues at positions 24 and 25 . The amino acid sequence of the mature polypeptide showed 35% identity and 52% similarity to the periplasmic phosphate-binding protein (PstS) from Escherichia coli, including three regions of much stronger homology which, by comparison with E . coli PstS, are directly involved in phosphate binding . Northern blot analysis revealed a pstS transcript of 1.2 kb in RNA extracted from cells grown in Pi-replete conditions and one of 1.4 kb in considerably increased abundance under Pi-depleted conditions . Homologues of the pstS gene were detected in other marine phycoerythrin-containing Synechococcus strains, but not in freshwater or halotolerant species.

Mol Microbiol, 1993 Oct, 10(1), 171 - 9
Proteolytic cleavage at arginine residues within the hydrophilic disulphide loop of the Escherichia coli Shiga-like toxin I A subunit is not essential for cytotoxicity; Burgess BJ et al.; Escherichia coli Shiga-like toxin I is a type II ribosome-inactivating protein composed of an A subunit with RNA-specific N-glycosidase activity, non-covalently associated with a pentamer of B subunits possessing affinity for galabiose-containing glycolipids . The A subunit contains a single intrachain disulphide bond encompassing a hydrophilic sequence containing two trypsin-sensitive arginine residues . By analogy with other bacterial toxins it has been proposed that proteolytic nicking, deemed essential for a cytotoxic effect, occurs within this disulphide-bonded loop to generate the A1 and A2 fragments . Reduced A1 is then believed to translocate an internal membrane to inactivate protein synthesis in the cytosol . In this report, the disulphide-loop arginines of the SLT I A subunit were mutated to block the specific proteolysis presumed to occur . However, the mutant generated remained an effective toxin having similar catalytic activity to wild-type toxin and only a marginally reduced cytotoxicity towards cultured cells . We conclude that the disulphide-loop arginine residues are not the unique and essential processing sites previously assumed, but that processing may occur at alternative accessible sites to compensate for loss of target sites within the loop.

Mol Microbiol, 1993 Oct, 10(1), 157 - 70
Analysis of the transfer region of the Streptomyces plasmid SCP2; Brolle DF et al.; pIJ903, a bifunctional derivative of the 31.4 kb low-copy-number, conjugative Streptomyces plasmid SCP2*, was mutagenized in Streptomyces lividans using Tn4560 . Mutant plasmids differing in their transfer frequencies, chromosome mobilization abilities, pock formation, and complementation properties were isolated . The mutations defined five transfer-related genes, traA, traB, traC, traD and spd, clustered in a region of 9 kb . The deduced sequences of the putative TraA and TraB proteins showed no overall similarity to known protein sequences, but the phenotype of traA mutant plasmids and sequence motifs in the putative TraA protein suggested that it might be a DNA helicase.

Mol Microbiol, 1993 Oct, 10(2), 407 - 20
Identification and characterization of stationary phase-inducible genes in Escherichia coli; Weichart D et al.; During transition into stationary phase a large set of proteins is induced in Escherichia coli . Only a minority of the corresponding genes has been identified so far . Using the lambda placMu system and a plate screen for carbon starvation-induced fusion activity, a series of chromosomal lacZ fusions (csi::lacZ) was isolated . In complex medium these fusions were induced either during late exponential phase or during entry into stationary phase . csi::lacZ expression in minimal media in response to starvation for carbon, nitrogen and phosphate sources and the roles of global regulators such as the alternative sigma factor sigma s (encoded by rpoS), cAMP/CRP and the relA gene product were investigated . The results show that almost every fusion exhibits its own characteristic pattern of expression, suggesting a complex control of stationary phase-inducible genes that involves various combinations of regulatory mechanisms for different genes . All fusions were mapped to the E . coli chromosome . Using fine mapping by Southern hybridization, cloning, sequencing and/or phenotypic analysis, csi-5, csi-17, and csi-18 could be localized in osmY (encoding a periplasmic protein), glpD (aerobic glycerol-3-phosphate dehydrogenase) and glgA (glycogen synthase), respectively . The other fusions seem to specify novel genes now designated csiA through to csiF . csi-17(glpD)::lacZ was shown to produce its own glucose-starvation induction, thus illustrating the intricacies of gene-fusion technology when applied to the study of gene regulation.

Mol Microbiol, 1993 Oct, 10(2), 341 - 50
A lowered concentration of cAMP receptor protein caused by glucose is an important determinant for catabolite repression in Escherichia coli; Ishizuka H et al.; A decreased intracellular concentration of cAMP is insufficient to account for catabolite repression in Escherichia coli . We show that glucose lowers the amount of cAMP receptor protein (CRP) in cells . A correlation exists between CRP and beta-galactosidase levels in cells growing under various conditions . Exogenous cAMP completely eliminates catabolite repression in CRP-overproducing cells, while it does not fully reverse the effect of glucose on beta-galactosidase expression in wild-type cells . When the CRP concentration is reduced by manipulating the crp gene, beta-galactosidase expression decreases in proportion to the concentration of CRP . These findings indicate that the lowered concentration of CRP caused by glucose is one of the major factors for catabolite repression . We propose that glucose causes catabolite repression by lowering the intracellular levels of both CRP and cAMP.

Mol Microbiol, 1993 Oct, 10(2), 245 - 51
The galactose regulon of Escherichia coli; Weickert MJ et al.; Galactose transport and metabolism in Escherichia coli involves a multicomponent amphibolic pathway . Galactose transport is accomplished by two different galactose-specific transport systems . At least four of the genes and operons involved in galactose transport and metabolism have promoters containing similar regulatory sequences . These sequences are recognized by at least three regulators, Gal repressor (GalR), Gal isorepressor (GalS) and cAMP receptor protein (CRP), which modulate transcription from these promoters . The negative regulators, GalR and GalS, discriminate between utilization of the high-affinity (regulated by GalS) and low-affinity (regulated by GalR) transport systems, and modulate the expression of genes for galactose metabolism in an overlapping fashion . GalS is itself autogenously regulated and CRP dependent, while the gene for GalR is constitutive . The gal operon encoding the enzymes for galactose metabolism has two promoters regulated by CRP in opposite ways; one (P1) is stimulated and the other (P2) inhibited by CRP . Both promoters are strongly repressed by GalR but weakly by GalS . All but one of the constituent promoters of the gal regulon have two operators . The gal regulon has the potential to coordinate galactose metabolism and transport in a highly efficient manner, under a wide variety of conditions of galactose availability.

Mol Microbiol, 1993 Oct, 10(2), 225 - 31
Control and function of lysyl-tRNA synthetases: diversity and co-ordination; Nakamura Y et al.; Lysyl-tRNA synthetases are synthesized from two distinct genes in Escherichia coli, lysS (constitutively) and lysU (inducibly); however, the physiological significance and the differential control mechanism of these two genes have been a long-standing puzzle . Recent studies have successfully uncovered a significant control mechanism of lysU expression, which involves the leucine-responsive regulatory protein (Lrp) and a translational enhancer element called 'downstream box' . Moreover, it is likely that there is a mechanism underlying co-ordinate expression of lysU with other genes outside the leucine-Lrp regulon under harsh conditions such as low pH and anaerobiosis . A possible mechanism of lysyl-tRNA synthetase expression and function is reviewed.

Microb Pathog, 1993 Oct, 15(4), 283 - 91
Expression of a truncated guanylate cyclase (GC-C), a receptor for heat-stable enterotoxin of enterotoxigenic Escherichia coli, and its dimer formation in COS-7 cells; Hirayama T et al.; A fragment of guanylate cyclase C (GC-C) of about 1.7 k bp corresponding to amino acids 1-553 spanning the extracellular and transmembrane domains and a portion of the intracellular region was amplified using template cDNA prepared from rat intestinal cells by the polymerase chain reaction method . The cloned 1.7 k bp fragment was inserted into the mammalian expression vector pCGUT and the truncated GC-C expressed on the surface of COS-7 cells was demonstrated to bind heat-stable enterotoxin by photo affinity labeling with 125I-N-5-azidonitrobenzoyl-STh{5-19} . Analysis by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis showed that the truncated GC-C formed dimers on the surface of COS-7 cells . The intracellular region of GC-C was found not to be necessary for dimer formation by the GC-C . Comparison of the molecular weights of the truncated GC-C expressed in COS-7 cells and Escherichia coli suggested that the truncated GC-C was glycosylated in the mammalian expression system.

Vet Microbiol, 1993 Oct, 37(1-2), 101 - 14
A monoclonal antibody identifies 2134P fimbriae as adhesins on enterotoxigenic Escherichia coli isolated from postweaning pigs; Dean-Nystrom EA et al.; Fimbriae (pili) of enterotoxigenic Escherichia coli (ETEC), including K88, K99, 987P, and F41, are adhesins that facilitate intestinal colonization in neonatal pigs . K88 is also associated with some ETEC isolated from weaned pigs . Many ETEC isolates from weaned pigs do not express known adhesins and are termed 4P- . A novel bacterial adhesin, 2134P, was recently identified on two 4P- ETEC isolates from weaned pigs . In this study, we identified a 2134P-specific monoclonal antibody, mAb 6C7/C1, that blocked the binding of 2134P+ bacteria to intestinal epithelial cells . Indirect immunofluorescent antibody and immunoperoxidase assays using mAb 6C7/C1 confirmed that the 2134P adhesin is expressed in vivo by adherent bacteria in pigs challenge-exposed with 2134P+ ETEC . 2134P was detected on 31% of 189 postweaning diarrhea 4P- ETEC isolates from the National Animal Disease Center's culture collection by dot blot immunoperoxidase assays using mAb 6C7/C1 . We conclude that 2134P is a bacterial adhesin and is an important virulence attribute of some ETEC that cause diarrhea in weaned pigs.

Anim Genet, 1993 Oct, 24(5), 393 - 6
Evidence for linkage between K88ab, K88ac intestinal receptors to Escherichia coli and transferrin loci in pigs; Guerin G et al.; Segregation at the loci coding for the K88ab and K88ac small intestinal receptors to E . coli adhesins (K88abR, K88acR) and at the transferrin (TF) locus was studied in 38 pig families including 273 piglets . The TF locus showed a segregation deviation towards the B variant while each of the K88 receptors behaved as a single autosomal dominant gene . Recombinants between K88abR and K88acR provide evidence that they are under the control of two different loci . Thirty-two triple backcross families were selected to test linkage and estimate recombination rates (theta) . Our results demonstrate that the two K88 receptor loci are closely linked (theta = 0.02) with a maximum lod score value (Zm) of 46.0 . In addition, they are linked to the TF locus, theta = 0.14, Zm = 19.6 for the K88abR locus and theta = 0.16, Zm = 17.9 for the K88acR locus . The estimated recombination rates, smaller in males than in females, are consistent with the order TF-K88abR-K88acR . This linkage thus localizes the K88 loci, as the TF locus, on chromosome 13.

Anim Genet, 1993 Oct, 24(5), 333 - 8
A linkage group on pig chromosome 4 comprising the loci for blood group L, GBA, ATP1B1 and three microsatellites; Marklund L et al.; Restriction fragment length polymorphisms (RFLPs) were described for the porcine loci for beta-glucosidase (GBA) and the beta-polypeptide 1 of the Na+,K(+)-transporting ATPase (ATP1B1) . Linkage analyses using a three-generation pedigree provided evidence for the assignment of ATP1B1, GBA and two microsatellite loci (S0001 and S0067) to a previously described linkage group comprising the loci for blood group L (EAL) and an anonymous microsatellite (S0097) . The linear order of the six markers was determined with confidence by multipoint analyses and the length of the linkage group was estimated at 88cM . This linkage group was assigned to pig chromosome 4 on the basis of a previous physical localization of the ATP1B1 gene . In situ hybridization data for S0001 presented in this study were consistent with a localization on chromosome 4 and suggested a regional localization to 4p12-p13 . The present study reveals conflicting data concerning the genetic localization of the K88 loci controlling the expression of the receptors for the E . coli pilus antigens . One group has reported data suggesting a loose linkage between K88 and EAL, now mapped to chromosome 4, whereas two other groups have found linkage between K88 and the transferrin locus (TF), mapped to chromosome 13 by in situ hybridization.

J Biochem (Tokyo), 1993 Oct, 114(4), 468 - 72
Self-assembly of symbionin, a chaperonin of intracellular symbiont; Morioka M et al.; Symbionin, a homologue of Escherichia coli GroEL, which functions as a molecular chaperone in the aphid endosymbiont, exists as a double-doughnut structure, consisting of two rings of seven 63-kDa subunits . Symbionin had a more labile oligomeric structure than GroEL and completely disassembled into monomeric (1-mer) components in 3 M urea . The urea-dissociated symbionin self-assembled into 14-mer symbionin in a Mg-ATP dependent manner . When 1-mer symbionin was incubated in the presence of Mg-ATP, its reassembly proceeded hyperbolically with time . The yield of reassembled symbionin increased in response to increase in the initial concentration of 1-mer symbionin . The reassembled symbionin not only had the same molecular mass as native symbionin but also exhibited the same ATPase and phosphotransferase activity, suggesting that the correct three-dimensional structure was restored in vitro . While the self-assembly of symbionin was markedly stimulated by the presence of reassembled symbionin, it was not affected by the presence of native symbionin, which may suggest that native symbionin contains component(s) inhibitory to its chaperoning activity . As in the self-assembly of GroEL, that of symbionin was stimulated by the presence of GroES.

Mol Biochem Parasitol, 1993 Oct, 61(2), 159 - 69
Cloning of the triosephosphate isomerase gene of Plasmodium falciparum and expression in Escherichia coli; Ranie J et al.; A major supply of energy in the rapidly multiplying intraerythrocytic Plasmodium falciparum is from the glycolytic pathway . We have isolated the cDNA and genomic clones of the glycolytic enzyme, triosephosphate isomerase (TPI) by polymerase chain reaction (PCR) . Degenerate oligonucleotides obtained by reverse translation of conserved polypeptide sequences derived from TPIs of other organisms, were used to prime PCR on P . falciparum DNA . The P . falciparum TPI gene is interrupted by a single intron which divides the coding region into two exons . The coding region encodes a protein of 248 amino acids which is of the same size as TPIs from other organisms and shares 42-45% homology with other known eukaryotic TPIs . On comparison with human TPI the catalytic domain was found to be highly conserved, while significant variations occurred at the other regions in the protein sequence . The P . falciparum TPI gene was cloned into the expression vector pTrc99A and hyperexpressed as an unfused protein in Escherichia coli . The 28-kDa protein was shown to be catalytically active.

Enferm Infecc Microbiol Clin, 1993 Oct, 11(8), 437 - 40
{DNA probe for the detection of uropathogenic strains of P-fimbriated Escherichia coli}; Rodriguez E et al.; BACKGROUND: P fimbria is one of the main factors of virulence of the uropathogenic Escherichia coli strains thus developing methods for its detection is of interest . P fimbriation may manifest through associated characteristic hemagglutination patterns . Another way of directly detecting its expression is by the PF test consisting in a specific agglutination test with latex particles which have the specific receptor of the fimbria incorporated . METHODS: The two phenotypic techniques (hemagglutination pattern using human, bovine, and sheep erythrocytes, and the PF test) were compared with colony hybridization with a specific DNA probe (pap1) in 35 strains of uropathogenic E . coli . RESULTS: Eight of the 35 strains studied were positive for the PF test with 7 strains presenting mannose-resistant agglutination to human erythrocytes without agglutinating the other erythrocytes tried . However, with hybridization with the DNA probe the number of positives was higher (25/35) . CONCLUSIONS: The difference found in the number of positive strains may be due to the probe used corresponding to cluster pap, thus the use of a smaller more specific probe for fimbrial expression obtained from pap should be used given that the hybridization technique is easy to perform and is carried out in less time than phenotypic detection which requires long periods of culture prior to the test.

Curr Opin Biotechnol, 1993 Oct, 4(5), 564 - 72
Baculovirus systems for the expression of human gene products; Luckow VA; Significant advances in basic and applied biology have resulted from the use of baculovirus vectors for the expression of heterologous proteins in cultured insect cells and in insect larvae . The development of improved vectors has greatly facilitated the construction of recombinant baculoviruses, both by increasing the efficiency of identifying recombinant viruses and by reducing or eliminating the tedious steps used to purify the desired recombinant virus from its non-recombinant parent virus.

Curr Opin Biotechnol, 1993 Oct, 4(5), 520 - 5
Recent advances in heterologous gene expression in Escherichia coli; Olins PO et al.; Recent advances in protein expression in E . coli have focused primarily on the enhancement of protein quality . Problems in mRNA translation such as inefficient initiation, mistranslation, frame-shifting and frame-hopping can often be addressed by altering heterologous gene-coding sequences . Fusion technology can also be used to address problems in translational initiation, the authenticity of amino-terminal amino acids, in vivo protein activity and protein purification . Accessory molecules, such as chaperones, are increasingly used to enhance protein quality in vivo and in vitro . E . coli has recently gained wide use as a host both for the engineering of proteins with altered activities and for the creation of multi-functional hybrids.

Biotechnology (N Y), 1993 Oct, 11(10), 1166 - 70
High level production of hybrid potyvirus-like particles carrying repetitive copies of foreign antigens in Escherichia coli; Jagadish MN et al.; Synthesis in E . coli of native coat protein of Johnsongrass mosaic virus, and hybrid protein molecules containing foreign antigens, resulted in the intracellular formation of potyvirus-like particles (PVLPs) . The foreign antigens used were an octapeptide epitope from Plasmodium falciparum and a decapeptide hormone (luteinizing hormone releasing hormone) at the N- or at both N- and C-terminal regions of the coat protein molecule, and a full length protein antigen (Sj26-glutathione S-transferase of 26 kD from Schistosoma japonicum) replacing the N-terminal 62 amino acids of the coat protein . Electron microscopy of ultrathin sections of E . coli revealed that PVLPs resulting from coat protein molecules containing peptide fusions appeared in vast arrays of parallel strands within the cytoplasm sometimes extending the length of the cell and at times the cells were strung together, with "threads" of PVLPs appearing to connect individual bacterial cells . PVLPs resulting from the fusion of the 26 kD antigen Sj26 to coat protein were shorter and wider . The physical form of the high molecular weight PVLPs enabled purification by simple size exclusion column chromatography . The Sj26-PVLPs administered to mice without adjuvant elicited antibody responses comparable to monomeric Sj26 administered with Freund's Complete Adjuvant.

Biotechnology (N Y), 1993 Oct, 11(10), 1138 - 43
Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli; Schatz PJ; I describe a technique for screening peptide libraries of over 10(9) independent clones for substrates of peptide-modifying enzymes . The peptides, linked to their genetic material by the lac repressor, are exposed to the enzyme and then screened by affinity purification on a receptor specific for the modified product . The enzyme characterized, E . coli biotin holoenzyme synthetase, normally adds biotin to a specific lysine residue in complex protein domains . The 13 residue substrate identified by this library screening approach is much smaller than the 75 amino acid required sequence of the natural substrate, and can function at either end of a fusion protein . The sequence is quite distinct at some positions from that region of the natural substrates, presumably because the peptides have to mimic the folded structure formed by the natural substrate . This technique should be useful for mapping the substrate specificity of a variety of peptide-modifying enzymes . In addition, small peptide substrates that are enzymatically biotinylated at a single site should be useful for a variety of purposes in labeling, purification, detection, and immobilization of proteins.

Gut, 1993 Oct, 34(10), 1405 - 11
Electrogenic colonic ion transport in Hirschsprung's disease: reduced secretion to the neural secretagogues acetylcholine and iloprost; Hardy SP et al.; The effects of the abnormal innervation in Hirschsprung's disease on colonic ion transport were examined in vitro using Ussing chambers . The response of the mucosal/submucosal preparations to different secretagogues were investigated in aganglionic and ganglionic rectosigmoid and transverse colon from children with Hirschsprung's disease and compared with normally innervated colon from children with anorectal anomalies . Basal values were similar in aganglionic and ganglionic rectosigmoid colon . Neurally mediated secretion with iloprost (10(-6) M) and acetylcholine (900 and 9 microM) was considerably reduced in aganglionic colon compared with normally innervated ganglionic colon . The ganglionic colon proximal to the aganglionic colon also had a reduced response to acetylcholine despite a normal acetylcholinesterase staining pattern . The responses to Escherichia coli STa enterotoxin (50 MU/ml) and isobutylmethylxanthine (10(-3) M) were similar in ganglionic and aganglionic colon . The response to STa enterotoxin was not changed by the nerve blocking agent tetrodotoxin (10(-6) M) . The data show that colonocytes from aganglionic colon are capable of a normal secretory response if stimulated directly by cAMP or cGMP acting secretagogues but secretion in response to neurally mediated secretagogues is impaired . The hypertrophied acetylcholinesterase positive nerve fibres that infiltrate the aganglionic colon are likely to contribute to the reduced secretion to acetylcholine.

J Lab Clin Med, 1993 Oct, 122(4), 388 - 94
Inhibitors of nitric oxide synthase attenuate human neutrophil chemotaxis in vitro; Belenky SN et al.; Products released through the L-arginine/nitric oxide biosynthetic pathway regulate soluble guanyl cyclase activity, which in turn modulates polymorphonuclear leukocyte chemotaxis . We hypothesized that inhibitors of nitric oxide synthase attenuate polymorphonuclear leukocyte chemotaxis in vitro . To test this hypothesis, unstimulated polymorphonuclear leukocytes were pretreated with buffer or the nitric oxide synthase inhibitors NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester, and L-canavanine before being exposed to three structurally unrelated chemoattractants, N-formyl-methionyl-leucyl-phenylalanine, C5a des arginine, and leukotriene B4 . Polymorphonuclear leukocyte chemotaxis was quantified with a modified blind-well chamber technique . We found that L-NMMA and L-canavanine but not NG-nitro-L-arginine significantly attenuated polymorphonuclear leukocyte chemotaxis (p < 0.05) . L-Arginine but not D-arginine, the nitric oxide donor sodium nitroprusside, and 8-bromo-cyclic guanosine monophosphate restored polymorphonuclear leukocyte chemotaxis attenuated by L-NMMA . Chemotaxis of polymorphonuclear leukocytes primed with lipopolysaccharide (Escherichia coli 0127:B8) or phorbol-13-butyrate was also significantly attenuated by pretreatment with L-NMMA and L-canavanine . Consistent with these observations, intracellular concentrations of cyclic guanosine monophosphate in polymorphonuclear leukocytes was decreased by L-NMMA during exposure to N-formyl-methionyl-leucyl-phenylalanine . These data indicate that nitric oxide synthase inhibitors attenuate chemotaxis of unstimulated and primed polymorphonuclear leukocytes in vitro . We suggest that the L-arginine/nitric oxide biosynthetic pathway plays an important role in regulating polymorphonuclear leukocyte emigration in vivo.

Proc Natl Acad Sci U S A, 1993 Oct 1, 90(19), 8763 - 8
An operational RNA code for amino acids and possible relationship to genetic code; Schimmel P et al.; RNA helical oligonucleotides that recapitulate the acceptor stems of transfer RNAs, and that are devoid of the anticodon trinucleotides of the genetic code, are aminoacylated by aminoacyl tRNA synthetases . The specificity of aminoacylation is sequence dependent, and both specificity and efficiency are generally determined by only a few nucleotides proximal to the amino acid attachment site . This sequence/structure-dependent aminoacylation of RNA oligonucleotides constitutes an operational RNA code for amino acids . To a rough approximation, members of the two different classes of tRNA synthetases are, like tRNAs, organized into two major domains . The class-defining conserved domain containing the active site incorporates determinants for recognition of RNA mini-helix substrates . This domain may reflect the primordial synthetase, which was needed for expression of the operational RNA code . The second synthetase domain, which generally is less or not conserved, provides for interactions with the second domain of tRNA, which incorporates the anticodon . The emergence of the genetic from the operational RNA code could occur when the second domain of synthetases was added with the anticodon-containing domain of tRNAs.

Mutat Res, 1993 Oct, 297(3), 313 - 21
Some aspects of EMS-induced mutagenesis in Escherichia coli; Grzesiuk E et al.; AB2497 and its mutS and umuDC derivatives were EMS-treated at the stationary phase and specificity of mutation measured . It was found that: (i) in mutS+ cells EMS induces predominantly GC-->AT transitions (by supB or supE(oc) formation) and in mutS- cells mainly AT-->TA transversions (by supL(NG) formation); (ii) transversions of AT-->TA are umuDC-dependent and mutational specificity is biased towards AT-->GC transitions in mutS- umuDC- strains . When mutS- umuDC- cells were transfected with plasmids bearing umuD'C or umuDC genes, mutational specificity was again biased towards AT-->TA transversions; (iii) experiments with bacteria bearing umuC::lacZ or recA::lacZ fusions suggest that processing of UmuD-->UmuD' might be poorer in EMS-treated mutS- than in mutS+ cells.

Mutat Res, 1993 Oct, 294(3), 317 - 23
Analysis of mutations caused by DNA double-strand breaks produced by a restriction enzyme in shuttle vector plasmids propagated in ataxia telangiectasia cells; Tatsumi-Miyajima J et al.; Rejoining of DNA double-strand breaks (DSB) produced by a restriction endonuclease AvaI in the supF gene in a plasmid pZ189Ava, and mutations presumably due to the altered rejoinings were analyzed . After allowing the rejoining and replication of the plasmids in human cells originating from normal subjects and ataxia telangiectasia (AT) patients, the plasmids were retrieved and those containing mutated supF were screened in an indicator strain of Escherichia coli . The proportion of correctly rejoined plasmids was significantly lower in AT cells than in normal cells, suggesting that AT cells have lower fidelity in rejoining DSB . DNA sequencing of the mutated supF genes revealed that all mutations were deletions or insertions occurring exactly or closely at the rejoining site in both normal and AT cells . In AT cells, the majority of mutations were deletions, while deletions and insertions were evenly formed in normal cells . AT cells may be deficient in the mechanism to protect the broken ends of DNA strands from the exonucleolytic digestion.

Mutat Res, 1993 Oct, 294(3), 263 - 74
The first zinc-binding domain of UvrA is not essential for UvrABC-mediated DNA excision repair; Visse R et al.; Specific mutations in uvrA were introduced to analyze the role of the zinc-binding domains of the protein in DNA excision repair . Zinc-coordinating cysteines were substituted into non-coordinating serine or glycine residues . Mutations leading to changes in the second zinc-binding domain had a profound effect on UV survival in vivo; however these mutant proteins could not be isolated for in vitro analyses . Amino acid substitutions in the first zinc-binding domain had very little effect on UV survival in vivo . In vitro analyses showed that although this domain no longer coordinates zinc, ATPase activity, helicase activity, DNA binding, incision of damaged DNA and DNA repair synthesis appeared to be normal . Therefore it seems that the first zinc-binding domain of UvrA is not essential for DNA excision repair.

Mutat Res, 1993 Oct, 294(3), 223 - 34
mei-3, a recombination and repair gene of Neurospora crassa, encodes a RecA-like protein; Cheng R et al.; Neurospora crassa mei-3 is a mutant which exhibits meiotic and mitotic defects and mutagen sensitivity . Its defect is believed to be in recombination and repair . We have cloned the mei-3 gene from a N . crassa cosmid library of genomic DNA . Restriction fragment length polymorphism analysis determined the location of the cloned fragment was on chromosome one in approximately the same position that was previously reported for mei-3 by classical genetic methods . Deletion analysis showed the approximate coding region of mei-3 on the cloned genomic fragment . Northern blot analysis identified a 900-bp transcript . Sequencing revealed a 798-bp open reading frame with high coding preference which could encode a protein having a molecular weight of approximately 29,000 . The predicted protein product of mei-3 has significant identity to the Rad51 and Dmc1 proteins from Saccharomyces cerevisiae . The mei-3 gene and both yeast genes have significant primary sequence homology with RecA, a recombination protein identified in Escherichia coli . The results suggest RecA-like proteins involved in DNA recombination and repair are highly conserved in eukaryotes.

Mutat Res, 1993 Oct, 294(3), 215 - 22
Excision repair reduces doxorubicin-induced genotoxicity; Anderson RD et al.; LacI mutations induced by doxorubicin in a wild-type, uvr(A)BC repair-proficient E . coli strain were analyzed by DNA sequencing . These mutations were contrasted with mutations previously recovered from doxorubicin-treated uvrB- organisms in order to assess the role of excision repair in doxorubicin-induced genotoxicity . After a 30-min exposure of wild-type E . coli to 330 microM doxorubicin, survival was 34% and the overall lacI mutation frequency increased 1.8-fold to 340 x 10(-8) . The distribution of doxorubicin-induced mutants among subclasses of mutation involving the i-d and lac operator regions differed significantly between repair-proficient and -deficient strains . Distributional differences appeared to result both from a decrease in deletions involving the lac operator and an increase in base substitutions involving the i-d region in repair proficient organisms . However, elements of the doxorubicin-induced mutation spectrum in uvrB- E . coli are still discernable in wild-type organisms . These elements include the remarkable shift of 3'-deletion endpoints to palindromic sequence within the lac operator and the recovery of multiple isolates of T:A-->A:T transversions at position 96 in doxorubicin-treated cultures . These observations suggest that components of the uvr(A)BC nucleotide excision repair system function through a general mechanism prior to fixation of mutations to reduce, but not completely eliminate, the genotoxic effects of doxorubicin.

Mutat Res, 1993 Oct, 292(2), 175 - 85
The Escherichia coli galK2 papillation assay: its specificity and application to seven newly isolated mutator strains; Oller AR et al.; The Escherichia coli dnaE and dnaQ genes encode, respectively, the alpha (polymerase) and epsilon (proofreading) subunits of DNA polymerase III . Mutations in these genes resulting in mutator or antimutator phenotypes provide important tools to understand the mechanisms by which mutations occur . One way to isolate such strains is the use of papillation assays . We used one such assay based on the reversion of the galK2 allele in cells grown on MacConkey-Gal plates . Here, we describe the identification of the galK2 mutation and its possible reversion pathways, and the characterization of 7 mutators isolated using this system . 1 mutator resided in dnaE and 6 in dnaQ . Sequencing of the galK2 allele revealed a G.C-->T.A transversion at base pair 571 that changed a glu codon (GAA) to a stop codon (TAA) . The analysis of 319 revertants showed that a Gal+ phenotype can be achieved by A.T-->G.C transition, A.T-->T.A transversion and A.T-->C.G transversion . We characterized the mutator phenotypes of the newly isolated mutators by determining (i) their mutation frequencies to resistance to rifampicin and nalidixic acid in both wild-type and mutL backgrounds, (ii) their temperature sensitivity and medium dependence and (iii) their mutational specificity (by analyzing the nature of galK revertants) . Based on the genomic locations of their mutations, specificity of reversion pathways and magnitude of mutator effects, the mutators can be grouped into 3 classes . These classes may represent different mutational mechanisms that include defective base insertion, defective proofreading and interference with the postreplicative mismatch-repair system.

J Gen Virol, 1993 Oct, 74 ( Pt 10), 2171 - 9
Conformation-dependent recognition of baculovirus-expressed Epstein-Barr virus gp350 by a panel of monoclonal antibodies; Zhang PF et al.; The Epstein-Barr virus (EBV) major membrane protein, gp350, induces antibodies that neutralize virus infectivity in vitro and is a potential candidate for an EBV vaccine . Full-length EBV gp350 and five protein fragments, encompassing the entire protein sequence, were generated in a baculovirus expression system . The recombinant proteins were analysed using a panel of 14 monoclonal antibodies (MAbs) (13 prepared against native gp350 derived from virus-producing cells and one prepared against an Escherichia coli recombinant protein) . All 14 MAbs, including a virus-neutralizing antibody, reacted with the full-length recombinant gp350 in a dot blot immunoassay, but only four of the 14 MAbs reacted with polypeptides expressed by the five subclones, indicating that the full-length protein, but not the protein fragments, was antigenically similar to native gp350 . Treatment of the six recombinant proteins with peptide-N-glycosidase F (PNGase F) indicated that the full-length gp350 protein and the N-terminal fragment were glycosylated and that the four internally initiated polypeptides were not glycosylated . PNGase F treatment of the full-length glycosylated gp350 did not eliminate its reactivity with all of the 10 MAbs examined (including the neutralizing MAb) in a dot blot immunoassay; however, denatured glycosylated gp350 lost reactivity with all but four of the 14 MAbs when analysed by either dot blot or Western blot immunoassay . The data suggest that conformational epitopes are more important in recognition of gp350 by this panel of MAbs than glycosylation sites, and that the epitope on gp350 recognized by the neutralizing MAb is conformation- and not glycosylation-dependent.

J Bacteriol, 1993 Oct, 175(20), 6663 - 70
Expression of the Escherichia coli dnaX gene; Chen KS et al.; The Escherichia coli dnaX gene encodes both the tau and gamma subunits of DNA polymerase III . This gene is located immediately downstream of the adenine salvage gene apt and upstream of orf12-recR, htpG, and adk . The last three are involved in recombination, heat shock, and nucleotide biosynthesis, respectively . apt, dnaX, and orf12-recR all have separate promoters, and the first two are expressed predominantly from those separate promoters . However, use of an RNase E temperature-sensitive mutant allowed the detection of lesser amounts of polycistronic messengers extending from both the apt and dnaX promoters through htpG . Interestingly, transcription of the weak dnaX promoter is stimulated 4- to 10-fold by a sequence contained entirely within the dnaX reading frame . This region has been localized; at least a portion of the sequence (and perhaps the entire sequence) is located within a 31-bp region downstream of the dnaX promoter.

J Bacteriol, 1993 Oct, 175(19), 6293 - 8
Induction of EcoRII methyltransferase: evidence for autogenous control; Friedman S et al.; The cytosine analog 5-azacytidine kills Escherichia coli cells that carry plasmids expressing EcoRII DNA (cytosine 5)methyltransferase under control of its own promoter . We previously showed that this enzyme binds tightly to azacytidine-containing DNA in vitro and proposed that such binding is lethal in vivo . In support of this proposal, we now show that the enzyme sediments with the nucleoid of azacytidine-treated cells . Azacytidine treatment led to an increase in the amount of enzyme, and this increase required sequences in the ecoRIIM promoter region . Enzyme inducibility correlated with drug sensitivity: plasmids carrying the methyltransferase gene but lacking the wild-type promoter did not confer sensitivity . These results suggested that the ecoRIIM gene was under autogenous control . Transcriptional ecoRIIM'-lacZ fusions in E . coli were, therefore, constructed . They showed that expression from the ecoRIIM promoter was inhibited when EcoRII DNA (cytosine-5)methyltransferase was introduced into the cell in trans and inhibition was reversed by treating the cells with azacytidine . These results provide evidence that the expression of the ecoRIIM gene is under autogenous regulation and that cell death induced by azacytidine is due, in part, to the disruption of autoregulation.

EMBO J, 1993 Oct, 12(10), 3739 - 45
The topology of the brown adipose tissue mitochondrial uncoupling protein determined with antibodies against its antigenic sites revealed by a library of fusion proteins; Miroux B et al.; The uncoupling protein (UCP) of brown adipose tissue mitochondria is a specialized member of the family of evolutionarily related mitochondrial membrane transporters, which also includes the ADP/ATP translocator and the phosphate carrier . We have generated a library of bacterial clones randomly expressing short subsequences of the UCP fused to the MalE periplasmic protein of Escherichia coli . Anti-UCP sera were used to select clones expressing antigenic sequences of the UCP . Ten different fusion proteins representing eight non-overlapping subsequences of the UCP were obtained . The ability of fusion proteins to select antibodies directed against a short segment of the UCP was used to study the topological organization of the UCP in the inner mitochondrial membrane . Four different fusion proteins were used to determine the orientation of the N-terminal extremities of the first, second, third and fourth predicted alpha-helices of the UCP . This topological study together with previous data on the UCP provides an experimental basis for the predicted structure of the UCP and for other homologous carrier proteins.

Mutat Res, 1993 Oct, 289(2), 255 - 63
C/G to A/T transversions represent the main type of mutation induced by gamma-irradiation in double-stranded M13mp10 DNA in a nitrogen-saturated solution; Braun JE et al.; To get more insight into the possible mutagenic consequences of DNA damage induced by radiation-generated H radicals (.H), a nitrogen-saturated solution of double-stranded (ds) M13mp10 DNA in phosphate buffer was irradiated with gamma-rays . Under these conditions 55% of the DNA-damaging species consists of H radicals and 45% of OH radicals (.OH) . The mutations were investigated in a 144-bp mutational target sequence inserted into the lacZ alpha gene . A very specific mutation spectrum was obtained with respect to the type of mutations . Twenty out of the 28 radiation-induced mutations were C/G to A/T transversions; the remaining 8 mutations were 4 C/G to G/C transversions, 2 C/G to T/A transitions, one T/A to A/T transversion and only one -1 bp deletion . The mutations were rather randomly distributed along the 144-bp mutation target sequence with no clear mutational hot spots . When these results are compared with those we have obtained previously after irradiation of ds M13mp10 DNA under O2 (100% .OH) or N2O (90% .OH; 10% .H) (Hoebee et al., 1988, 1989), the data strongly suggest that H radicals may be responsible for the observed C/G to A/T transversions but not for -1 bp deletions.

Mutat Res, 1993 Oct, 289(2), 205 - 14
Mutagenic and recombinagenic effects of diethylstilbestrol quinone; Korah RM et al.; Estrogens are believed to be major contributors to many cancers of the human female genital tract, but the mechanism of their carcinogenic action is not well-understood . While a tumor-promoting role for estrogens is well-supported, whether they also act as tumor initiators has remained controversial . Here, we have sought to examine the mutagenic potential of diethylstilbestrol, a synthetic estrogen that is a powerful carcinogen in hamsters, and is suspected to be a human carcinogen . Phage M13 single-stranded DNA was treated in vitro with diethylstilbestrol quinone (DES Q: 1.25 mM) and transfected into Escherichia coli cells . DES Q treatment resulted in an apparent enhancement of mutagenesis in the LacZ(alpha) gene segment . DNA sequence analysis of LacZ(alpha) mutants obtained by transfection of DES Q-treated DNA revealed that the major effect of DES Q treatment has been a 6-fold elevation of recombination between the phage-borne LacZ(alpha) sequence and the LacZ delta M15 sequence on the E . coli fertility plasmid F . To confirm whether DES Q treatment is recombinagenic, we used an experimental system that allows the detection of recombination between a defective E . coli chromosomal LacY gene and a normal counterpart borne on a plasmid . Transfection of DES Q (0.06-12 mM) treated plasmid DNA showed significant enhancement (2-100-fold) in recombination, but not in mutagenesis . These results raise the possibility that estrogen quinones may induce recombinagenic DNA damage.

Mutat Res, 1993 Oct, 289(2), 181 - 6
Effect of DNA-repair enzymes on mutagenesis by oxygen free radicals; Reid TM et al.; Cytosine to thymine transitions are among the most common types of mutations produced by oxygen damage to DNA . One possible mechanism for these transitions is deamination of cytosine to uracil . Using both a forward mutation assay as well as a reversion assay specific for damage to cytosines we show that direct deamination to uracil does not play a significant role in mutagenesis induced by reactive oxygen free radicals . In contrast, lesions sensitive to repair by E . coli endonuclease III play a major role in oxidative mutagenesis as evidenced by the ability of endonuclease III to modulate the extent of mutagenesis that results from exposure of DNA to oxygen free radicals.

J Exp Med, 1993 Oct 1, 178(4), 1347 - 55
Lipopolysaccharides prime whole human blood and isolated neutrophils for the increased synthesis of 5-lipoxygenase products by enhancing arachidonic acid availability: involvement of the CD14 antigen; Surette ME et al.; Stimulation of heparinized blood with 1 microM formyl-methionyl-leucyl-phenylalanine (FMLP) resulted in the formation of < 30 pmol/ml plasma of 5-lipoxygenase (5-LO) products . The preincubation of blood with 1 microgram/ml of lipopolysaccharide (LPS) (Escherichia coli 0111-B4) for 30 min before stimulation with FMLP resulted in the accumulation of 250-300 pmol of 5-LO products per ml plasma . The major products detected were leukotriene B4 and (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid which were produced in equivalent amounts . The priming activity was detectable with as little as 1-10 ng LPS per ml blood and was optimal using 1-10 micrograms LPS/ml blood . The priming for 5-LO product synthesis was optimal after 20-30 min of preincubation with LPS and declined at preincubation times > 30 min . The priming effect of LPS was also observed using the complement fragment C5a or interleukin 8 as agonists . Polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells accounted for 80 and 20% of the synthesis of 5-LO products, respectively . The ability of LPS to prime isolated PMN was dependent on the presence of plasma and was inhibited by the anti-CD14 antibody IOM2, indicating a CD14-dependent priming mechanism . The priming of whole blood with tumor necrosis factor alpha (TNF-alpha) and LPS was additive and the presence of mononuclear cells did not enhance the ability of LPS to prime PMN, indicating that the priming activity of LPS is independent of LPS-induced TNF-alpha synthesis . The mechanism by which LPS enhance 5-LO product synthesis in PMN was investigated . Treatment of PMN with LPS strongly enhanced the release of arachidonic acid after stimulation with FMLP . The release of arachidonic acid was optimal 2-3 min after stimulation with FMLP, attaining levels 5-15-fold greater than those observed in unprimed cells stimulated with FMLP . These results demonstrate that LPS dramatically increases the ability of blood to generate 5-LO products, and support the putative role of leukotrienes in pathological states involving LPS.

Virology, 1993 Oct, 196(2), 731 - 8
Disruption of a salt bridge between Asp 488 and Lys 465 in HIV-1 reverse transcriptase alters its proteolytic processing and polymerase activity; Goobar-Larsson L et al.; The conserved aspartic acid residue 488 in the RNase H domain of HIV-1 reverse transcriptase (RT) was mutated to alanine . RT was expressed in Escherichia coli alone or with the entire pol-gene polyprotein consisting of proteinase, RT, and integrase and processed by the HIV-1 proteinase in the bacterial cell . Expression of mutant RT together with the proteinase resulted in an overproduction of RT p51 vs p66 . The mutation also altered the conformation of the RT p66/p51 heterodimer as shown by the loss of binding of monoclonal antibodies to mutant RT in ELISA . Crystallographic data shows that a salt bridge exists between Asp 488 and Lys 465 of RNase H which stabilizes the uncleavable form of RT p66, and that substitution of Asp for Ala would prevent the formation of this salt bridge . Our results indicate that disruption of this salt bridge through mutation of Asp 488 interferes with the conformational changes that regulate the limited processing of p66 to 51 by the virus proteinase . Homology data suggest that such a bridge may be present in other lentiviruses . The mutation introduced caused a moderate decrease in both the RNase H activity and the polymerase activity of RT, indicating that the proper folding of the RNase H domain of RT is necessary to achieve full polymerase activity.

Mol Microbiol, 1993 Oct, 10(1), 35 - 43
Structural and functional analyses of the FinP antisense RNA regulatory system of the F conjugative plasmid; van Biesen T et al.; The efficiency of conjugation of F-like plasmids is regulated by the FinOP fertility inhibition system . The transfer (tra) operon is under the direct control of the TraJ transcriptional activator which, in turn, is negatively regulated by FinP, an antisense RNA, and FinO, a 22 kDa protein . Recently, FinO has been shown to extend the chemical stability of FinP in vivo in the absence of traJ mRNA . The in vitro secondary structures of both the FinP and TraJ RNAs were determined by the use of single- and double-strand-specific nucleases; both RNAs were found to have double stem-loop structures that are complementary to each other and, therefore, FinP RNA and TraJ RNA have the potential to form a duplex with each other . This was verified by in vitro binding experiments . The reaction was shown to be biomolecular with an apparent rate constant (kapp) of 5 x 10(5)M-1s-1, a value that is similar to those found for other natural antisense RNA systems . Preliminary evidence for the in vivo formation of the FinP-TraJ RNA duplex was obtained by primer extension of the traJ mRNA; the presence of both FinO and FinP was required to cause a dramatic reduction in the steady-state level of traJ mRNA, perhaps as a result of RNase III degradation of the resulting RNA duplex.

Plant Physiol, 1993 Oct, 103(2), 565 - 73
Identification, cDNA cloning, and gene expression of soluble starch synthase in rice (Oryza sativa L.) immature seeds; Baba T et al.; Three forms of soluble starch synthase were resolved by anion-exchange chromatography of soluble extracts from immature rice (Oryza sativa L.) seeds, and each of these forms was further purified by affinity chromatograph . The 55-, 57-, and 57-kD proteins in the three preparations were identified as candidates for soluble starch synthase by western blot analysis using an antiserum against rice granule-bound starch synthase . It is interesting that the amino-terminal amino acid sequence was identical among the three proteins, except that the 55-kD protein lacked eight amino acids at the amino terminus . Thus, these three proteins are products of the same gene . The cDNA clones coding for this protein have been isolated from an immature rice seed library in lambda gt11 using synthetic oligonucleotides as probes . The deduced amino acid sequence of this protein contains a lysine-X-glycine-glycine consensus sequence for the ADP-glucose-binding site of starch and glycogen synthases . Therefore, we conclude that this protein corresponds to a form of soluble starch synthase in immature rice seeds . The precursor of the enzyme contains 626 amino acids, including a 113-residue transit peptide at the amino terminus . The mature form of soluble starch synthase shares a significant but low sequence identity with rice granule-bound starch synthase and Escherichia coli glycogen synthase . However, several regions, including the substrate-binding site, are highly conserved among these three enzymes . Blot hybridization analysis demonstrates that the gene encoding soluble starch synthase is a single-copy gene in the rice genome and is expressed in both leaves and immature seeds . These results suggest that soluble and granule-bound starch synthases play distinct roles in starch biosynthesis of plant.

J Protein Chem, 1993 Oct, 12(5), 525 - 31
Cysteine 17 of recombinant human granulocyte-colony stimulating factor is partially solvent-exposed; Arakawa T et al.; Oh-eda et al . have shown instability of granulocyte-colony stimulating factor (G-CSF) upon storage above pH 7.0 {J . Biol . Chem . (1990) 265, 11,432-11,435} . To clarify the mechanism of this instability, the accessibility of a free cysteinyl residue at position 17 for disulfide exchange reaction was examined using a sulfhydryl reagent . The results show that the cysteine is partially solvent-exposed in both glycosylated and nonglycosylated forms, suggesting that the exposure of the cysteine plays a critical role in the instability of the protein . This is supported by the facts that at low pH where the cysteine is protonated, both proteins have much greater stability and that a Cys17-->Ser analog is extremely stable at neutral pH and 37 degrees C . It was observed that the rate of sulfhydryl titration is slower for the glycosylated form than for the nonglycosylated form, suggesting that the cysteine residue is less solvent-exposed for the former protein or that the pKa is somewhat more basic . In either case, the carbohydrate appears to affect the reactivity of the sulfhydryl group through steric hindrance or alteration in local conformation . Both the glycosylated and nonglycosylated proteins showed essentially identical conformation as determined by circular dichroism, fluorescence, and infrared spectroscopy . Unfolding of these two proteins, induced either by guanidine hydrochloride or by pH, showed an identical course, indicating comparable conformational stability . Contribution of conformational changes to the observed instability at higher pH is unlikely, since little difference in fluorescence spectrum occurs between pH 6.0 and 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Avian Dis, 1993 Oct-Dec, 37(4), 1105 - 12
Cloning and partial sequence analysis of a Mycoplasma synoviae DNA fragment encoding epitopes shared with the major adhesin P1 protein of Mycoplasma pneumoniae; Morsy MA et al.; Polyclonal antibodies specific for the adhesin P1 protein of Mycoplasma pneumoniae were used to screen an expression library of M . synoviae genomic DNA constructed in the expression vector lambda gt11 . Following several cycles of immunoscreening using the anti-P1 antiserum, a lambda gt11 recombinant clone containing 3.9 kilobase pairs (kbp) of M . synoviae DNA was identified and isolated from the expression library . Expression of the recombinant clone (designated MS-1) in Escherichia coli Y1089 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the crude E . coli lysates revealed the presence of two novel proteins . Two antibodies that recognize the adhesin polypeptide--chicken anti-M . synoviae antibodies and anti-P1 antiserum--reacted with both proteins on immunoblots . Partial sequence analysis of the M . synoviae DNA in clone MS-1 and computer comparison of the predicted amino-acid sequences with existing protein sequence files revealed homology with the adhesin P1 protein of M . pneumoniae.

Vaccine, 1993 Oct, 11(13), 1341 - 6
Induction of cytolytic and antibody responses using Plasmodium falciparum repeatless circumsporozoite protein encapsulated in liposomes; White K et al.; Plasmodium circumsporozoite (CS) protein-induced antibody and T-cell responses are considered to be important in protective immunity . Since the key repeat determinant of the CS protein may actually restrict the recognition of other potential T- and B-cell sites, a modified Plasmodium falciparum CS protein lacking the central repeat region, RLF, was expressed in Escherichia coli . On purification, RLF was encapsulated into liposomes {L(RLF)} and used for the in vivo induction of cytolytic T lymphocytes (CTL) and antibodies . Immunization of B10.Br (H-2k) mice with L(RLF), but not with RLF, induced CD8+ CTL specific for the P . falciparum CS protein CTL epitope, amino acid residues 368-390 . Anti-L(RLF) serum reacted with antigens on intact sporozoites and inhibited sporozoite invasion of hepatoma cells . Antibody specificity studies in New Zealand White rabbits revealed new B-cell sites localized in amino acid residues 84-94, 91-99, 97-106 and 367-375 . Although the mechanisms by which liposomes enhance cellular and humoral immune responses remain unknown, liposome-formulated vaccines have been well tolerated in humans; hence, their use in vaccines, when efficacy depends on antibody and CTL responses, may be broadly applicable.

Vaccine, 1993 Oct, 11(13), 1310 - 5
Peptide sequences with strong stimulatory activity for lymphoid cells: implications for vaccine development; Russell-Jones GJ et al.; Seven peptides derived from the bacterial major outer-membrane protein TraT were synthesized and then tested in lymphoproliferative assays using lymphoid cells from a variety of animals that had been immunized with the native TraT molecule in saline . A hierarchical pattern of responsiveness to the peptides was observed in the four animal species studied and in particular three of the peptides (T2, T4 and T6) showed very strong responses in all species . The 'universality' of the TraT-derived peptides was confirmed by studying the responsiveness of lymphoid cells obtained from the peripheral blood of twenty clinically normal human donors . Thus, following a secondary in vitro immunization with TraT-pulsed human peripheral blood mononuclear cells, responsiveness to TraT and to the TraT-derived peptides was observed in the cultures derived from all twenty donors . Taken together, our findings imply that the putative T-cell epitope peptides (T2, T4 and T6) could be employed as carriers in subunit vaccines and thereby help to overcome the unresponsiveness observed in animals and humans as a result of MHC restriction.

Comput Appl Biosci, 1993 Oct, 9(5), 551 - 61
Automatic display of RNA secondary structures; Muller G et al.; A set of programs written in C language with the GL library and under UNIX has been developed for generating compact, pleasant and non-overlapping displays of secondary structures of ribonucleic acids . The first program, rnasearch, implements a new search procedure that dynamically rearranges overlapping portions of the two-dimensional drawing while preserving clear and readable displays of the two-dimensional structure . The algorithm is fast (the execution time for the command rnasearch is 38.6 s for the 16S rRNA of Escherichia coli with 1542 bases), accepts outputs from two-dimensional prediction programs and therefore allows for rapid comparison between the various two-dimensional folds generated . A second program, rnadisplay, allows the graphical display of the computed two-dimensional structures on a graphics workstation . Otherwise, it is possible to obtain a paper output of the two-dimensional structure by using the program print2D which builds a Postscript file . Moreover the two-dimensional drawing can be labelled for representing data coming from chemical modifications and/or enzymatic cleavages . Application to a few secondary structures such as RNaseP, 5S rRNA and 16S rRNA are given.

Arch Oral Biol, 1993 Oct, 38(10), 903 - 10
The fate of genetically marked human oral keratinocytes in vitro; Garlick JA et al.; The fate of the progeny of human oral gingival keratinocytes was mapped in stratified epithelial tissues in vitro by following the expression of a marker gene in genetically related clones . Oral epithelial progenitor cells were genetically marked at high efficiency by transducing them with a retrovirus vector that carried the gene for a histochemically detectable product, Escherichia coli beta-galactosidase (beta-gal) . These cells were then grown in submerged cultures and on collagen rafts at the air-liquid interface to demonstrate the distribution of genetically marked cells in a differentiating tissue in vitro . The dynamics of transduced cells showed that clonally related cells were arranged in discrete units of labelled cells and these clusters were defined as 'clonal proliferation units' . The size and configuration of these units were related to the proliferative potential and differentiating capacity of the cell that was initially transduced . This model demonstrates the relation between clonally related cells and tissue architecture for oral keratinocytes in vitro.

Bioorg Khim, 1993 Oct, 19(10), 981 - 8
{Computerized structural analysis of O-specific polysaccharides O1A, O1B, and O1C from Escherichia coli}; Nifant'ev NE et al.; A computer evaluation of 13C-NMR data for the title polysaccharides based on the monosaccharide and methylation analysis data led to the structure of the repeating unit of the O1A polysaccharide as well as to several probable structures of the O1C polysaccharide, of which the correct one was inferred by means of a single NOE experiment . The analysis of the spectrum of the O1B polysaccharide was unsuccessful, due to the presence in its structure of the fragment alpha-L-Rha-(1-->2)-alpha-D-Gal-(1-->3)-D-GlcNAc with the terminal (1-->2)-linkage, whose spectral data could not be calculated by additive schemes using only glycosylation effects . However in reevaluation of the O1B spectral data by taking into account the deviations from additivities of the chemical shifts values in spectra of the related trisaccharides, to reveal the most probable structure of the O1B's repeating unit . {formula: see text}

New Microbiol, 1993 Oct, 16(4), 323 - 32
Classification of Italian isolates of Borrelia burgdorferi into three genomic groups; Cinco M et al.; In this study we investigated the genotypic characteristics of some locally isolated strains of B . burgdorferi by three different methodologies: restriction endonuclease analysis (REA), Southern blot hybridization with whole DNAs from Borrelia strains and Southern blot hybridization with rRNA 16 + 23S genes derived from E . coli . REA fingerprintings were evaluated by cluster analysis, according to the principles of numerical taxonomy . The genomas of the locally isolated strains were compared with borreliae originating from different countries of Europe, including Sweden and with the American reference strain B31 . Among the European strains, some already described by Baranton (Baranton et al., 1992) as representatives of different genomic groups Borrelia sensu stricto and Borrelia garinii were used . By the different techniques the isolates were included in three genomic groups which could correspond to the three genospecies identified by Baranton, namely B . burgdorferi sensu stricto, B . garinii and B . group VS461: in fact two strains were included in a homogeneous group, probably corresponding to the VS461 genomic group, together with other European borreliae; one isolate was included in a group consisting of B31 and some other European strains already described as belonging to Borrelia burgdorferi in sensu stricto . Finally two isolates were ascribed to a third genomic group probably corresponding to the genospecies indicated as Borrelia garinii . These findings indicate that a small number of Borrelia strains isolated from a very restricted area can be genetically heterogeneous.

J Cardiovasc Pharmacol, 1993 Oct, 22(4), 550 - 6
Antishock effect of U-67,590A, methylprednisolone suleptanate, associated with restoration of lowered vascular reactivity in endotoxemic rats; Nomura S et al.; We wished to investigate the modulating effects of a glucocorticoid on mortality and sustained hypotension in endotoxemic rats in conjunction with in vitro study of responses of isolated aorta to KCl, norepinephrine (NE), and acetylcholine (ACh) . Endotoxemia was induced by intravenous (i.v.) bolus injection of 50 mg/kg Escherichia coli endotoxin in rats, resulting in high mortality . Pretreatment with U-67,590A, methylprednisolone suleptanate, at a dose of 9 mg/kg resulted in 100% survival; the survival rate of saline-treated controls was 21% . Isolated rat aorta that had been treated with endotoxin for 4 h showed decreases in contractile responses to KCl and NE and in relaxing response to ACh . Similar attenuation of contractile responsiveness was observed in endothelium-denuded preparations . Addition of endotoxin to the in vitro tissue bath did not inhibit the responses in a 4-h period . Pretreatment with U-67,590A inhibited the late gradual decrease in blood pressure (BP) but not the early hypotensive response to endotoxin . The responses to KCl or NE of aorta isolated 4 h after the endotoxin injection remained suppressed in U-67,590A-treated rats but were restored in 24 h . These results suggest that endotoxemia impairs the endothelium moderately, but this does not account for either reduced reactivity of vascular smooth muscle to vasoconstrictor agents or the sustained hypotension . The steroid inhibits endotoxemia-induced mortality and sustained hypotension.

Leuk Lymphoma, 1993 Oct, 11(3-4), 249 - 62
Cytotoxicity of a recombinant diphtheria toxin-granulocyte colony-stimulating factor fusion protein on human leukemic blast cells; Chadwick DE et al.; Granulocyte colony-stimulating factor (G-CSF) is a potent stimulator of the growth of normal and malignant hematopoietic cells and synergizes with other factors such as interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) . The action of G-CSF is mediated through a specific membrane receptor, however it is not clear if all of the effects of G-CSF are direct or indirect . As a step towards addressing this problem, a recombinant diphtheria toxin (DT)-related human G-CSF fusion protein has been constructed and purified from E . coli . The 70,000 dalton chimeric protein has immunologic determinants characteristic of both DT and G-CSF . At high concentrations, DAB486-G-CSF is cytotoxic towards G-CSF-dependent OCI/AML1 cells, but not factor independent OCI/AML3 cells; colony formation by G-CSF-responsive leukemic blasts from a patient with acute myeloblastic leukemia (AML) was also inhibited . The G-CSF fusion toxin displayed ADP-ribosyltransferase activity in a cell-free system . Genetic conjugation of G-CSF to an enzymatically inactive DT mutant, CRM197, resulted in a 200-fold reduction in the ability of G-CSF to stimulate normal bone marrow colony formation . These results suggest that fusion of G-CSF to DT sequences interferes with some of the activity but not the specificity of the ligand binding domain of the molecule . Nevertheless, DAB486-G-CSF may be included with the increasing number of other toxin-hormone fusion proteins whose toxicity is directed towards specific receptor-bearing cells, and may represent a novel approach towards the study and treatment of leukemia.

Plant J, 1993 Oct, 4(4), 717 - 25
DIP: a member of the MIP family of membrane proteins that is expressed in mature seeds and dark-grown seedlings of Antirrhinum majus; Culianez-Macia FA et al.; DiP, a gene from Antirrhinum majus, which encodes a protein with striking homology to other integral membrane proteins, was cloned . The gene was specifically expressed in mature seeds and during seedling germination, particularly in cotyledons of seedlings grown in the dark . The deduced product, called DiP, for dark intrinsic protein, shows strong homology with the MIP family of channel transporters which include; the bovine major intrinsic protein (MIP), the Escherichia coli glycerol facilitator (GIpF), the peribacteroid nodulin-26 (Nod26), and the tonoplast protein from kidney bean (TIP) . DiP is most similar to other plant members of this family, and in particular to the tobacco protein TobRB7 which is expressed specifically in roots . However, the expression pattern of diP suggests that its product is functionally more similar to the tonoplast intrinsic protein from kidney bean since it is most highly expressed in the cotyledons of germinating seedlings, before the cells undergo expansion growth and become photosynthetic.

Protein Expr Purif, 1993 Oct, 4(5), 465 - 72
Isolation and characterization of three recombinant human granulocyte colony stimulating factor His-->Gln isoforms produced in Escherichia coli; Lu HS et al.; This report demonstrates that three variant isoforms of recombinant methionyl human granulocyte colony stimulating factor are present in small quantities in the crude preparation solubilized from Escherichia coli inclusion bodies . These isoforms were separated from the main form of the factor during purification and further isolated by a series of cationic exchange chromatographic separations . They exhibit full in vitro biological activity and have slightly lower pI's . Structural characterization of the intact proteins and their isolated peptides by sequence determination and mass spectrometric analysis revealed that they are methionyl granulocyte colony stimulating factors having a His-->Gln replacement at sequence position 53, 157, or 171, respectively . The specific His-->Gln change suggests the occurrence of mistranslation during protein synthesis . These variant forms are chromatographically separable during purification and are not detectable in the final purified form of the factor.

Protein Expr Purif, 1993 Oct, 4(5), 445 - 55
Purification of an active TATA-binding protein-containing factor using a monoclonal antibody that recognizes the human TATA-binding protein; Chatterjee PK et al.; The human TATA-binding protein was expressed in Escherichia coli as a fusion with an N-terminal hexahistidine sequence, partially purified, and used to raise monoclonal antibodies . More than 50 hybridoma clones producing antibodies that reacted in immunoblot assays with HeLa cell TATA-binding protein and its bacterially synthesized derivative were identified . All antibodies examined recognized epitopes within the N-terminal 159 amino acids of the human TATA-binding protein . Further characterization of one monoclonal antibody, MTBP-6, established that it immunoprecipitates both native HeLa cell TATA-binding protein and TATA-binding protein extracted from cells in the presence of 0.5% SDS . Antibody MTBP-6 immunoprecipitates of native, human cell TATA-binding protein contained the TATA-binding protein and additional polypeptides . Immunoprecipitation of both the TATA-binding protein and several additional polypeptides was specifically blocked by bacterially synthesized, hexahistidine-tagged TATA-binding protein, suggesting that MTBP-6 can efficiently recognize the TATA-binding protein in TFIID and other complexes . Consistent with this conclusion, immunoaffinity chromatography on antibody MTBP-6 permitted purification, in active form, of a TATA-binding protein-containing factor required for transcription by RNA polymerase III . These properties suggest that MTBP-6 will be a useful reagent for the purification and characterization of the multiple TBP-containing complexes present in human cells.

Nature, 1993 Sep 30, 365(6445), 464 - 8
Crystal structure of the DsbA protein required for disulphide bond formation in vivo; Martin JL et al.; Proteins that contain disulphide bonds are often slow to fold in vitro because the oxidation and correct pairing of the cysteine residues is rate limiting . The folding of such proteins is greatly accelerated in Escherichia coli by DsbA, but the mechanism of this rate enhancement is not well understood . Here we report the crystal structure of oxidized DsbA and show that it resembles closely the ubiquitous redox protein thioredoxin, despite very low sequence similarity . An important difference, however, is the presence of another domain which forms a cap over the thioredoxin-like active site of DsbA . The redox-active disulphide bond, which is responsible for the oxidation of substrates, is thus at a domain interface and is surrounded by grooves and exposed hydrophobic side chains . These features suggest that DsbA might act by binding to partially folded polypeptide chains before oxidation of cysteine residues.

Nature, 1993 Sep 30, 365(6445), 454 - 6
Skeletal muscle myosin light chains are essential for physiological speeds of shortening; Lowey S et al.; In muscle each myosin head contains a regulatory light chain (LC2) that is wrapped around the head/rod junction, and an alkali light chain that is distal to LC2 (ref . 1) . The role of these light chains in vertebrate skeletal muscle myosin has remained obscure . Here we prepare heavy chains that are free of both light chains in order to determine by a motility assay whether the light chains are necessary for movement . We find that removal of light chains from myosin reduces the velocity of actin filaments from 8.8 microns s-1 to 0.8 microns s-1 without significantly decreasing the ATPase activity . Reconstitution of myosin with LC2 or alkali light chain increases filament velocity to intermediate rates, and readdition of both classes of light chains fully restores the original sliding velocity . We conclude that even though the light chains are not essential for enzymatic activity, light-chain/heavy-chain interactions play an important part in the conversion of chemical energy into movement.

Nature, 1993 Sep 30, 365(6445), 448 - 51
Generating loss-of-function phenotypes of the fushi tarazu gene with a targeted ribozyme in Drosophila; Zhao JJ et al.; The ability to isolate gene sequences and analyse their expression patterns has generated demand for mutations created to assess their biological functions . In Drosophila melanogaster this can be achieved by traditional mutagenesis, but this is time-consuming, labour-intensive and not always successful . Moreover, the functions of genes that are expressed several times during development are often obscured in the later stages because of disruptions caused by the absence of early gene function . Here we propose a new strategy to create conditional knock-out mutations using a targeted heat-inducible ribozyme . Ribozymes are catalytic RNA molecules that specifically cleave RNAs and are potentially useful for studying gene function during animal development because the expression of critical regulatory genes is usually low and their function is often dosage-dependent . The ribozyme can be delivered to a specific region or at a particular developmental stage using a region-specific or inducible promoter . The Drosophila fushi tarazu (ftz) gene is a good candidate for testing this approach . We generated transgenic flies carrying a ribozyme against the ftz gene . The two developmental phases of ftz function can be distinguished by timed induction of the ribozyme . Activation of the ribozyme in the blastoderm disrupts the ftz seven-stripe pattern and produces ftz-like pair-rule defects in larvae . The involvement of ftz in neurogenesis was verified by activation of the ribozyme during the early phase of formation of the central nervous system.

Nature, 1993 Sep 30, 365(6445), 412 - 9
Association between proto-oncoprotein Rel and TATA-binding protein mediates transcriptional activation by NF-kappa B; Kerr LD et al.; The c-Rel protein is able to associate in vitro and in vivo with the TATA-binding protein (TBP) of the TFIID complex . Coexpression of TBP with c-Rel augments transactivation from the kappa B site in Drosophila Schneider cells . DNA-binding mutants of TBP not only fail to cooperate, but they repress transactivation by c-Rel . There may be a direct communication between kappa B enhancer binding proteins and basal transcription factors which leads to enhanced transcription.

Gene, 1993 Sep 30, 132(1), 143 - 8
The glutamate dehydrogenase-encoding gene of the hyperthermophilic archaeon Pyrococcus furiosus: sequence, transcription and analysis of the deduced amino acid sequence; Eggen RI et al.; Glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon, Pyrococcus woesei, has been isolated, characterized and found to be very similar if not identical to the recently purified GDH from P . furiosus . Using a polymerase chain reaction, based on the N-terminal amino acid sequences of GDH, the P . furiosus gdh gene was identified, cloned into Escherichia coli and sequenced . The transcription start point of gdh has been mapped 1 nucleotide upstream from the ribosome-binding site . Using antiserum raised against purified GDH, expression of gdh was observed in E . coli . The deduced primary sequence of the P . furiosus GDH has been compared to various bacterial, archaeal and eukaryal GDHs and showed a high degree of similarity (32-52%).

Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1415 - 21
Histidine residues are involved in translocation-coupled ATP hydrolysis by the Sec-A protein; Tokuda H et al.; Treatment of SecA, an essential component of the protein translocation machinery of Escherichia coli, with a histidine-specific reagent, diethylpyrocarbonate, caused significant abolition of its translocation-coupled ATPase (translocation ATPase) activity, which requires a presecretory protein and membrane vesicles, whereas its endogenous ATPase (SecA-ATPase) activity was enhanced by a factor of 2 . Diethylpyrocarbonate-treated SecA exhibited an absorption maximum at 240 nm due to the formation of N-carbethoxyhistidine . Upon the modification of about 5 of the total 22 histidine residues in the SecA molecule, both the abolition of its translocation ATPase activity and the enhancement of its SecA-ATPase activity occurred . Intact and modified SecA exhibited similar affinities for ATP, proOmpA and membranes, whereas Vmax of the translocation ATPase activity was significantly lower in the case of the modified SecA . ATP had no effect on the modification of SecA . Taken together, these results indicate that histidine residues susceptible to diethylpyrocarbonate are essential for the translocation ATPase, but not directly involved in the binding of ATP, proOmpA and membranes . A possible reason for the abolition of translocation ATPase is discussed.

Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1386 - 93
cDNA cloning, expression in Escherichia coli and purification of human 6-pyruvoyl-tetrahydropterin synthase; Ashida A et al.; cDNA clones for human 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the biosynthetic pathway of tetrahydrobiopterin, were isolated from a human Molt-4 cell cDNA library by cross-hybridization with a rat cDNA . One cDNA clone contained the entire coding sequence of 435 base pairs . The cDNA was expressed in Escherichia coli using the expression vector pMAL as a fusion protein with maltose-binding protein . After affinity purification through its maltose-binding protein domain, the fusion protein was digested by factor Xa at a specific cleavage site inserted between the domains . The main product was a protein species with a native molecular mass of 90 kDa and a subunit molecular mass of 17 kDa, and the molecular masses and its kinetic properties were similar to those of the human enzyme purified from the liver.

Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1237 - 44
Fluorescence properties of the three tyrosine residues in the ribose-binding protein; Kim D et al.; Three tyrosine residues but no tryptophan exist in the ribose-binding protein (RBP) of Escherichia coli . In order to assess the contribution of each tyrosine to the fluorescence properties, mutants were constructed by site-directed mutagenesis to replace tyrosines at 32, 115, and 261 by phenylalanines . The mutant proteins were functional as confirmed by in vivo tactic response and by their ability to bind to ribose . The fluorescence emission spectra of the native proteins purified from the various tyrosine mutants were measured from emission scans with a peak at 303 nm . The tyrosines, at positions 32, 115, and 261, contribute 10.0, 69.6, and 23.4%, respectively, to the total intensity of fluorescence . In completely unfolded polypeptide, these tyrosines have almost the same intensities of fluorescence, indicating that the fluorescence from tyrosines at 32 and 261 are considerably quenched in the folded, native protein.

Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1211 - 7
Cloning and functional expression of a Schistosoma japonicum cDNA homologous to the enolase gene family; Waine GJ et al.; A cDNA encoding the complete open reading frame of Schistosoma japonicum enolase has been cloned . The 1494bp cDNA (C30) was isolated from a S . japonicum cDNA expression library immunoscreened with hyperimmune rabbit sera raised against soluble adult S . japonicum proteins . The ORF encodes a protein of 434 amino acids exhibiting 72% identity to human, murine, and rat enolases, and 62% identity to Saccharomyces cerevisiae enolase . The inferred molecular mass of the protein is 47,251 Daltons, similar to that reported for the enolases of other species . In vitro translation of C30 also generated a protein of 47kDa . After subcloning and expression, the recombinant protein was purified by affinity chromatography under non-denaturing conditions and shown to exhibit functional enolase enzymatic activity.

Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1174 - 83
Efficient adenovirus-mediated gene transfer into human blood monocyte-derived macrophages; Haddada H et al.; The efficiency of gene transfer into human blood monocyte-derived macrophages has been evaluated using a replication-defective adenovirus vector harboring a lac Z gene of E . coli as a reporter gene . Whereas, no beta-galactosidase activity was found in freshly infected purified monocytes, 40% to 80% of infected macrophages which derived from these monocytes showed a beta-galactosidase activity, 2 to 4 days after infection and lasted for at least 3 weeks . Moreover, beta-galactosidase activity was found in infected monocyte/macrophages 7 days after their injection into a human tumor preestablished in nude mice . These data indicate that it is possible to transfer and stably express a gene of potential therapeutical function into human monocyte-derived macrophages using an adenovirus vector.

Gene, 1993 Sep 30, 132(1), 89 - 94
Characterization and sequence of a 33-kDa enterohemolysin (Ehly 1)-associated protein in Escherichia coli; Stroeher UH et al.; The nucleotide sequence of the 2.1-kb EcoRI-AccI fragment of the enterohemolysin (Ehly)-associated plasmid, pEO21, has been determined . A third of this sequence encodes a 29.6-kDa protein, and coincides with the location of Tn1725 insertions which inactivate the production of Ehly . A protein of similar size (33-35 kDa) was found to be produced in large amounts by JM83{pEO21} and found to be immunologically related to the approximately 65-kDa protein made by the parental strain, C3888 . DNA sequences coding for the 29.6-kDa protein of phi C3888, now designated Ehly 1, were found only in some enterohemolytic Escherichia coli indicating the existence of multiple, genetically different Ehlys.

Gene, 1993 Sep 30, 132(1), 21 - 31
Gene organization and primary structure of a ribosomal RNA gene cluster from Streptomyces griseus subsp . griseus; Kim E et al.; The Streptomyces griseus subsp . griseus KCTC 9080 genome contains six rRNA-encoding gene (rDNA) clusters . One rDNA cluster (rrnE), contained in an 8.7-kb BamHI fragment, was cloned and sequenced . The rDNA were arranged in the order 16S-23S-5S, and separated by small intergenic spacers . No tRNA-encoding sequences were found in the spacer regions . The lengths of the mature 16S, 23S and 5S rRNAs were 1528, 3120 and 120 nucleotides (nt), respectively . The G + C content of the gene cluster was lower than that of the chromosomal DNA . In general, the primary and secondary structures of the three rRNAs showed good agreement with those from other Streptomyces species . However, in comparison with Escherichia coli, two noticeable changes (mismatches and deletions) and two large insertions were identified in the 16S and 23S rRNAs, respectively . On the other hand, regions showing considerable heterogeneity, even within the genus Streptomyces, were found in both rRNAs . Putative primers and processing signals showing high sequence similarity to those from other Streptomyces species were located in the region upstream from the 5' end of the mature 16S rRNA . A potential hairpin loop structure reminiscent of a Rho-independent terminator was located just downstream from the 5S rRNA . A considerable degree of sequence conservation and variation within rDNA gene clusters was revealed in this study, both at the infra- and suprageneric levels.

Biochim Biophys Acta, 1993 Sep 29, 1170(1), 62 - 71
Early catalytic steps of Euglena gracilis chloroplast type II fatty acid synthase; Worsham LM et al.; Euglena gracilis is a very ancient eukaryote whose chloroplast acquisition and evolution has been independent of higher plants . The organism in unique in possessing two de novo fatty acid synthases, a true multienzyme complex of great size in the cytosol and a plastid-localized type II fatty acid synthase composed of discrete enzymes and acyl carrier protein (ACP) . The enzymology of the early steps of fatty acid biosynthesis differed in the Euglena type II fatty acid synthase compared to those of Escherichia coli and plants . The enzymes of Euglena participating in both priming and elongation reactions to form a new carbon-carbon bond were acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and beta-ketoacyl-ACP synthase I . The effects of inhibitors on the three different enzymes were noted . All carbon-carbon bond formation was inhibited by cerulenin . Although neither fatty acid biosynthesis nor any of the isolated enzymes were sensitive to diisopropylphosphofluoridate, the three Euglena enzymes studied were sensitive to different sulfhydryl-alkylating agents . Acetyl-ACP supported fatty acid biosynthesis as effectively as did comparable amounts of ACPSH and acetyl-CoA . There was no evidence for a beta-ketoacyl-ACP synthase III for priming such as has been reported in type II fatty acid synthase of higher plants and bacteria . The roles of the acetyl-CoA-ACP transacylase and beta-ketoacyl-ACP synthase I appear to be unique in the type II fatty acid synthase of Euglena . Acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and beta-ketoacyl-ACP synthase I were separated from one another and shown to have different molecular weights.

Biochemistry, 1993 Sep 28, 32(38), 9985 - 93
Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments; Hawkins M et al.; ADF (actin depolymerizing factor) is an M(r) 19,000 actin-binding protein present in many vertebrate tissues and particularly abundant in neuronal cells . We have cloned human ADF and here show it to be identical in sequence to porcine destrin . Human ADF expressed in Escherichia coli behaves like native ADF from porcine brain . It binds to G-actin at pH 8 with a 1:1 stoichiometry and Kd approximately 0.2 microM, thereby sequestering monomers and preventing polymerization . It does not cosediment with F-actin at this pH, but severs actin filaments in a calcium-insensitive manner . The severing activity is only about 0.1% efficient . By contrast, at pH values below 7, ADF binds to actin filaments in a highly cooperative manner and at a 1:1 ratio to filament subunits . When the pH is raised to 8.0, the decorated filaments are rapidly severed and depolymerized.

Biochemistry, 1993 Sep 28, 32(38), 9944 - 59
Novel zinc finger motif in the basal transcriptional machinery: three-dimensional NMR studies of the nucleic acid binding domain of transcriptional elongation factor TFIIS; Qian X et al.; Transcriptional elongation provides a key control point in the regulation of eukaryotic gene expression . Here we describe homonuclear and 15N-heteronuclear 3D NMR studies of the nucleic acid binding domain of human transcriptional elongation factor TFIIS . This domain contains a Cys4 Zn(2+)-binding site with no homology to previously characterized Cys4, Cys6, or Cys2-His2 Zn fingers . Complete 1H and 15N NMR resonance assignment of a 50-residue TFIIS peptide-Zn2+ complex is obtained . Its solution structure, as determined by distance geometry/simulated annealing (DG/SA) calculations, exhibits a novel three-stranded antiparallel beta-sheet (designated the Zn ribbon) . Analogous sequence motifs occur in a wide class of proteins involved in RNA or DNA transactions, including human basal transcriptional initiation factor TFIIE . A three-dimensional model of the TFIIE Cys4 domain is obtained by DG-based homology modeling . The role of the TFIIS Zn ribbon in the control of eukaryotic transcriptional elongation is discussed.

Biochemistry, 1993 Sep 28, 32(38), 10165 - 9
Effects of intra- and intersubunit hydrogen bonds on the R-T transition in human hemoglobin as studied with alpha 42(C7) and beta 145(HC2) mutations; Togi A et al.; To clarify the effects of specific inter- and intrasubunit hydrogen bonds on the R-T transition in human hemoglobin (Hb A), the recombination reaction of carbon monoxide with artificial mutant Hbs was measured and analyzed . One of the hydrogen bonds we focused on is formed between Tyr-42 alpha and Asp-99 beta in the alpha 1-beta 2 interface of Hb A, which is one of the hydrogen bonds characteristic of the T state . Hb His-42 alpha, in which Tyr-42 alpha is replaced by His to perturb this hydrogen bond, showed that the ligand-free R to T transition rate was decreased by 20-fold compared with that for Hb A . This mutation caused the destabilization of the transition state in the R to T quaternary structure change by about 7 kJ mol-1, indicating that the hydrogen bond between Tyr-42 alpha and Asp-99 beta plays a definite role in the R-T transition as well as in stabilization of the equilibrium T state . Hb Phe-145 beta, in which Tyr-145 beta is replaced by Phe and the intrasubunit hydrogen bond between Tyr-145 beta and Val-98 beta is lacking, also showed a slow R-T transition rate as observed in Hb His-42 alpha . The published crystallographic data suggest that this intrasubunit hydrogen bond stabilizes the transition state by reducing the freedom of motion of the C-terminus of the beta subunit and, thereby, facilitates the R-T transition.

Biochemistry, 1993 Sep 28, 32(38), 10159 - 64
Catalase HPII of Escherichia coli catalyzes the conversion of protoheme to cis-heme d; Loewen PC et al.; Catalase HPII from aerobically grown Escherichia coli normally contains heme d but cultures grown with poor or no aeration produce HPII containing a mixture of heme d and protoheme IX . The protoheme component of HPII from anaerobically grown cells is converted into heme d during treatment of the purified enzyme with hydrogen peroxide . It is concluded that heme d found in catalase HPII is formed by the cis-hydroxylation of protoheme in a reaction catalyzed by catalase HPII using hydrogen peroxide as a substrate . The distal His128 residue of HPII is absolutely required for the protoheme to heme d conversion . Two mutant enzymes, Ala128 and Asn128, are catalytically inactive and contain only protoheme, which is unaffected by hydrogen peroxide treatment . The Asn201 residue is not an absolute requirement for heme conversion . The mutant enzyme Ala201 contains predominantly heme d and is partially active . However, insertion of a histidyl residue to give the His201 enzyme interferes with the heme conversion reaction . This mutant form is isolated as a protoheme enzyme with limited activity, and a reversible conversion to a heme d-like species occurs in vitro in the presence of continuously generated hydrogen peroxide.

Biochemistry, 1993 Sep 28, 32(38), 10140 - 9
Recombinant bovine heart mitochondrial F1-ATPase inhibitor protein: overproduction in Escherichia coli, purification, and structural studies; Van Heeke G et al.; A synthetic gene coding for the inhibitor protein of bovine heart mitochondrial F1 adenosine triphosphatase was designed and cloned in Escherichia coli . Recombinant F1-ATPase inhibitor protein was overproduced in E . coli and secreted to the periplasmic space . Biologically active recombinant F1-ATPase inhibitor protein was recovered from the bacterial cells by osmotic shock and was purified to near homogeneity in a single cation-exchange chromatography step . The recombinant inhibitor protein was shown to inhibit bovine mitochondrial F1-ATPase in a pH-dependent manner, as well as Saccharomyces cerevisiae mitochondrial F1-ATPase . Thorough analysis of the amino acid sequence revealed a potential coiled-coil structure for the C-terminal portion of the protein . Experimental evidence obtained by circular dichroism analyses supports this prediction and suggests F1I to be a highly stable, mainly alpha-helical protein which displays C-terminal alpha-helical coiled-coil intermolecular interaction.

Biochemistry, 1993 Sep 28, 32(38), 10027 - 35
Properties of interacting aspartic acid and lysine residues in the lactose permease of Escherichia coli; Sahin-Toth M et al.; The side chains of the interacting pair Asp237(helix VII)-Lys358(helix XI) or Asp240(helix VII)-Lys319(helix X) in the lactose permease of Escherichia coli were extended by replacement with Glu and/or Arg or by site-specific derivatization of single-Cys replacement mutants . Iodoacetic acid was used to carboxymethylate Cys, or methanethiosulfonate derivatives {Akabas, M . H., Stauffer, D . A., Xu, M., & Karlin, A . (1992) Science 258, 307} were used to attach negatively charged ethylsulfonate or positively charged ethylammonium groups . Replacement of Asp237 with Glu, carboxymethyl-Cys, or sulfonylethylthio-Cys yields active permease with Lys or Arg at position 358 . Similarly, the permease tolerates replacement of Lys358 with Arg or ammonioethylthio-Cys with Asp or Glu at position 237 . Remarkably, moreover, permease with Lys, Arg, or ammonioethylthio-Cys in place of Asp237 is highly active when Lys358 is replaced with Asp or Glu, in agreement with the conclusion that the polarity of the charge interaction can be reversed without loss of activity {Sahin-Toth, M., Dunten, R . L., Gonzalez, A., & Kaback, H . R . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 10547} . In contrast, replacement of Asp240 with Glu abolishes lactose transport, and permease with carboxymethyl-Cys, at position 240 is inactive when paired with Lys319, but it exhibits significant activity with Arg319 . Interestingly, sulfonylethylthio-Cys substitution for Asp240 also results in significant transport activity . Permease with Arg or ammonioethylthio-Cys in place of Lys319 exhibits high activity with Asp240 as the negative counterion, but no lactose transport is observed when either of these modifications is paired with Glu240.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Sep 28, 32(38), 10150 - 8
The conserved residues glutamate-37, aspartate-100, and arginine-269 are important for the structural stabilization of Escherichia coli aspartate transcarbamoylase; Baker DP et al.; Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits . The X-ray structure of this enzyme indicates that the side chains of His-41, Asp-100, and Asp-90 from one catalytic chain form interactions with the side chains of Glu-37, Arg-65, and Arg-269, respectively, from an adjacent catalytic chain . In order to determine whether these interactions are important for the structural stabilization of the enzyme and/or homotropic and heterotropic effects, four mutant versions of aspartate transcarbamoylase, Glu-37-->Ala, Asp-100-->Asn, Asp-100-->Ala, and Arg-269-->Ala, were created by site-specific mutagenesis . The Glu-37-->Ala holoenzyme exhibits essentially wild-type behavior with respect to homotropic cooperativity and heterotropic regulation by ATP and CTP . The Glu-37-->Ala catalytic subunit exhibits a half-life of inactivation at 69 +/- 0.5 degrees C of 4.9 min, as compared to 5.8 min for the wild-type catalytic subunit . The Asp-100-->Asn and Asp-100-->Ala holoenzymes are slightly more active than the wild-type holoenzyme, exhibit 1.4-fold and 1.8-fold reductions in the aspartate concentration at half the maximal specific activity, respectively, and show increased affinities for ATP and CTP . Both the Asp-100-->Asn and Asp-100--> Ala catalytic subunits exhibit a 2-fold reduction in the half-life of inactivation at 69 +/- 0.5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Sep 28, 32(38), 10254 - 62
Nature of the ribosomal mRNA track: analysis of ribosome-binding sites containing different sequences and secondary structures; Ringquist S et al.; The ribosomal mRNA track was investigated by toeprinting 30S ribosomes, in the presence or absence of tRNA, using a variety of different ribosome-binding sites . We found that: (1) the ribosome, by itself, recognizes the mRNA translational initiation site; (2) the ribosomal mRNA track makes extensive contact with mRNA independent of tRNA and the start codon; (3) ribosome-mRNA complexes are less stable than complexes containing tRNA; and (4) toeprinting can be used to analyze the contour of the ribosomal mRNA track, yielding information on its "height" as well as its "length" dimension . Examination of several ribosome-binding sites, including those containing very stable secondary structure, indicated that the "height" of the mRNA track is quite roomy, while the nucleotide distance between the site of Shine-Dalgarno annealing, the P site, and the 3'-edge of the mRNA track is fixed . The data suggest a mechanism for tethering regulatory elements to the ribosome during translation.

Biochemistry, 1993 Sep 28, 32(38), 10102 - 8
SH2 domain proteins as high-affinity receptor tyrosine kinase substrates; Sierke SL et al.; Activation of a growth factor receptor tyrosine kinase (RTK) is accompanied by a rapid autophosphorylation of the receptor on tyrosine residues . Receptor activation has been shown to promote the association of signal-transducing proteins containing SH2 domains (second domain of src homology) . These receptor-associated proteins can, in turn, be phosphorylated by the RTK, an event which presumably regulates their activities . It has been suggested that SH2 domains in signal-transducing proteins target these proteins as substrates of the activated RTK . To test this hypothesis, recombinant proteins were generated that contained tyrosine phosphorylation sites of the erbB3 receptor and/or the SH2 domain of c-src . Incorporation of the SH2 domain led to a decrease in KM and an increase in Vmax for the substrate . The KM determined for one chimeric SH2/erbB3 substrate was among the lowest reported for epidermal growth factor RTK substrates . Experiments with a truncated kinase lacking C-terminal autophosphorylation sites indicated that the reduction in KM for these substrates was mediated by interactions between the substrate SH2 domain and phosphotyrosine residues of the RTK . These interactions could also inhibit RTK activity . These results demonstrate that the SH2 domain can effectively target substrates to a RTK and that SH2 domain proteins can regulate RTK activity.

J Immunol Methods, 1993 Sep 27, 165(1), 59 - 66
Particulate nitrocellulose as a solid phase for protein immobilization in immuno-affinity chromatography; Hammerl P et al.; The use of nitrocellulose paper as a solid phase matrix for protein immobilization and its application in immunoaffinity chromatography is described . Pieces of nitrocellulose paper were frozen in liquid nitrogen and ground to a powder (NCP) which was then fractionated according to particle size by repeated sedimentation/resuspension cycles in water . The flow properties of different NCP fractions were compared with those of Sephadex G-50 . Protein binding capacity and binding dynamics were investigated in a model system with bovine serum albumin (BSA) in phosphate buffered saline . Applications of the material are illustrated by batch chromatography for the removal of anti-carrier protein antibodies from a hapten antiserum and by affinity purification of a hapten-specific antibody fraction using NaSCN elution from haptenated NCP . Furthermore, the applicability of the material in column chromatography is demonstrated by elution of a monospecific fraction of anti-fluorescein antibodies from a hapten/carrier mixed bed column by excess of soluble hapten . The results demonstrate that NCP chromatography appears to be a cheap and useful alternative to many other chromatographic media used for protein immobilization.

FEBS Lett, 1993 Sep 27, 331(1-2), 91 - 5
Carboxy-terminal processing of the large subunit of {NiFe} hydrogenases; Menon NK et al.; Two electrophoretic forms of the large subunit of the soluble periplasmic {NiFe} hydrogenase from Desulfovibrio gigas have been detected by Western analysis . The faster moving form co-migrates with the large subunit from purified, active enzyme . Amino acid sequence and composition of the C-terminal tryptic peptide of the large subunit from purified hydrogenase revealed that it is 15 amino acids shorter than that predicted by the nucleotide sequence . Processing of the nascent large subunit occurs by C-terminal cleavage between His536 and Val537, residues which are highly conserved among {NiFe} hydrogenases . Mutagenesis of the analogous residues, His582 and Val583, in the E . coli hydrogenase-1 (HYD1) large subunit resulted in significant decrease in processing and HYD1 activity.

FEBS Lett, 1993 Sep 27, 331(1-2), 19 - 24
Nucleotide and negatively charged lipid-dependent vesicle aggregation caused by SecA . Evidence that SecA contains two lipid-binding sites; Breukink E et al.; SecA which is an overall acidic protein was found to induce an increase in the turbidity of a solution of vesicles consisting of negatively charged phospholipids . This increase was found to be due to an aggregation of the vesicles mediated by SecA . The SecA-mediated vesicle aggregation was not found for zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine and showed a large dependence on both temperature and ionic strength . Furthermore it was shown that ATP and to a lesser extent ADP+Pi were able to reduce the SecA-mediated vesicle aggregation, while no effect could be seen for a non-hydrolysable ATP analog AMP-PNP . Using the steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene present in 1,2-dioleoyl-sn-glycero-3-phosphoglycerol vesicles we could show that SecA inserts in the bilayer . Monolayer studies confirmed that SecA is able to cause close contact between two membranes and gave a direct insight into the different types of lipid-protein interactions involved . From our results we propose that the SecA monomer possesses two lipid-binding sites which in the functional dimer conformation are responsible for the SecA-mediated vesicle aggregation.

FEBS Lett, 1993 Sep 27, 331(1-2), 173 - 6
Selective inhibition of Colorado potato beetle cathepsin H by oryzacystatins I and II; Michaud D et al.; The use of oryzacystatins I and II, two cysteine proteinase inhibitors naturally produced in rice grains, represents an attractive way for the control of Coleoptera insect pests . The present study was done to analyze the inhibitory effect of recombinant oryzacystatins produced in Escherichia coli as fusion proteins against digestive proteinases of the major pest Colorado potato beetle (Leptinotarsa decemlineata Say) . Both inhibitors had a significant effect on total proteolytic activity, but maximal inhibitions ranged from 20 to 80% for pHs varying from 5.0 to 7.0, respectively . This pH-dependent efficiency of plant cystatins was due to the selective inactivation of potato beetle cathepsin H, as demonstrated by the use of inhibitors with different specificities against cathepsins B and H . These results demonstrate the importance of having an adequate knowledge of insect proteinases specifically recognized by the inhibitors to be used in pest control strategies.

J Biol Chem, 1993 Sep 25, 268(27), 20037 - 45
Enhanced oxidation of aniline derivatives by two mutants of cytochrome c peroxidase at tryptophan 51; Roe JA et al.; Two hyperactive mutants of cytochrome c peroxidase (CCP), W51F and W51A, catalyze the enhanced oxidation of a number of substituted anilines . The reaction of CCP compound ES with mesidine is biphasic, while similar reactions using compound II give monophasic kinetics . These data, in addition to the ratio of the Fe4+ = O and free-radical species observed during steady-state turnover, indicate that reduction of the Trp-191 free radical of compound ES is more rapid than the reduction of the Fe4+ = O species . Transient kinetics were examined for the oxidation of eight mono-substituted anilines by CCP, W51F, and W51A . Each of the aniline derivatives were oxidized by the mutants at rates that exceeded that of the wild-type enzyme, and the rate constant for m-chloroaniline was 400-fold faster for W51F than for wild-type CCP . Variations in the rate constants for the different substrates follow a linear free-energy relationship using the Hammet substituent effect parameter sigma +, implicating electron transfer from the aniline ring in the transition state . For aniline oxidation, the free energy of activation is 3 kcal/mol lower for the mutants than for wild-type CCP, and this is due primarily to an increase in the activation entropy . These results indicate that the enhanced kinetics of W51F and W51A result from a generalized increase in enzyme reactivity characterized by an exo-entropic transition state such as dissociation of bound H2O from the Fe4+ = O center.

J Biol Chem, 1993 Sep 25, 268(27), 20386 - 91
Heterodimer formation between Escherichia coli Rep and UvrD proteins; Wong I et al.; DNA helicases catalyze the essential process of unwinding duplex DNA to form the single-stranded DNA intermediates required for DNA metabolic processes including replication, recombination, and repair . Most cells, possibly all, encode multiple helicases that function selectively in different processes, although some helicases can complement each other in vivo . Thus, although Escherichia coli can survive mutations or deletions of either the uvrD gene (encoding Helicase II) or the rep gene (encoding Rep helicase) separately, deletion of both rep and uvrD genes is lethal (Washburn, B . K., and Kushner, S . R . (1991) J . Bacteriol . 173,2569-2575) . The Rep and UvrD polypeptides share approximately 40% sequence homology, and we have previously shown that both form homodimeric species and that the Rep homodimer appears to be the functionally active helicase . We report here that these two proteins can also interact in vitro to form a heterodimer . The heterodimer appears to be energetically more stable than the Rep homodimer but less stable than the UvrD homodimer under our conditions . The observation of Rep/UvrD heterodimer formation in vitro opens up the intriguing possibility that the heterodimer may play a physiologically important role that is distinct from the role of the Rep or UvrD homodimers . Therefore, considerations of the role of either protein in DNA metabolic processes, such as replication and repair, must include a potential role for a Rep/UvrD heterodimer.

J Biol Chem, 1993 Sep 25, 268(27), 20327 - 34
Nebulin as an actin zipper . A two-module nebulin fragment promotes actin nucleation and stabilizes actin filaments; Chen MJ et al.; Nebulin is a family of giant muscle proteins (700-900 kDa) that interact with actin to form composite thin filaments in the skeletal muscle sarcomere . This modular protein is composed predominantly of repeating sequence modules of 31-38 residues . To understand the minimum size and number of sequence modules that are required for actin interaction, we studied the behavior of a highly soluble two-module nebulin fragment ND8 that was expressed in Escherichia coli . By fluorescence spectroscopy with pyrenyl-actin and co-sedimentation assays, we observed the following . 1) ND8 greatly accelerated actin nucleation, especially in a buffer that is suboptimal for actin nucleation . The presence of ND8 abolished the lag phase of actin polymerization and increased the net extent of steady state polymerization, thereby reducing the critical concentration of actin polymerization . 2) ND8 reduced the rate of actin depolymerization and might increase the rate of elongation . 3) Cytochalasin E, which caps both ends of actin filaments, inhibited the effect of ND8 on actin polymerization and caused the depolymerization of actin-ND8 complexes . These data suggest that ND8 interacts with actin in such a fashion that it stabilizes the actin nuclei and slows the depolymerization from the ends of actin filaments . 4) The binding stoichiometry of ND8 to F-actin, as estimated by co-sedimentation assays, is 1 to 2 mol of ND8 to 1 mol of actin with an apparent dissociation constant of 20 to 40 microM . Our data suggest that nebulin-actin interaction promotes actin nucleation and stabilizes preformed actin filaments, both of which are desirable attributes of a length-regulating template for actin filaments of the skeletal muscle . Each nebulin molecule may contain as many as 100-200 actin binding domains to form a zipper-like nebulin/actin composite filament.

J Biol Chem, 1993 Sep 25, 268(27), 20305 - 11
Hydrophobic amino acids in the retinal-binding pocket of bacteriorhodopsin; Greenhalgh DA et al.; The hydrophobic amino acids Met-20, Val-49, Ala-53, Met-118, Gly-122, and Met-145 are believed to be among the amino acids that form the retinal-binding pocket in bacteriorhodopsin . We have now replaced the above amino acids, one at a time, and report on the effects of these replacements (M20A/E, V49A/L, A53G, M118A/E, G122C, and M145A/E) on the properties of bacteriorhodopsin . With the exception of Met-20, replacements at all of the other positions resulted in (i) altered rates of in vitro chromophore formation that ranged from 20-fold faster to 45-fold slower than wild-type bacteriorhodopsin, (ii) blue shifts in the visible spectra of up to 80 nm, and (iii) caused changes in the retinal isomer compositions of the mutant chromophores . Specific effects were also observed in the photocycles of the Met-118, Met-145, and Val-49 mutants, suggesting that these 3 amino acids have important roles in light transduction by bacteriorhodopsin . These results are discussed together with previous studies on the effects of amino acid replacements in the retinal-binding pocket of bacteriorhodopsin.

J Biol Chem, 1993 Sep 25, 268(27), 20170 - 4
Site-directed mutagenesis of the dual translational initiation sites of the clpB gene of Escherichia coli and characterization of its gene products; Park SK et al.; The heat shock protein ClpB in Escherichia coli is a protein-activated ATPase and consists of two proteins with sizes of 93 and 79 kDa . By polymerase chain reaction-aided site-directed mutagenesis, both the proteins have been shown to be encoded by the same reading frame of the clpB gene, the 93-kDa protein (ClpB93) from the 5'-end AUG translational initiation site and the 79-kDa protein (ClpB79) from the 149th codon (an internal GUG start site) . Both the purified ClpB93 and ClpB79 proteins behave as tetrameric complexes with a very similar size of about 350 kDa upon gel filtration on a Superose-6 column . Both appear to be exclusively localized to the cytosol of E . coli . Both show inherent ATPase activities and have an identical Km of 1.1 mM for ATP . The ATPase activity of ClpB93 is as markedly stimulated by proteins, including casein and insulin, as that of wild-type ClpB, but the same proteins show little or no effect on ClpB79 . Because ClpB79 lacks the 148 N-terminal sequence of ClpB93 but retains the two consensus sequences for adenine nucleotide binding, the N-terminal portion appears to contain a site(s) or domain(s) responsible for protein binding . Furthermore, ClpB79 is capable of inhibiting the casein-activated ATPase activity of ClpB93 in a dose-dependent manner but without any effect on its inherent ATPase activity . In addition, ClpB93 mixed with differing amounts of ClpB79 behave as tetrameric molecules, although its protein-activated ATPase activity is gradually reduced . These results suggest that tetramer formation between ClpB93 and ClpB79 may be responsible for the inhibition of the activity.

J Biol Chem, 1993 Sep 25, 268(27), 20126 - 33
Specific placement of tryptophan in the catalytic sites of Escherichia coli F1-ATPase provides a direct probe of nucleotide binding: maximal ATP hydrolysis occurs with three sites occupied; Weber J et al.; Residue beta Y331 of Escherichia coli F1-ATPase is known from previous affinity labeling, mutagenesis, and lin-benzo-ADP binding experiments to interact directly with the adenine moiety of substrates bound in catalytic sites . Here we mutagenized beta Y331 to tryptophan . Mutant cells grew well on succinate or limiting glucose; purified mutant F1 had kappa cat/Km and lin-benzo-ADP binding characteristics similar to wild type . Fluorescence from beta W331 residues exhibited a maximum at 349 nm, indicating a polar environment in unoccupied sites . ATP, ADP, or AMPPNP caused virtually complete quenching of beta W331 fluorescence, so that the fluorescence of mutant F1 with occupied catalytic sites resembled that of wild-type enzyme . Therefore the beta W331 fluorescence provided a direct probe of nucleotide binding to catalytic sites under true equilibrium conditions . We measured ATP binding and hydrolysis in parallel experiments and found that occupancy of one or two catalytic sites per F1 molecule did not yield significant rates of hydrolysis while occupancy of all three sites yielded Vmax rates . Km(ATP) was similar to Kd3, the Kd for ATP binding to the third catalytic site . We also measured AMPPNP and ADP binding parameters . For ADP, the "on" rate at the first catalytic site was much faster (> or = 5 x 10(5) M-1 s-1) than seen previously by centrifuge column procedures, although the Kd was not much changed . For AMPPNP, the "on" rate at the first site was 2 orders of magnitude less than for ADP or ATP, and the Kd was similar to that for ADP.

Nucleic Acids Res, 1993 Sep 25, 21(19), 4604 - 9
Dam methyltransferase from Escherichia coli: sequence of a peptide segment involved in S-adenosyl-methionine binding; Wenzel C et al.; DNA adenine methyltransferase (Dam methylase) has been crosslinked with its cofactor S-adenosyl methionine (AdoMet) by UV irradiation . About 3% of the enzyme was radioactively labelled after the crosslinking reaction performed either with (methyl-3H)-AdoMet or with (carboxy-14C)-AdoMet . Radiolabelled peptides were purified after trypsinolysis by high performance liquid chromatography in two steps . They could not be sequenced due to radiolysis . Therefore we performed the same experiment using non-radioactive AdoMet and were able to identify the peptide modified by the crosslinking reaction by comparison of the separation profiles obtained from two analytical control experiments performed with 3H-AdoMet and Dam methylase without crosslink, respectively . This approach was possible due to the high reproducibility of the chromatography profiles . In these three experiments only one radioactively labelled peptide was present in the tryptic digestions of the crosslinked enzyme . Its sequence was found to be XA-GGK, corresponding to amino acids 10-14 of Dam methylase . The non-identified amino acid in the first sequence cycle should be a tryptophan, which is presumably modified by the crosslinking reaction . The importance of this region near the N-terminus for the structure and function of the enzyme was also demonstrated by proteolysis and site-directed mutagenesis experiments.

Nucleic Acids Res, 1993 Sep 25, 21(19), 4577 - 85
A novel method for the determination of post-transcriptional modification in RNA by mass spectrometry; Kowalak JA et al.; A method is described for the detection, chemical characterization and sequence placement of post-transcriptionally modified nucleotides in RNA . Molecular masses of oligonucleotides produced by RNase T1 hydrolysis can be measured by electrospray mass spectrometry with errors of less than 1 Da, which provides exact base composition, and recognition of modifications resulting from incremental increases in mass . Used in conjunction with combined liquid chromatography-mass spectrometry and gene sequence data, modified residues can be completely characterized at the nucleoside level, and assigned to sequence sites within oligonucleotides defined by selective RNase cleavage . The procedures are demonstrated using E.coli 5S rRNA, in which all RNase T1 fragments predicted from the rDNA sequence are identified solely on the basis of their molecular masses, and using E.coli 16S rRNA for analysis of post-transcriptional modification, including placement of 3-methyluridine at position 1498 . The principles described are generally applicable to other covalent structural modifications of RNA which produce a change in mass, such as those resulting from editing, photochemical cross-linking, or xenobiotic modification.

Nucleic Acids Res, 1993 Sep 25, 21(19), 4563 - 8
Specific binding of MobA, a plasmid-encoded protein involved in the initiation and termination of conjugal DNA transfer, to single-stranded oriT DNA; Bhattacharjee MK et al.; MobA protein, encoded by the broad host-range plasmid R1162, is required for conjugal mobilization of this plasmid . The protein is an essential part of the relaxosome, and is also necessary for the termination of strand transfer . In vitro, MobA is a nuclease specific for one of the two DNA strands of the origin of transfer (oriT) . The protein can cleave this strand at the same site that is nicked in the relaxosome, and can also ligate the DNA . We show here that purified MobA protein forms a complex that is specific for this single oriT strand . The complex is unusually stable, with a half-life of approximately 95 min, is not disrupted by hybridization with the complementary strand, and reforms rapidly after boiling . Both the inverted repeat within oriT, and the eight bases between this repeat and the site cleaved by MobA, are required for binding by the protein . Mutations reducing base complementarity between the arms of the inverted repeat also decrease binding . This effect is partially suppressed by second-site mutations restoring complementarity . These results parallel the effects of these mutations on termination . Footprinting experiments with P1 nuclease indicate that the DNA between the inverted repeat and the nick site is protected by MobA, but that pairing between the arms of the repeat, which occurs in the absence of protein, is partially disrupted . Our results suggest that termination of strand transfer during conjugation involves tight binding of the MobA protein to the inverted repeat and adjacent oriT DNA . This complex positions the protein for ligation of the ends of the transferred strand, to reform a circular plasmid molecule.

Nucleic Acids Res, 1993 Sep 25, 21(19), 4491 - 8
Efficient method for constructing comprehensive murine Fab antibody libraries displayed on phage; Orum H et al.; We have developed efficient methodologies for construction and expression of comprehensive phage display libraries of murine Fab antibody fragments in E . coli cells . Our methods optimize several critical steps of the polymerase chain reaction (PCR) amplification of transcripts of the re-arranged immunoglobulin genes and of their subsequent assembly and expression: Firstly, we have designed exhaustive sets of PCR primers of low degeneracy for the amplification of transcripts of the Fab region of the heavy and light-chain genes . These primers proved effective in amplification of Fab gene fragments from a large panel of hybridoma cell lines of different specificity and family sub-type . Secondly, we have developed a 'jumping PCR' technique that effectively assembled and recombined the amplified heavy and light-chain gene fragments into a bi-cistronic operon . Thirdly, we have constructed expression vectors for insertion of the combinatorial Fab gene-cassette in fusion with a truncated version of the phage surface protein, gIIIp . The heavy chain and the light chain-gIII fusion are transcribed as a polycistronic mRNA from the lacZ promoter and efficient transcriptional control is provided by wildtype lacI present on the vector . The utility of the system was demonstrated by isolating several antigen-binding clones from hybridomas and libraries made from immunized mice.

Nucleic Acids Res, 1993 Sep 25, 21(19), 4476 - 82
The 5' flanking sequence negatively modulates the in vivo expression and in vitro transcription of a human tRNA gene; Tapping RI et al.; The consequences of altering the 5' flanking region of a human amber suppressor tRNA(ser) gene on phenotypic expression in vivo and transcription in vitro was examined by constructing a series of upstream deletion and substitution mutants . The resulting tDNA variants were examined for functional tRNA expression in vivo, by measuring suppression of a nonsense mutation in the Escherichia coli chloramphenicol acetyltransferase (cat) gene in co-transfection assays, and for transcriptional activity in vitro using HeLa cell nuclear extracts . Mutant genes in which the 18 nucleotides 5' proximal to the coding region were deleted and replaced with heterologous sequences were 2 to 5 fold more active in vivo in comparison to the wild type gene . There was a strong, but not exclusive, correlation between the levels of nonsense suppression observed in vivo and transcriptional activity in vitro . In certain cases, introduction of an oligonucleotide encompassing this 18 nucleotide element upstream of more active tRNA genes reduced both the levels of suppression and template activity . These results indicate that the immediate 5' contiguous sequence of this tRNA gene negatively modulates expression both in vivo and in vitro.

Nucleic Acids Res, 1993 Sep 25, 21(19), 4435 - 43
A deletion mutant of the type IC restriction endonuclease EcoR1241 expressing a novel DNA specificity; Abadjieva A et al.; We have developed a complementation assay which allows us to distinguish between mutations affecting subunit assembly and mutations affecting DNA binding in the DNA recognition subunit (HsdS) of the multimeric restriction endonuclease EcoR1241 . A number of random point mutations were constructed to test the validity of this assay . Two of the mutants produced were found to be truncated polypeptides that were still capable of complementation with the EcoR1241 Hsd subunits to give an active restriction enzyme of novel DNA specificity . The N-terminal variable domain (responsible for recognition of GAA from the EcoR1241 recognition sequence GAAnnnnnnRTCG) and the spacer region (central conserved region) is intact in both of these mutants . One of these mutant genes (hsdS(delta 50) has been cloned as an active Mtase . Purification of the Mtase proved to be difficult because the complex is weak . However, Mtase activity was obtained from a soluble cell extract, and this allowed us to determine the DNA recognition sequence of the Mtase to be GAAnnnnnnnTTC . This recognition sequence is an inverted repeat of 5'-end of the EcoR1241 recognition sequence . This suggests that the mutant Mtase is assembled from two inverted HsdS(D50) subunits, possibly held together by the HsdM subunits.

J Biol Chem, 1993 Sep 25, 268(27), 20578 - 83
Amino acid sequence and function of the light subunit of rat kidney gamma-glutamylcysteine synthetase; Huang CS et al.; The heavy subunit (M(r), 72,614) of rat kidney gamma-glutamylcysteine synthetase, the enzyme that catalyzes the first step of glutathione (GSH) synthesis, mediates the catalytic activity of this enzyme and its feedback inhibition by GSH . There is evidence that the light subunit has a regulatory function (Huang, C.-S., Chang, L.-S., Anderson, M.E., and Meister, A . (1993) J . Biol . Chem . 268, 19675-19680) . In the present work the cDNA for the light subunit was isolated, sequenced, and expressed in Escherichia coli . The cDNA was found to code for a protein of 274 amino acid residues (M(r) 30, 548) . Recombinant holoenzyme was obtained by co-expression of the heavy and light subunits and by mixing of the separately expressed proteins . These recombinant holoenzyme preparations exhibit catalytic and GSH feedback inhibitory properties that are virtually identical to those of the isolated holoenzyme . These studies establish that the light subunit is an integral part of the enzyme and that the light and heavy subunits, are coded for separately . Possibly significant similarity of sequence of amino acids was found between the light subunit and E . coli gamma-glutamylcysteine synthetase, which is a single polypeptide.

J Biol Chem, 1993 Sep 25, 268(27), 20343 - 51
Human Clara cell 10-kDa protein is the counterpart of rabbit uteroglobin; Mantile G et al.; Human Clara cell 10-kDa protein has been suggested to be a counterpart of rabbit uteroglobin, an immunomodulatory and antiinflammatory secretory protein . Since this human protein is not readily available in substantial quantity for detailed characterization of its biochemical, biological, and pharmacological properties, we sought to express it in Escherichia coli in order to study its structure-function relationship . However, bacterial overproduction of homodimeric proteins with interchain disulfide bonds, such as Clara cell 10-kDa protein, was thought to be impossible until we achieved expression of native uteroglobin (Miele, L., Cordella-Miele, E., and Mukherjee, A.B . (1990) J . Biol . Chem . 265, 6427-6435) . Here, we report high level production of recombinant native dimeric human Clara cell 10-kDa protein in E . coli and its characterization . Recombinant human Clara cell 10-kDa protein forms its disulfide bonds within the bacterial cytoplasm . The purified protein possesses two of the most important activities characteristic of uteroglobin: (i) it is an excellent substrate of transglutaminase, and (ii) it is a potent inhibitor of porcine pancreatic and, more importantly, human synovial phospholipase A2 . These results demonstrate that human Clara cell 10-kDa protein and rabbit uteroglobin have very similar biochemical properties . Our data suggest that this protein may possess immunomodulatory and antiinflammatory activities of potential physiological and pharmacological importance.

Cell, 1993 Sep 24, 74(6), 1021 - 31
Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion; Bennett RJ et al.; E . coli RuvC protein resolves Holliday junctions during genetic recombination and postreplication repair . Using small synthetic junctions, we show that junction recognition is structure-specific and occurs in the absence of metal cofactors . In the presence of Mg2+, Holliday junctions are resolved by the introduction of symmetrically related nicks at the 3' side of thymine residues . The nicked duplex products are repaired by the action of DNA ligase . Within the RuvC-Holliday junction complex, the DNA is distorted such that 2 of the 4 strands become hypersensitive to hydroxyl radical attack . The ionic requirements of binding, hydroxyl radical sensitivity, and strand cleavage indicate three distinct steps in the mechanism of RuvC-mediated Holliday junction resolution: structure-specific recognition, DNA distortion, and sequence-dependent cleavage.

Nature, 1993 Sep 23, 365(6444), 364 - 8
Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing; Moore MJ et al.; Excision of introns from nuclear precursors to messenger RNAs (pre-mRNAs) by the spliceosome requires two distinct phosphodiester transfer (transesterification) reactions: exchange of a 3'-5' for a 2'-5' bond in the first step (lariat formation) and exchange of one 3'-5' phosphodiester for another in the second step (exon ligation) . We report here determination of the stereochemical course of each step using splicing substrates that contained a chiral phosphorothioate . This has provided strong evidence that both steps occur as single 'in-line' SN2 nucleophilic displacement reactions, analogous to the mechanism of group I self-splicing introns . Additionally, because both steps are strongly inhibited by the RP phosphorothioate diastereomer, but not by SP, the spliceosome probably shifts between two active sites in catalysis of the two steps . Chemical and stereochemical similarities suggest that the catalytic site for the second step of spliceosomal processing is related to that of group I self-splicing introns.

Nature, 1993 Sep 23, 365(6444), 343 - 7
Assembly and function of a quaternary signal transduction complex monitored by surface plasmon resonance; Schuster SC et al.; We have used surface plasmon resonance biosensor technology to monitor the assembly and dynamics of a signal transduction complex which controls chemotaxis in Escherichia coli . A quaternary complex formed which consisted of the response regulator CheY, the histidine protein kinase CheA, a coupling protein CheW and a membrane-bound chemoreceptor Tar . Using various experimental conditions and mutant proteins, we have shown that the complex dissociates under conditions that favour phosphorylation of CheY . Direct physical analysis of interactions among proteins in this signal transduction pathway provides evidence for a previously unrecognized binding interaction between the kinase and its substrate . This interaction may be important for enhancing substrate specificity and preventing 'crosstalk' with other systems . The approach is generally applicable to furthering our understanding of how signalling complexes transduce intracellular messages.

Mol Cell Biochem, 1993 Sep 22, 126(2), 115 - 24
Altered topoisomerase activities may be involved in the regulation of DNA supercoiling in aerobic-anaerobic transitions in Escherichia coli; Cortassa S et al.; This study uncovers a new mechanism of regulation of DNA supercoiling operative in vivo upon an aerobic-anaerobic transition in Escherichia coli . Exponentially growing aerobic batch cultures were subjected to a shift to anaerobic conditions . The ratio {ATP}/{ADP} remained essentially constant at 8.5 in the aerobic culture and after a transition to anaerobiosis while DNA supercoiling increased noticeably upon anaerobiosis . This result indicated that the mechanism of regulation of DNA supercoiling by the {ATP}/{ADP} ratio was not operative . The increase in DNA supercoiling was followed by a large decrease in the DNA-relaxing activity of topoisomerase I while gyrase activity remained relatively constant . This decrease in the activity of topoisomerase I is likely to be responsible for the increase in DNA supercoiling.

Biochemistry, 1993 Sep 21, 32(37), 9866 - 73
Coordination of vanadyl(IV) cation in complexes of S-adenosylmethionine synthetase: multifrequency electron spin echo envelope modulation study; Zhang C et al.; S-Adenosylmethionine (AdoMet) synthetase requires two freely dissociable divalent cations for activity, and its activity is greatly stimulated by certain monovalent cations (K+, Tl+) . Omission of the native Mg2+ cations prevents enzyme-catalyzed reactions, although the substrates and products still bind . Vanadyl (oxovanadium, VO2+) serves as a convenient paramagnetic probe of the substrate-independent, divalent cation binding site in the enzyme . In the present study, multifrequency electron spin echo envelope modulation (ESEEM) is employed to explore the cation's coordination sphere in several functionally relevant complexes . In the substrate complex enzyme.VO2+.ATP.methionine.K+, an equatorially coordinating 14N ligand is found, with Aiso = 4.3 MHz . Selective 15N labeling of the epsilon-amino nitrogens of all lysine residues in the protein reveals that lysine is the source of the ligand . A lysine 14N ligand is also observed in the intermediate complex enzyme.VO2+.AdoMet.PPPi.K+, with Aiso = 4.8 MHz, and in the initial product complex enzyme.VO2+.AdoMet.PPi.K+ . In the subsequent product, enzyme.VO2+.AdoMet.K+ (formed upon dissociation of PPi), the methionyl amino nitrogen of AdoMet coordinates VO2+ (Aiso = 5.3 MHz), and the lysine ligand is lost . In each complex, the monovalent cation activator can be changed from K+ to Tl+ or Na+ with no effect on the ESEEM spectra . Combination of the ESEEM data from this study with previous CW data {Markham, G . D . (1984) Biochemistry 23, 470-478} leads to identification of three of the equatorial ligands to VO2+ and places constraints upon the identity of the fourth ligand, in both the substrate and product complexes . A hypothetical outline of changes in the metal coordination scheme during the reaction is presented, based upon these results.

Biochemistry, 1993 Sep 21, 32(37), 9845 - 50
Autocatalytic generation of dopa in the engineered protein R2 F208Y from Escherichia coli ribonucleotide reductase and crystal structure of the dopa-208 protein; Aberg A et al.; The mutant form Phe-208-->Tyr of the R2 protein of Escherichia coli ribonucleotide reductase contains an intrinsic ferric-Dopa cofactor with characteristic absorption bands at 460 and ca . 700 nm {Ormo, M., de Mare, F., Regnstrom, K., Aberg, A., Sahlin, M., Ling, J., Loehr, T . M., Sanders-Loehr, J., & Sjoberg, B . M . (1992) J . Biol . Chem . 267, 8711-8714} . The three-dimensional structure of the mutant protein, solved to 2.5-A resolution, shows that the Dopa is localized to residue 208 and that it is a bidentate ligand of Fe1 of the binuclear iron center of protein R2 . Nascent apoR2 F208Y, lacking metal ions, can be purified from overproducing cells grown in iron-depleted medium . ApoR2 F208Y is rapidly and quantitatively converted to the Dopa-208 form in vitro by addition of ferrous iron in the presence of oxygen . Other metal ions (Cu2+, Mn2+, Co2+) known to bind to the metal site of wild-type apoR2 do not generate a Dopa in apoR2 F208Y . The autocatalytic generation of Dopa does not require the presence of a tyrosine residue at position 122, the tyrosine which in a wild-type R2 protein acquires the catalytically essential tyrosyl radical . It is proposed that generation of Dopa initially follows the suggested reaction mechanism for tyrosyl radical generation in the wild-type protein and involves a ferryl intermediate, which in the case of the mutant R2 protein oxygenates Tyr 208 . This autocatalytic metal-mediated reaction in the engineered R2 F208Y protein may serve as a model for formation of covalently bound quinones in other proteins.

Biochemistry, 1993 Sep 21, 32(37), 9754 - 62
Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique; Bakin A et al.; A new technique has been developed for the facile location of pseudouridylate (psi) residues in any RNA molecule . The method uses two known modification procedures which in combination uniquely identify U residues which have been converted into psi . The first procedure involves reaction of all U-like and G-like residues with N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide p-tosylate (CMC), followed by alkaline removal of all CMC groups except those linked to the N3 of psi . This stops reverse transcription, resulting in a gel band which identifies the U residue . The second procedure is uridine-specific hydrazinolysis which cleaves the RNA chain at all U residues and produces a gel band upon reverse transcription . psi residues, being resistant to hydrazinolysis, are not cleaved and do not stop reverse transcription . This leads to the absence of a band at psi residues . The combined method can also distinguish psi from 5-methyluridine, 4-thiouridine, uridine-5-oxyacetic acid, and 2-thio-5-methylaminomethyluridine as shown by treating rRNA and tRNA species known to contain these modified bases at defined sites . By this procedure, four new sites for psi in Escherichia coli 23S RNA were discovered, and one was disproven . The four new sites are at positions 2457, 2504, 2580, and 2605 . The erroneous site is at position 2555 . These four new psi residues, which are all in or within 2-3 residues of the peptidyltransferase ring, are thus in a position to play a functional and/or structural role at the peptidyltransferase center.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Sep 21, 32(37), 9701 - 8
Mutagenesis of structural half-cystine residues in human thioredoxin and effects on the regulation of activity by selenodiglutathione; Ren X et al.; A human thioredoxin cDNA was modified to optimize Escherichia coli expression and subcloned into the plasmid pACA, a vector for T7 RNA polymerase-directed expression . The substitution of structural (noncatalytic) half-cystines in human thioredoxin (hTrx) was made by site-directed mutagenesis . The recombinant wild-type (wt) hTrx and its mutant C61S, C72S, and C61S/C72S were expressed and purified to homogeneity . Characterization of the wt and mutant hTrx was done with respect to redox activity with thioredoxin reductase (TR), tryptophan fluorescence, and effects of incubation with GS-Se-SG, which is believed to be the major metabolite of inorganic selenium compounds in mammalian tissues . The Km and kcat of wild-type hTrx for human placenta thioredoxin reductase (HP-TR) at pH 7.0 were 2.0 microM and 2800 min-1, respectively . The mutant proteins C61S, C72S, and C61S/C72S had Km and kcat values similar to those of the wt thioredoxin . Tryptophan fluorescence measurements showed that the wt and mutant proteins had similar stability to a denaturing agent . Incubation of fully reduced thioredoxin with 0.1 molar equivalent of GS-Se-SG resulted in continued oxidation of SH groups . After 3.5 h only 0.5 of initially 4.6 SH groups/thioredoxin remained . With the oxidized protein, a pronounced lag phase in thioredoxin reductase-dependent insulin disulfide reduction was present . Disulfide-linked dimers of the protein were present . The results clearly showed that noncatalytic cysteine residues in hTrx were oxidized accompanied by dimerization and inactivation . The activities of the mutant proteins C72S and C61S/C72S were unchanged after 3 h of incubation with GS-Se-SG . No dimer appeared of the C72S thioredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Sep 21, 32(37), 9553 - 62
Expression and characterization of a structural and functional domain of the mannitol-specific transport protein involved in the coupling of mannitol transport and phosphorylation in the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli; Robillard GT et al.; The mannitol-specific transport protein in Escherichia coli, EIImtl, consists of three structural and functional domains: a hydrophilic EIII-like domain (the A domain); a hydrophobic transmembrane domain (the C domain); and a second hydrophilic domain (the B domain) which connects the A and C domains together . The A domain contains the first phosphorylation site, His554, while the B domain contains the second phosphorylation site, Cys384 . The phosphoryl group which is needed for the active transport of mannitol is sequentially transferred from P-enolpyruvate via the two phosphorylation sites to mannitol bound to the substrate binding site . In this paper, the expression, purification, and initial characterization of the B domain, IIBmtl, are described . Oligonucleotide-directed mutagenesis was used to produce an amber stop codon (TAG) and HindIII restriction site in a flexible loop between the B and A domains in the subcloned gene fragment coding for IIBAmtl (van Weeghel et al., 1991c) . The gene fragment coding for IIBmtl was then subcloned behind strong promoters, located in two different expression/mutagenesis vectors, which directed the expression of the 15.3-kDa polypeptide in Escherichia coli . The domain was purified from E . coli crude cell extracts by using Q-Sepharose Fast Flow, S-Sepharose Fast Flow, and hydroxylapatite column steps . This purification procedure resulted in 1 mg of pure IIBmtl/g of cell, wet weight . The purified B domain was analyzed in vitro for its catalytic activity with membranes containing the phosphorylation site mutant form of EIImtl, C384S, and with the transmembrane domain, IICmtl . The B domain, together with purified IIA, was able to restore the P-enolpyruvate-dependent phosphorylation activity of the membrane-bound C domain . Steady-state mannitol phosphorylation kinetics at saturating EI, HPr, and IIAmtl yielded an apparent Km of P-IIBmtl for IICmtl of 200 microM and an apparent Vmax of 71 nmol of mtl-P min-1 mg of membrane protein)-1 . This Vmax value is comparable to that of wild-type EIImtl measured under the same experimental conditions.

J Theor Biol, 1993 Sep 21, 164(2), 239 - 44
Sequestration of origins of chromosome replication in Escherichia coli by lipid compartments: the pocket hypothesis; Norris V; After initiation of chromosome replication in Escherichia coli, the newly replicated, hemi-methylated origins of replication are exempt from both re-initiation and methylation for up to a third of the generation time . In the model proposed here, the exemption from methylation is attributed to the phospholipid preferences of proteins binding hemi-methylated origins to membrane; these proteins associate with unsaturated phosphatidylethanolamine which forms structures with small radii of curvature; hence, a lipid compartment or "pocket" forms around the origin making it inaccessible to cytoplasmic enzymes such as Dam methyltransferase . Since autolysins and peptidoglycan-synthesizing enzymes are sensitive to lipid composition, peptidoglycan synthesis is stimulated around this compartment and creates structures obtained in membrane fractionation that specifically bind hemi-methylated origins.

FEBS Lett, 1993 Sep 20, 330(3), 329 - 33
Cloning and expression of novel isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine heart; Vidal H et al.; Distinct 6-phosphofructo-2-kinase (PFK-2)/fructose 2,6-bisphosphatase (FBPase-2) cDNAs were cloned from bovine heart, showing that PFK-2/FBPase-2 gene B, which contains 16 exons, codes for at least five mRNAs . Three of them (B1, B2, B4) could encode the 58,000-M(r) isozyme . In B2 mRNA, exon 15 encodes four more residues than in B1 . In B4 mRNA, exon 15 encodes six more residues than in B1, but exon 16 (20 residues) is missing . B3 mRNA corresponds to the 54,000-M(r) isozyme . It lacks exon 15 and also differs from the other mRNAs in the 5' noncoding region . B5 mRNA encodes a truncated form . When expressed in E . coli, the recombinant isoforms corresponding to all these mRNAs except B5 exhibited PFK-2 activity.

J Mol Biol, 1993 Sep 20, 233(2), 322 - 4
Crystallization and preliminary X-ray diffraction studies of recombinant barley lectin and pro-barley lectin; Wright CS et al.; Barley lectin (BL) and its precursor form (proBL), synthesized and over-expressed in Escherichia coli, have been crystallized under conditions identical to those used for the closely related lectin wheat germ agglutinin . These lectins are members of the Gramineae family and possess a unique disulfide-rich domain structure . The pro-lectin polypeptides are extended by 15 amino acid residues at the carboxy terminus . This pro-peptide, which is proteolytically removed as the mature lectin is deposited in the vacuoles, is thought to function as a targeting signal for molecular sorting . Crystals of BL and proBL are well ordered and belong to space groups C222(1) and P2(1)2(1)2(1) . The unit cell dimensions for BL and proBL are a = 51.9 A, b = 73.7 A, c = 89.3 A (one monomer per asymmetric unit), and a = 45.2 A, b = 70.5 A, c = 111.6 A (two monomers per asymmetric unit), respectively . Diffraction patterns on precession photographs of BL crystals are closely similar to those of mature wheat germ agglutinin crystals, suggesting similar crystal packing and correct conformation of this recombinant protein in terms of the four structural domains and 16 disulfide bridges.

J Mol Biol, 1993 Sep 20, 233(2), 203 - 18
Some base substitutions in the leader of an Escherichia coli ribosomal RNA operon affect the structure and function of ribosomes . Evidence for a transient scaffold function of the rRNA leader; Theissen G et al.; Recently, we reported on the creation of a systematic series of C to T transition mutations, located between 19 and 45 nucleotides upstream of the mature 16 S RNA 5' end of a complete, plasmid encoded ribosomal RNA operon . We showed that some of these base transitions have pronounced effects on the growth phenotype of mutant cells, and on the stability of their 16 S RNA as well as on the association capability of their ribosomal subunits . From these observations we concluded that the mutated leader region has post-transcriptional functions in the biogenesis of ribosomes . To further substantiate our conclusions we have now analyzed the growth phenotypes of some leader mutants in more detail, and show here that they are temperature dependent . Furthermore, we have isolated ribosomal RNA, 70 S ribosomes and ribosomal subunits from wild-type and mutant strains and subjected them to a detailed structural and functional analysis . We show that processing and maturation of the 16 S RNA is not altered as a consequence of the base transitions in the leader . In contrast, comparison of the protein composition of wild-type and mutant 30 S particles by two-dimensional gel electrophoresis revealed specific differences . Wild-type 30 S subunits, which are not tightly bound to 50 S, are lacking many ribosomal proteins, while the same fraction of ribosomes from mutant cells has an approximately complete r-protein set, and instead contains some additional non-ribosomal proteins . The translational activity of mutant and wild-type total ribosome preparations was analyzed in vitro . Ribosomes from slowly growing mutants show a significantly reduced in vitro translational activity, which is caused by the 30 S subunits . In contrast, the defects in association reside mainly in the 50 S subunits . Our results demonstrate that some base substitutions in the leader of an Escherichia coli rRNA operon affect the structure and function of ribosomes, although the mutated region is not part of the particles finally formed . This finding implies that at least part of the leader region assists the structure formation of functional 30 S subunits, before it is cut away and discarded . We argue that the rrn leader thus fulfills the functional criteria of a transient molecular scaffold.

J Mol Biol, 1993 Sep 20, 233(2), 191 - 202
Dissection of the DNA-binding domain of Xenopus laevis TFIIIA . Quantitative DNase I footprinting analysis of specific complexes between a 5 S RNA gene fragment and N-terminal fragments of TFIIIA containing three, four or five zinc-finger domains; Hansen PK et al.; Recombinant zinc finger proteins corresponding to N-terminal fragments of Xenopus laevis transcription factor IIIA (TFIIIA) comprising three, four and five fingers produced in Escherichia coli as cleavable hybrid proteins were shown to form specific stoichiometric complexes with DNA fragments containing the internal control region (ICR) of a 5 S RNA gene . The ordered set of DNase I footprints of each of the three proteins on the ICR comprise a nested set of footprints extending upstream from its 3' end (position +96 relative to start of the mature transcript) 20 bp, 20 bp or 34 bp into the ICR, respectively . Quantitative analysis of the footprinting data provided firm evidence that the DNase I footprint, and hence the structure, of the authentic TFIIIA:ICR complex in this region is fully and precisely accounted for by the N-terminal three fingers binding within the +77 to +96 region plus the pair of fingers 4 and 5, both required to extend the footprint upwards from the +77 to the +63 position . A structural interpretation of this set of new footprinting data in view of previous results and data is presented and discussed in terms of a refined model in which the protein-DNA interaction between the ICR and the three N-terminal fingers corresponds closely to that observed in the homologous three-finger zif268:DNA complex, whereas the basic mode of protein-DNA interaction, in which the pair of fingers 4 and 5 is engaged in forming the TFIIIA:ICR complex is of an entirely different, albeit not yet understood nature . To allow assessment of our model in terms of potential specificity-determining H-bonding patterns, a molecular model of the complex between the three-finger TFIIIA fragment and the ICR was constructed, using the zif268:DNA co-ordinates . Eight out of the nine amino acid residues, which according to our model are suitably located for forming hydrogen bonds with the bases, are potential H-bond acceptors or donors.

FEBS Lett, 1993 Sep 20, 330(3), 265 - 9
Extracellular secretion of STa heat-stable enterotoxin by Escherichia coli after fusion to a heterologous leader peptide; Sanchez J et al.; The mature 19-amino acid STa heat-stable enterotoxin of E . coli has a preceding peptide of 53 amino acids which contains two domains called Pre (aa 1-19) and Pro (aa 20-53) sequences, proposed to be essential for extracellular toxin release by this host . The Pro sequence, however, has been proven not be indispensable for this process since Pro deletion mutants secrete STa . To find out if Pre and/or other unremoved natural STa flanking sequences are responsible for toxin secretion in those mutants we genetically fused mature STa directly to the leader peptide of the periplasmic E . coli heat-labile enterotoxin B-subunit (LTB) . Expression of this gene fusion resulted in extracellular secretion of biologically active STa by E . coli independently of natural STa neighboring genetic sequences . Moreover, these results suggest that STa might be able to gain access to the extracellular milieu simply upon its entry into the E . coli periplasm once guided into this compartment by the LTB leader peptide . To test if extracellular secretion in this fashion might be extended to other disulfide bond-rich small peptides, the 13 amino acid conotoxin GI and a non-enterotoxic STa-related decapeptide were cloned . None of the two peptides was found in culture supernatants, in spite of high structural homology to the toxin . Failure to be secreted most likely leads to degradation as peptides were also not detected in bacterial sonicates . We hypothesize that cysteine-rich peptides must have an amino acid length and/or number of disulfide bridges closer to those in STa for them to follow this toxin secretory pathway in E . coli.

Gene, 1993 Sep 15, 131(2), 287 - 92
Cloning and expression in Escherichia coli of the cDNA encoding human cardiac troponin I; Armour KL et al.; We have cloned and sequenced the human cardiac troponin I (cTnI)-encoding cDNA with the aim of expressing the cDNA in Escherichia coli . The cDNA was successfully expressed as a fusion product with beta-galactosidase and as an unfused protein . Both polypeptides were recognised by an anti-human cTnI antibody.

Gene, 1993 Sep 15, 131(2), 167 - 73
A Caenorhabditis elegans act-4::lacZ fusion: use as a transformation marker and analysis of tissue-specific expression; Stone S et al.; A plasmid vector that serves as a dominant marker for isolating transformed animals in Caenorhabditis elegans has been constructed as a translational fusion of the C . elegans act-4 gene (encoding actin) and the Escherichia coli lacZ gene . This gene fusion can be used as a marker in transformation rescue experiments in any fertile strain of C . elegans . Progeny of animals injected with the act-4::lacZ fusion vector are stained histochemically with XGal, and transformants turn blue . The internal eggs of stained animals remain viable, allowing recovery of the transformed strain . When the act-4::lacZ vector is co-injected with an unselected plasmid with which it shares some sequence homology, most transformants that are recovered by screening for expression of the act-4::lacZ fusion contain both plasmids . Production of active beta Gal in animals transformed with the act-4::lacZ gene fusions appears to be limited to certain tissues . A chimeric gene that contains the 5' and 3' regions of act-4 is expressed strongly in the body-wall muscles, vulval muscles, and spermathecae . Addition of the internal portion of act-4, including the protein-coding region and introns, to this chimeric gene leads to additional lacZ expression in the pharynx.

Biochem Biophys Res Commun, 1993 Sep 15, 195(2), 643 - 53
Augmentation of protein production by a combination of the T7 RNA polymerase system and ubiquitin fusion: overproduction of the human DNA repair protein, ERCC1, as a ubiquitin fusion protein in Escherichia coli; Koken MH et al.; This article presents the development of a set of new expression vectors for overproduction of proteins in Escherichia coli . The vectors, pETUBI-ES1, 2 and 3, allow in-frame cloning of any sequence with the ubiquitin gene driven by the strong T7f10 promoter . Combination of the T7 expression system with ubiquitin fusion appears to have a synergistic effect on protein overproduction . Large amounts of stable RNA are produced by T7 RNA polymerase, and fusion of ubiquitin to the N-terminus of target proteins seems to confer more efficient translation, better folding or protection against proteolytic degradation . The ubiquitin part can be utilized for purification of the fusion protein, after which it can be easily removed from the fusion product by ubiquitin-specific proteases . The advantage of combining both systems is demonstrated by the synthesis of large quantities (up to 40-50% of the total protein) of the human ERCC1 protein that hitherto was refractory to overproduction in various other E . coli and yeast expression systems.

J Biol Chem, 1993 Sep 15, 268(26), 19739 - 43
Vitamin D receptor zinc finger region binds to a direct repeat as a dimer and discriminates the spacing number between each half-site; Nishikawa J et al.; 1 alpha,25-Dihydroxyvitamin D3, the most active metabolite of vitamin D3, is a multifunctional agent . The actions of 1 alpha,25-dihydroxyvitamin D3 are mediated through its receptor that activates the specific genes in a ligand-dependent manner . In order to investigate the details of DNA binding properties of vitamin D receptor, we have developed the overexpression and purification system of vitamin D receptor DNA binding domain . The purified peptide could specifically bind to the osteopontin-derived vitamin D responsible element (VDRE) but not to the osteocalcin and the calbindin D-9k-derived VDREs, as determined by bandshift analysis . The osteopontin VDRE contains a direct repeat of GGTTCA motif separated by 3 nucleotides, whereas the osteocalcin and calbindin D-9k VDREs have inadequate direct repeat . Further analyses using synthetic oligonucleotides revealed that vitamin D receptor DNA binding domain could discriminate the spacing number between the consensus steroid-responsible element motif and had different affinities to direct repeats that consisted of various related sequences . These studies give insight into ways in which vitamin D receptor mediates the signal of 1 alpha,25-dihydroxyvitamin D3.

J Biol Chem, 1993 Sep 15, 268(26), 19681 - 9
Expression and purification of functional human 17 alpha-hydroxylase/17,20-lyase (P450c17) in Escherichia coli . Use of this system for study of a novel form of combined 17 alpha-hydroxylase/17,20-lyase deficiency; Imai T et al.; Enzymatically active human 17 alpha-hydroxylase cytochrome P450 (P450c17) has been expressed in and purified from Escherichia coli . The cDNA containing modifications within the amino-terminal eight codons which are favorable for expression in E . coli, as well as codons for 4 histidine residues at the carboxyl terminus, was placed in the pCWori+ expression vector . The modified human P450c17 was detected spectrophotometrically (400 nmol of P450c17/liter culture) and was found to be integrated into E . coli membranes . This previously inaccessible human P450 was purified to electrophoretic homogeneity (10.7 nmol of P450/mg) from solubilized bacterial membranes using two sequential chromatographic steps, nickel nitrilotriacetate followed by hydroxylapatite . The expected enzymatic activities of human P450c17 were reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase, giving turnover numbers of 8.0 nmol/min/nmol P450 for pregnenolone, 6.5 nmol/min/nmol P450 for progesterone, 0.06 nmol/min/nmol P450 for 17 alpha-hydroxypregnenolone, and no detectable activity for 17 alpha-hydroxyprogesterone . This system was utilized to study the molecular basis of a novel form of combined 17 alpha-hydroxylase, 17,20-lyase deficiency resulting from compound heterozygous mutations, a missense point mutation Tyr64(TAT)--> Ser (TCT), and an Ile112 duplication (ATCATC) . Upon expression of these mutant proteins in E . coli, the Tyr64 mutant has 15% of the wild type 17 alpha-hydroxylase activity, whereas the Ile112 duplication shows no activity, results consistent with the observed clinical phenotype.

Biochem J, 1993 Sep 15, 294 ( Pt 3), 667 - 74
High-level expression and in vitro mutagenesis of a fibrillogenic 109-amino-acid C-terminal fragment of Alzheimer's-disease amyloid precursor protein; Gardella JE et al.; We amplified DNA encoding the 3' 109 codons of Alzheimer's-disease amyloid precursor protein (APP) inclusive of the beta protein (A beta) and cytoplasmic domains from cDNA using oligonucleotide primers designed to facilitate cloning into the T7 expression vector pT7Ad23K13 . We also modified this construct to generate recombinant molecules incorporating two recently described APP mutants by site-directed mutagenesis . Both native C109 (deletion construct inclusive of the C-terminal 109 residues of APP) and constructs with a single mutation at codon 642 (T-->G, resulting in a substitution of glycine for valine) or a double mutation at codons 595 (G-->T, substituting asparagine for lysine) and 596 (A-->C, substituting leucine for methionine) were expressed in Escherichia coli to levels of 5-20% of total bacterial protein after induction . The major constituent of expressed C109 protein had an apparent molecular mass of 16-18 kDa by SDS/PAGE and appeared to be the full-length construct by size and N-terminal microsequencing . Also present was a 4-5 kDa species that co-purified with C109, constituting only approximately 1% of expressed protein, which was revealed by Western-blot analysis with antibodies specific for A beta epitopes and after biotinylation of purified recombinant C109 . This fragment shared N-terminal sequence with, and appeared to arise by proteolysis of, full-length C109 in biosynthetic labelling experiments . C109 spontaneously precipitated after dialysis against NaCl or water, and with prolonged (> 20 weeks) standing was found by electron microscopy to contain a minor (< 5%) fibrillar component that was reactive with antibodies to a C-terminal epitope of APP . Recombinant C109 appears to duplicate some of the biochemical and physicochemical properties of C-terminal A beta-inclusive fragments of APP that have been found in transfected cells, brain cortex and cerebral microvessels.

Biochem J, 1993 Sep 15, 294 ( Pt 3), 639 - 44
Biochemical characterization of the molecular interaction between recombinant basic fibroblast growth factor and a recombinant soluble fibroblast growth factor receptor; Caccia P et al.; The extracellular domain of human fibroblast growth factor receptor (XC-FGF-R) was expressed in Escherichia coli . The protein was purified to homogeneity and the interaction with basic fibroblast growth factor (bFGF), its physiological ligand, was examined . Using resins on which bFGF was reversibly bound, we analysed the characteristics of the binding between XC-FGF-R and immobilized bFGF . We also investigated the stoichiometry of the binding between XC-FGF-R and recombinant human bFGF (rhbFGF) applying non-denaturing gel electrophoresis, chemical cross-linking followed by SDS/PAGE, and gel-filtration chromatography . In cross-linking and gel-filtration chromatography experiments, a 1:1 complex between rhbFGF and XC-FGF-R was observed . The complex was separated from the non-complexed proteins using non-denaturing PAGE in the presence of 0.1% Triton X-100 . The band corresponding to the complex was recognized by specific antibodies directed against bFGF and its receptor, blotted on poly(vinylidene difluoride) membranes and submitted to sequence and amino acid analysis . The data obtained from these determinations confirmed the formation of a 1:1 complex between rhbFGF and XC-FGF-R.

Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8732 - 6
Molecular cloning and characterization of rho, a ras-related small GTP-binding protein from the garden pea; Yang Z et al.; The rho proteins, members of the ras superfamily of small GTP-binding proteins, play a central role in the modulation of cellular functions involving the actin cytoskeleton such as in the establishment of cell polarity and morphology . As a first step in elucidating signal transduction pathways leading to processes mediated by the actin cytoskeleton in plants, we initiated cloning and characterization of rho proteins from pea . One rho-related, partial cDNA clone of 167 bp was isolated utilizing a polymerase chain reaction-based cloning strategy, using degenerate primers that correspond to conserved domains within the rho proteins . A full-length cDNA was isolated by screening a pea cDNA library using the 167-bp cDNA as a probe . The Rho1Ps cDNA contains an open reading frame encoding a polypeptide (Rho1Ps) of 197 amino acids that shows 45-64% sequence identity to members of the rho family and about 30% identity to other members of the ras superfamily . In addition to the nucleotide-binding and GTPase domains, Rho1Ps shares conserved residues and motifs unique to the rho proteins . Purified Rho1Ps protein expressed in Escherichia coli retains specific GTP-binding activity . These data indicate that Rho1Ps encodes a small GTP-binding protein of the rho family . The Rho1Ps transcript is expressed in all organs of pea seedlings, being more abundant in root tips and apical buds . DNA gel blot analyses show that the rho proteins in pea are encoded by a multigene family.

Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8648 - 52
Heat shock of Escherichia coli increases binding of dnaK (the hsp70 homolog) to polypeptides by promoting its phosphorylation; Sherman MY et al.; The "molecular chaperone", dnaK, is induced in Escherichia coli upon heat shock and promotes ATP-dependent refolding or degradation of damaged proteins . When cells were grown at 25 degrees C and disrupted, a small fraction of the dnaK bound to affinity columns containing unfolded polypeptides (e.g., a fusion protein named CRAG or casein) and could be dissociated by ATP-Mg2+ . After shifting cells to 42 degrees C for 30 min, up to 5-fold more dnaK bound to these columns than after growth at 25 degrees C . This enhanced binding capacity was reversed after shifting cells back to 25 degrees C . It resulted from a covalent modification, which decreases dnaK's electrophoretic mobility and isoelectric point . This modification appears to be phosphorylation; after treatment with phosphatases, the ATP-eluted dnaK resembled the predominant form in electrophoretic and binding properties . In addition, after incubating cells with {32P}orthophosphate at 42 degrees C, the 32P-labeled dnaK bound quantitatively to the CRAG column, unlike the nonlabeled protein . Thus, the phosphorylated dnaK is a special form of the chaperone with enhanced affinity for unfolded proteins . Its accumulation at high temperatures may account for dnaK's function as the "cellular thermometer."

Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8557 - 61
Isoprenylation of the plant molecular chaperone ANJ1 facilitates membrane association and function at high temperature; Zhu JK et al.; We demonstrate that ANJ1, a higher plant homolog of the bacterial molecular chaperone DnaJ, is a substrate in vitro for protein farnesyl- and geranylgeranyl-transferase activities present in cell extracts of the plant Atriplex nummularia and yeast Saccharomyces cerevisiae . Isoprenylation did not occur when cysteine was replaced by serine in the CAQQ motif at the carboxyl terminus of ANJ1, indicating that this sequence functions as a CaaX consensus sequence for polyisoprenylation (where C is cysteine, a is an aliphatic residue, and X is any amino acid residue) . Substitution of leucine for the terminal glutamine did not result in the expected geranylgeranylation as occurs with mammalian proteins containing a carboxyl-terminal leucine . Unlike the wild-type ANJ1, neither of the proteins containing these amino acid substitutions could functionally complement the yeast temperature-sensitive mutant mas5 . Farnesylation enhanced the association of ANJ1 with A . nummularia microsomal membranes . Electrophoretic mobility of ANJ1 from the plant indicated that the protein is isoprenylated in vivo.

Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8552 - 6
Stromal factor plays an essential role in protein integration into thylakoids that cannot be replaced by unfolding or by heat shock protein Hsp70; Yuan J et al.; The light-harvesting chlorophyll a/b protein (LHCP) is an integral thylakoid membrane protein . It is made in the cytosol as a precursor (pLHCP), imported into chloroplasts, and subsequently integrated into thylakoids . Integration of pLHCP into thylakoids requires a stromal protein factor that functions in part to maintain the solubility and integration competence of pLHCP . Recently, it was reported that unfolded pLHCP was sufficient for integration and that the stromal factor, identified as the plastid Hsp70, was required only to prevent pLHCP refolding {Yalovsky, S., Paulsen, H., Michaeli, D., Chitnis, P . R . & Nechushtai, R . (1992) Proc . Natl . Acad . Sci . USA 89, 5616-5619} . Our studies, using more rigorous criteria for integration, show that unfolded pLHCP is not sufficient; stromal factor is an absolute requirement for integration . Furthermore, experiments with purified Hsp70 as well as Hsp70-depleted stromal extract demonstrate that Hsp70 is not the stromal factor . These results plus the finding that pLHCP diluted out of urea is relatively stable as a substrate for integration point to an additional role for the stromal factor in targeting and/or membrane translocation.

Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8387 - 91
Crystal structure of human immunodeficiency virus (HIV) type 2 protease in complex with a reduced amide inhibitor and comparison with HIV-1 protease structures; Tong L et al.; The crystal structure of HIV-2 protease in complex with a reduced amide inhibitor {BI-LA-398; Phe-Val-Phe-psi (CH2NH)-Leu-Glu-Ile-amide} has been determined at 2.2-A resolution and refined to a crystallographic R factor of 17.6% . The rms deviation from ideality in bond lengths is 0.018 A and in bond angles is 2.8 degrees . The largest structural differences between HIV-1 and HIV-2 proteases are located at residues 15-20, 34-40, and 65-73, away from the flap region and the substrate binding sites . The rms distance between equivalent C alpha atoms of HIV-1 and HIV-2 protease structures excluding these residues is 0.5 A . The shapes of the S1 and S2 pockets in the presence of this inhibitor are essentially unperturbed by the amino acid differences between HIV-1 and HIV-2 proteases . The interaction of the inhibitor with HIV-2 protease is similar to that observed in HIV-1 protease structures . The unprotected N terminus of the inhibitor interacts with the side chains of Asp-29 and Asp-30 . The glutamate side chain of the inhibitor forms hydrogen bonds with the main-chain amido groups of residues 129 and 130.

J Immunol, 1993 Sep 15, 151(6), 3324 - 36
Ligand specificity of purified complement receptor type three (CD11b/CD18, alpha m beta 2, Mac-1) . Indirect effects of an Arg-Gly-Asp (RGD) sequence; Van Strijp JA et al.; We have purified CR3 to homogeneity by affinity chromatography on C3bi-Sepharose and elution with EDTA . C3bi-coated erythrocytes bound to this purified CR3, and binding was dependent on the concentration of both C3bi and CR3, as well as on temperature and the presence of divalent cations . Moreover, binding could be blocked by mAb against CR3 or C3bi and could be enhanced by the addition of integrin modulating factor-1 . We used the purified CR3 to test whether several putative ligands of CR3 directly bound the receptor . The interaction of purified CR3 with fibrinogen, filamentous hemagglutinin of Bordetella pertussis, lipophosphoglycan and glycoprotein 63 of Leishmania mexicana, and lipopolysaccharide from Escherichia coli was confirmed . However the interaction of CR3 with zymosan or its major component, beta-glucan, was not observed in these assays . Previous studies showed that binding of C3bi to PMN could be blocked with Arg-Gly-Asp (RGD) containing peptides and were interpreted to indicate that the RGD sequence in C3bi interacts directly with CR3 . We found, however, that RGD containing peptides were unable to block the interaction of C3bi with purified CR3, yet retained the ability to block binding of C3bi to cells . We conclude that RGD-peptides do not directly bind CR3, but instead indirectly effect CR3 function . Inasmuch as the effect of RGD-peptides could be mimicked with antibodies against leukocyte response integrin, we suggest that RGD-peptides may bind to leukocyte response integrin on polymorphonuclear leukocytes and influence CR3 activity via this receptor.

J Biol Chem, 1993 Sep 15, 268(26), 19889 - 95
Complementation of transport-deficient mutants of Escherichia coli alpha-hemolysin by second-site mutations in the transporter hemolysin B; Zhang F et al.; Hemolysin B (HlyB) is a membrane-bound transport protein composed of an amino-terminal multiple membrane-spanning portion followed by a conserved ATP binding sequence . Together with the inner membrane protein HlyD and the outer membrane protein TolC, HlyB is responsible for transport of the 107-kDa toxin HlyA from the cytoplasm, across both membranes of the cell envelope of Escherichia coli, directly to the medium . We have used a mutational approach to investigate a postulated interaction between HlyA and HlyB . We have isolated transport-deficient mutants of HlyA altered in the C-terminal signal sequence and used one of these, a deletion of 29 amino acids, to select compensatory mutants in the transporter protein HlyB . Fifteen mutants located at six different sites, all mapping within the amino-terminal multiple membrane-spanning domain of HlyB, were identified . All of the mutations are clustered into three groups located close to the predicted inner face of the cytoplasmic membrane . We propose that these locations are close to sites on HlyB that interact with the C-terminal signal sequence of HlyA . This interaction is likely to involve either binding of HlyA to HlyB or activation of the transport mechanism . The compensatory mutants also display different patterns of specificity in terms of their ability to transport different HlyA mutants . The fact that point mutations are able to compensate for drastic changes in the signal sequence of HlyA suggests that substrate specificity of transporters such as HlyB may shift dramatically during evolutionary history . This could account for the diversity of substrates observed for the ABC transporter superfamily in nature.

J Biol Chem, 1993 Sep 15, 268(26), 19858 - 65
UDP-N-acetylglucosamine acyltransferase of Escherichia coli . The first step of endotoxin biosynthesis is thermodynamically unfavorable; Anderson MS et al.; UDP-N-acetylglucosamine acyltransferase of Escherichia coli catalyzes the reaction, UDP-GlcNAc + R-3-hydroxymyristoyl-ACP--> UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc + ACP . Using Matrex Gel Green A and heparin-agarose, we have purified the enzyme to near homogeneity from a strain that overproduces it 474-fold . The subunit molecular mass determined by SDS-gel electrophoresis is approximately 30 kDa, consistent with results of previous radiolabeling experiments in mini-cells . The amino-terminal sequence (Met-Ile-Asp-Lys-Ser-Ala-Phe-Val-His-Pro) and the amino acid composition of the purified protein are consistent with DNA sequencing (Coleman, J., and Raetz, C . R . H . (1988) J . Bacteriol . 170, 1268-1274) . At saturating concentrations of the second substrate, the apparent Km values for UDP-GlcNAc and R-3-hydroxymyristoyl-ACP are 99 and 1.6 microM, respectively . There is an absolute requirement for the R-3-hydroxy moiety of the fatty acyl-ACP substrate; myristoyl-ACP binds effectively (IC50 = 2 microM) but is inactive (< 0.01%) as an alternate substrate . The most remarkable feature of the reaction is its unfavorable equilibrium constant, Keq approximately equal to 0.01, which is not predicted by model S-->O acyl transfer reactions . Thus, although UDP-GlcNAc acyltransferase catalyzes the first unique step of lipid A biosynthesis, it is the second enzyme (the deacetylase) that commits the substrates to this pathway . The specific activity of the deacetylase is elevated approximately 5-fold when lipid A synthesis is inhibited.

J Biol Chem, 1993 Sep 15, 268(26), 19802 - 9
The role of the carboxyl-terminal tail in human O6-methylguanine DNA methyltransferase substrate specificity and temperature sensitivity; Morgan SE et al.; The human O6-methylguanine DNA methyltransferase (MGMT) repairs O6-methylguanine (O6-MG) in DNA at a much lower rate than the Escherichia coli Ada protein, and only MGMT repairs the altered base, O6-benzylguanine (O6-BG) . The diversity in DNA repair properties between MGMT and Ada may be a result of divergent amino acid sequences outside their common proline-cysteine-histidine-arginine-valine (PCHRV) acceptor site . One notable sequence difference is an MGMT 28-amino acid carboxyl-terminal tail which is highly conserved among all mammalian alkyltransferases . The role of this tail sequence in substrate specificity was assessed by expressing full-length MGMT and Ada proteins, and mutant MGMT proteins lacking either 10 or 28 amino acids from the carboxyl terminus, as GST fusion proteins in alkyltransferase-deficient E . coli cells, and comparing rates of repair of O6-MG containing DNA and O6-BG by these fusion proteins at 4 degrees C and 37 degrees C . The MGMT carboxyl-terminal tail was not required for repair of O6-MG in DNA at 37 degrees C although the deletion of this tail sequence reversibly inhibited the ability of MGMT to repair O6-MG in DNA at 4 degrees C . Therefore, the absence of this region affects the ability of the protein to repair O6-MG in DNA at lower temperatures . Furthermore, removal of the tail sequence from MGMT decreased the rate of O6-BG repair 5-fold . We conclude that the 28-amino acid carboxyl-terminal MGMT tail, while not required for activity, modulates the rate of MGMT repair at reduced temperatures and plays a role in substrate specificity.

J Biol Chem, 1993 Sep 15, 268(26), 19710 - 6
Expression, purification, and kinetic characterization of recombinant human adenylosuccinate lyase; Stone RL et al.; Adenylosuccinate adenosine 5'-monophosphate lyase (EC 4.3.2.2; ASL) catalyzes two distinct reactions in adenosine 5'-monophosphate (AMP) biosynthesis . A S413P mutation in ASL segregates with mental retardation in an affected family (Stone, R . L., Aimi, J., Barshop, B . A., Jaeken, J., Van den Berghe, G., Zalkin, H., and Dixon, J . E . (1992) Nature Genet . 1, 59-63) . ASL and S413P ASL have been expressed, purified, and kinetically characterized . Lowering the Escherichia coli growth temperature to 25 degrees C and the concentration of inducer, isopropyl-1-thio-beta,D-galactopyranoside, to 40 microM was necessary for synthesis of soluble, tetrameric enzymes . The recombinant enzymes were purified to homogeneity using anion exchange chromatography followed by chromatography on Blue 2A Sepharose . At pH 7.0 and 25 degrees C, the kcat for cleavage of 5-amino-4-imidazole-N-succinocarboxamide ribotide (SAI-CAR) by ASL was 90 s-1 with a Km of 2.35 microM . The kcat for adenylosuccinate (SAMP) cleavage was 97 s-1 with a Km of 1.79 microM . The catalytic mechanism involved one general base catalyst (pK alpha = 6.4) and one general acid catalyst (pK alpha = 7.5) . ASL follows an ordered uni-bi reaction mechanism with fumarate released first . 5-Amino-4-imidazolecarboxamide ribotide (AICAR) and AMP were competitive with SAICAR and SAMP (Ki{AICAR} = 11.3 microM; Ki{AMP} = 9.2 microM), whereas fumarate inhibited noncompetitively (Kii = 2.3 mM, Kis = 2.8 mM) . The competitive inhibition by AICAR and AMP suggests a single active site that binds both SAICAR and SAMP . The kinetic constants at pH 7.0, 25 degrees C and the kcat/Km versus pH profiles for ASL and S413P ASL were very similar . These results are consistent with S413P being a structural rather than a catalytic defect.

J Biol Chem, 1993 Sep 15, 268(26), 19626 - 31
Ring finger motif of Arabidopsis thaliana COP1 defines a new class of zinc-binding domain; von Arnim AG et al.; The COP1 gene of Arabidopsis thaliana encodes a protein mediating the switch between the two developmental pathways utilized in light and darkness . A cysteine-rich motif identified the COP1 protein as a member of a group of regulatory proteins which share the amino acid motif Cys-X-X-Cys-loop I-Cys-X-His-X-X-Cys-X-X-Cys-loop II-Cys-X-X-Cys (ring finger) . Although this new class of cysteine-rich motifs has been proposed to bind metal ions, no direct evidence supporting this has been presented . By analyzing the COP1 protein expressed in Escherichia coli, we demonstrate here that each COP1 molecule can bind up to two zinc atoms . The two zinc ions are bound with different affinities . One is tightly bound and resistant to urea and EDTA, whereas the other one is labile under those conditions . It is further shown that deletion of the ring finger motif abolishes the metal-binding capacity of COP1 . We conclude that the ring finger motif constitutes a zinc-coordinating element distinct from previously characterized zinc-binding domains.

J Biol Chem, 1993 Sep 15, 268(26), 19610 - 7
A plant glutamate decarboxylase containing a calmodulin binding domain . Cloning, sequence, and functional analysis; Baum G et al.; Molecular procedures have been applied to isolate plant calmodulin-binding proteins . A petunia cDNA expression library was screened with 35S-labeled recombinant calmodulin as a probe, and a cDNA coding for a Ca(2+)-dependent calmodulin-binding protein was isolated . The deduced amino acid sequence of the petunia protein (500 amino acid residues, 58 kDa) has 67% overall amino acid sequence similarity to glutamate decarboxylase (GAD) from Escherichia coli (466 amino acid residues, 53 kDa) . The recombinant protein expressed in E . coli cells displays GAD activity, i.e . catalyzes the conversion of glutamic acid to gamma-aminobutyric acid and binds calmodulin, whereas E . coli GAD does not bind calmodulin . The calmodulin binding domain in the petunia GAD was mapped by binding truncated forms of GAD immobilized on nitrocellulose membranes to recombinant petunia 35S-calmodulin as well as to biotinylated bovine calmodulin and by binding truncated forms of GAD to calmodulin-Sepharose columns . The calmodulin binding domain in petunia GAD is part of a carboxyl end extension that is not present in E . coli GAD . Polyclonal antibodies raised against the recombinant petunia GAD detect a single protein band from plant extracts of gel mobility identical to that of the recombinant GAD . Moreover, the plant protein binds calmodulin in vitro . This is the first report of the isolation of a GAD gene from plants and of a calmodulin-binding GAD from any organism . Our results raise the possibility that intracellular Ca2+ signals via calmodulin are involved in the regulation of gamma-aminobutyric acid synthesis in plants.

J Biol Chem, 1993 Sep 15, 268(26), 19574 - 80
Purification and characterization of recombinant Bet v I, the major birch pollen allergen . Immunological equivalence to natural Bet v I; Ferreira FD et al.; Pollen from trees of the order Fagales (e.g . birch, alder, hazel, oak, and hornbeam) are a major cause of Type I allergies observed in early spring . Previously, we reported the cloning and sequencing of Bet v I, the major birch pollen allergen, which showed high sequence similarities to a family of plant pathogen-activated genes (Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O., and Breitenbach, M . (1989) EMBO J . 8, 1935-1938) . Here, we present the results on the expression, purification, and characterization of recombinant Bet v I produced in Escherichia coli as fusion and non-fusion protein, respectively . The purified recombinant proteins were analyzed to verify purity and structural integrity, and their immunological properties were compared to those of Bet v I isolated from birch pollen (natural Bet v I) . Immunoblot analyses showed that the recombinant proteins are specifically recognized by monoclonal antibodies raised against natural Bet v I as well as by IgE from birch pollen-allergic patients . However, enzyme-linked immunosorbent assays revealed a decreased IgE-binding activity of the recombinant fusion Bet v I compared to the non-fusion and natural Bet v I proteins, which probably results from conformational changes due to the fusion tail . Recombinant non-fusion Bet v I was equivalent to natural Bet v I with respect to IgE-binding properties, the ability to induce in vitro proliferation of allergen-specific T-cell clones, and the ability to release histamine from basophils derived from birch pollen-allergic patients.

J Biol Chem, 1993 Sep 15, 268(26), 19422 - 30
LE-ACS4, a fruit ripening and wound-induced 1-aminocyclopropane-1-carboxylate synthase gene of tomato (Lycopersicon esculentum) . Expression in Escherichia coli, structural characterization, expression characteristics, and phylogenetic analysis; Lincoln JE et al.; ACC (1-aminocyclopropane-1-carboxylic acid) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene and is encoded by a highly divergent multigene family in tomato (Rottmann, W . H., Peter, G . F., Oeller, P . W., Keller, J . A., Shen, N . F., Nagy, B . P., Taylor, L . P., Campbell, A . D., and Theologis, A . (1991) J . Mol . Biol . 222, 937-961) . Two members of the family, LE-ACS2 and LE-ACS4, are induced during fruit ripening and upon treatment of mature green fruits with exogenous ethylene (C2H4) in a dose-dependent manner . Both genes are superinduced by wounding of pericarp tissue during various stages of ripening . The wound-induced accumulation of LE-ACS2 mRNA is more rapid and greater than that of LE-ACS4 . Both mRNAs accumulate in the absence of protein synthesis, suggesting that their induction is a primary response to the inducer . The LE-ACS4 gene was isolated and structurally characterized . The function of the LE-ACS4 protein (53,509 Da, pI 5.4) was verified by expression experiments in Escherichia coli . The promoters of LE-ACS2 and LE-ACS4 contain potential cis-acting regulatory elements responsible for induction by ethylene, wounding, and anaerobiosis . In addition, elements for binding the transcriptional factors EmBP1, GBF-1, and OCSBF-1 are also present . Phylogenetic analysis of 20 ACC synthases from dicots and monocots indicate that the LE-ACS2 and LE-ACS4 proteins belong to an unique sublineage that includes an additional member of the tobacco family, NT-ACS1 . The divergence of this sublineage is a relatively recent event in the evolution of ACC synthase protein.

J Biol Chem, 1993 Sep 15, 268(26), 19398 - 402
Identity of Escherichia coli tRNA(Cys) determined by nucleotides in three regions of tRNA tertiary structure; McClain WH; The in vivo aminoacylation specificity of tRNA(Cys) was studied with mutants of opal and amber suppressor tRNAs in Escherichia coli . This specificity depends not only on nucleotides in the acceptor end and anticodon of tRNA(Cys), but also on nucleotides in the dihydrouridine stem and loop and the variable loop that interact, forming the complex core of tRNA tertiary structure.

J Biol Chem, 1993 Sep 15, 268(26), 19384 - 91
Identification of an uncleavable targeting signal in the 70-kilodalton spinach chloroplast outer envelope membrane protein; Wu C et al.; A cDNA clone encoding a cognate 70-kDa heat shock protein from the spinach chloroplast outer envelope (SCE70) was recently characterized (Ko, K., Bornemisza, O., Kourtz, L., Ko, Z . W., Plaxton, W . C., and Cashmore, A . R . (1992) J . Biol . Chem . 267, 2986-2993) . Initial studies revealed that SCE70 is targeted to the chloroplast outer envelope membrane without further processing . To determine whether SCE70 possesses a "targeting domain," we tested the targeting ability of SCE70 proteins with various carboxyl- and amino-terminal deletions . Carboxyl-terminal deletions of up to 60% of the protein had no apparent effect on the targeting ability of SCE70 . Amino-terminal deletions abolished targeting to the chloroplast except when the extreme NH2-terminal 48-amino acid sequence was retained . We further assessed the chloroplast-targeting ability of the NH2-terminal 48 amino acids by fusing to the foreign protein, mouse dihydrofolate reductase (DHFR) . The resulting fusion protein, SCE70-DHFR, was localized to the outer envelope membrane of isolated chloroplasts . SCE70-DHFR exhibited targeting characteristics similar to native SCE70 . The targeting of SCE70-DHFR was inhibited effectively by anti-SCE70 antibodies . Immunoprecipitation and chemical cross-linking experiments revealed that SCE70-DHFR is targeted to the same complex as SCE70 in the chloroplast envelope . These results suggest that the extreme NH2 terminus of SCE70 is required for directing SCE70 to a destination in the chloroplast outer envelope membrane, possibly through assembling the polypeptide into a protein complex.

J Biol Chem, 1993 Sep 15, 268(26), 19358 - 63
Functions of potA and potD proteins in spermidine-preferential uptake system in Escherichia coli; Kashiwagi K et al.; Functions of potA and -D proteins in the spermidine-preferential uptake system, which consists of potA, -B, -C, and -D proteins, were studied . Spermidine uptake activity was lost when the gene for potA or potD protein was disrupted, and transformation of the cells with potA or potD gene recovered the uptake activity . PotD protein was found to bind spermidine with a 3.2 microM dissociation constant . Spermidine uptake by membrane vesicles prepared from Escherichia coli DR112 containing the genes for potA, -B, and -C proteins was strongly dependent on the addition of potD protein, and its optimal concentration was 5 microM when 10 microM spermidine was used as substrate . The ATP dependence of spermidine uptake was examined with the atp mutant of E . coli . The uptake was completely dependent on ATP . When the membrane potential was extinguished by carbonyl cyanide m-chlorophenylhydrazone, the uptake activity was decreased by 60% even if ATP existed . This suggests that the membrane potential is also involved in the spermidine uptake . ATP was found to bind to potA protein . In the spermidine transport-deficient mutant E . coli NH1596, valine 135 of potA protein, which is located between two consensus amino acid sequences for nucleotide binding, was replaced by methionine . Although the amount of mutated potA protein expressed in E . coli cells was the same as that of normal potA protein and the mutated protein was membrane-associated, no significant spermidine uptake was observed . The results taken together indicate that potA and -D proteins are absolutely necessary for spermidine uptake in conjunction with the two channel forming proteins (potB and -C).

J Biol Chem, 1993 Sep 15, 268(26), 19267 - 73
Probing the role of the carboxyl-terminal region of ferredoxin-NADP+ reductase by site-directed mutagenesis and deletion analysis; Orellano EG et al.; The carboxyl-terminal region of plant ferredoxin-NADP+ reductases is formed by an invariant alpha-helix/loop/beta-strand, culminating in a conserved tyrosine that displays extensive interaction with the prosthetic group FAD . We have investigated the potential role of the terminal region in reductase function, by introducing mutations and deletions on pea ferredoxin-NADP+ reductase overexpressed in Escherichia coli . Replacement of the terminal tyrosine by tryptophan, phenylalanine, serine, and glycine resulted in a 2.2-, 2.0-, 22-, and 302-fold reduction, respectively, in kcat for the diaphorase reaction, whereas elimination of the tyrosine caused a 846-fold decrease in kcat . Km values were largely unaffected by the substitutions . Similar results were obtained when the mutants were assayed for cytochrome c reduction, indicating that aromaticity is the most important factor to the function of the tyrosine in catalysis . The presence of the phenol ring at the carboxyl-terminal position of wild-type reductase is important, but not an absolute requirement for enzyme function or FAD assembly . Deletion of the alpha-helix/beta-strand region prevented reductase proper folding in the bacterial host, while shortening of the terminal region by splicing 3 amino acids at the beginning of the alpha-helix produced a moderately soluble reductase, devoid of FAD and enzymatic activity.

J Biol Chem, 1993 Sep 15, 268(26), 19204 - 9
Assembly of the primosome of DNA replication in Escherichia coli; Allen GC Jr et al.; Assembly of the Escherichia coli primosome requires six proteins, PriA, PriB, PriC, DnaB, DnaC, and DnaT, acting at a primosome assembly site (pas) on an SSB-coated single-stranded (ss) DNA . Assembly is initiated by interactions of PriA and PriB with ssDNA and the pas . PriC, DnaB, DnaC, and DnaT then act on the PriA-PriB-DNA complex to yield the primosome . In the primosome, the dATPase (ATPase) of PriA becomes hyper-activated . In addition, the assembled primosome appears to block the pas, preventing it from activating additional PriA molecules . Either ATP alone or dATP in combination with GTP is sufficient for primosome assembly, while ATP or GTP provides for its maintenance during isolation . These nucleotide requirements can be reconciled with the need for ATP or dATP for DnaB-DnaC complex formation and hydrolysis of ATP or GTP by DnaB when it binds ssDNA . Such isolated primosomes contain a dATPase, the hallmark of PriA, and a GTPase indicative of DnaB . Further studies indicate that the isolated primosome contains the PriB replication activity in addition to PriA and DnaB.

Thromb Res, 1993 Sep 15, 71(6), 479 - 86
Enhanced endothelial tissue factor but normal thrombomodulin in endotoxin-treated rabbits; Semeraro N et al.; Exposure of cultured endothelial cells to bacterial endotoxin induces an enhancement of cell procoagulant activity (PCA) and a simultaneous reduction of thrombomodulin activity (TM) . We evaluated the effect of endotoxin on the expression of both endothelial PCA and TM in vivo, in rabbits . Animals were given a single i.v . injection of endotoxin (E . coli 0111:B4 LPS, W, 10-200 micrograms/kg); the thoracic aorta was harvested after 2 or 4 hours and placed in an ad hoc device to expose the endothelial surface only . Endotoxin treatment resulted in a dose-dependent increase of endothelial PCA (p < 0.001, at 100 micrograms/kg or more), which was totally dependent on factor VII and thus identified as tissue factor . In contrast, endothelial TM activity, as measured by the rate of thrombin-induced protein C activation, was similar in control and endotoxemic rabbits, even when the animals were given two injections (50 micrograms/kg, 24 h apart), or a continuous infusion (40 micrograms/kg/h during 4 hours) of endotoxin . To explore the effect of endotoxin on TM activity at the microcirculation level, we measured the extent of protein C activation in vivo, induced by a continuous infusion of low doses of thrombin (1 NIH U/kg/min for 60 min) . Again, endotoxin administration was not associated with significant changes in TM-dependent protein C activation, as assessed by the anticoagulant activity present in a barium citrate plasma eluate obtained at the end of thrombin infusion . Although reduction of TM during persistent endotoxemia cannot be definitively excluded, our data support a major role of endothelial PCA in LPS-induced coagulative changes.

Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8571 - 5
Decatenation activity of topoisomerase IV during oriC and pBR322 DNA replication in vitro; Peng H et al.; Topoisomerase IV (Topo IV), encoded by parC and parE, is required for partition of the daughter chromosomes in Escherichia coli . This enzyme is likely responsible for decatenating the linked daughter chromosomes after replication . In this report, we have examined the action of Topo IV in both pBR322 and oriC DNA replication reconstituted in vitro with purified proteins . Gyrase fails to decatenate the linked daughter molecules under any condition in the oriC system and at physiological salt concentrations in the pBR322 system, whereas Topo IV stimulates generation of monomer product DNA by 7- to 10-fold . Topo IV-catalyzed decatenation of isolated multiply linked DNA dimers was relatively insensitive to salt; it proceeded at 14% of the maximal rate even in the presence of 800 mM potassium glutamate . In contrast, decatenation in vitro by gyrase was inhibited completely under these conditions . Pulse-chase analysis indicated that Topo IV-catalyzed resolution of linked daughter DNA molecules occurred prior to completion of DNA replication, such that multiply linked daughter molecules did not arise . These results suggest that during DNA replication, gyrase acts primarily to relieve accumulated positive supercoiling and Topo IV acts to segregate the daughter chromosomes.

J Biol Chem, 1993 Sep 15, 268(26), 19675 - 80
Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase; Huang CS et al.; gamma-Glutamylcysteine synthetase (rat kidney), which catalyzes the first step of GSH synthesis, can be dissociated into subunits (M(r) 73,000 and 27,700) by native gel electrophoresis after treatment with dithiothreitol (DTT); the heavy subunit, which exhibits catalytic activity and feedback inhibition by GSH (Seelig, G . F., Simondsen, R . P., and Meister, A . (1984) J . Biol . Chem . 259, 9345-9347), was cloned and sequenced (Yan, N., and Meister, A . (1990) J . Biol . Chem . 265, 1588-1593) . Here, the cDNA for the heavy sub unit was expressed in Escherichia coli, and the recombinant enzyme was separated from E . coli gamma-glutamylcysteine synthetase and purified . The recombinant enzyme and the isolated heavy subunit have much lower affinity for glutamate and higher sensitivity to GSH inhibition than the holoenzyme, suggesting that the heavy subunit alone would not be very active in vivo . A GSH analog, gamma-Glu-alpha-aminobutyryl-Gly (ophthalmic acid), inhibits only slightly, but inhibits much more after treatment of the holoenzyme with DTT . In contrast, ophthalmic acid inhibits the recombinant and isolated heavy subunit enzymes substantially without DTT treatment . We conclude that (a) the light subunit has a regulatory function affecting the affinity of the enzyme for glutamate and GSH and (b) feedback inhibition by GSH involves reduction of the enzyme and also competition between GSH and glutamate for the glutamate site.

J Biol Chem, 1993 Sep 15, 268(26), 19346 - 51
Refolding of yeast enolase in the presence of the chaperonin GroE . The nucleotide specificity of GroE and the role of GroES; Kubo T et al.; GroE, a chaperonin protein from Escherichia coli, facilitates the folding of numerous proteins by binding to protein-folding intermediates and suppressing aggregation (Gething, M., and Sambrook, J . (1992) Nature 355, 33-45) . The specific mechanism of GroE-facilitated folding involves numerous interactions between GroEL, GroES, the refolding protein, and ATP . In the present study, we have probed the molecular characteristics of the refolding reaction of yeast enolase in the presence of GroE . We have found that (a) GroEL interacts specifically with enolase during the folding reaction, resulting in folding arrest; (b) the release of partially folded molecules of enolase from the GroE complex may be mediated by the addition of nucleotides other than ATP (ADP, CTP, and UTP); and (c) GroES is required for enolase to be released from GroEL in the presence of ADP, CTP, and UTP but not required in the presence of ATP . The nucleotide binding mechanism of GroEL and the specific role of GroES during the refolding reaction are discussed in detail.

J Biol Chem, 1993 Sep 15, 268(26), 19254 - 9
Cloning and characterization of the gene that encodes acetyl-coenzyme A carboxylase in the alga Cyclotella cryptica; Roessler PG et al.; The gene that encodes acetyl-coenzyme A carboxylase (ACCase; EC 6.4.1.2) in the eukaryotic alga Cyclotella cryptica has been isolated and cloned, representing the first time that a full-length gene for this enzyme has been isolated from a photosynthetic organism . The gene contains a 447-base pair intron that is located near the putative translation initiation codon and a 73-base pair intron that is located slightly upstream from the region that encodes the biotin binding site of the enzyme . The gene encodes a polypeptide that is predicted to be composed of 2089 amino acids and to have a molecular mass of 230 kDa . The deduced amino acid sequence exhibits strong similarity to the sequences of animal and yeast ACCases in the biotin carboxylase and carboxyltransferase domains . There is less sequence similarity in the biotin carboxyl carrier protein domain, although the highly conserved Met-Lys-Met of the biotin binding site is present . The amino terminus of the predicted ACCase sequence has characteristics of a signal sequence, suggesting that the enzyme is imported into chloroplasts via the endoplasmic reticulum, as has been shown to be the case for certain nuclear-encoded proteins that are transported into the chloroplasts of the diatom Phaeodactylum tricornutum . Southern blot analyses suggest that a single copy of this gene is present in C . cryptica.

Structure, 1993 Sep 15, 1(1), 35 - 50
The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP conformation; Kjeldgaard M et al.; BACKGROUND: Elongation factor Tu (EF-Tu) is a GTP-binding protein that is crucial for protein biosynthesis . In the GTP form of the molecule, EF-Tu binds tightly to aminoacyl-tRNA, forming a ternary complex that interacts with the ribosomal acceptor site . During this interaction, GTP is hydrolyzed, and EF-Tu.GDP is ejected . RESULTS: The crystal structure of EF-Tu from Thermus aquaticus, complexed to the GTP analogue GDPNP, has been determined at 2.5 A resolution and compared to the structure of Escherichia coli EF-Tu.GDP . During the transition from the GDP (inactive) to the GTP (active) form, domain 1, containing the GTP-binding site, undergoes internal conformational changes similar to those observed in ras-p21 . In addition, a dramatic rearrangement of domains is observed, corresponding to a rotation of 90.8 degrees of domain 1 relative to domains 2 and 3 . Residues that are affected in the binding of aminoacyl-tRNA are found in or near the cleft formed by the domain interface . CONCLUSION: GTP binding by EF-Tu leads to dramatic conformational changes which expose the tRNA binding site . It appears that tRNA binding to EF-Tu induces a further conformational change, which may affect the GTPase activity.

J Immunol, 1993 Sep 15, 151(6), 3171 - 9
Analysis and partial epitope mapping of human T cell responses to Trypanosoma cruzi cysteinyl proteinase; Arnholdt AC et al.; Human infection with Trypanosoma cruzi (Chagas' disease) is usually accompanied by humoral and cellular immune responses to GP57/51, a major antigen that was recently identified as a prominent cysteinyl proteinase (cruzipain) . The PBMC responses of 11 chronic chagasic patients and the properties of anti-cruzipain T cell lines are reported herein . GP57/51, isolated from Y strain epimastigotes (n-cruzipain) or the recombinant protein expressed in E . coli (r-cruzipain), elicited proliferative responses of variable intensity from the patient's PBMC . T cell lines were then generated using each of these antigens . These lines, which always carried the CD4+ phenotype, were reciprocally stimulated by n-cruzipain or r-cruzipain, the responses to the former being usually stronger . The analysis of cytokine production suggested that Th1-like subsets dominate the patient's responses: IFN-gamma was consistently induced on stimulation with either n-cruzipain or r-cruzipain . In contrast, IL-4 was present in very small concentrations or was undetectable . We then sought to define T cell epitopes of cruzipain using synthetic peptides spanning portions of the central (catalytic) domain and COOH-terminal extension . From a panel of 11 peptides, only one 33 mer peptide (P214) elicited a strong proliferative response on anti-cruzipain T cell lines, the intensity being comparable to that induced by r-cruzipain . Conversely, T cell lines started with P214 were responsive to either n-cruzipain or r-cruzipain, the proliferative responses again being accompanied by IFN-gamma production, but not IL-4 . Interestingly, P214 is located in a conserved region of the catalytic domain of cruzipain, hence may propitiate opportunities for cross-recognition of other members of the papain superfamily . Fine epitope mapping should reveal whether structurally similar regions of host thiol-cathepsins can be potential targets for cross-reactive T cell responses during chronic human infection.

J Biol Chem, 1993 Sep 15, 268(26), 19185 - 7
Active center rearrangement in RNA polymerase initiation complex; Mustaev A et al.; His1237 in the beta subunit of Escherichia coli RNA polymerase marks the "5' face" of the active center since it can be cross-linked to the gamma-phosphate of the priming substrate . It is demonstrated that RNA chains up to 9 nucleotides in length can be synthesized using His1237-cross-linked nucleotide as a primer . Thus, a substantial mass of RNA can be accommodated in the active center between His1237 and the site of catalysis that remains juxtaposed to the growing 3' end . The apparent "filling" of the active center with RNA precedes promoter clearance and suggests a mechanism of coupling between catalysis and saltatory translocation of RNA polymerase.

Biochemistry, 1993 Sep 14, 32(36), 9486 - 91
Expression and identification of p90 as the murine mitochondrial glycerol-3-phosphate acyltransferase; Yet SF et al.; Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step in glycerolipid biosynthesis . Mitochondrial GPAT, unlike the microsomal isozyme, prefers saturated fatty acids as a substrate . We have recently reported cloning of a cDNA to an unidentified 6.8-kb mRNA by a differential hybridization . The mRNA contains an open reading frame of 827 amino acids (p90) with 30% sequence homology in a 300 amino acid stretch to Escherichia coli GPAT . The 6.8-kb mRNA was induced dramatically when fasted mice were refed a high-carbohydrate diet . Here, we have expressed the open reading frame as trpE fusion proteins and used them to generate antibodies . The antibodies recognized a polypeptide of 90 kDa (p90) when the 6.8-kb cDNA sequence was used for in vitro transcription and translation . By Western blot analysis using these antibodies, we detected p90 in mitochondrial fractions of liver, and the p90 level was increased by refeeding . The increase in the p90 level correlated with the increase in mitochondrial GPAT activity . Moreover, p90 was not detectable in 3T3-L1 preadipocytes but markedly increased during adipose conversion . This increase was consistent with the 11-fold increase we observed in N-ethylmaleimide (NEM)-resistant mitochondrial GPAT activity during adipocyte differentiation . In addition, we have expressed p90 in CHO cells by stable transfection . The transfected genes in both correct and reverse orientations produced distinct 3.9-kb transcripts owing to the truncation of a part of the noncoding regions of the endogenous 6.8-kb mRNA before insertion into the pMSXND vector . The transfected CHO cells were treated with 2-aminopurine, an agent that increases expression of exogenous genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Sep 14, 32(36), 9310 - 6
Structure of isocitrate dehydrogenase with isocitrate, nicotinamide adenine dinucleotide phosphate, and calcium at 2.5-A resolution: a pseudo-Michaelis ternary complex; Stoddard BL et al.; The structure of isocitrate dehydrogenase (IDH) with a bound complex of isocitrate, NADP+, and Ca2+ was solved at 2.5-A resolution and compared by difference mapping against previously determined enzymatic complexes . Calcium replaces magnesium in the binding of metal-substrate chelate complex, resulting in a substantially reduced turnover rate . The structure shows the following: (i) A complete, structurally ordered ternary complex (enzyme, isocitrate, NADP+, and Ca2+) is observed in the active site, with the nicotinamide ring of NADP+ exhibiting a specific salt bridge with isocitrate . The binding of the cofactor nicotinamide ring is dependent on this interaction . (ii) Isocitrate is bound by the enzyme with the same interactions as those found for the magnesium/substrate binary complex, but the entire molecule is shifted in the active site by approximately 1 A in order to accommodate the larger metal species and to interact with the nicotinamide ring . The distances from isocitrate to the bound calcium are substantially longer than those previously found with magnesium . (iii) NADP in the Escherichia coli IDH has a novel binding site and conformation as compared to previously solved dehydrogenases . (iv) The orientation and interactions of the nicotinamide ring with the substrate are consistent with the stereospecificity of the enzyme-catalyzed reaction.

Biochemistry, 1993 Sep 14, 32(36), 9302 - 9
Kinetic mechanism of Escherichia coli isocitrate dehydrogenase; Dean AM et al.; The kinetic mechanism of the NADP-dependent isocitrate dehydrogenase of Escherichia coli was investigated using initial steady-state kinetic analyses . Kinetic coefficients, obtained using natural and alternative substrates with the wild-type and two mutant enzymes (S113L and S113N), suggest that the forward reaction {the oxidative decarboxylation of (2R,3S)-isocitrate by NADP} of the wild-type enzyme is a steady-state random mechanism, with catalysis more rapid than product release . The mechanism of the wild-type enzyme becomes rapid-equilibrium random when an alternative substrate {(2R)-malate or NAD} is used . The mutant enzymes always display rapid-equilibrium random kinetics, and for each enzyme the apparent dissociation constant of each substrate from the binary complex {Kia = E.A/(EA)} is similar to its apparent dissociation constant from the Michaelis complex {Ka = (EB).A/(EAB)}, which suggests that the binding of one substrate is independent of the binding of the second . When the wild-type enzyme catalyzes the forward reaction, the apparent dissociation constant, KiIso, is equal to its equilibrium dissociation constant, KdIso, determined from equilibrium binding studies . However, the apparent dissociation constant of the cofactor, KiNADP, is far smaller than its equilibrium dissociation constant, KdNADP . This is consistent with the proposed mechanism, because simulations show that when catalysis is steady-state and product release is rate-limiting, KiNADP and KNADP will be far smaller than KdNADP, while KiIso and KIso remain similar to KdIso . Product inhibition studies support the steady-state random mechanism of the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Sep 14, 32(36), 9296 - 301
Isolation of bovine kidney leucine aminopeptidase cDNA: comparison with the lens enzyme and tissue-specific expression of two mRNAs; Wallner BP et al.; Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates . Leucine aminopeptidase (LAP) from bovine lens is the best characterized aminopeptidase and the only LAP for which the amino acid sequence was determined by protein sequencing . Using this sequence information, we isolated a bovine kidney LAP cDNA and compared its deduced amino acid sequence to the published amino acid sequence for bovine lens LAP . Overall, the sequences are highly conserved . However, several differences are observed . The kidney LAP cDNA indicates a 26 amino acid extension at the amino terminus which is not found in the mature purified lens LAP . The cDNA also indicates an additional octapeptide in the C-terminal region which was not indicated in the published lens LAP amino acid sequence but which was required for best fit of crystallographic data regarding bovine lens LAP . Several other single amino acid changes were also noted . Levels of LAP transcripts were examined in bovine lens and kidney tissue as well as in cultured lens cells . Lens epithelial tissue showed only one LAP transcript (2.4 kb) whereas two transcripts (2.0 and 2.4 kb) were observed in cultured lens cells derived from epithelial tissue and in kidney tissue . Using Northern blot analysis, we correlated LAP mRNA levels with previously determined changes of LAP activity in aging lens tissue and in progressively passaged lens epithelial cells which were used to simulate aging in vitro . No differences were found in LAP mRNA levels in epithelial tissue from old and young lenses.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Sep 14, 32(36), 9290 - 5
Assignment of enzymatic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli; Drummond JT et al.; Cobalamin-dependent methionine synthase catalyzes methyl group transfer from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine, and the cobalamin prosthetic group serves as an intermediate methyl carrier . Enzyme possessing cobalamin in the cobalt(II) oxidation state is inactive, and this form is activated by one-electron reduction coupled to methylation by S-adenosylmethionine (AdoMet) . The enzyme from Escherichia coli has been divided into separable fragments by limited proteolysis with trypsin, and the contribution of each of these fragments to substrate binding and catalysis has been evaluated . The 37.7-kDa carboxyl-terminal domain binds AdoMet, and this was demonstrated through covalent modification with radiolabeled AdoMet during ultraviolet irradiation . Following reductive activation with AdoMet, the enzyme was digested with trypsin and a 98.4-kDa amino-terminal fragment was isolated . It retained at least 70% of the activity of the intact enzyme and must therefore possess determinants sufficient for the binding of methyltetrahydrofolate and homocysteine, as well as residues required for catalysis . However, when the cobalamin was oxidized to the cob(II) alamin state, the 98.4-kDa fragment could not be reductively remethylated with AdoMet . A purified, 28-kDa domain within the 98.4-kDa fragment retained bound cobalamin and therefore must play a central role in catalysis, but the isolated 28-kDa domain retained no catalytic activity . Because AdoMet binds to a different domain of the protein than methyltetrahydrofolate and homocysteine, the enzyme probably uses conformational flexibility to allow the cobalamin access to the required methyl donor or acceptor at the appropriate time in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Sep 14, 32(36), 9282 - 9
Electrospray mass spectrometric analysis of the domains of a large enzyme: observation of the occupied cobalamin-binding domain and redefinition of the carboxyl terminus of methionine synthase; Drummond JT et al.; Cobalamin-dependent methionine synthase from Escherichia coli catalyzes the methylation of homocysteine to form methionine, using methyltetrahydrofolate as the primary methyl donor . We have used electrospray mass spectrometry as a powerful tool for characterizing separable fragments obtained by proteolysis of this monomeric 136.1-kDa enzyme . A central 28.0-kDa domain, reported to bind the cobalamin, has been purified to homogeneity in 30% yield . We were able to detect the domain with bound cobalamin by electrospray mass spectrometry at neutral pH . Mass analysis of a 37.2-kDa carboxyl-terminal domain was grossly inconsistent with either of the two amino acid sequences from previously published DNA sequences . We then used electrospray mass spectrometry to analyze peptides generated by a lysyl endoproteolytic digest of a C-terminal fragment, and we have constructed a peptide map that accounts for > 95% of the peptide mass derived from this domain . The correct translational end of this protein (27 residues downstream from the previously predicted ultimate residue) has been established, and sequence conflicts within the two published DNA sequences have been resolved (GenBank Accession Number J04975) . Resequencing the DNA near the carboxyl terminus ruled out a frameshifted reading of the DNA and suggested that a cytosine had twice been incorrectly inserted late in the reading frame . The strategies reported here for sequence confirmation, localization of coenzyme-binding regions, and identification of chemically modified peptides within a large protein are potentially applicable to the characterization of many other proteins.

Biochemistry, 1993 Sep 14, 32(36), 9268 - 73
A fluorescence spectroscopic study of substrate-induced conformational changes in glutaminyl-tRNA synthetase; Bhattacharyya T et al.; Glutaminyl-tRNA synthetase from Escherichia coli is a member of a subgroup of aminoacyl-tRNA synthetases that do not catalyze ATP-PPi exchange in the absence of the cognate tRNA . Such behavior suggests conformational changes upon substrate binding . Two different fluorescent probes, pyrenylmaleimide and acrylodan, were used to specifically label a nonessential sulfhydryl group of GlnRS . Conformational changes induced by substrates were studied using glutaminyl-tRNA synthetase labeled with these two environment-sensitive probes . ATP was shown to cause a significant conformational change that alters the mode of binding to tRNA(Gln) to GlnRS . The alteration of the salt sensitivity pattern of tRNA(Gln) binding to GlnRS by ATP supports this . Binding of tRNA(Gln) causes a conformational change that may be different in nature for the ATP/GlnRS complex and free GlnRS . Hydrodynamic parameters deduced from fluorescence polarization studies and the use of a noncovalent probe indicate that the ATP-induced conformational change may not be global in character.

Biochemistry, 1993 Sep 14, 32(36), 9274 - 81
Substitution of glutamine for glutamic acid-58 in Escherichia coli thymidylate synthase results in pronounced decreases in catalytic activity and ligand binding; Zapf JW et al.; The recent determination of the crystal structure of Escherichia coli thymidylate synthase (TS) {Matthews et al . (1989) J . Mol . Biol . 205, 449-454} has implicated the glutamic acid residue at position 58 in a mechanistic role which could involve the interaction of its gamma-carboxyl side chain with the nucleotide substrate and/or the folate cofactor . The site-specific mutagenesis of Glu-58 to Gln-58 in E . coli TS provided the opportunity to explore its functional role in activity and binding . When profiled by the spectrophotometric and tritium release assays, the 370- and 760-fold decreases, respectively, in kcat and the elevated Km values for the Gln-58 mutant enzyme indicated a significant involvement of Glu-58 in substrate binding and turnover . The apparent dissociation constant for the covalent FdUMP-enzyme binary complex was 30 microM, which is 5-fold higher than that found for the wild-type enzyme, while the inhibitory ternary complex apparent dissociation constants for FdUMP and CH2H4folate for the Gln-58 enzyme were 10- and 60-fold higher, respectively, than those for the wild-type enzyme under saturating conditions . The extent of covalent FdUMP binding to the Gln-58 enzyme was reduced from 1.5 to 0.7 per dimer in the inhibitory ternary complex but only from 0.7 to 0.5 per dimer in the binary complex of the Gln-58 enzyme . The usual 2.1-fold enhancement of FdUMP binding to wild-type TS in the presence of CH2H4folate was not observed for the Gln-58 enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1993 Sep 13, 330(2), 137 - 40
Three-dimensional structure of a barnase-3'GMP complex at 2.2A resolution; Guillet V et al.; Barnase has been co-crystallized at neutral pH with its natural product the 3'-guanylic acid . The X-ray structure was solved by molecular replacement methods and refined to a final R-factor of 18.7% . The protein folding is essentially the same as that of the native form . The base recognition site is almost identical to that of the homologous binase-3'GMP complex, but the nucleotide is bound in a productive binding mode for a substrate with a syn glycosyl torsion angle allowing the general base Glu73 to hydrogen bond with the 2'O of the nucleotide as is assumed in the classical catalytic mechanism . The two molecules of the asymmetric unit form a dimer and the positions of the two nucleotides partially mimic the interaction of the RNA with the enzyme, one of the inhibitors being located in a secondary subsite.

FEBS Lett, 1993 Sep 13, 330(2), 133 - 6
Modification by site-directed mutagenesis of the specificity of Erythrina corallodendron lectin for galactose derivatives with bulky substituents at C-2; Arango R et al.; Examination of the three-dimensional structure of Erythrina corallodendron lectin (ECorL) in complex with a ligand (lactose), the first of its kind for a Gal/GalNAc-specific lectin {(1991) Science 254, 862-866}, revealed the presence of a hydrophobic cavity, surrounded by Tyr108 and Pro134-Trp135, which can accommodate bulky substituents such as acetamido or dansylamido (NDns) at C-2 of the lectin-bound galactose . Comparison of the primary sequence of ECorL with that of soybean agglutinin, specific for galactose and its C-2 substituted derivatives, and of peanut agglutinin, specific for galactose only, showed that in soybean agglutinin, Tyr108 is retained, and Pro134-Trp135 is replaced by Ser-Trp, whereas in peanut agglutinin, the former residue is replaced by Thr and the dipeptide by Ser-Glu- Tyr-Asn . Three mutants of ECorL were therefore constructed: L2, in which Pro134-Trp135 was replaced by Ser-Glu-Tyr-Asn; Y108T, in which Tyr108 was replaced by Thr and the double mutant L2; Y108T . They were expressed in Escherichia coli, as done for recombinant ECorL {(1992) Eur . J . Biochem . 205, 575-581} . The mutants had the same hemagglutinating activity as native or rECorL . Their specificity for galactose, GalNAc and Me beta GalNDns was examined by inhibition of hemagglutination and of the binding of the lectin to immobilized asialofetuin; in addition, their association constants with Me alpha GalNDns and Me beta GalNDns were measured by spectrofluorimetric titration . The results showed that Y108T had essentially similar specificity as the native and recombinant lectins . The affinity of L2 and L2;Y108T for galactose was also the same as ECorL, but they had a lower affinity for GalNAc and markedly diminished affinity for the dansyl sugars (up to 43 times, or 2 kcal, less) . This appears to be largely due to steric hindrance by the two additional amino acids present in the cavity region in these mutants . Our findings also provide an explanation for the inability of PNA to accommodate C-2-substituted galactose derivatives at its primary subsite.

Biochim Biophys Acta, 1993 Sep 13, 1178(3), 302 - 6
Recombinant expression and partial characterization of the human formyl peptide receptor; Lala A et al.; FMLP-receptor DNA was expressed in Escherichia coli . The expressed product could specifically bind FMLP . This is the first-reported expression of a functional FMLP receptor in Escherichia coli . We confirm that receptor glycosylation is not essential for ligand binding . A deletion mutant did not bind FMLP, suggesting that the deleted portion plays a role in ligand binding.

Science, 1993 Sep 10, 261(5127), 1454 - 7
Cloning of an M . tuberculosis DNA fragment associated with entry and survival inside cells; Arruda S et al.; Mycobacterium tuberculosis infects one-third of the world's human population . This widespread infection depends on the organism's ability to escape host defenses by gaining entry and surviving inside the macrophage . DNA sequences of M . tuberculosis have been cloned; these confer on a nonpathogenic Escherichia coli strain an ability to invade HeLa cells, augment macrophage phagocytosis, and survive for at least 24 hours inside the human macrophage . This capacity to gain entry into mammalian cells and survive inside the macrophage was localized to two distinct loci on the cloned M . tuberculosis DNA fragment.

Biochemistry, 1993 Sep 7, 32(35), 8987 - 93
Molecular cloning and expression of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase and sequence similarity with protein phosphatase 2C; Lawson JE et al.; After many unsuccessful attempts to detect cDNA encoding the catalytic subunit of bovine pyruvate dehydrogenase phosphatase (PDPc) in bovine cDNA libraries, an approach based on the polymerase chain reaction (PCR) was undertaken . Overlapping DNA fragments were generated by PCR from bovine genomic DNA and from cDNA synthesized from total RNA with synthetic oligonucleotide primers on the basis of experimentally determined amino acid sequences . The DNA fragments were subcloned and sequenced . The complete cDNA is 1900 base pairs in length and contains an open reading frame of 1614 nucleotides encoding a putative presequence of 71 amino acid residues and a mature protein of 467 residues with a calculated M(r) of 52,625 . Hybridization analysis showed a single mRNA transcript of about 2.0 kilobases . Comparison of the deduced amino acid sequences of the mitochondrial PDPc and the rat cytosolic protein phosphatase 2C indicates that these protein serine/threonine phosphatases evolved from a common ancestor . The mature form of PDPc was coexpressed in Escherichia coli with the chaperonin proteins groEL and groES . The recombinant protein (rPDPc) was purified to near homogeneity . Its activity toward the bovine 32P-labeled pyruvate dehydrogenase complex was Mg(2+)-dependent and Ca(2+)-stimulated and comparable to that of native bovine PDP . An active, truncated form of rPDPc, with M(r) approximately 45,000, was produced in variable amounts during growth of cells and/or during the purification procedure.

Biochemistry, 1993 Sep 7, 32(35), 9189 - 98
Luminescence studies with trp repressor and its single-tryptophan mutants; Eftink MR et al.; Time-resolved and steady-state fluorescence, low-temperature phosphorescence, and optically detected magnetic resonance (ODMR) measurements have been made to resolve the luminescence contributions of the two intrinsic tryptophan residues in the subunits of trp aporepressor from Escherichia coli . Assignments of spectral information have been confirmed by use of the single-tryptophan mutants W19F and W99F . Solute fluorescence quenching studies show that both Trp19 and Trp99 are exposed to acrylamide and iodide, with Trp99 being the more exposed . Time-resolved and steady-state fluorescence measurements show Trp19 to have a bluer emission, a longer mean fluorescence decay time, a higher quantum yield, and essentially no independent rotational motion with respect to the protein . Trp99 is found to have a redder emission, a shorter mean fluorescence decay time, a lower quantum yield, and a significant degree of rotational freedom . Phosphorescence studies show a clear resolution of 0-0 vibronic transitions for each type of residue, with maxima at 407 and 415 nm that are assigned to Trp19 and Trp99, respectively . ODMR measurements show the zero-field splitting parameters to be quite characteristically different for each tryptophan residue . The existence of resonance energy transfer from Trp19 to Trp99, in the wild-type protein, is indicated by three types of data: comparison of the long-lived decay time (attributed to Trp19) in the absence (W99F) and presence (wild type) of the acceptor Trp99, comparison of the fluorescence quantum yield of the wild-type and mutant proteins, and deviations from the expected phosphorescence intensities for Trp19 and Trp99 in the absence of energy transfer.

Biochemistry, 1993 Sep 7, 32(35), 9181 - 8
Simple centrifugation method for efficient pelleting of both small and large unilamellar vesicles that allows convenient measurement of protein binding; Tortorella D et al.; Separation of unilamellar model membrane vesicles from external solution is often an important step in quantitation of vesicle bound or entrapped materials . An efficient method that allows pelleting of both small and large model membrane vesicles by centrifugation is described in this report . In this method streptavidin is added to vesicles containing a trace amount of biotinylated lipid . The resulting aggregation allows pelleting of the vesicles using an ordinary high-speed centrifuge . Control experiments show that the addition of streptavidin does not induce substantial vesicle fusion or leakage of substances trapped in the internal aqueous compartment of the vesicles . The method can accommodate different phospholipid compositions and lipid concentrations . Experiments with proteins that switch between hydrophilic and hydrophobic states show that the method can readily be used to monitor protein binding to vesicles.

Biochemistry, 1993 Sep 7, 32(35), 9115 - 24
Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences; Ikeda RA et al.; The compatible plasmids pKGP1-1 and pCM-X# will confer chloramphenicol resistance to Escherichia coli harboring the two plasmids if the T7 RNA polymerase produced from pKGP1-1 can recognize the T7 promoter carried on pCM-X# and transcribe the CAT gene that is cloned behind the promoter {Ikeda et al . (1992) Biochemistry 31, 9073-9080} . When E . coli harbor pKGP1-1 and a pCM-X# plasmid that carries a point mutation in the T7 promoter that destroys promoter activity (an inactive pCM-X#), the T7 RNA polymerase will not utilize the T7 promoter point mutant, will not produce CAT, and will not induce chloramphenicol resistance . The selection of mutants of T7 RNA polymerase that exhibit altered promoter recognition was pursued by randomly mutagenizing pKGP1-1 with aqueous hydroxylamine, cotransforming E . coli with the mutagenized pKGP1-1 and a mixture of seven different inactive pCM-X# plasmids, and isolating and characterizing the RNA polymerase that was present in those colonies that exhibited chloramphenicol resistance . It was established that E . coli harboring the mutant plasmid pKGP-HA1mut4 and an inactive pCM-X# are chloramphenicol-resistant and that the mutation responsible for the expression of CAT from the inactive pCM-X# plasmid is a G to A transition at nucleotide 664 of T7 gene 1 that converts glutamic acid (222) to lysine . Apparently this mutation expands the range of T7 promoter sequences that can be utilized by the enzyme . The mutant T7 RNA polymerase, GP1(Lys222), utilizes all seven inactive T7 promoter point mutants more efficiently than wild-type T7 RNA polymerase both in vivo and in vitro . Furthermore, the correlation of in vivo and in vitro promoter utilization suggests that the restoration of chloramphenicol resistance in the cotransformed E . coli results from the ability of GP1(Lys222) to initiate transcription from T7 promoter point mutants that are normally inactive.

Gene, 1993 Sep 6, 131(1), 119 - 24
Cloning of a sensory-kinase-encoding gene that belongs to the two-component regulatory family from the cyanobacterium Synechococcus sp . PCC7942; Nagaya M et al.; A screening method employing Escherichia coli was adopted to clone a sensory-kinase (SK)-encoding gene directly from a phylogenetically distant species, the phototrophic cyanobacterium Synechococcus sp . PCC7942 . From the Synechococcus chromosomal DNA, we searched for DNA clones which are able to complement phenotypically not only an E . coli envZ mutant for the expression of ompC, but also an E . coli phoR/creC mutant for the expression of alkaline phosphatase . These E . coli genes are known to encode SK . A 0.75-kb DNA fragment was thus cloned under the control of the E . coli lac promoter carried on an E . coli plasmid vector . A larger DNA fragment encompassing an entire open reading frame was then cloned and its complete nucleotide (nt) sequence determined . The nt sequence corresponds to a gene that encodes a 43,280-Da protein of 387 amino acids with a high degree of homology to the bacterial SK . Thus, we succeeded in cloning a SK-encoding gene, which most likely functions in signal transduction in Synechococcus sp . PCC7942 . Hence, the gene was designated sasA (Synechococcus adaptive-response SK A) . The purified SasA protein was demonstrated in vitro to undergo autophosphorylation.

FEBS Lett, 1993 Sep 6, 330(1), 61 - 5
Efficient processing and export of human growth hormone by heat labile enterotoxin chain B signal sequence; Ghorpade A et al.; The heat-labile enterotoxin chain B (LTB) signal sequence was used for the processing and export of human growth hormone (hGH) . The protein was completely processed and exported across the cell membrane to accumulate in the periplasmic space in Escherichia coli . The human growth hormone cDNA was cloned as a PCR amplified fragment under the control of tac promoter and translationally fused to the LTB signal sequence . The rate of processing of hGH under the control of the LTB signal sequence was equal to or more than the rate of induction of expression, indicating efficient processing . The receptor binding activity of the processed periplasmic protein was established in a radio receptor assay.

FEBS Lett, 1993 Sep 6, 330(1), 105 - 9
Observation of inter-subunit nuclear Overhauser effects in a dimeric protein . Application to the Arc repressor; Burgering MJ et al.; For the structure determination of symmetric protein dimers it is necessary to distinguish between intra- and inter-subunit NOEs . A method is presented to measure selectively the inter-subunit NOEs using uniform 15N and 13C isotope labelling . This is accomplished by double filtered 2D NOE experiments on mixtures of native protein with isotope-labeled protein . The method has been applied to the Arc repressor and allows the characterization of virtually all proton-proton NOEs in terms of their intra- or inter-subunit nature.

Gene, 1993 Sep 6, 131(1), 87 - 91
Isolation of Escherichia coli mutants lacking methylcytosine-dependent restriction systems for cloning extensively methylated frog virus 3 DNA; Dy L et al.; Many bacterial strains possess methylation-dependent restriction systems (MDRS) that demonstrate methylcytosine-dependent restriction endonuclease activity for the dinucleotide sequence, dCpdG . This makes these strains unsuitable for cloning methylated DNA . Some commercially available bacterial cells are recommended for cloning DNA fragments with methylated cytosines and adenines, e.g., Escherichia coli DH5-alpha MCR . Our attempts to clone frog virus 3 (FV3) DNA, which has the highest degree of cytosine methylation ever reported, using DH5-alpha MCR cells, were not successful . This and other observations suggested the existence of additional MDRS that have not yet been eliminated from DH5-alpha MCR cells . In order to isolate a mutant from this bacterial strain that is suitable to clone highly methylated FV3 DNA, we transformed these cells with a recombinant pUC19 plasmid containing a methylated 1.4-kb genomic DNA fragment from FV3, and selected for ampicillin (Ap) resistance . Three such attempts yielded only one colony that contained a fully methylated 1.4-kb FV3 genomic DNA fragment . Furthermore, plasmid-cured Ap-sensitive colonies originating from this clone were isolated and have been successfully employed to clone the highly methylated FV3 genomic DNA fragment.

J Biol Chem, 1993 Sep 5, 268(25), 18626 - 32
Identification of phosphorylation sites in the recombinant catalytic subunit of cAMP-dependent protein kinase; Yonemoto W et al.; The catalytic subunit of cAMP-dependent protein kinase expressed in Escherichia coli is a phosphoprotein . By in vivo labeling with {32Pi}orthophosphate, the sites of phosphorylation were identified as Ser-10, Ser-139, Thr-197, and Ser-338 . Two of these sites, Thr-197 and Ser-338, are found in the mammalian enzyme (Shoji, S., Titani, K., Demaille, J . G., and Fischer, E . H . (1979) J . Biol . Chem . 254, 6211-6214) . The predominant isoform is phosphorylated at Ser-10, Ser-338, and Thr-197 . The isoforms cannot be readily interconverted by in vitro autophosphorylation, suggesting that the phosphates are relatively stable once the mature protein is assembled . Unlike the mammalian enzyme, the recombinant enzyme is not myristylated at its animo terminus . By coexpressing the catalytic subunit and N-myristyl transferase, the recombinant catalytic subunit is myristylated, and, under these conditions, phosphorylation at Ser-10 is reduced . The fact that recombinant catalytic subunit mutants that are enzymatically impaired are not phosphorylated in vivo indicates that the phosphorylation of the catalytic subunit observed in E . coli is due to autophosphorylation . Whether this process is intramolecular or intermolecular cannot be distinguished . Although autophosphorylation accounts for the modification of the catalytic subunit when it is expressed in E . coli, there may be heterologous protein kinases that are responsible for its in vivo phosphorylation when the enzyme is expressed in eukaryotic cells.

J Biol Chem, 1993 Sep 5, 268(25), 18485 - 90
Protonation of arginine 57 of Escherichia coli ornithine transcarbamoylase regulates substrate binding and turnover; Goldsmith JO et al.; The amino acid residue Arg57 of Escherichia coli ornithine transcarbamoylase is located in the carbamoyl phosphate-binding domain of the enzyme . This residue has been implicated to be critical for efficient carbamoylation and is linked to an induced-fit protein isomerization elicited by the lead substrate carbamoyl phosphate . To elucidate its role in substrate binding and catalysis, Arg57 has been substituted by one of two amino acids, glycine and histidine, varying in size and charge . Elimination of the positive charge and steric bulk on residue 57 with an arginine-to-glycine substitution results in an enzyme which binds its substrates in a random order (Kuo, L . C., Miller, A . W., Lee, S., and Kozuma, C . (1988) Biochemistry 27, 8823-8832) . Replacement of Arg57 by histidine provides a substantial portion of the steric bulk at this residue position and also brings the pKa of this residue into an experimentally observable window . Kinetic and pH titration experiments reveal that when His57 is deprotonated the enzyme binds its substrates randomly . However, when His57 is protonated the enzyme observes the obligatory substrate binding order as seen for the wild type . Both the Gly57 and His57 point mutants are incapable of undergoing the carbamoyl phosphate-induced protein conformational changes apparent in the wild type . These results reveal that the induced-fit isomerization of ornithine transcarbamoylase does not contribute to the order in which the substrates bind . A comparison of the reaction schemes and pH profiles of the wildtype and His57 enzymes further indicates that protonation of residue 57 facilitates formation of the binary enzyme-carbamoyl phosphate complex and augments the turnover rate of the reaction . Together with steady-state kinetic parameters derived in terms of microscopic rate constants, our results provide additional support to our earlier suggestion (Zambidis, I . and Kuo, L . C . (1990) J . Biol . Chem . 265, 2620-2623) that the turnover rate of the wild-type ornithine transcarbamoylase in the forward reaction is largely dictated by the rate of the carbamoyl phosphate-induced isomerization.

J Biol Chem, 1993 Sep 5, 268(25), 18411 - 4
Direct identification of the primary nucleophile of thioredoxin f; Brandes HK et al.; Thioredoxin, by virtue of the proximal active-site sulfhydryls (Trp-Cys-Gly-Pro-Cys), catalyzes thiol-disulfide exchange with specific target enzymes . Considerable data (chemical modification, spectroscopic, and crystallographic) have implicated the cysteinyl residue nearest the N terminus of thioredoxin as the primary nucleophile; however, direct proof has been lacking . Proof is now provided by characterization of site-directed mutants of thioredoxin f with respect to activation of chloroplastic fructose-1,6-bisphosphatase (FBPase) . The C49S mutant retains the capacity to activate FBPase, whereas the C46S mutant is totally lacking in this regard . Based on kinetics of FBPase activation, wild-type and C49S thioredoxins exhibit half-saturation values of 0.9 and 4 microM, respectively . Lack of activation by C46S is not because of failure to interact with FBPase, for it exhibits a Ki of 5 microM in competition with wild-type thioredoxin . Therefore, in the normal thioredoxin-catalyzed reduction pathway, Cys-46 is the nucleophile required to attack the disulfide of the substrate and Cys-49 serves to cleave the mixed disulfide intermediate, thus allowing for the release of oxidized thioredoxin and the reduced target enzyme.

J Biol Chem, 1993 Sep 5, 268(25), 18403 - 6
The signal anchor sequence of mitochondrial Mas70p contains an oligomerization domain; Millar DG et al.; pOMD29 is a mitochondrial precursor protein that contains the NH2-terminal signal anchor sequence of Mas70p fused to dihydrofolate reductase . The signal anchor mediates insertion of pOMD29 into the outer mitochondrial membrane in the Nin-Ccyto orientation . Following import in vitro, pOMD29 was chemically cross-linked, via a unique cysteine residue adjacent to the signal anchor (residue 34), to form a product that was approximately twice the size of pOMD29 . The cross-linked product was a dimer of pOMD29, as judged by the following . 1) It exhibited the same charge:mass ratio as pOMD29 . 2) Formation of radioactive cross-linked product containing 35S-labeled pOMD29 was stimulated by co-import with unlabeled pOMD29 . 3) Co-import of pOMD29 with a modified pOMD29 that contains two copies of dihydrofolate reductase resulted in formation of the predicted homo- and heterodimers . Cross-linking of pOMD29 was unaffected by concentrations of methotrexate that lock the dihydrofolate reductase moiety into its native monomeric conformation, indicating that oligomerization was mediated by the signal anchor rather than by the cytosolic domain of pOMD29 . The predicted transmembrane core of the signal anchor sequence contains structural motifs similar to those found in a variety of signal-transducing cell surface receptors that dimerize through their transmembrane segments.

J Mol Biol, 1993 Sep 5, 233(1), 59 - 66
Two mutant RecA proteins possessing pH-dependent strand exchange activity exhibit pH-dependent presynaptic filament formation; Pinsince JM et al.; In previous studies it was shown that the mutant RecA proteins, {G160N}RecA and {H163A}RecA, are unable to catalyze ATP-dependent DNA strand exchanges at pH 7.5, but are active at pH 6.0 to 6.8 . Here, we have used electron microscopy to follow the assembly of these mutant proteins onto single-stranded DNA at pH 7.5 and pH 6.2 . In the absence of ATP, the filaments formed by the mutant proteins were similar to those formed by the wild-type protein, at both pH 7.5 and pH 6.2 . In the presence of ATP, however, the filaments formed by the wild-type protein at pH 7.5 were extended and were stable in the presence of saturating SSB protein, whereas the filaments formed by the mutant proteins were shorter and unstable in the presence of SSB protein . At pH 6.2, in contrast, the filaments formed by the mutant proteins in the presence of ATP were of the same contour length as the wild-type RecA protein filaments and were stable in the presence of SSB protein . In the presence of the non-hydrolyzable ATP analog, ATP gamma S, and SSB protein, the mutant proteins formed full-length filaments at pH 7.5 that had a helical periodicity identical with that of the wild-type filaments (and characteristic of the strand exchange-active open conformational state); if SSB protein was omitted, the mutant protein filaments still exhibited the open helical periodicity, but were shorter and of highly variable length, presumably because of an improper threading of the ssDNA into the mutant filament . To account for these results, we propose that: (1) the mutant proteins are unable to isomerize efficiently to the open conformational state at pH 7.5 in the presence of ATP, but are able to do so in the presence of ATP gamma S; this indicates that the mechanistic defect is related to ATP hydrolysis rather than ATP binding; and (2) the mutant proteins are able to isomerize to the open conformational state in the presence of ATP at pH 6.2, indicating that protonation of the mutant filaments is sufficient to relieve the mechanistic deficiency.

J Mol Biol, 1993 Sep 5, 233(1), 25 - 42
Dissection of the his leader pause site by base substitution reveals a multipartite signal that includes a pause RNA hairpin; Chan CL et al.; A key feature of transcriptional attenuation in some amino acid biosynthetic operons is a transcriptional pause that occurs immediately after synthesis of the first leader transcript secondary structure . Both RNA secondary structure and downstream DNA sequence are important for pausing at these sites; however, the precise RNA structures involved and the relative contribution of other RNA and DNA bases to pausing are unknown . We studied the effects of base substitutions upstream from the his leader pause site (immediately prior to addition of G103) to determine how nucleic acid sequences and RNA structure contribute to pausing . By testing compensatory base substitutions, we found that pausing depended in part on an RNA secondary structure containing a five base-pair stem and eight nucleotide loop, which we call the his pause RNA hairpin . The his pause hairpin forms 11 nucleotides upstream from the paused transcript 3' end and thus corresponds to only the upper portion of the larger his A:B leader transcript secondary structure . Some base substitutions in the ten nucleotides between the pause hairpin and the 3' end of the transcript increased pausing, whereas others decreased pausing . However, compensatory substitutions that restored pairing of these bases in the lower portion of the A:B secondary structure did not alter these effects . Changing the 3'-terminal nucleotide of the transcript (U102) altered both the position and strength of pausing . Thus, in addition to the downstream DNA sequence, three distinct segments of nucleic acid upstream from the nucleotide-addition site in the transcription complex contribute to pausing in different ways: the pause RNA hairpin, the 3'-proximal region of transcript or DNA template, and the 3'-terminal nucleotide . We suggest that electrostatic interaction between the pause hairpin and RNA polymerase, rather than disruption of an RNA:DNA heteroduplex, delays elongation at the his leader pause site.

J Mol Biol, 1993 Sep 5, 233(1), 179 - 82
Preliminary crystallographic studies on the D15 5' to 3' exonuclease from phage T5; Ceska TA et al.; The D15 exonuclease from phage T5 has been crystallized from 35% (w/v) ammonium sulfate by the hanging drop vapor diffusion technique . The crystals grow in tetragonal space group P4(1)22 or P4(3)22 with cell dimensions a = b = 79.2 A and c = 138.0 A . The crystals diffract to 2.5 A and are suitable for X-ray structure determination.

J Mol Biol, 1993 Sep 5, 233(1), 177 - 8
Crystallization and preliminary X-ray crystallographic analysis of the soybrean proglycinin expressed in Escherichia coli; Utsumi S et al.; Glycinin is one of the dominant storage proteins of soybean seeds . Soybean proglycinin expressed in Escherichia coli has been crystallized from Tris.HCl buffer (pH 7.6) by the dialysis equilibrium method . The crystals belong to the tetragonal system, space group P4(1) or P4(3), with unit cell dimensions of a = b = 115.2 A, and c = 147.1 A . The asymmetric unit contains three molecules of proglycinin, with crystal volume per protein mass (Vm) of 3.05 A3/Da and solvent content of 58.4% by volume . The crystals diffract X-rays to a resolution limit of at least 2.9 A and are resistant to X-ray radiation damage . They appear to be suitable for X-ray structure analysis.

J Mol Biol, 1993 Sep 5, 233(1), 16 - 24
Replicatively active complexes of DnaA protein and the Escherichia coli chromosomal origin observed in the electron microscope; Crooke E et al.; DnaA protein and the Escherichia coli chromosomal origin (oriC) form an initial complex at an early stage in the initiation of DNA replication . We have used electron microscopy to determine which structure among the several formed in the reconstitution of this multicomponent system is the replicatively active complex . One distinctive structure could be correlated with activity and localized to oriC, whilst several others could not . Formation of an open complex in the next stage of initiation was accompanied by the presence of a structure similar in size and shape to that of the functional initial complex . Whereas the initial complex was observed with either ATP or the ADP-forms of DnaA protein, only the ATP-form was effective in producing the open complex . Mutagenesis of several DNA sequence elements in oriC, known to be important for replication, was employed to determine the effects of these alterations on formation of the initial complex . As judged by electron microscopy and by functional assays, the region containing the four 9-mer dnaA boxes proved to be essential for the formation of the initial complex, while the three contiguous AT-rich 13-mers, known sites for opening of oriC, were not.

J Mol Biol, 1993 Sep 5, 233(1), 1 - 6
The 3' codon context effect on UAG suppressor tRNA is different in Escherichia coli and human cells; Phillips-Jones MK et al.; We have compared the effect of 3' context on the efficiency of nonsense suppressor tRNAs in Escherichia coli and human cells . Plasmids containing amber (UAG) termination codons were constructed in the vector pRSV beta gal by oligonucleotide insertion at an N-terminal location in a lacZ fusion . A family of identical vectors was prepared with either A, C, G or U as the first 3' base following the stop codon . These derivatives of pRSV beta gal were expressed in E . coli as stable plasmids, or transiently in human 293 cell tissue culture . Nonsense suppression was monitored using enzyme assays for beta-galactosidase . In E . coli the efficiency of a plasmid-borne bacterial tRNA(trp) UAG suppressor varied A > G > C = U . When the same lacZ reporter vectors were cotransfected with a human tRNA(ser) UAG suppressor plasmid into human cells, context effects of a different nature were detected . Double reciprocal analysis of dose-response experiments were used to show that the efficiency of suppression varied C > G > U = A . The discovery of different codon context effects on nonsense suppression in human cells suggest that the interaction between mammalian tRNAs or release factors and their target codons may have different characteristics from those in bacteria.

J Biol Chem, 1993 Sep 5, 268(25), 18936 - 42
Characterization of the bifunctional cytochrome c reductase-processing peptidase complex from potato mitochondria; Emmermann M et al.; In potato, cytochrome c reductase, a protein complex of the respiratory chain, exhibits processing activity toward mitochondrial precursor proteins . One of the two cooperating components of the processing peptidase was shown to be identical with subunit III of the complex . Here we report that two additional proteins of the complex (subunit I and II) share 40-50% sequence identity with the processing enhancing protein, the other component of the processing enzyme from fungi and mammals . Thus the composition and structure of the complex integrated processing peptidase seems to be different from its fungal and mammalian counterparts . Cytochrome c reductase from potato is extraordinarily stable, and separation of subunit III from the complex leads to aggregation of the remaining subcomplex and irreversible loss of processing activity . Expression of the three high molecular weight subunits of the complex allowed purification of each individual protein . Neither the individual subunits nor their combinations are active in in vitro processing assays suggesting that they may need the structural support of the complex for activity . In contrast to mitochondrial processing peptidases from other organisms, the purified potato enzyme is active in the presence of high salt (above 1 M NaCl) and works efficiently without addition of metal ions . These data indicate that potato cytochrome c reductase is a bifunctional protein complex with unique features . Possibly, there is a more general evolutionary relationship between cytochrome c reductases and mitochondrial processing peptidases than hitherto assumed.

J Biol Chem, 1993 Sep 5, 268(25), 18898 - 904
Myristoylation of hippocalcin is linked to its calcium-dependent membrane association properties; Kobayashi M et al.; Hippocalcin, a recently identified Ca(2+)-binding protein of the recoverin family exclusively expressed in the hippocampus, has a primary structure containing three putative Ca(2+)-binding sites (EF-hands) and a possible NH2-terminal myristoylation site . 45Ca blots demonstrated that every three EF-hand domains, expressed as fusion proteins in Escherichia coli, bind Ca2+, indicating that hippocalcin binds 3 mol of Ca2+/mol of protein . To determine whether hippocalcin is myristoylated, hippocalcin mRNA was translated in vitro in the presence of {3H}myristic acid . 3H label was resistant to hydroxylamine treatment, and replacement of NH2-terminal glycine with alanine prevented 3H label incorporation, indicating that in vitro translated hippocalcin covalently bound {3H}myristic acid at the NH2-terminal glycine . In vitro translated hippocalcin is quantitatively myristoylated, as evidenced by an electrophoretic mobility shift of {35S}methionine-labeled protein on two-dimensional gels . Native hippocalcin comigrated precisely with the in vitro translated hippocalcin on two-dimensional gels, suggesting that native hippocalcin is myristoylated . Native and in vitro translated hippocalcins, but not non-myristoylated mutagenic (Gly1-Ala1) hippocalcin, displayed Ca(2+)-dependent membrane association, indicating that myristoylation participates in its Ca(2+)-dependent membrane association properties . In vitro translated hippocalcin bound to phospholipid vesicles somewhat, however, phospholipid association was insufficient for its membrane association properties, suggesting that the NH2-terminal myristoyl moiety on hippocalcin interacts with lipid bilayers and facilitates interaction with other membrane proteins.

J Biol Chem, 1993 Sep 5, 268(25), 18866 - 74
Purification, characterization, and biosynthesis of margatoxin, a component of Centruroides margaritatus venom that selectively inhibits voltage-dependent potassium channels; Garcia-Calvo M et al.; A novel peptidyl inhibitor of K+ channels has been purified to homogeneity from venom of the new world scorpion Centruroides margaritatus . The primary structure of this 39-amino-acid peptide, which we term margatoxin (MgTX), was determined by amino acid compositional analysis and peptide sequencing . Margatoxin potently inhibits binding of radiolabeled charybdotoxin (ChTX) to voltage-activated channels in brain synaptic plasma membranes . Like ChTX, MgTX blocks the n-type current of human T-lymphocytes (Kv1.3 channel), but compared to ChTX, is 20-fold more potent (half-block at approximately 50 pM), has a slower dissociation rate, and has no effect on calcium-activated channels . To demonstrate that these characteristics are due solely to the purified toxin, recombinant MgTX was expressed in Escherichia coli as part of a fusion protein . After cleavage and folding, purified recombinant MgTX displayed the same properties as native peptide . Replacement of the COOH-terminal histidine residue of MgTX with asparagine resulted in a peptide with a 10-fold reduction in potency . This was due to a faster apparent dissociation rate, suggesting that the COOH-terminal amino acid may play an important role in the binding of MgTX to the Kv1.3 channel . MgTX displays significant sequence homology with previously identified K+ channel inhibitors (e.g . ChTX, iberiotoxin, noxiustoxin, and kaliotoxin) . However, given its potency and unique selectivity, MgTX represents an especially useful tool with which to study the physiologic role of Kv1.3 channels.

J Biol Chem, 1993 Sep 5, 268(25), 18726 - 33
The amino-terminal 29 amino acids of cytochrome P450 2C1 are sufficient for retention in the endoplasmic reticulum; Ahn K et al.; Cytochromes P450 are inserted into and anchored to the endoplasmic reticulum (ER) membrane by a hydrophobic signal sequence at the NH2 terminus . To determine whether the NH2-terminal sequence might also have an ER retention function, the NH2-terminal 29 amino acids of cytochrome P450 2C1, with and without an additional 29 amino acids containing an N-glycosylation site, were fused either to a soluble cytoplasmic protein, Escherichia coli beta-galactosidase, or to a secreted protein, E . coli alkaline phosphatase, and the hybrid proteins were expressed in COS1 cells . Subcellular fractionation indicated that both the beta-galactosidase and alkaline phosphatase hybrid proteins cosedimented with marker enzymes for ER membranes, and localization by immunofluorescent staining was consistent with an ER location . Hybrid proteins with the NH2-terminal glycosylation site were glycosylated in COS1 cells, and the carbohydrate moiety was sensitive to endoglycosidase H digestion, providing further evidence that the proteins were retained in the ER . In vitro studies of membrane insertion of the alkaline phosphatase hybrid indicated that fusion to alkaline phosphatase hybrid indicated that fusion to alkaline phosphatase did not alter the topological properties of the cytochrome P450 NH2-terminal sequence . In addition, alkaline phosphatase fused to the extracellular and transmembrane domains of epidermal growth factor receptor was transported to the plasma membrane in COS1 cells, which establishes that alkaline phosphatase as a cytoplasmic domain does not prevent transport from the ER . These observations indicate that the large cytoplasmic domain of cytochrome P450 is not required for retention in the ER and suggest that a specific sequence or structure within the NH2-terminal 29 amino acids functions as an ER retention signal.

J Biol Chem, 1993 Sep 5, 268(25), 18710 - 6
Mutational study of Streptomyces tyrosinase trans-activator MelC1 . MelC1 is likely a chaperone for apotyrosinase; Chen LY et al.; The melanin operon (melC) of Streptomyces antibioticus contains two genes, melC1 and melC2 (apotyrosinase) . Our previous studies indicated that MelC1 forms a transient binary complex with the downstream apotyrosinase MelC2 to facilitate the incorporation of copper ion and the secretion of tyrosinase . In this study, we investigated the role of histidine residues in the function of MelC1 by examining a series of substitution or deletion mutants . Of eight mutants only the substitution of His-117 with Asp in the mutant M-117D rendered the complete abolishment of the intracellular tyrosinase activity in both Streptomyces and Escherichia coli . Replacement of His-102 by Leu in the mutant M-102L also caused a 64-70% reduction of tyrosinase activity in Streptomyces and E . coli . These two mutations also affected the secretion of both MelC1 and MelC2 proteins . In vitro copper activation of the purified MelC1.MelC2 binary complex from these two mutants regained only 20-30% tyrosinase activity of the wild type . Biochemical characterization of the tyrosinases from these two mutants revealed that they were different in several aspects . The intracellular tyrosinase activity in M-117D, but not in M-102L, could be partially reactivated by copper ion or by the cell extract containing MelC1 . The copper content and the specific activity of the tyrosinase purified from the culture supernatant from M-117D were only 40% of those in wild type and M-102L . Additionally, fast protein liquid chromatography analysis indicated that in these two mutants the copper activation process was defective, very likely due to the incompetent MelC1.MelC2 binary complex formed: reduced association in M-117D and elevated association in M-102L . Furthermore, the conformation of MelC2 in the binary complex or in the mature enzyme form in wild type could be differentiated by the proteinase K digestion pattern, and so did the conformation of MelC2 found in those of M-102L, but not in M-117D mutant . Taken together, our results demonstrate that MelC1 is indispensable in the incorporation of copper ion into MelC2 apotyrosinase via a transient, competent binary complex formation, during which a conformational transition of MelC2 has occurred . This strongly suggests that MelC1 is a chaperone for the apotyrosinase MelC2.

J Biol Chem, 1993 Sep 5, 268(25), 18701 - 9
Placement of dinitrophenyl-modified ribosomal proteins in totally reconstituted Escherichia coli 30 S subunits . Localization of proteins S6, S13, S16, and S18 by immune electron microscopy; Montesano-Roditis L et al.; Purified Escherichia coli ribosomal proteins S6, S13, S16, and S18 were dinitrophenylated at their amino termini and/or at one or more internal lysine residues . Each dinitrophenyl protein was then separately incorporated into reconstituted small ribosomal subunits . Modified proteins were localized on the 30 S subunit surface by electron microscopy of reconstituted subunits complexed with antibodies to dinitrophenol (DNP) . DNP protein S13 was placed on the subunit head above the platform and on the surface that faces the large subunit . DNP-S18 was localized to the subunit platform below the tip and in a region associated with binding to 50 S subunits . DNP proteins S6 and S16 were both localized near the junction of the subunit body and platform; DNP-S6 was available to antibody in 70 S ribosomes and was placed on the cytoplasm-facing side of the subunit in an area that overlaps the platform and body of the particle . DNP-S16 in 70 S ribosomes was not bound by antibody . It was localized to the 30 S body near its junction with the platform and on the surface facing the 50 S particle . The results complement and clarify data obtained using other approaches.

J Biol Chem, 1993 Sep 5, 268(25), 18604 - 9
NADPH-sulfite reductase from Escherichia coli . A flavin reductase participating in the generation of the free radical of ribonucleotide reductase; Coves J et al.; Protein R2, the small subunit of ribonucleotide reductase of Escherichia coli, contains an essential free radical localized to tyrosine 122 of its polypeptide chain . When this radical is scavenged by hydroxyurea, the enzyme is transformed into an inactive form, metR2 . E . coli contains a NAD(P)H:flavin oxidoreductase, named Fre, absolutely required for the regeneration of the radical and the activation of metR2 into R2 . Consequently, an E . coli mutant strain lacking an active fre gene is more sensitive to hydroxyurea during growth, demonstrating the physiological protective function of Fre from the loss of the radical . However, this gene is not essential, and we found that E . coli contains a second tyrosyl radical generating activity, also residing in a flavin reductase . The enzyme has been purified 200-fold to homogeneity and found to be identical to sulfite reductase . Pure sulfite reductase has the ability to catalyze the reduction of free riboflavin, FMN, or FAD by NADPH and thus, as Fre, to transfer electrons to the iron center of metR2, a key step during the activation reaction.

J Biol Chem, 1993 Sep 5, 268(25), 18481 - 4
Utilization of conformational flexibility in enzyme action-linkage between binding, isomerization, and catalysis; Goldsmith JO et al.; An intimate relationship between protein conformational changes and catalysis has often been suggested . The present study employs ligand-induced ultraviolet difference spectra and kinetic parameters determined for Escherichia coli ornithine transcarbamoylase and its site-specific mutants to evaluate the linkage between binding, isomerization, and reaction rate . For the wild-type enzyme, the lead substrate carbamoyl phosphate introduces a large difference absorbance in the enzyme upon binding (delta epsilon max approximately 1,800 M-1 cm-1; Miller, A . W., and Kuo, L . C . (1990) J . Biol . Chem . 265, 15023-15027) . The spectrum is the same in lineshape as that produced by the bisubstrate analog N-(phosphonacetyl)-L-ornithine and is 80% as intense . Both substrate and analog cause gross protein conformational rearrangements as evident by swift and severe cracking of enzyme crystals in their presence . For the mutants, the difference spectra actuated by the substrate are the same in lineshape as that of the wild type but vary in intensity . A wide range of substrate affinity and steady-state kinetic constants are also observed for the mutants . When the binding energy of carbamoyl phosphate and the activation energy for transcarbamoylation are calculated for the wild-type and mutant enzymes, they are found to be inversely correlated to the intensity of protein difference absorbance elicited by the lead substrate . Together with analyses of steady-state kinetic parameters derived for various plausible reaction schemes, the experimental data suggest that carbamoyl phosphate induces the committed isomerization in ornithine transcarbamoylase for transition state binding . Our results provide a unique demonstration that an induced-fit isomerization, triggered by binding, either controls or contributes significantly to the rate of an enzyme-catalyzed reaction.

J Biol Chem, 1993 Sep 5, 268(25), 18673 - 8
Cloning of ornithine delta-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis; Delauney AJ et al.; Proline prototrophy was restored to an Escherichia coli proBA proline auxotroph by ornithine and a mothbean (Vigna aconitifolia) cDNA expression library . This novel strategy, "trans-complementation," allowed isolation of a cDNA encoding ornithine delta-aminotransferase (delta-OAT) . This enzyme transaminates ornithine to glutamic-gamma-semialdehyde (GSA), thereby bypassing the block in GSA synthesis from glutamate in the proBA mutant . The identity of the mothbean enzyme was confirmed by its high sequence homology to mammalian and yeast delta-OATs as well as to a family of bacterial and fungal omega-aminotransferases and an absence of significant homology to various alpha-aminotransferases . The V . aconitifolia OAT cDNA encodes a polypeptide of 48.1 kDa . The native enzyme expressed in E . coli appears to be a monomer with Km of 2 mM for ornithine and 0.75 mM for alpha-ketoglutarate . Levels of mRNA in V . aconitifolia for delta 1-pyrroline-5-carboxylate synthetase (P5CS) and delta-OAT, the two key enzymes for proline synthesis, were monitored under different physiological conditions . Salt stress and nitrogen starvation induced P5CS mRNA levels and depressed OAT mRNA levels . Conversely, OAT mRNA level was elevated in plants supplied with excess nitrogen while the P5CS mRNA level was reduced . These data suggest that the glutamate pathway is the primary route for proline synthesis in plants during conditions of osmotic stress and nitrogen limitation whereas the ornithine pathway assumes prominence under high nitrogen input.

J Biol Chem, 1993 Sep 5, 268(25), 18549 - 53
Partial functional mapping of the human interleukin-8 type A receptor . Identification of a major ligand binding domain; Hebert CA et al.; We have previously demonstrated that a basic amino acid residue of interleukin (IL)-8, namely Arg-6, is critical for the binding of IL-8 to its receptor . We reasoned that this residue is likely to be poised to directly interact with a counterpart acidic residue on the receptor . To identify this key residue, we systematically mutated to Ala all acidic residues present on the ligand accessible surface of IL-8 receptor type A . Using this strategy, we demonstrate that two residues which are present in extracellular loop 3 of the receptor, namely Glu-275 and Arg-280, are critical for ligand binding . In addition, we show that although Asp-11 is critical for ligand binding, a conservative mutation of Asp-11 to Glu or a substitution of Asp-11 with Lys (the residue found at position 11 in IL-8 receptor type B) does not affect the Kd of the receptor/ligand interaction . These data suggest that Lys-11 recruits a new and favorable interaction with IL-8 (analogous to that of IL-8 receptor type B with IL-8) or that the cavity created by mutating Asp-11 to Ala is particularly deleterious . Finally, we discuss fluorescence-activated cell sorter staining data which support the hypothesis that the N-terminal region and the extracellular loop 3 of the receptor may lie in close proximity of one another and constitute a major binding domain for IL-8.

J Biol Chem, 1993 Sep 5, 268(25), 18696 - 700
Incorporation of dinitrophenyl derivatives of proteins S6, S13, S16, and S18 into the 30 S subunit of Escherichia coli ribosomes by total reconstitution; Olah TV et al.; This is the third paper in a series (Olah, T . V., Olson, H . M., Glitz, D . G., and Cooperman, B . S . (1988) J . Biol . Chem . 263, 4795-4800; Olson, H . M., Olah, T., Cooperman, B . S., and Glitz, D . G . (1988) J . Biol . Chem . 263, 4801-4806) describing the use of 2,4-dinitrophenyl (DNP) derivatives of Escherichia coli 30 S ribosomal proteins to locate the positions of these proteins within the 30 S subunit by immune electron microscopy . In it we describe the derivatization of proteins S6, S13, S16, and S18 with {3H}2,4-dinitrofluorobenzene, identify the nature of the derivatized amino acids within each protein, and demonstrate that each DNP protein, denoted DNP-Sx, can be taken up into a reconstituted 30 S subunit when added to a reconstitution mixture containing 16 S rRNA and total 30 S protein depleted in Sx . We further demonstrate that each DNP-Sx binds within the 30 S subunit in a position identical or similar to that of the unmodified Sx protein, as judged by its meeting one or more of the following three criteria: (i) unmodified Sx competes with the uptake of DNP-Sx into 30 S subunits; (ii) DNP-Sx restores functional activity to those single protein omission reconstitution particles lacking full activity; (iii) DNP-Sx induces the uptake of proteins into 30 S subunits that depend on the presence of Sx . The fourth paper in this series (Montesano-Roditis, L., McWilliams, R., Glitz, D . G., Olah, T . V., Perrault, A . R., and Cooperman, B . S . (1993) J . Biol . Chem . 268, 18701-18709), which follows this one, describes the localization of the DNP-Sx proteins within the 30 S subunit by immune electron microscopy.

Biochim Biophys Acta, 1993 Sep 3, 1202(1), 82 - 6
Substitution of a haem-iron axial ligand in flavocytochrome b2; Miles CS et al.; The importance of haem-iron axial coordination in flavocytochrome b2 (L-lactate: cytochrome-c oxidoreductase) has been examined by replacing one of the ligating histidines, His-43, with methionine . The His-43-->Met mutation (H43M) results in a distinct colour change from red in the wild-type enzyme to green in the mutant enzyme . The electronic absorption spectrum indicates that only approx . 5% of the haem binding sites are occupied . There is no evidence of any absorption band at 695 nm (characteristic of methionine ligation) suggesting that methionine does not act as an axial ligand in the mutant enzyme . The H43M-mutant enzyme shows a band around 640-650 nm which is usually associated with high-spin ferric-haem proteins, either five coordinate or with a weak-field ligand in the sixth position . The EPR spectrum of the H43M-enzyme at 7 K shows a g-value near 6.0, indicating that the haem-iron is high-spin in contrast to its low-spin state in the wild-type enzyme . The His-43-->Met mutation has only a small effect on the lactate dehydrogenase activity of the enzyme as measured with ferricyanide as external electron acceptor, but greatly reduces its cytochrome-c reductase activity.

Biochim Biophys Acta, 1993 Sep 3, 1202(1), 107 - 12
Altered protein synthesis in a cell-free system exposed to a sinusoidal magnetic field; Goodman EM et al.; This report describes a new approach for examining weak extremely low frequency (ELF) electric and magnetic field interactions with living systems that exploits a cell-free transcription/translation system derived from Escherichia coli . Using two-dimensional polyacrylamide gel electrophoresis we previously had determined that the level of the alpha subunit of RNA polymerase in intact E . coli was elevated by exposure to weak ELF magnetic fields . In this paper, plasmids containing the alpha, or both the beta,beta' subunits of the RNA polymerase from E . coli were placed into a cell-free expression system . When this transcription/translation system was exposed to a 72-Hz sinusoidal magnetic field in the range 0.07 to 1.1 mT (rms) for periods of 5 min to 1 h, expression was enhanced . Weaker fields must be applied longer to produce an effect . For 10 min of field exposure, the threshold for an effect is 0.1 mT . These experiments demonstrate that an intact membrane is not an absolute requirement for transducing magnetic bio-effects.

Biochim Biophys Acta, 1993 Sep 3, 1202(1), 1 - 6
Site-directed mutagenesis of human tissue transglutaminase: Cys-277 is essential for transglutaminase activity but not for GTPase activity; Lee KN et al.; Transglutaminases (EC 2.3.2.13) catalyze an acyl-transfer reaction between peptide-bound glutamine residues and primary amines, including the epsilon-amino group of lysine residues in protein . Purified human erythrocyte transglutaminase was found to have another activity, i.e., GTP hydrolysis . Treatment of the enzyme with iodoacetamide, a cysteine-directed reagent, caused a 94% loss of TGase activity within 8 min, but no significant loss of GTPase activity . Cys-277, a known residue which is selectively modified by iodoacetamide, was replaced with Ser by site-directed mutagenesis to assess the role of the Cys-277 in the transglutaminase/GTPase activities . Wild-type cDNA, coding for human endothelial cell transglutaminase, and its C277S-mutated cDNA were cloned into a plasmid vector that contained a promoter from phage T7, and then expressed in Escherichia coli . The wild-type recombinant enzyme was indistinguishable from human erythrocyte transglutaminase in mobility on a SDS-polyacrylamide gel, immunoreactivity and catalytic activities for transglutaminase and GTPase . However, the recombinant enzyme was not blocked at the N-terminal alanine residue, as is the case in the naturally occurring erythrocyte enzyme . The C277S mutant enzyme showed no transglutaminase activity, but had Km and kcat values for GTPase activity that were comparable to those of wild-type recombinant and natural erythrocyte enzymes . These results demonstrate that Cys-277 is essential for transglutaminase activity, but not for GTPase activity, and that N-terminal blocking of tissue-type transglutaminase is not critical for either transglutaminase or GTPase activities.

Nature, 1993 Sep 2, 365(6441), 79 - 82
Inhibition of DNA replication factor RPA by p53; Dutta A et al.; The tumour suppressor p53 specifically interferes with the onset of S phase . The mechanism of the growth suppression action of the protein is unclear, though recent evidence points to transcriptional activation and repression functions of the protein . A competing hypothesis suggests that p53 interacts with the DNA replication apparatus and directly interferes with DNA replication . The major evidence for this hypothesis is that p53 interacts with the simian virus 40 (SV40)-encoded protein T antigen and interferes with the ability of T antigen to unwind the SV40 origin of DNA replication, and recruit DNA polymerase alpha to the replication initiation complex . Here we report that p53 physically interacts with and inhibits the function of a cellular DNA replication factor, the single-stranded DNA-binding protein complex RPA.

Carbohydr Res, 1993 Sep 2, 247, 255 - 62
Structural studies of the Escherichia coli O127 O-antigen polysaccharide; Widmalm G et al.; The O-specific side-chain of the lipopolysaccharide from Escherichia coli O127a:H- (O127a:4932-53) has been investigated using 2D NMR spectroscopy, methylation analysis, and partial solvolysis with anhydrous hydrogen fluoride as the principal methods . It is concluded that the polysaccharide is composed of tetrasaccharide repeating-units having the following structure . -->2)-alpha-L-Fucp-(1-->2)-beta-D-Galp-(1-->3)-alpha-D-GalpNAc-(1- ->3)-alpha-D- GalpNAc-(1--> The polysaccharide contains approximately one mole of O-acetyl groups per repeating unit distributed over several positions.

Cancer Biochem Biophys, 1993 Sep, 13(4), 239 - 44
Microwave induced alteration in the neuron specific enolase gene expression; Verma M et al.; Exposure of pNGE7, a recombinant clone containing the coding and regulatory sequences for the expression of neuron specific enolase gene, cells to electromagnetic radiations (915 MHz, 16 Hz AM, SAR 0.05 mW/kg) resulted in the elevation of neuron specific enolase (NSE), a diagnostic marker for neuron and lung cancer . Using ion-exchange chromatography we separated the neuron specific enolase activity from the non-neuronal enolase (NNE) activity and observed an alteration in the expression of neuron specific enolase and non-neuronal enolase . The clinical applications of the present studies have been discussed.

Mol Gen Genet, 1993 Sep, 240(3), 455 - 60
Evidence from directed mutagenesis that positively charged amino acids are necessary for interaction of nitrogenase with the {2Fe-2S} heterocyst ferredoxin (FdxH) from the cyanobacterium Anabaena sp., PCC7120; Schmitz S et al.; Sequence comparison of the heterocyst-type ferredoxin (FdxH) from Anabaena 7120 and type-1 ferredoxins (PetF) from the same organism and other cyanobacteria revealed a group of positively charged residues characteristic for FdxH . Molecular modeling showed that these basic amino acids are clustered on the surface of FdxH . The corresponding domain of PetF contained acidic or nonpolar residues instead . To identify amino acids that are important for interaction with nitrogenase, we generated site-directed mutations in the fdxH gene and assayed the in vitro activity of the resulting recombinant proteins isolated from Escherichia coli . In addition to the point mutants, two chimeric proteins, FdxH:PetF and PetF:FdxH, were constructed containing the 58 N-terminal amino acids of one ferredoxin fused to the 40 C-terminal amino acids of the other . Exchange of lysines 10 and 11 of FdxH for the corresponding residues of PetF (glutamate 10 and alanine 11) resulted in a ferredoxin with greatly decreased affinity to nitrogenase . This indicates an important function of these basic amino acids in interaction with dinitrogenase reductase (NifH) from Anabaena . In addition we checked the reactivity of the recombinant ferredoxins with ferredoxin-NADP+ oxidoreductase (FNR) and photosystem I . The experiments with both the chimeric and point mutated ferredoxins showed that the C-terminal part of this protein determines its activity in NADP+ photoreduction.

Mol Gen Genet, 1993 Sep, 240(3), 450 - 4
Photolyase-dimer-DNA complexes and exclusion stimulation in Escherichia coli: depolarization of the plasma membrane; Li BH et al.; Using cells that overproduce DNA photolyase, we found that UV irradiation (3 J/m2) efficiently inactivates accumulation of methylthiogalactoside (TMG) when RexAB proteins of phage lambda are present . The effect requires both formation of photolyase-dimer-DNA (PDD) complexes and expression of the RexAB proteins . It is reversed completely by a flash of visible light if given immediately after UV and becomes irreversible after post-UV incubation for about 15 min . Inactivation is significant after only 5 min of post-UV incubation, is accompanied by a loss of previously accumulated TMG, and does not require de novo protein synthesis . Passive transport of O-nitrophenylgalactoside by inactivated cells is typical of energy-depleted membranes . We suggest that PDD complexes mimic a developmental intermediate of phage superinfection and stimulate formation of the RexB membrane channel recently proposed by others to explain classical "exclusion" . This suggestion is supported by additional data showing an inactivation of colony-forming ability by exclusion stimulation and an inability of PDD complexes to inactivate accumulation of TMG if RexB is present in larger relative amounts than RexA (a detail characteristic of exclusion stimulated by phage superinfection).

Mol Gen Genet, 1993 Sep, 240(3), 395 - 402
TdcA, a transcriptional activator of the tdcABC operon of Escherichia coli, is a member of the LysR family of proteins; Ganduri YL et al.; The tdcB and tdcC genes of the tdcABC operon of Escherichia coli encode threonine dehydratase and a threonine-serine permease, respectively . These proteins are involved in transport and metabolism of threonine and serine during anaerobic growth . In this study, we functionally characterized tdcA, which encodes a 35 kDa polypeptide consisting of 312 amino acid residues . Non-polar and partially polar mutations introduced into tdcA drastically reduced the expression of the genes down-stream from tdcA . Complementation studies using single-copy chromosomal integrants of a tdcB-lacZ fusion harboring an in-frame deletion of tdcA with chromosomal or plasmid-borne tdcA+ in trans showed complete restoration of tdc operon expression in vivo . The amino acid sequence at the amino-terminal end of TdcA revealed a significant homology to the helix-turn-helix motifs of typical DNA binding proteins . Sequence alignment of TdcA with LysR also showed considerable sequence similarity throughout their entire lengths . Our results suggest that TdcA is related to the LysR family of proteins by common ancestry and, based on its functional role in tdc expression, belongs to the LysR family of transcriptional activators.

Mol Gen Genet, 1993 Sep, 240(3), 339 - 47
Osmoregulation of the fatty acid receptor gene fadL in Escherichia coli; Higashitani A et al.; The fadL gene of Escherichia coli codes for an outer membrane protein that is involved in the uptake of long-chain fatty acids . Uptake is regulated by environmental osmolarity, and decreases when the cells are grown under conditions of high osmolarity . A temperature-sensitive mutant that requires fatty acid for growth at 42 degrees C was unable to grow at the high temperature even in the presence of fatty acid if the medium contained 10% sucrose . Promoter activity of the fadL gene in vivo was repressed by high osmolarity in a FadR repressor null mutant . Furthermore, in vitro transcription of the fadL gene was strongly repressed by the addition of OmpR and EnvZ proteins . The results of gel retardation and DNase I protection experiments indicated that OmpR, after incubation with the protein kinase EnvZ, specifically binds to at least four sites around the fadL promoter, two upstream and two downstream from the transcriptional start site . These results suggest that transcription of the fadL gene is osmotically regulated by the OmpR-EnvZ two-component system.

Mol Gen Genet, 1993 Sep, 240(3), 307 - 14
Specific chromosomal sites enhancing homologous recombination in Escherichia coli mutants defective in RNase H; Nishitani H et al.; To clone new replication origin(s) activated under RNase H-defective (rnh-) conditions in Escherichia coli cells, whole chromosomal DNA digested with EcoRI was to with a Kmr DNA fragment and transformed into an rnh- derivative host . From the Kmr transformants, we obtained eight kinds of plasmid-like DNA, each of which contained a specific DNA fragment, termed "Hot", derived from the E . coli genome . Seven of the Hot DNAs (HotA-G) mapped to various sites within a narrow DNA replication termination region (about 280 kb), without any particular selection . Because Hot DNA could not be transformed into a mutant strain in which the corresponding Hot region had been deleted from the chromosome, the Hot DNA, though obtained as covalently closed circular (ccc) DNA, must have arisen by excision from the host chromosome into which it had initially integrated, rather than by autonomous replication of the transformed species . While Hot DNA does not have a weak replication origin it does have a strong recombinational hotspot active in the absence of RNase H . This notion is supported by the finding that Chi activity was present on all Hot DNAs tested and no Hot-positive clone without Chi activity was obtained, with the exception of a DNA clone carrying the dif site.

Metabolism, 1993 Sep, 42(9), 1190 - 4
Evidence that inhibition of muscle amino acid uptake during endotoxemia is not mediated by glucocorticoids; Zamir O et al.; Sepsis and endotoxemia are associated with increased muscle protein breakdown and inhibited amino acid uptake . Glucocorticoids are important for the regulation of muscle protein breakdown in catabolic conditions; in contrast, the role of glucocorticoids in the regulation of muscle amino acid transport during sepsis or endotoxemia is not known . The present study was designed to test the role of glucocorticoids in the regulation of muscle amino acid uptake during endotoxemia . Amino acid transport, determined as uptake of 3H-alpha-aminoisobutyric acid (AIB) by incubated soleus muscles in vitro, was reduced by approximately 40% 2 hours after intraperitoneal (IP) injection of 10 micrograms/kg endotoxin in rats . Administration of 5 mg/kg of the glucocorticoid receptor antagonist RU 38486 2 hours before endotoxin injection did not affect the inhibition of amino acid uptake . In vitro addition of plasma from endotoxemic rats to incubated rat soleus muscles inhibited amino acid uptake by approximately 30% . This effect of endotoxic plasma also was noted when muscles were from rats that had been treated with RU 38486 and when RU 38486 was present in the incubation medium . Results confirm previous reports of reduced muscle amino acid transport during endotoxemia and of the presence of a circulating factor that inhibits muscle amino acid uptake in this condition . Data suggest that inhibited muscle amino acid transport during endotoxemia is not regulated by glucocorticoids.

Gastroenterol Clin North Am, 1993 Sep, 22(3), 609 - 22
Escherichia coli diarrhea; Cantey JR; E . coli diarrheal disease is becoming ever more complicated as more and more pathogenic mechanisms are identified . E . coli strains remain the major causes of infectious diarrhea worldwide . Presumptive diagnosis based on clinical and laboratory criteria is practical for strains known to be important in the United States . Specific diagnosis is not currently feasible outside of research centers . Therapy, when indicated, shortens the duration of illness . Research is proceeding rapidly at the molecular level and may lead to new diagnostic and therapeutic approaches in the near future.

FEMS Microbiol Lett, 1993 Sep 1, 112(2), 141 - 6
Brucella group 3 outer membrane proteins contain a heat-modifiable protein; Gamazo C et al.; Brucella melitensis and B . ovis outer membrane blebs contained a protein displaying a temperature-dependent molecular mass upshift from 25 kDa to 30 kDa . A fraction of the protein tightly bound to LPS did not show the molecular mass upshift which was also blocked by exposure of the protein to Zwittergent 314 . The B . melitensis heat-modifiable protein and Escherichia coli OmpA shared antigenic determinants . These data indicate that the Brucella group 3 outer membrane proteins belonged to the OmpA family of proteins.

Clin Sci (Lond), 1993 Sep, 85(3), 289 - 93
Human pyruvate dehydrogenase complex as an autoantigen in primary biliary cirrhosis; Palmer JM et al.; 1 . The sera of more than 90% of patients with primary biliary cirrhosis contain antimitochondrial antibodies which react with the E2 component of the pyruvate dehydrogenase complex, identified as the major autoantigen in primary biliary cirrhosis . All previous studies in this area have utilized protein derived from animal tissue or have used recombinant human pyruvate dehydrogenase complex E2 expressed in Escherichia coli . 2 . We report the preparation and characterization of native pyruvate dehydrogenase complex and pyruvate dehydrogenase complex E2 from human heart tissue and its application in studies of immune reactivity with the sera of patients with primary biliary cirrhosis . 3 . The immune reactivity of sera from patients with primary biliary cirrhosis versus the bovine and human E2/X components of pyruvate dehydrogenase complex was indistinguishable in both immunoblotting and the more sensitive e.l.i.s.a . 4 . These findings suggest that the reactivity of sera from patients with primary biliary cirrhosis against the major autoantigen of the disease is a property of that antigen, independent of its human or bovine origin . Furthermore, this justifies the use of bovine pyruvate dehydrogenase complex in past and future work on primary biliary cirrhosis antibody reactivity.

Circ Shock, 1993 Sep, 41(1), 40 - 7
Age-related differences in responses to endotoxin infusion in unanesthetized piglets; Li JX et al.; Newborn endotoxic shock syndrome is associated with high morbidity and mortality, yet presents with different clinical manifestations than in older patients . To determine the influence of age on hemodynamic and metabolic responses to endotoxin, we developed a chronically instrumented endotoxic shock model using eight 1-3-day-old and seven 2-3-week-old piglets . Three days after surgery, 10 mg/kg of endotoxin was infused intravenously over 10 min in the younger group, and 5-10 mg/kg was given to the older animals . Two older piglets died immediately after infusion of 5 mg/kg of endotoxin, and five of the seven died within 4 hr, while all eight younger animals lived longer than 4 hr . Pulmonary artery pressure increased significantly after endotoxin in both groups, and there were no differences between groups . Systemic artery pressure and cardiac index fell by 44 +/- 10% and 70 +/- 15%, respectively, 5 min after endotoxin infusion in the older group, while these values did not change significantly in the younger group . Endotoxin infusion also caused greater elevation in pulmonary vascular resistance index in the older animals . In the later phase, which began 30 min after endotoxin, both groups displayed systemic hypotension and pulmonary hypertension, and the groups did not differ from one another in this regard . With progression of endotoxic shock, more severe metabolic acidosis developed in the older animals than in the younger animals . Plasma thromboxane B2 levels in the older group were about double those in younger piglets . Plasma 6-keto-PGF1 alpha and TNF alpha levels in both groups were similar and were significantly increased in the later phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Chem Biol Interact, 1993 Sep, 88(2-3), 155 - 73
Effects of nickel ions on polymerase activity and fidelity during DNA replication in vitro; Snow ET et al.; Nickel is a genotoxic carcinogen . However, the mechanisms of nickel-induced genotoxicity are not well understood . We have investigated the effects of Ni2+ ions on DNA polymerase activity and the fidelity of DNA replication in vitro . The effect of Ni2+ on different DNA polymerases is quite variable . The amount of enzyme inhibition and degree of alteration in replication fidelity induced by Ni2+ are dependent both on the polymerase and its associated 3'-5' exonuclease activity . Some polymerases, such as E . coli DNA polymerase I, AMV reverse transcriptase and human DNA polymerase alpha, can utilize Ni2+ as a weak substitute for Mg2+ during DNA replication . Other polymerases are very sensitive to inhibition by Ni2+ and the IC50 can vary by an order of magnitude . T4 polymerase is relatively insensitive to inhibition by Ni2+, although the sensitivity is enhanced in the absence of added Mg2+, and Ni preferentially inhibits the 3'-5' exonuclease function of T7 DNA polymerase . The fidelity and processivity of DNA polymerases may be either increased or decreased by Ni ions in a polymerase dependent manner . The inhibition DNA polymerase activity and altered replication fidelity may contribute significantly to Ni-induced mutagenesis and genotoxicity in vivo.

Protein Sci, 1993 Sep, 2(9), 1472 - 81
Role of the C-terminus in the activity, conformation, and stability of interleukin-6; Ward LD et al.; Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H) . The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent . The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6 . Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis . These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding . A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed . Some of these changes involved residues widely separated in the primary structure . For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity . Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable . These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.

Protein Sci, 1993 Sep, 2(9), 1441 - 51
Recombinant human erythropoietin (rHuEPO): cross-linking with disuccinimidyl esters and identification of the interfacing domains in EPO; Haniu M et al.; Several amino groups of recombinant human erythropoietin are selectively cross-linked by specific cross-linkers including disuccinimidyl suberate or dithiobis(succinimidyl propionate) . Intramolecular cross-linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the cross-linkers, which possess an 11-12-A length of a spacer between two reacting groups . Intramolecularly cross-linked peptides obtained suggest that several amino groups in erythropoietin (EPO) are positioned at a distance of near 12 A in the solution state . These interfacing amino groups include Lys 20-Lys 154, Lys 45-Lys 140, Lys 52-Lys 154, Lys 52-Lys 140, and Ala 1-Lys 116 . A comparison of the cross-linking results between nonglycosylated EPO and glycosylated EPO suggests that both proteins retain high similarity regarding protein conformation . These results fit a structural model similar to that of human growth hormone, in which four alpha-helical bundles and a long stretch of beta-sheet structure are involved in the active protein.

Protein Sci, 1993 Sep, 2(9), 1411 - 28
Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein; Chao H et al.; To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two-dimensional (2D) NMR studies was produced in Escherichia coli . A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase-directed expression vector . The 67-amino acid-long gene product differed from the natural AFP by inclusion of an N-terminal methionine but was indistinguishable in activity . The NMR spectra of this AFP were complicated by cis-trans proline isomerization from the C-terminal sequence YPPA . Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP . This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy . Nine beta-strands are paired to form two triple-stranded antiparallel sheets and one double-stranded antiparallel sheet . Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP . These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1 . In contrast to the replacement of the C-terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity.

Plant Cell, 1993 Sep, 5(9), 1011 - 27
Suppressors of trp1 fluorescence identify a new arabidopsis gene, TRP4, encoding the anthranilate synthase beta subunit; Niyogi KK et al.; Suppressors of the blue fluorescence phenotype of the Arabidopsis trp1-100 mutant can be used to identify mutations in genes involved in plant tryptophan biosynthesis . Two recessive suppressor mutations define a new gene, TRP4 . The trp4 mutant and the trp1-100 mutant are morphologically normal and grow without tryptophan, whereas the trp4; trp1-100 double mutant requires tryptophan for growth . The trp4; trp1-100 double mutant does not segregate at expected frequencies in genetic crosses because of a female-specific defect in transmission of the double mutant genotype, suggesting a role for the tryptophan pathway in female gametophyte development . Genetic and biochemical evidence shows that trp4 mutants are defective in a gene encoding the beta subunit of anthranilate synthase (AS) . Arabidopsis AS beta subunit genes were isolated by complementation of an Escherichia coli anthranilate synthase mutation . The trp4 mutation cosegregates with one of the genes, ASB1, located on chromosome 1 . Sequence analysis of the ASB1 gene from trp4-1 and trp4-2 plants revealed different single base pair substitutions relative to the wild type . Anthranilate synthase alpha and beta subunit genes are regulated coordinately in response to bacterial pathogen infiltration.

Plant Mol Biol, 1993 Sep, 22(6), 937 - 43
Characterization of pea chloroplastic carbonic anhydrase . Expression in Escherichia coli and site-directed mutagenesis; Provart NJ et al.; A cDNA encoding the mature, chloroplast-localized carbonic anhydrase in pea has been expressed in E . coli . The enzyme is fully active and yields of up to 20% of the total soluble protein can be obtained from the bacteria . This expression system was used to monitor the effects of site-directed mutagenesis of seven residues found within conserved regions in the pea carbonic anhydrase amino acid sequence . The effects of these modifications are discussed with respect to the potential of various amino acids to act as sites for zinc coordination or intramolecular proton shuttles.

Plant Mol Biol, 1993 Sep, 22(6), 1039 - 46
Destabilization of pea lectin by substitution of a single amino acid in a surface loop; Hoedemaeker FJ et al.; Legume lectins are considered to be antinutritional factors (ANF) in the animal feeding industry . Inactivation of ANF is an important element in processing of food . In our study on the stability of Pisum sativum L . lectin (PSL), a conserved hydrophobic amino acid (Val103) in a surface loop was replaced with alanine . The mutant lectin, PSL V103A, showed a decrease in unfolding temperature (Tm) by some 10 degrees C in comparison with wild-type (wt) PSL, and the denaturation energy (delta H) is only about 55% of that of wt PSL . Replacement of an adjacent amino acid (Phe104) with alanine did not result in a significant difference in stability in comparison with wt PSL . Both mutations did not change the sugar-binding properties of the lectin, as compared with wt PSL and with PSL from pea seeds, at ambient temperatures . The double mutant, PSL V103A/F104A, was produced in Escherichia coli, but could not be isolated in an active (i.e . sugar-binding) form . Interestingly, the mutation in PSL V103A reversibly affected sugar-binding at 37 degrees C, as judged from haemagglutination assays . These results open the possibility of production of lectins that are active in planta at ambient temperatures, but are inactive and possibly non-toxic at 37 degrees C in the intestines of mammals.

Biol Mass Spectrom, 1993 Sep, 22(9), 524 - 36
Structural characterization of the cyanelle peptidoglycan of Cyanophora paradoxa by 252Cf plasma desorption mass spectrometry and fast atom bombardment/tandem mass spectrometry; Pittenauer E et al.; A strategy for the structural characterization of the four major NaBH4-reduced peptidoglycan monomers derived from muramidase-digested peptidoglycan from the cyanelles of the flagellate Cyanophora paradoxa Korschikoff is described . Initial molecular weight determination of these glycopeptides was performed by positive and negative ion plasma desorption mass spectrometry . Due to the presence of two pairs of disaccharide tripeptide and disaccharide tetrapeptide monomers differing in mass by 112 units, respectively, an as yet unknown peptidoglycan modification either at the carbohydrate or at the peptide moiety was assumed . beta-Elimination of the disaccharide unit from the unreduced peptidoglycan monomers yielded the corresponding (modified) N1-lactyltripeptides and -tetrapeptides, respectively . These peptides, N-terminally blocked with lactic acid, unambiguously showed the modification to be located on the peptide moiety . By positive ion fast atom bombardment/hybrid tandem mass spectrometry of the reduced peptidoglycan monomers as well as of the corresponding deglycosylated monomers (= N1-lactylpeptides) the modification was determined to be linked to the glutamic acid moiety . Based on combined data from plasma desorption mass spectrometry, tandem mass spectrometry, accurate mass measurement and amino acid analysis of the acid hydrolysate after derivatization with o-phthaldialdehyde by high-performance liquid chromatography we could establish the structure of the modification as N-acetylputrescine . Finally, the confirmation of the linkage of the glutamic acid to diaminopimelic acid via the gamma-COOH was based on the presence of a-type peptide backbone fragment ions in the positive ion plasma desorption mass spectra of the modified N1-lactylpeptides.

J Bacteriol, 1993 Sep, 175(18), 5984 - 92
Expression of the capsular K5 polysaccharide of Escherichia coli: biochemical and electron microscopic analyses of mutants with defects in region 1 of the K5 gene cluster; Bronner D et al.; The gene cluster of the capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three regions responsible for polymerization and surface expression have been defined (I.S . Roberts, R . Mountford, R . Hodge, K . B . Jann, and G . J . Boulnois, J . Bacteriol . 170:1305-1330, 1988) . Region 1 has now been sequenced, and five open reading frames (kpsEDUCS) have been defined (C . Pazzani, C . Rosenow, G . J . Boulnois, D . Bronner, K . Jann, and I . S . Roberts, J . Bacteriol . 175:5978-5983, 1993) . In this study, we characterized region 1 mutants by immunoelectron microscopy, membrane-associated polymerization activity, cytoplasmic CMP-2-keto-3-deoxyoctonate (KDO) synthetase activity, and chemical analysis of their K5 polysaccharides . Certain mutations within region 1 not only effected polysaccharide transport (lack of region 1 gene products) but also impaired the polymerization capacity of the respective membranes, reflected in reduced amounts of polysaccharide but not in its chain length . KDO and phosphatidic acid (phosphatidyl-KDO) substitution was found with extracellular and periplasmic polysaccharide and not with cytoplasmic polysaccharide . This and the fact that the K5 polysaccharide is formed in a kpsU mutant (defective in capsule-specific K-CMP-KDO synthetase) showed that CMP-KDO is engaged not in initiation of polymerization but in translocation of the polysaccharide.

Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 8023 - 7
Time-resolved EPR studies with DNA photolyase: excited-state FADH0 abstracts an electron from Trp-306 to generate FADH-, the catalytically active form of the cofactor; Kim ST et al.; Photolyase repairs UV-induced cyclobutane-pyrimidine dimers in DNA by photoinduced electron transfer . The enzyme isolated from Escherichia coli contains 5,10-methenyltetrahydrofolate, which functions as the light-harvesting chromophore, and fully reduced flavin adenine dinucleotide (FAD), which functions as the redox catalyst . During enzyme preparation, the flavin is oxidized to FADH0, which is catalytically inert . Illumination of the enzyme with 300- to 600-nm light converts the flavin to the fully reduced form in a reaction that involves photooxidation of an amino acid in the apoenzyme . The results of earlier optical studies had indicated that the redox-active amino acid in this photoactivation process was tryptophan . We have now used time-resolved electron paramagnetic resonance (EPR) spectroscopy to investigate the photoactivation reaction . Excitation of the flavin-radical-containing inactive enzyme produces a spin-polarized radical that we identify by 2H and 15N labeling as originating from a tryptophan residue, confirming the inferences from the optical work . These results and Trp-->Phe replacement by site-directed mutagenesis reveal that flavin radical photoreduction is achieved by electron abstraction from Trp-306 by the excited-state FADH0 . Analysis of the hyperfine couplings and spin density distribution deduced from the isotopic-labeling results shows that the product of the light-driven redox chemistry is the Trp-306 cation radical . The results strongly suggest that the active form of photolyase contains FADH- and not FADH2.

J Bacteriol, 1993 Sep, 175(17), 5585 - 94
Sequencing and characterization of a gene cluster encoding the enzymes for L-rhamnose metabolism in Escherichia coli; Moralejo P et al.; The sequencing of the EcoRI-HindIII fragment complementing mutations in the structural genes of the L-rhamnose regulon of Escherichia coli has permitted identification of the open reading frames corresponding to rhaB, rhaA, and rhaD . The deduced amino acid sequences gave a 425-amino-acid polypeptide corresponding to rhamnulose kinase for rhaB, a 400-amino-acid polypeptide corresponding to rhamnose isomerase for rhaA, and a 274-amino-acid polypeptide corresponding to rhamnulose-1-phosphate aldolase for rhaD . Transcriptional fusions of the three putative promoter regions to lacZ showed that only the rhaB leader region acted as a promoter, as indicated by the high beta-galactosidase activity induced by rhamnose, while no significant activity from the rhaA and rhaD constructions was detected . The rhaB transcription start site was mapped to -24 relative to the start of translation . Mutations in the catabolic genes were used to show that L-rhamnose may directly induce rhaBAD transcription.

J Bacteriol, 1993 Sep, 175(17), 5559 - 65
Cooperation of the prs and dnaA gene products for initiation of chromosome replication in Escherichia coli; Sakakibara Y; A new Escherichia coli mutant allele, named dnaR, that causes thermosensitive initiation of chromosome replication has been identified to be an allele of the prs gene, the gene for phosphoribosylpyrophosphate synthetase (Y . Sakakibara, J . Mol . Biol . 226:979-987, 1992; Y . Sakakibara, J . Mol . Biol . 226:989-996, 1992) . The dnaR mutant became temperature resistant by acquisition of a mutation in the dnaA gene that did not affect the intrinsic activity for the initiation of replication . The suppressor mutant was capable of initiating replication from oriC at a high temperature restrictive for the dnaR single mutant . The thermoresistant DNA synthesis was inhibited by the presence of the wild-type dnaA allele at a high but not a low copy number . The synthesis was also inhibited by an elevated dose of a mutant dnaR allele retaining dnaR activity . Therefore, thermoresistant DNA synthesis in the suppressor mutant was dependent on both the dnaA and the dnaR functions . On the basis of these results, I conclude that the initiation of chromosome replication requires cooperation of the prs and dnaA products.

J Bacteriol, 1993 Sep, 175(17), 5460 - 8
Mutations in the alpha subunit of RNA polymerase that affect the regulation of porin gene transcription in Escherichia coli K-12; Sharif TR et al.; The two-component regulatory system consisting of OmpR and EnvZ controls the differential expression of major outer membrane porin proteins OmpF and OmpC of Escherichia coli K-12 . We have isolated and characterized two mutations in rpoA, the gene encoding the alpha subunit of RNA polymerase, that decrease the expression of OmpF . These mutations have a number of properties that distinguish them from previously isolated rpoA mutations that affect porin expression . The rpoA203 mutation decreases the expression of porin genes ompF and ompC and also decreases the expression of the malE and phoA genes . In contrast, rpoA207 decreases the expression of ompF but does not affect ompC, malE, or phoA transcription . Our results suggest that mutations at various positions in the alpha subunit may affect the OmpR-dependent transcription of ompF and ompC differently and may be useful for analyzing the mechanism underlying their differential expression in response to medium osmolarity.

J Bacteriol, 1993 Sep, 175(17), 5375 - 83
Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12; Iida A et al.; We isolated a transposon Tn10 insertion mutant of Escherichia coli K-12 which could not grow on MacConkey plates containing D-ribose . Characterization of the mutant revealed that the level of the transketolase activity was reduced to one-third of that of the wild type . The mutation was mapped at 63.5 min on the E . coli genetic map, in which the transketolase gene (tkt) had been mapped . A multicopy suppressor gene which complemented the tkt mutation was cloned on a 7.8-kb PstI fragment . The cloned gene was located at 53 min on the chromosome . Subcloning and sequencing of a 2.7-kb fragment containing the suppressor gene identified an open reading frame encoding a polypeptide of 667 amino acids with a calculated molecular weight of 72,973 . Overexpression of the protein and determination of its N-terminal amino acid sequence defined unambiguously the translational start site of the gene . The deduced amino acid sequence showed similarity to sequences of transketolases from Saccharomyces cerevisiae and Rhodobacter sphaeroides . In addition, the level of the transketolase activity increased in strains carrying the gene in multicopy . Therefore, the gene encoding this transketolase was designated tktB and the gene formerly called tkt was renamed tktA . Analysis of the phenotypes of the strains containing tktA, tktB, or tktA tktB mutations indicated that tktA and tktB were responsible for major and minor activities, respectively, of transketolase in E . coli.

J Bacteriol, 1993 Sep, 175(17), 5350 - 8
Insertion and deletion mutations in the repA4 region of the IncFII plasmid NR1 cause unstable inheritance; Jiang T et al.; Mutants of IncFII plasmid NR1 that have transposons inserted in the repA4 open reading frame (ORF) are not inherited stably . The repA4 ORF is located immediately downstream from the replication origin (ori) . The repA4 coding region contains inverted-repeat sequences that are homologous to the terC inverted repeats located in the replication terminus of the Escherichia coli chromosome . The site of initiation of leading-strand synthesis for replication of NR1 is also located in repA4 near its 3' end . Transposon insertions between ori and the right-hand terC repeat resulted in plasmid instability, whereas transposon insertions farther downstream did not . Derivatives that contained a 35-bp frameshift insertion in the repA4 ORF were all stable, even when the frameshift was located very near the 5' end of the coding region . This finding indicates that repA4 does not specify a protein product that is essential for plasmid stability . Examination of mutants having a nest of deletions with endpoints in or near repA4 indicated that the 3' end of the repA4 coding region and the site of leading-strand initiation could be deleted without appreciable effect on plasmid stability . Deletion of the pemI and pemK genes, located farther downstream from repA4 and reported to affect plasmid stability, also had no detectable effect . In contrast, mutants from which the right-hand terC repeat, or both right- and left-hand repeats, had been deleted were unstable . None of the insertion or deletion mutations in or near repA4 affected plasmid copy number . Alteration of the terC repeats by site-directed mutagenesis had little effect on plasmid stability . Plasmid stability was not affected by a fus mutation known to inactivate the termination function . Therefore, it appears that the overall integrity of the repA4 region is more important for stable maintenance of plasmid NR1 than are any of the individual known features found in this region.

J Neurochem, 1993 Sep, 61(3), 845 - 51
Regional distribution and quantitative measurement of the phosphoinositidase C-linked guanine nucleotide binding proteins G11 alpha and Gq alpha in rat brain; Milligan G; Levels of the guanine nucleotide binding proteins G11 alpha and Gq alpha, which produce receptor regulation of phosphoinositidase C, were measured immunologically in 13 regions of rat central nervous system . This was achieved by immunoblotting membranes from these regions with antisera (CQ series) that identify these two polypeptides equally, following separation of the membranes using sodium dodecyl sulphate-polyacrylamide gel electrophoresis conditions that can resolve Gq alpha and G11 alpha . In all regions examined, Gq alpha was more highly expressed than G11 alpha . Ratios of levels of Gq alpha to G11 alpha varied between the regions from 5:1 to 2:1 . Quantitative measurements of the levels of Gq alpha and G11 alpha in each region were obtained by comparison with known amounts of purified liver Gq alpha and G11 alpha and with E . coli expressed recombinant Gq alpha . Areas that expressed Gq alpha highly included olfactory bulb (930 ng/mg of membrane protein), frontal cortex (700 ng/mg of membrane protein), parietal occipital cortex (670 ng/mg of membrane protein), caudate putamen (1,003 ng/mg of membrane protein), hippocampus (1,045 ng/mg of membrane protein), hypothalamus (790 ng/mg of membrane protein), and cerebellum (950 ng/mg of membrane protein) . More modest levels were observed in thalamus (450 ng/mg of membrane protein), pituitary (480 ng/mg of membrane protein), optic chiasma (330 ng/mg of membrane protein), and spinal cord (350 ng/mg of membrane protein) . G11 alpha was more evenly expressed with values ranging from about 170 ng/mg of membrane protein in spinal cord and optic chiasma to close to 300 ng/mg of membrane protein in regions expressing high levels of Gq alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

J Virol, 1993 Sep, 67(9), 5450 - 62
The ps/hr gene (B5R open reading frame homolog) of rabbitpox virus controls pock color, is a component of extracellular enveloped virus, and is secreted into the medium; Martinez-Pomares L et al.; Wild-type rabbitpox virus (RPV) produces red hemorrhagic pocks on the chorioallantoic membranes (CAMs) of embryonated chicken eggs . Like the crmA (SPI-2) gene of cowpox virus, disruption of the RPV ps/hr gene results in a mutant which produces white pocks on the CAMs . An examination of the properties of the RPV(ps/hr) mutant in cell culture also reveals a significantly reduced host range, defined as the inability to form plaques, compared with wild-type virus . One of several cell types on which RPV(ps/hr) mutants fail to produce plaques is chicken embryo fibroblasts, cells which have been traditionally used to propagate spontaneously arising white pock mutants isolated from CAMs . The inability of the RPV(ps/hr) mutant to form plaques in chicken embryo fibroblasts correlates with a failure of a low multiplicity of infection to spread to neighboring cells and to form extracellular enveloped virus (EEV), although the formation and yields of infectious intracellular naked virus appear relatively normal . The gene product of the ps/hr gene, initially synthesized as a 45-kDa glycoprotein, is found as a component of EEV, but not intracellular naked virus, and as a smaller, secreted soluble protein of 35 kDa . Production of the secreted 35-kDa protein was found to be independent of any viral morphogenesis, suggesting two distinct pathways for release of the ps/hr gene product from the cell, i.e., as a component of the EEV particle and as a separately secreted glycoprotein.

J Virol, 1993 Sep, 67(9), 5206 - 15
cdc2 phosphorylation of threonine 124 activates the origin-unwinding functions of simian virus 40 T antigen; McVey D et al.; Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro . We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication . We find that the hexamer assembly, helicase, DNA polymerase alpha-binding, and transcriptional-autoregulation functions are independent of phosphorylation of threonine 124 . In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does . Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin . We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA . Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication . We also show that DNase footprinting and KMnO4 modification assays are not as stringent as immunoprecipitation and origin-dependent strand displacement assays for detecting defects in the origin-binding and -unwinding functions of T antigen . Differences in the assays may explain discrepancies in previous reports on the role of T-antigen phosphorylation in DNA binding.

J Gen Virol, 1993 Sep, 74 ( Pt 9), 1927 - 31
Complete nucleotide sequence of the genome of an apple isolate of apple chlorotic leaf spot virus; Sato K et al.; The complete nucleotide sequence of the genome of an apple isolate of apple chlorotic leaf spot virus (ACLSVA) was determined . The genome is 7552 nucleotides excluding the poly(A) tail and contains three open reading frames (ORFs 1, 2 and 3), encoding proteins with M(r) values of 216,503 (216.5K), 50,453 (50.4K) and 21,394 (21.4K), respectively . Nucleotide sequence comparisons between ACLSV-A and the previously sequenced ACLSV from plum (ACLSV-P) showed that the sequence identity at the nucleotide level was 79.8% . Amino acid sequence identities of ORFs 1 and 2 between both isolates were 88.4% and 79.9%, respectively . The 21.4K protein encoded by ORF 3 of ACLSV-A had an amino acid sequence identity of 88.6% with the 28.3K protein encoded by ORF 3 of ACLSV-P . Immunoblot analysis of the 21.4K protein expressed in Escherichia coli showed that this protein is the coat protein of ACLSV-A.

J Clin Invest, 1993 Sep, 92(3), 1412 - 7
Role of the eaeA gene in experimental enteropathogenic Escherichia coli infection; Donnenberg MS et al.; Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infant diarrhea in developing countries . Recently eaeA, a gene necessary for the characteristic intimate attachment of EPEC to epithelial cells in tissue culture, was described . We conducted a randomized, double-blind study to determine the role of the eaeA gene in human EPEC infection . 11 adult volunteers ingested 2 x 10(10) colony-forming units of O127:H6 EPEC strain E2348/69, and an equal number received the same dose of an isogenic eaeA deletion mutant constructed from E2348/69 . Volunteers were monitored for the development of diarrhea, fever, and systemic and gastrointestinal complaints . Diarrhea developed in all 11 volunteers who received E2348/69 and in 4 of 11 who received the mutant (P = 0.002) . Fever was more common in recipients of the wild-type strain (P = 0.024) . Stool volumes were lower in recipients of the mutant . All volunteers seroconverted to E2348/69 LPS, but the geometric mean peak titers of serum IgG and IgA in recipients of the mutant were lower than those of recipients of the wild-type strain . IgA against LPS was detected in the jejunal fluid of six of six recipients of E2348/69 and 5/6 recipients of the mutant . This study unambiguously assigns a role for eaeA as an EPEC virulence gene, but the residual diarrhea seen in recipients of the mutant indicates that other factors are involved.

J Bacteriol, 1993 Sep, 175(18), 6056 - 8
The desA gene of the cyanobacterium Synechocystis sp . strain PCC6803 is the structural gene for delta 12 desaturase; Wada H et al.; The desA gene of the cyanobacterium Synechocystis sp . strain PCC6803 was expressed in Escherichia coli, which does not contain any fatty acid desaturase . The product of the desA gene catalyzed the desaturation of fatty acids at the delta 12 position . This result demonstrates that desA is the structural gene for a delta 12 desaturase.

J Bacteriol, 1993 Sep, 175(18), 6049 - 51
A method for selection of mutations at the tdk locus in Escherichia coli; Summers WC et al.; Mutants of Escherichia coli which are resistant to 5-fluorodeoxyuridine all have mutations which map at a single locus at 27.5 min on the genetic map of E . coli . Extracts prepared from each mutant were deficient in thymidine kinase activity measured in vitro . Simple selective conditions which allowed detection of one mutant in the presence of 10(7) wild-type bacteria were found . These results show that loss of thymidine kinase activity is the usual mechanism for 5-fluorodeoxyuridine resistance and that all such mutations occur at the locus previously designated tdk.

J Bacteriol, 1993 Sep, 175(18), 6046 - 8
The pSC101 par locus alters protein-DNA interactions in vivo at the plasmid replication origin; Ingmer H et al.; We report here direct evidence that mutations in the par locus affect protein-DNA interactions in vivo at the replication origin of plasmid pSC101 . Concomitant with par-mediated plasmid stabilization, two sites in the origin region show an altered methylation pattern as detected by in vivo footprinting with dimethyl sulfate . One site is located near an integration host factor-binding sequence adjacent to the first of three direct repeats known to be involved in the initiation of pSC101 replication; the second site is within the third direct repeat.

J Bacteriol, 1993 Sep, 175(18), 6018 - 27
The Escherichia coli DNA polymerase III holoenzyme contains both products of the dnaX gene, tau and gamma, but only tau is essential; Blinkova A et al.; The replicative polymerase of Escherichia coli, DNA polymerase III, consists of a three-subunit core polymerase plus seven accessory subunits . Of these seven, tau and gamma are products of one replication gene, dnaX . The shorter gamma is created from within the tau reading frame by a programmed ribosomal -1 frameshift over codons 428 and 429 followed by a stop codon in the new frame . Two temperature-sensitive mutations are available in dnaX . The 2016(Ts) mutation altered both tau and gamma by changing codon 118 from glycine to aspartate; the 36(Ts) mutation affected the activity only of tau because it altered codon 601 (from glutamate to lysine) . Evidence which indicates that, of these two proteins, only the longer tau is essential includes the following . (i) The 36(Ts) mutation is a temperature-sensitive lethal allele, and overproduction of wild-type gamma cannot restore its growth . (ii) An allele which produced tau only could be substituted for the wild-type chromosomal gene, but a gamma-only allele could not substitute for the wild-type dnaX in the haploid state . Thus, the shorter subunit gamma is not essential, suggesting that tau can be substitute for the usual function(s) of gamma . Consistent with these results, we found that a functional polymerase was assembled from nine pure subunits in the absence of the gamma subunit . However, the possibility that, in cells growing without gamma, proteolysis of tau to form a gamma-like product in amounts below the Western blot (immunoblot) sensitivity level cannot be excluded.

J Bacteriol, 1993 Sep, 175(18), 5993 - 6001
Minimal essential origin of plasmid pSC101 replication: requirement of a region downstream of iterons; Sugiura S et al.; The minimal replication origin (ori) of the plasmid pSC101 was defined as an about 220-bp region under the condition that the Rep (or RepA) protein, a plasmid-encoded initiator protein, was supplied in trans . The DnaA box is located at one end of ori, as in other plasmids, like mini-F and P1 . The other border is a strong binding site (IR-1) of Rep which is palindromic sequence and lies in an about 50-bp region beyond the repeated sequences (iterons) in ori . This IR-1 is located just upstream of another strong Rep binding site (IR-2), the operator site of the structure gene of Rep (rep), but its function has not been determined . The present study shows that the IR-1 sequence capable of binding to Rep is essential for plasmid replication with a nearly normal copy number . Furthermore, a region between the third iteron and IR-1 is also required in a sequence-specific fashion, since some one-base substitution in this region inactivate the origin function . It is likely that the region also is a recognition site of an unknown protein . Three copy number mutations of rep can suppress any one-base substitution mutation . On the other hand, the sequence of a spacer region between the second and the third iterons, which is similar to that of the downstream region of the third iteron, can be changed without loss of the origin function . The requirement of the region downstream of iterons in pSC101 seems to be unique among iteron-driven plasmid replicons.

J Bacteriol, 1993 Sep, 175(18), 5824 - 8
Chloramphenicol induces the transcription of the major cold shock gene of Escherichia coli, cspA; Jiang W et al.; A downshift in temperature or exposure of cells to certain inhibitors of translation has been shown to induce the synthesis of cold shock proteins in Escherichia coli . We characterized the induction of the major cold shock protein (CS7.4, the product of the cspA gene) of E . coli in response to a shift from 37 to 15 degrees C and in response to the addition of chloramphenicol at 15 degrees C . A pulse-labeling assay and primer extension experiments indicated that the cold shock treatment resulted in a transient increase in the level of the cspA transcript and a transient induction of CS7.4, while the addition of chloramphenicol resulted in a constitutive increase in the level of cspA transcript and constitutive production of CS7.4 . The addition of rifamycin immediately following the temperature downshift or along with the addition of chloramphenicol repressed the transcription of cspA as well as the induced production of CS7.4 . Furthermore, changes in the cspA mRNA level were coincident with changes in CS7.4 synthesis . These results indicate that the expression of cspA induced by cold shock and chloramphenicol is at the level of transcription but not at the level of translation . Measurement of the half-life revealed that the cspA mRNA induced by chloramphenicol was more stable than that induced by cold shock.

J Bacteriol, 1993 Sep, 175(18), 5785 - 90
Behavioral responses of Escherichia coli to changes in temperature caused by electric shock; Shi W et al.; The behavioral response of Escherichia coli to electric shock in 10(-2) M potassium phosphate plus 10(-4) M potassium EDTA was studied . When presented with a 150-V/cm electric shock that lasted 250 ms, the bacteria at first exclusively ran, then exclusively tumbled, and finally returned to their original running and tumbling . This response is due to increased temperature caused by the electric shock, i.e., to thermotaxis, and it is mediated by the chemotaxis machinery . A more severe electric shock, 150 V/cm for 550 ms, caused cells to tumble immediately, and then they went back to their original running and tumbling . The mechanism of that response is unknown since, unlike known thermotaxis, it does not require the chemotaxis machinery.

J Bacteriol, 1993 Sep, 175(18), 5769 - 77
Integration host factor is required for anaerobic pyruvate induction of pfl operon expression in Escherichia coli; Sirko A et al.; The expression of the pyruvate formate-lyase gene (pfl) is induced by anaerobic growth, and this is increased further by growth in pyruvate . Previous work has shown that anaerobic induction is strongly dependent on the activator FNR and partially dependent on a second transcription factor, ArcA, while pyruvate induction only required FNR . Anaerobic and pyruvate regulation both require the presence of a 5' nontranslated regulatory sequence which spans approximately 500 bp of DNA . A mobility shift assay was developed to identify proteins that bind to this regulatory region . Several binding activities were separated by heparin agarose chromatography, and one of these activities was characterized and shown to be integration host factor (IHF) . Mobility shift and DNase I footprinting experiments defined a single IHF binding site in the pfl promoter-regulatory region . With pfl-lacZ fusions, it could be shown that introduction of a himD mutation abolished pyruvate-dependent induction of anaerobic expression in vivo . The same result was observed when the pfl IHF binding site was mutated . In addition, the partial anaerobic induction of expression found in an fnr strain was completely blocked in an fnr himD double mutant and in an fnr IHF binding site double mutant . Taken together, these data suggest that IHF is necessary for both pyruvate induction and the anaerobic induction mediated by ArcA.

Exp Parasitol, 1993 Sep, 77(2), 155 - 61
Ascaris suum: cloning of a cDNA encoding phosphoenolpyruvate carboxykinase; Geary TG et al.; A cDNA encoding phosphoenolpyruvate carboxykinase (PEPCK) from Ascaris suum was cloned by complementation of a strain of Escherichia coli deficient in PEPCK and malic enzyme . The product of this cDNA was enzymatically similar to a recombinant PEPCK obtained from Haemonchus contortus by the same method . Comparison of the predicted amino acid sequence of A . suum PEPCK with other PEPCKs showed that this enzyme is most closely related to the H . contortus enzyme . The two nematode enzymes share considerable homology in regions thought to be functionally involved in substrate binding and catalysis, some of which distinguish the nematode enzymes from PEPCKs from other organisms . This analysis suggests a structural explanation for the kinetic differences seen between nematode and vertebrate PEPCKs and supports the hypothesis that nematode PEPCK is a target for selective inhibition.

Eur J Biochem, 1993 Sep 1, 216(2), 579 - 86
A glutathione S-transferase with glutathione-peroxidase activity from Arabidopsis thaliana . Molecular cloning and functional characterization; Bartling D et al.; A full-length cDNA clone for a novel glutathione S-transferase was isolated from Arabidopsis thaliana and characterized . The cDNA encodes a polypeptide of 218 amino acids with a calculated molecular mass of 24,363 Da . The sequence was most related to the theta class within the glutathione-S-transferase superfamily of enzymes . The protein encoded by the cDNA was functionally expressed and enzymically active in Escherichia coli; glutathione-S-transferase activity with the standard enzyme substrate 1-chloro-2,4-dinitrobenzene was demonstrated (apparent Km, 10 mM; apparent Km for glutathione, 0.08 mM) . The enzyme is substrate specific and did not use several electrophilic reduced-glutathione acceptor molecules for conjugation . However, it efficiently catalyzed the conversion of 13-hydroperoxy-9,11,15-octadecatrienoic acid (Km, 0.67 mM) as well as 13-hydroperoxy-9,11-octadecadienoic acid (Km, 0.79 mM) to the corresponding hydroxy derivatives with concomitant formation of oxidized glutathione . The enzyme did not use H2O2 as substrate . Thus, the cloned A . thaliana enzyme functions as glutathione peroxidase and, in the plant cell, may be involved in the removal of reactive organic hydroperoxides, such as the products of lipid peroxidation . The enzyme is structurally and enzymatically, however, unrelated to the selenium-containing glutathione peroxidases . Enzymic and immunoblotting data suggest that the A . thaliana enzyme is soluble and constitutively expressed in vegetative rosettes, but is under developmental control during the transition to bolting and flowering.

Eur J Biochem, 1993 Sep 1, 216(2), 539 - 48
Cloning and nucleotide sequence of the gcv operon encoding the Escherichia coli glycine-cleavage system; Okamura-Ikeda K et al.; P-protein, H-protein and T-protein of the glycine cleavage system have been purified from Escherichia coli . Their N-terminal amino acid sequences were determined, and a set of oligonucleotide probes was designed for gene cloning . The nucleotide sequence of a fragment of DNA around the 62-min region of the E . coli chromosome, containing genes for the components of the glycine-cleavage system has been determined . The sequence includes three structural genes encoding T-protein (363 amino acids, 40013 Da), H-protein (128 amino acids, 13679 Da) and P-protein (956 amino acids, 104240 Da) . These genes are named gcvT, gcvH and gcvP, respectively . They are organized in the above-mentioned order on the same strand of DNA with short intercistronic sequences . The presence of a potential promoter preceding gcvT and a typical rho-independent terminator sequence following gcvP indicated that the three genes constitute a single operon . Each component of the E . coli glycine-cleavage system exhibits considerable amino acid sequence similarity with the animal and plant counterparts . When the plasmid containing the gcv operon was transfected in E . coli cells, the gene products of gcvT, gcvH and gcvP were overexpressed under the direction of the promoter of the gcv operon . However, bacteria harboring the plasmid that contained the gcv operon without the promoter region and the 5' terminal portion of gcvT failed to overexpress any of the three components.

Eur J Biochem, 1993 Sep 1, 216(2), 519 - 25
Acylation of porcine pancreatic phospholipase A2 influences penetration and substrate head-group binding, depending on the position of the acylated lysine in the enzyme molecule; Lugtigheid RB et al.; A porcine pancreatic phospholipase A2 mutant was constructed in which all nine lysines were replaced by arginines . The mutant displayed 68% residual activity on micellar zwitterionic substrates, indicating that lysines are not absolutely required for the catalytic action of the enzyme . Likewise, mutants with one single lysine present either at position 56, located close to the entrance of the active site, or at position 108, remote from the active site, were constructed . Selective acylation of Lys56 with acyl chains of two, eight or fourteen carbon atoms resulted in increased activities on 1,2-dioctanoylglycero-3-phosphocholine micelles . Moreover, acylation strongly influenced the affinity for these micelles, as was evidenced by an up to 60-fold increase in apparent Km . The kinetic properties of the (acylated) mutants were studied with the monolayer technique . Pre-steady-state kinetics showed that penetration into monomolecular layers composed of 1,2-didodecanoylglycero-3-phosphocholine was faster for acylated Lys56 derivatives than for non-acylated enzyme . The acylated enzymes were also capable of penetrating densely packed lipid films . This effect increased with increasing acyl chain length . The observed velocities in the steady state were similar for acylated and non-acylated Lys56 mutants . In contrast, no changes in the kinetic properties were observed after acylation of Lys108, located on the posterior part of the protein . Therefore, the effects observed upon acylation of Lys56 are probably specific . Apart from an increase in hydrophobicity, acylation of Lys results in charge neutralization . The latter effect was studied with a mutant in which Gln instead of Lys was present at position 56 . The activity of this mutant on micellar substrates is higher than that of the parent Lys56, whereas its affinity for micelles is slightly improved . Therefore, whereas the charge at position 56 mainly influences the activity, the hydrophobicity of the introduced acyl chain mainly determines the affinity for aggregated lipids.

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