J Biochem (Tokyo), 1991 Jan, 109(1), 171 - 7 Studies on Phe-228 and Leu-307 recombinant mutants of porcine kidney D-amino acid oxidase: expression, purification, and characterization; Miyano M et al.; Two recombinant mutants of porcine kidney D-amino acid oxidase {EC 1.4.3.3, DAO}, in which Tyr(228) and His(307) are replaced with Phe and Leu, respectively, have been expressed in Escherichia coli and purified to apparent homogeneity . The molecular size and amino-terminal sequence of the two mutants were the same as those of the native DAO . Kinetic analysis revealed that the Michaelis constants of the Phe-228 and Leu-307 mutants for D-alanine were 71- and 10-fold and the inhibition constants for benzoate, a potent competitive inhibitor, were 1,189- and 18-fold greater than those of the native DAO, respectively . The maximum velocities of the Phe-228 and Leu-307 mutants were 66 and 58% that of the native DAO . The kinetically estimated dissociation constant of the Leu-307 mutant for FAD was 28-fold greater than that of the native DAO, whereas the value of the Phe-228 mutant was comparable to that of the native DAO . The Leu-307 mutant and the recombinant wild-type DAO were inactivated by D-propargylglycine (D-PG), a suicide substrate . However, the Phe-228 mutant was resistant to the inactivation . Absorption peaks of the Phe-228 mutant were blue-shifted about 10 nm from the corresponding peaks of the wild-type DAO, and the oxidized form was fully reduced by D-alanine without appearance of the purple intermediate.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Microbiol, 1991 Jan, 5(1), 61 - 70 987P fimbrial gene identification and protein characterization by T7 RNA polymerase-induced transcription and TnphoA mutagenesis; Schifferli DM et al.; The 987P fimbrial gene cluster has been previously cloned as a 12 kb fragment from prototype strain 987 . Gene products encoded by the whole clone were analysed by utilizing an in vivo system based on the induction of transcription by T7 RNA polymerase . The sensitivity of this technique permitted us to identify new proteins involved in 987P fimbriation . In total, eight proteins were detected, their genes (fasA to fasH) were mapped and their orientation of transcription determined . Several of the gene products demonstrated typical properties of exported proteins . Precursor and processed forms could be correlated after inhibiting protein transport with ethanol . The detection of enzymatically active fusion proteins after TnphoA (Tn5IS50L::phoA) mutagenesis supported and complemented these results . One protein encoded by the 12kb fragment was found not to be related to fimbriation but rather the product of the STla gene, identified as a component of a Tn1681-like transposon. Mol Microbiol, 1991 Jan, 5(1), 19 - 22 Molecular chaperones and protein translocation across the Escherichia coli inner membrane; Kumamoto CA; Proteins that are able to translocate across biological membranes assume a loosely folded structure . In this review it is suggested that the loosely folded structure, referred to here as the 'pre-folded conformation', is a particular structure that interacts favourably with components of the export apparatus . Two soluble factors, SecB and GroEL, have been implicated in maintenance of the pre-folded conformation and have been termed 'molecular chaperones' . Results suggest that SecB may be a chaperone that is specialized for binding to exported protein precursors, while GroEL may be a general folding modulator that binds to many intracellular proteins. FEMS Microbiol Lett, 1991 Jan 1, 61(1), 31 - 8 Sequence analysis of the Legionella micdadei groELS operon; Hindersson P et al.; A 2.7 kb DNA fragment encoding the 60 kDa common antigen (CA) and a 13 kDa protein of Legionella micdadei was sequenced . Two open reading frames of 57,677 and 10,456 Da were identified, corresponding to the heat shock proteins GroEL and GroES, respectively . Typical -35, -10, and Shine-Dalgarno heat shock expression signals were identified upstream of the L . micdadei groEL gene . Further upstream, a poly-T region, also a feature of the sigma 32-regulated Escherichia coli groELS heat shock operon, was found . Despite the high degree of homology of the expression signals in E . coli and L . micdadei, Western blot analysis with an L . micdadei specific anti-groEL antibody did not reveal a significant increase in the amount of the GroEL protein during heat shock in L . micdadei or in the recombinant E . coli expressing L . micdadei GroEL. Pediatr Infect Dis J, 1991 Jan, 10(1), 15 - 9 Escherichia coli in patients with renal scarring: genotype and phenotype of Gal alpha 1-4Gal beta-, Forssman- and mannose-specific adhesins; Plos K et al.; The frequency of Escherichia coli with Gal alpha 1-4Gal beta-specific adhesins is reduced among children who develop renal scars . The adhesion-negative phenotype may be due to the absence of the pap DNA sequences which encode these adhesins or to a phase variation event induced by in vitro culture . In the present study the frequency of pap and pil homologous DNA was determined by dot blot analysis with probes specific for the respective sequence using E . coli strains from children with recurrent pyelonephritis with and without renal scarring . The frequency of pap was 79% in the strains isolated from the nonscarring group compared with 39% in the strains from the scarring group (P less than 0.001) . The Gal alpha 1-4Gal beta phenotype was expressed by 89% of the pap-positive strains from the nonscarring group compared with 71% in the scarring group (P less than 0.05) . In addition 13 of 77 of the pap-positive E . coli strains agglutinated sheep erythrocytes but not the Gal alpha 1-4Gal beta latex beads; a reaction attributed to reactivity with the Forssman glycolipid . DNA sequences homologous with pil were found in 95% of all strains and there was no significant difference between the nonscarring and the scarring groups . The low frequency of Gal alpha 1-4Gal beta specific strains in the scarring group was therefore due to the absence of pap-homologous DNA sequences and to a reduced rate of phenotypic expression among pap-positive scarring strains . There was no support for a relationship between type 1 fimbriae and renal scarring. Int J Biochem, 1991, 23(2), 161 - 7 Anionic phospholipids in the control of the membrane surface potential in Escherichia coli: their influence on transport mechanisms; Cerbon J et al.; 1 . Phosphatidylserine (PS)-rich Escherichia coli cells were utilized to investigate the role of anionic phospholipids on membrane surface potential and their effect upon active transport mechanisms . 2 . It was found that: 3 . The transport of inorganic phosphate and glutamate, which depends upon cations, was increased (Km decreases) in PS-rich cells as compared to normal cells . 4 . The reduction of the negative surface potential by MgCl2 or by the cationic local anesthetic procaine, brought about a decrement in the uptake of both substrates . 5 . When the negative surface potential of the PS-rich cells was reduced, the Km returned back to the values found in normal cells . 6 . A direct correlation between the ratio anionic/zwitterionic phospholipids, negative surface potential and increment in the initial rate of transport was found. EMBO J, 1991 Jan, 10(1), 153 - 62 Direct interaction of the Polycomb protein with Antennapedia regulatory sequences in polytene chromosomes of Drosophila melanogaster; Zink B et al.; The Polycomb (Pc) gene is responsible for the elaboration and maintenance of the expression pattern of the homeotic genes during development of Drosophila . In mutant Pc- embryos, homeotic transcripts are ectopically expressed, leading to abdominal transformations in all segments . From this it was suggested that PC+ acts as a repressor of homeotic gene transcription . We have mapped the cis-acting control sequences of the homeotic Antennapedia (Antp) gene regulated by Pc . Using Antp P1 and P2 promoter fragments linked to the E . coli lacZ reporter gene we show different expression patterns of beta-galactosidase (beta-gal) in transformed Pc+ and Pc- embryos . In addition we are able to visualize by immunocytochemical techniques on polytene chromosomes the direct binding of the Pc protein to the transposed cis-regulatory promoter fragments . However, short Antp P1 promoter constructs which are--due to position effects--ectopically activated in salivary glands, do not reveal a Pc binding signal. Int J Radiat Biol, 1991 Jan, 59(1), 145 - 51 Is N2O-dependent damage 'fixed' by action of the recA locus? Ewing D, Guilfoil DS. Nitrous oxide (N2O) is often a radiation sensitizer of procaryotic cells, although it has little or no effect in recA- strains of E . coli and Saccharomyces . Here we test the hypothesis that N2O-dependent damage is in itself not lethal, but that lethality occurs when this damage is incorrectly repaired by the recA+ strains . Because sensitization by N2O requires the radiolytic production of H2O2, the hypothesis can be tested with reagent H2O2 . If the 'inaction' of the recA locus prevents N2O sensitization in the recA- strains, then those strains should not be sensitized by reagent H2O2 . Our data show that all these strains, including the recA+ parent, are efficiently sensitized by reagent H2O2 in N2 and also in N2O; thus or data do not support that hypothesis . We propose instead that the correlation between N2O sensitization and the recA- genotype occurs because of the inherent anoxic sensitivity of the recA- strains; the doses used to assay survival, and thus to test for sensitization by N2O, are simply too low to produce sufficient H2O2 for sensitization to occur. Infect Immun, 1991 Jan, 59(1), 91 - 6 Characterization and identification of a porcine small intestine mucus receptor for the K88ab fimbrial adhesin; Metcalfe JW et al.; The ability of Escherichia coli K-12(K88ab) to adhere to immobilized porcine small intestine mucus was examined . E . coli K-12(K88ab) but not the isogenic E . coli K-12 strain was found to adhere readily to immobilized crude mucus but not to bovine serum albumin . The adhesion of E . coli K-12(K88ab) was inhibited in a specific fashion by anti-K88 antiserum . Adhesion was also inhibited by pretreatment of receptor-containing crude mucus preparations with sodium metaperiodate or proteolytic enzymes . Removal of glycolipids from crude mucus by chloroform-methanol extraction did not affect the ability of E . coli K-12(K88ab) to bind to mucus preparations . Adsorption of crude mucus preparations with K88ab fimbriae but not type 1 fimbriae resulted in the removal of K88-specific receptors . Analysis of the pelleted fimbriae-receptor complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, together with gel filtration chromatography of crude mucus preparations, suggest that the K88-specific receptor present in porcine small intestine mucus is a 40- to 42-kDa glycoprotein. Nucleic Acids Symp Ser, 1991, (24), 99 - 102 Design and applications of single and double stranded DNA analogs; Shabarova ZA; Chemical ligation method for assembling modified DNA-duplexes as well as solid support method and postsynthetic modification in aqueous media was used to synthesize a variety of single and double stranded oligonucleotide derivatives . Novel oligodeoxynucleotides with cluster or alternating sugar modifications and probes resistant to cell nuclease degradation were obtained . The use of such probes for effective and regiospecific hybridase RNA-hydrolysis is provided . Effective synthesis of new DNA duplexes containing modified and chemical cleavable internucleotide bonds was developed . A novel type of affinity reagent was suggested for covalent attachment of substrate to active site nucleophiles in the restriction/modification systems. Orig Life Evol Biosph, 1991-92, 21(4), 225 - 42 Possible prebiotic significance of polyamines in the condensation, protection, encapsulation, and biological properties of DNA; Baeza I et al.; Some properties of DNA condensed with spermidine have been compared with the properties of DNA condensed with Co3+(NH3)6 to determine whether condensation of DNA with these trivalent cations protects DNA against the action of DNase I and increases transcription and encapsulation of DNA into liposomes . It was shown that DNA condensed with Co3+(NH3)6 was resistant to the action of the endonuclease DNase I such as DNA condensed with spermidine was . However, DNA condensed with Co3+(NH3)6 was significantly less active in transcription with the E . coli RNA polymerase than DNA-spermidine condensed forms . In addition, it was demonstrated that both compacted forms of DNA were more efficiently encapsulated into neutral liposomes; however, negatively, charged liposomes were scarcely formed in the presence of DNA condensed with Co3+(NH3)6 . These experiments and the well documented properties of polyamines increasing the resistance to radiations and hydrolysis of nucleic acids, as well as their biological activities, such as replication, transcription, and translation, together with the low concentration of Co3+ in the environment, lead us to propose spermidine as a plausible prebiotic DNA condensing agent rather than Co3+ and the basic proteins proposed by other authors . Then, we consider the possible role and relevance of the polyamine-nucleic acids complexes in the evolution of life. Braz J Med Biol Res, 1991, 24(4), 359 - 63 Expression of an active foot-and-mouth disease virus RNA polymerase in Escherichia coli; Brindeiro RM et al.; The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained . Two oligonucleotides of 66 base pairs were used to rebuild the 5' end of the gene and to introduce the first methionine codon . The expression of the active polymerase in E . coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid. J Hepatol, 1991, 13 Suppl 4, S146 - 51 Characterization of a hepatitis A virus strain suitable for vaccine production; Fineschi N et al.; A novel isolate of hepatitis A virus, obtained from a clinical sample, has been adapted to grow on cultured human diploid cells . Growth and purification parameters have been optimized to obtain conditions suitable for the development of an inactivated vaccine . The entire viral genome was molecularly cloned, and the gene encoding the VP3 capsid protein was expressed in Escherichia coli . The resulting recombinant VP3 was used to obtain rabbit antisera which recognize the denatured protein in purified virion preparations . Nucleotide sequencing data are presented and compared to known sequences of different strains. Antisense Res Dev, 1991 Fall, 1(3), 229 - 42 Sequence-specific alkylation of dsDNA with derivatives of pyrimidine oligonucleotides conjugated to 2-chloroethylamine groups; Brossalina EB et al.; Reaction of homopyrimidine oligonucleotides bearing a 5'-terminal alkylating aromatic 2-chloroethyl-amino group with a bovine papilloma vector expressing human interferon-gamma was investigated . The oligonucleotide derivatives bound to corresponding homopurine-homopyrimidine sequences in dsDNA and alkylated guanosine residues at these sites in the purine strand of the target . The alkylated DNA can be cleaved at the modified residues . At pH 5.4, the reaction was highly specific to the target sequences; at pH less than 5, some nonspecific reactions were observed at the sequences partially complementary to the oligonucleotides . Elongation of the linker between the alkylating group and the oligonucleotide phosphate increased the alkylation efficiency . Repeated treatment of the DNA with gradually increased concentrations of the reagent resulted in quantitative modification of the target guanosines. Z Kardiol, 1991, 80 Suppl 7, 87 - 90 The L-arginine-NO pathway and cyclic GMP in the vessel wall; Stoclet JC; Nitric oxide (NO) formation from L-arginine and subsequent activation of a soluble guanylate cyclase accounts for the effect of the endothelium derived relaxing factor (EDRF) . Cyclic GMP produced in smooth muscle cells induces relaxation through a mechanism which involves cyclic GMP kinase, but has not yet been entirely elucidated . Experiments with specific inhibitors of the different cyclic nucleotide phosphodiesterases (PDEs) suggest that a cyclic GMP-inhibited PDE which selectively hydrolyzes cyclic AMP, called PDE III, might also be involved in the relaxing mechanism of cyclic GMP . In arteries removed from endotoxemic rats or exposed to E . coli endotoxin, an extra-endothelial production of NO or a NO-like relaxing factor is induced in smooth muscle cells . Evidence that this phenomenon may be important in endotoxin shock is provided by experiments in which vascular reactivity is restored to control level by inhibitors of NO production in endotoxemic rats . These findings show that the L-arginine-NO pathway and cyclic GMP play a major role in regulating vascular contractility in physiological and pathological conditions. J Ocul Pharmacol, 1991 Fall, 7(3), 227 - 41 The role of endogenous eicosanoids in rabbit-intraocular inflammation; Kulkarni PS; The role of endogenously released eicosanoids in intraocular inflammation was assessed in two rabbit models . The models were: (1) paracentesis in which only breakdown of blood-aqueous barrier (BAB) occurs and (2) uveitis induced by endotoxin in which the disruption of the BAB and polymorphonuclear leukocyte infiltration are the predominant events . Indomethacin (a specific cyclooxygenase inhibitor) applied topically inhibited both the disruption of the BAB and increased levels of aqueous humor 6-keto-Prostaglandin (PG)F1 alpha . However, indomethacin and flurbiprofen applied topically and BWA4C or BWA218C (both selective lipoxygenase inhibitors) given parenterally, did not inhibit BAB response in endotoxin-induced uveitis . The cyclooxygenase inhibitors attenuated PGE2 release into aqueous humor . The 5-lipoxygenase inhibitors reduced the PMN infiltration as well as LTB4 release into aqueous humor . However, myeloperoxidase activity (an index for PMN chemotaxis) in iris-ciliary body was not affected by these drugs . Furthermore, concentrations of LTB4 in aqueous humor after paracentesis and uveitis-induced by endotoxin were similar, although in the former model there was no leukocyte infiltration, but in the latter model this leukocyte response was predominant . The results of this study suggest that locally released autocoids may not initiate ocular inflammation and other mediators such as cytokines may be involved in the inflammatory responses of the rabbit eye . We tried to detect IL-1 activity in aqueous humor following endotoxin . However, we could not detect the presence of IL-1-like activity, possibly because endotoxin also releases PGs, which inhibit IL-1 bioassay. Microbiol Immunol, 1991, 35(10), 863 - 70 Japanese encephalitis virus fusion protein with protein A expressed in Escherichia coli confers protective immunity in mice; Srivastava AK et al.; A complementary DNA (cDNA) that codes C-terminal, one-third of envelope glycoprotein (E) and N-terminal 65 amino acids of NS1 protein of Japanese encephalitis (JE) virus was inserted into Escherichia coli expression vector pRIT2T . The inserted gene was expressed as a fusion protein with protein A, and the expressed protein was intraperitoneally injected into mice . The immunized mice produced anti-JE antibodies measured by the hemagglutination-inhibition and neutralization tests as well as ELISA and were protected from the lethal challenge of JE virus by intraperitoneal inoculation. Methods Enzymol, 1991, 208, 458 - 68 Probing information content of DNA-binding sites; Stormo GD; An information content analysis of protein-binding sites gives a quantitative description of the specificity of the protein, independent of the mechanism of specificity . It gives useful information about the total specificity of the protein and about the individual positions within the binding sites . Information content is consistent with both thermodynamic and statistical analyses of specificity . When applied to a collection of known binding sites, the description provided may be limited by the sample size or by unknown constraints on those sites . Experimental procedures to determine the information content can give much more reliable measures . A large number of functional sites can be obtained from a much larger pool of randomized potential sites . Quantitative assays for the activity of different sites can be easily incorporated into the analysis, thereby increasing its sensitivity . Both in vitro and in vivo experiments are amenable to information content analysis. Nephron, 1991, 58(3), 276 - 82 Leukotriene B4 and tumor necrosis factor release from leukocytes: effect of peritoneal dialysate; Jorres A et al.; The effect of peritoneal dialysate on the capacity of peripheral blood polymorphonuclear (PMNL) and mononuclear leukocytes (MNC) to release leukotriene B4 (LTB4) and tumor necrosis factor alpha (TNF alpha) was investigated in vitro . Following density gradient separation, aliquots of 5 x 10(6) PMNL or MNC were incubated in peritoneal dialysis fluid containing 1.5% glucose or Hanks' buffer (= control) for 1-2 h at 37 degrees C . TNF alpha and LTB4 production was stimulated with Escherichia coli lipopolysaccharide (LPS) and calcium ionophore A23187, respectively . MNC incubated in buffer and LPS produced (mean +/- SD) 1,006 +/- 522 pg TNF alpha/5 x 10(6) cells; no significant amounts of TNF alpha were detectable in the presence of dialysate . An inhibition of TNF alpha release was also observed in MNC exposed to bicarbonate-buffered dialysates (pH 7.40) and 4.25% and 1.5% glucose solution with physiologic osmolality . Incubation of PMNL in Hanks' buffer followed by A23187 stimulation led to production of 29.1 +/- 19.2 ng LTB4/5 x 10(6) cells, whereas glucose-incubated cells were refractory to ionophore stimulation (less than 0.1 ng LTB4/5 x 10(6) cells) . The failure of dialysate-exposed leukocytes to release inflammatory mediators in response to adequate stimuli may contribute to the impairment of cellular host defense in the setting of continuous ambulatory peritoneal dialysis. Biosens Bioelectron, 1991, 6(3), 233 - 7 Biosensors based on membrane transport proteins; Kiefer H et al.; We propose a novel class of biosensors based on membrane bound receptors or transport proteins as the sensing element . The protein is incorporated in a planar lipid bilayer which covers the transducer . The transducer may detect an electric current, a voltage, or a change in fluorescence . A prototype lactose sensor is presented which consists of a quartz slide covered by a lipid membrane containing the protein lactose permease from Escherichia coli . This protein is a lactose/H+ cotransporter, hence lactose in the external medium initiates lactose/H+ cotransport across the lipid membrane . This leads to a rise in proton concentration in the small volume between the lipid membrane and the quartz surface which can be detected by a pH-sensitive fluorescence dye. Bioconjug Chem, 1991 Jan-Feb, 2(1), 57 - 66 Photolytic cleavage of DNA by nitrobenzamido ligands linked to 9-aminoacridines gives DNA polymerase substrates in a wavelength-dependent reaction; Nielsen PE et al.; A series of reagents containing 3- or 4-nitrobenzamido ligands tethered to 9-aminoacridine via variable-length linkers have been prepared and their properties as photochemical DNA cleavers (photonucleases) examined . When irradiated with approximately 300-nm light, where the nitrobenzamido ligand can absorb, they cleave DNA in an oxygen-independent reaction presumably involving oxygen transfer from the nitro group to the deoxyribose units of the DNA backbone (Nielsen et al., 1988b) . This reaction is pH independent and only slightly affected by the linker length, and the DNA fragments are not substrates for DNA polymerase . When approximately 420-nm light is used, were only the 9-aminoacridinyl ligands absorb, the DNA cleavage is also oxygen-independent but pH dependent, requires DNA saturation with the reagent (base pair:reagent less than or equal to 2), and is most efficient with the longer linkers . The cleavage is specific for guanine residues and results in 5'-phosphate termini and heterogeneous (more than four products) 3'-termini . One of the products is presumably 3'-hydroxy since DNA photocleaved with nitrobenzamido acridine reagents and 420-nm radiation are substrates for DNA polymerase in a nick translation assay as well as for the Klenow fragment . An electron-transfer mechanism is suggested. Genetica, 1991, 84(1), 23 - 9 Unusual codon bias occurring within insertion sequences in Escherichia coli; Lawrence JG et al.; The large open reading frames of insertion sequences from Escherichia coli were examined for their spatial pattern of codon usage bias and distribution of rarely used codons . There is a bias in codon usage that is generally lower toward the terminal ends of the coding regions, which is reflected in the occurrence of an excess of nonpreferred codons in the 3' portions of the coding regions as compared with the 5' portions . In contrast, typical chromosomal genes have a lower codon usage bias toward the 5' ends of the coding regions . These results imply that the selective forces reflected in codon usage bias may differ according to position within the coding sequence . In addition, these constraints apparently differ in important ways between genes contained in insertion sequences and those in the chromosome. Biochem Int, 1991 Jan, 23(2), 317 - 26 Chemical synthesis and expression of a gene coding for bovine liver phosphotyrosine-protein phosphatase; Raugei G et al.; A chemically synthesized polynucleotide sequence coding for bovine liver low molecular weight acid phosphatase (which possesses phosphotyrosine-protein phosphatase activity) has been cloned in a E . coli expression vector . The recombinant protein retains correct affinity for substrate and inhibitors but shows a reduced specific activity. Free Radic Res Commun, 1991, 12-13 Pt 1, 59 - 66 Superoxide production by respiring membranes of Escherichia coli; Imlay JA et al.; O2- production by homogenates and isolated membranes of E . coli has been examined . Approximately one-fourth of the O2- generated by extracts in the presence of NAD (P) H is attributable to the membranes . The autoxidizable membrane component is a member of the respiratory chain, since O2- production is NADH-specific, amplified by cyanide, and absent from membranes lacking the respiratory NADH dehydrogenase . Other respiratory substrates (succinate, 1-phosphoglycerol, D-lactate, and L-lactate) supported O2-production at efficiencies between 3 and 30 O2- released per 10,000 electrons transferred, under conditions of substrate saturation . Membranes from quinoneless mutants quantitatively retain the ability to evolve O2-, indicating that the dehydrogenases are the sites of O2- production . Relative O2- production was greater at low substrate concentrations, probably reflecting the facilitation of unpairing of electrons that may occur when enzymes with multiple redox centers are only partially reduced . Respiration rate, cell volume, rates of membraneous and cytosolic O2- production, and SOD levels were used to calculate a steady-state concentration of O2- between 10(-10) and 10(-9) M in well-fed, aerobic, SOD-proficient cells. Free Radic Res Commun, 1991, 12-13 Pt 1, 221 - 7 Novel iron complexes behave like superoxide dismutase in vivo; Nagano T et al.; Novel iron and copper complexes having tris{N-(5-methyl-2-pyridylmethyl)-2-aminoethyl}amine (5MeT-PAA), tris{N-(3-methyl-2-pyridylmethyl)-2-aminoethyl}amine (3MeTPAA), tris{N-(5-methoxycarbonyl-2-pyridylmethyl)-2-aminoethyl}amine (TNAA), tris{(2-thienylmethyl)-2-aminoethyl}amine (TTAA), tris{(2-furylmethyl)-2-aminoethyl}amine (TFAA) or tris{(2-imidazoyl)-2-aminoethyl}amine (TIAA) as ligand, were synthesized to examine the superoxide dismutase (SOD) activity . The concentrations of Fe-3MeTPAA and Fe-TIAA equivalent to 1 unit of SOD (IC50) were 0.5 microM and 1.0 microM, respectively . Fe-3MeTPAA and Fe-TIAA had higher SOD activity than other Fe and Cu complexes and protected Escherichia coli cells from paraquat toxicity . In case of using tris{N-(6-methyl-2-pyridylmethyl)-2-aminoethyl}amine (6MeTPAA) as ligand, the Fe complex could not be obtained, which may be due to the steric hindrance of 6-methyl substituent . Generally, Cu complexes had low SOD activity, compared with Fe complexes, and could not suppress paraquat toxicity. Res Microbiol, 1991 Jan, 142(1), 37 - 45 Utilization of exogenous glucose-1-phosphate as a source of carbon or phosphate by Escherichia coli K12: respective roles of acid glucose-1-phosphatase, hexose-phosphate permease, phosphoglucomutase and alkaline phosphatase; Pradel E et al.; The periplasmic acid glucose-1-phosphatase (G-1-Pase) encoded by gene agp is necessary for the growth of Escherichia coli in a minimal medium containing glucose-1-phosphate (G-1-P) as the sole source of carbon . From a mutant in which the agp gene was inactivated, suppressors were isolated which recovered the ability to utilize G-1-P as carbon source . The mutants constitutively expressed hexose phosphate permease activity (encoded by uhpT) . The mutation involved mapped in the uhp region and, unlike those of wild-type strains, bacteria of the suppressed strains required phosphoglucomutase (pgm), to grow on G-1-P . Surprisingly, in a minimal medium deprived of inorganic phosphate, uhpT+ bacteria lacking the two enzymes, alkaline-phosphatase (phoA) and glucose-1-phosphatase (agp), could utilize G-1-P as the sole source of phosphate, and also as both the sole phosphate and carbon source provided the integrity of pgm and of uhpT was conserved . Although glucose-6-phosphate, the inducer of UhpT permease, was not present in the medium, the activity of uhpT was greatly stimulated by inorganic phosphate depletion . This phosphate-starvation-induced bypass of G-1-Pase by UhpT + Pgm systems shows that agp is essential for G-1-P assimilation as a carbon source only in a high-phosphate medium, a result in agreement with the lack of agp regulation by inorganic phosphate. Res Microbiol, 1991 Jan, 142(1), 29 - 36 Are appR and katF the same Escherichia coli gene encoding a new sigma transcription initiation factor? Touati E, Dassa E, Dassa J, Boquet PL, Touati D. The phenotype of Escherichia coli appR pleiotropic mutants has been compared with that of mutants in the katF gene, which lies in the same region and controls expression of catalase HPII (katE) and exonuclease III (xth) . All the described characters of appR mutants--reduced pH 2.5 acid phosphatase level, overexpression of alkaline phosphatase and ability of crp or cya mutants to utilize some CAP + cAMP-dependent carbon sources--were reproduced by a katF:: Tn10 insertion . In all cases, the wild-type phenotype was restored by the presence of a plasmid-borne katF+ gene . Conversely, spontaneous appR mutants were hypersensitive to H2O2 to the same degree as katF mutants . We conclude that the appR gene is identical to katF, which encodes a putative new sigma factor (Mulvey and Loewen, 1989). J Neurosci Methods, 1991 Jan, 36(1), 91 - 103 A system, using neural cell lines, to characterize HSV-1 vectors containing genes which affect neuronal physiology, or neuronal promoters; Geller AI; Among the potential uses of defective herpes simplex virus (HSV-1) vectors are to study neuronal physiology, neuronal gene regulation, and to perform gene therapy of neuronal diseases . The prototype HSV-1 vector, pHSVlac, stably expresses Escherichia coli beta-galactosidase from the HSV-1 immediate early (IE) 4/5 promoter in cultured rat peripheral and CNS neurons, and in neurons in the adult rat brain . The LacZ gene and the IE 4/5 promoter in pHSVlac can be replaced with genes which affect neuronal physiology or cellular promoters, respectively . A system is required to characterize these HSV-1 vectors; cultured neurons, a mixture of different kinds of neurons and glia, cannot be used . In contrast, neural cell lines represent a homogenous population of neural cells available in virtually unlimited quantities . A system, using neural cell lines, to characterize HSV-1 vectors carrying other genes or promoters is now reported: First, 4 assays are described to detect HSV-1 vector DNA, RNA transcribed from the vector, and to quantitate beta-galactosidase expression . Second, 8 cell lines derived from rodents, primates, and humans were infected with pHSVlac virus and shown to express beta-galactosidase . The cell lines tested included adrenergic and cholinergic mouse neuroblastoma cells, rat pheochromocytoma cells, rodent pituicytes, and human neuroblastoma cells . Infection of these cell lines should prove useful for characterizing HSV-1 vectors with molecular and biochemical assays . Third, differentiated rat pheochromocytoma and mouse neuroblastoma cells, which resemble neurons, were infected with pHSVlac virus and shown to stably express beta-galactosidase . Infection of these cells should be useful for determining the effect of various HSV-1 vectors on neuronal physiology . Thus, HSV-1 vectors containing various genes or promoters can be characterized using the system described in this study. Arch Microbiol, 1991, 155(4), 341 - 7 Ferrous iron dependent nitric oxide production in nitrate reducing cultures of Escherichia coli; Brons HJ et al.; L-Lactate-driven ferric and nitrate reduction was studied in Escherichia coli E4 . Ferric iron reduction activity in E . coli E4 was found to be constitutive . Contrary to nitrate, ferric iron could not be used as electron acceptor for growth . "Ferric iron reductase" activity of 9 nmol Fe2+ mg-1 protein min-1 could not be inhibited by inhibitors for the respiratory chain, like Rotenone . Quinacrine, Actinomycin A, or potassium cyanide . Active cells and L-lactate were required for ferric iron reduction . The L-lactate-driven nitrate respiration in E . coli E4 leading to the production of nitrite, was reduced to about 20% of its maximum activity with 5 mM ferric iron, or to about 50% in presence of 5 mM ferrous iron . The inhibition was caused by nitric oxide formed by a purely chemical reduction of nitrite by ferrous iron . Nitric oxide was further chemically reduced by ferrous iron to nitrous oxide . With electron paramagnetic resonance spectroscopy, the presence of a free {Fe2(+)-NO} complex was shown . In presence of ferrous or ferric iron and L-lactate, nitrate was anaerobically converted to nitric oxide and nitrous oxide by the combined action of E . coli E4 and chemical reduction reactions (chemodenitrification). SAAS Bull Biochem Biotechnol, 1991 Jan, 4, 22 - 6 Regulatory sequences controlling short chain fatty acid metabolism in Escherichia coli; Chen CY et al.; Acetoacetate in Escherichia coli is metabolized via the combined enzymatic action of a CoA-transferase and a thiolase . Growth of E . coli on short chain fatty acids such as butyrate and valerate is also predicated upon the expression of these enzymes . The genes for these enzymes (atoDAB) are arranged in an operon and are coordinately transcribed in response to the inducer acetoacetate . A positive regulatory element, the product of the atoC gene, regulates expression of the operon . The atoC gene lies adjacent to the atoDAB operon and all the ato genes have been cloned as a single 6.2 kbp restriction fragment (kindly provided by Dr . Lauren Sallus Jenkins) . We have isolated a series of mutant E . coli strains with altered regulatory properties that are either inducible by an alternate substrate, or that show constitutive expression of the atoDAB genes . The -10 and -35 regions upstream of the atoDAB operon poorly match consensus sequences . In addition, the transcriptional start is preceded by a catabolite activator protein binding site (CAP site), as well as a putative binding site for the atoC gene product as represented by a region of dyad symmetry. Appl Microbiol Biotechnol, 1991 Jan, 34(4), 488 - 94 Optimizing the promoter and ribosome binding sequence for expression of human single chain urokinase-like plasminogen activator in Escherichia coli and stabilization of the product by avoiding heat shock response; Surek B et al.; The expression of recombinant single-chain urokinase-like plasminogen activator (rscuPA) in Escherichia coli was optimized by fusing the puk gene to different promoters and ribosome binding sequences . Comparison of the tac, trp and lambda PL promoters showed that expression was maximal under tac control . Variation in the ribosome binding sequence and its distance to the AUG start codon yielded a further slight improvement of expression . The largest increase in rscuPA expression was achieved by variations in the host strain and growth conditions . In E . coli DG75 grown at 37 degrees C maximal expression was achieved 30 min after induction and decreased gradually until 240 min after induction . Growth at 30 degrees C yielded maximal expression 60 min after induction and resulted in reduced activity at longer times . Western blot analysis of the products showed that degradation of rscuPA was much larger at 37 degrees C than at 30 degrees C . Using E . coli CAG630 carrying the htpR mutation, which avoids heat shock response, for expression of rscuPA eliminated the instability of the product at both temperatures . Expression in this strain was even more efficient than in E . coli JM101 carrying the lon mutation . It is concluded that induction of the general heat-shock response in E . coli must be avoided to obtain stabilization of rscuPA . This drastically improves the overall yield of rscuPA from recombinant E . coli strains. J Biotechnol, 1991 Jan, 17(1), 19 - 33 Intracellular and extracellular production of proteins in Aspergillus under the control of expression signals of the highly expressed Aspergillus nidulans gpdA gene; Punt PJ et al.; The expression in Aspergillus is described of genes, coding for intracellular and extracellular proteins controlled by the promoter region of the constitutively and efficiently expressed glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) of Aspergillus nidulans . Both the homologous gpdA and the heterologous Escherichia coli beta-galactosidase (lacZ) and beta-glucuronidase (uidA) genes could be expressed intracellularly at levels as high as 10-25% of total soluble protein . Efficient extracellular production of A . niger glucoamylase could be achieved with a fusion-gene containing the region of the glucoamylase gene coding for the mature protein preceded by a synthetic fungal signal sequence . Extracellular production of a heterologous protein, E . coli beta-glucuronidase, with such a fusion was much less efficient . Only very low levels of beta-glucuronidase were detected in the culture fluid, whereas considerable enzyme activity was detected in the mycelium. Bioprocess Technol, 1991, 12, 163 - 81 Purification of secreted recombinant proteins from Escherichia coli; Le HV et al.; Secretion systems engineered for the expression of heterologous protein in E . coli provide several advantages for subsequent isolation of purified product . Proteins released from the periplasmic space, which represent a small fraction (i.e., 4-10%) of total cell protein, can readily be separated from other cellular proteins by centrifugation of the remaining cellular debris or cross-flow ultrafiltration . The starting material derived from secretion systems is generally of higher purity than comparable material produced from strains expressing cytoplasmically for systems exhibiting similar expression levels . The available evidence suggests that recombinant proteins derived from the periplasm are generally, but not always (44-46), soluble in a nonaggregated form . Consequently, simple purification protocols can be effectively employed for producing homogeneous product with a high yield . The majority of the secreted recombinant proteins reviewed in this chapter were purified by simple one- or two-step chromatography procedures . High-resolution techniques such as reversed phase HPLC were found necessary only in cases where the secreted polypeptides were contaminated with proteolytic degradation variants, e.g., hirudin (51) and beta-endorphin (22) . The fact that a high level of biological activity has been shown to be characteristic of purified recombinant proteins secreted into the periplasmic space suggests the presence of a native conformation stabilized by the expected disulfide linkages . Intramolecular disulfide bonds most probably form either as the polypeptide is translocated through the cytoplasmic membrane into the periplasm or within the periplasmic compartment, which has a higher oxidation potential than that found in the cytoplasm (57) . Studies performed with hGH (31) and muIL-2 (35) provide excellent examples of differences observed in protein folding and disulfide bond formation between heterologous proteins expressed in the cytoplasmic and periplasmic compartments . Thus, hGH and muIL-2 extracted from the cytoplasm of E . coli have been characterized as high molecular weight disulfide-bonded oligomers . It is likely that oligomerization occurs as the polypeptides are released from the reducing environment of the cytoplasm . In contrast, secreted hGH and muIL-2 extracted from the periplasm of E . coli by osmotic shock displayed the properties of a property folded native protein with correct disulfide pairing . In the case of muIL-2 only a small residual fraction (approximately 15%) of the purified secreted protein exhibited incomplete oxidation of cysteine (35) . Secretion of heterologous proteins into the periplasm prevents their exposure to the action of proteases located in the cytoplasm of E . coli (58) . The smaller polypeptides such as somatostatin (59), IGF-1 (46), and hEGF (54) are known to be particularly susceptible to intracellular degradation.(ABSTRACT TRUNCATED AT 400 WORDS) Biochem Biophys Res Commun, 1990 Dec 31, 173(3), 1375 - 81 Human thioredoxin reactivity-structure/function relationship; Jacquot JP et al.; The reactivity of human thioredoxin (HTR) was tested in several reactions . HTR was as efficient as E . coli or plant and algal thioredoxins when assayed with E . coli ribonucleotide reductase or for the reduction of insulin . On the other hand, HTR was poorly reduced by NADPH and the E . coli flavoenzyme NADPH thioredoxin reductase as monitored in the DTNB reduction test . When reduced with dithiothreitol (DTT), HTR was much less efficient than thioredoxin m and thioredoxin f, the respective specific thioredoxins for the chloroplast enzymes NADP-malate dehydrogenase (NADP-MDH) and fructose 1,6 bisphosphatase (FBPase) . Finally, HTR could be used in the photoactivation of NADP-MDH although less efficiently than thioredoxin m, proving nevertheless that it can be reduced by the iron sulfur enzyme ferredoxin thioredoxin reductase in the presence of photoreduced ferredoxin . Based on sequence comparisons, it was expected that HTR would display a reactivity similar to chloroplast thioredoxin f rather than to thioredoxin m . However the observed behavior of FTR did not exactly fit this prediction . The results are discussed in relation to the structural data available for the proteins. Eur J Biochem, 1990 Dec 27, 194(3), 853 - 61 Over-production, purification and properties of the uridine-diphosphate-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase from Escherichia coli; Michaud C et al.; The UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase was over-produced and purified from two plasmid-harbouring strains of Escherichia coli . The first strain, E . coli JM83(pHE5), gave a 15-fold over-production relative to parental strain . The enzyme could be partially purified (8.8-fold) by ion-exchange chromatography . With the second strain, E . coli JM83(pMLD25), a very strong over-production was obtained, since the enzyme represented about 20% of the cytoplasmic proteins . Purification yielded 77% protein homogeneity . However, the enzymatic activity, which was very unstable, was lost during the purification procedure . Several properties of the enzyme were studied . The enzyme gave maximal activity around pH 8 . The isoelectric point was 5.2 . The activity was increased by potassium phosphate . Reverse and exchange reactions could be catalysed . The N-terminal sequence of the protein was determined and correlated with the nucleotide sequence of the murE gene . The actual initiation codon was assigned. Eur J Biochem, 1990 Dec 27, 194(3), 845 - 52 Construction, expression and unexpected regulatory properties of a tropomyosin mutant with a 31-residue deletion at the C-terminus (exon 9); Bartegi A et al.; The cDNA coding for human skeletal muscle beta-tropomyosin was expressed in Escherichia coli to produce an unacetylated beta-tropomyosin . This cDNA was deleted from the sequence corresponding to the exon 9 and expressed in E . coli to produce an unacetylated beta-tropomyosin mutant lacking the C-terminal residues 254-284 . The main structural and functional properties of the two isolated proteins, designated tropomyosin-1 and des-(254-284)-tropomyosin, respectively, were characterized in comparison with those of the genuine rabbit skeletal muscle alpha beta-tropomyosin . The folding and thermal stability of the three tropomyosins were indistinguishable . Tropomyosin-1, but not des-(254-284)-tropomyosin, was polymerized in the presence of troponin and did bind to actin in the presence of the troponin complex . Despite its weak binding to actin, des-(254-284)-tropomyosin displayed a regulatory function in the presence of troponin with a marked activation of the actomyosin subfragment-1 ATPase in the presence of Ca2+ and low concentrations of subfragment-1 . The data were interpreted in the light of the allosteric models of regulation and suggest the involvement of the sequence coded by exon 9 in the stabilization by tropomyosin of the off state of the thin filament. Eur J Biochem, 1990 Dec 27, 194(3), 731 - 7 The functional and structural roles of residues Gln114 and Glu117 in elongation factor Tu; Harmark K et al.; The effects of substituting residues Gln114 by Glu and Glu117 by Gln, both situated in the vicinity of the guanine-nucleotide-binding pocket, were investigated in the isolated N-terminal domain (G domain) of elongation factor Tu with respect to the binding of the substrate GDP/GTP, GTPase activity and stability . The major change in the interaction with the guanine nucleotides is a lower affinity for GTP and a reduced GTPase activity when Gln114 is substituted by Glu . This mutation also abolishes most of the selective effects on the GTPase activity induced by the different monovalent cations . Substitution of Glu117 by Gln does not affect the interaction with the guanine nucleotides or the GTPase activity of the G domain in an essential way, but it reduces the stability towards denaturation of the G-domain.GDP complex . Our results therefore suggest, that Gln114 is involved in keeping a functional conformation of the guanine-nucleotide-binding pocket, whereas Glu117 participates in the regulation of the overall conformation of the G domain . Neither of these two residues appears to play a role in the actual GTPase mechanism. Eur J Biochem, 1990 Dec 27, 194(3), 799 - 804 Effector-dependent conformational changes in protein kinase C gamma through epitope mapping with inhibitory monoclonal antibodies; Cazaubon S et al.; Three monoclonal antibodies (mAb) directed against the regulatory domain of the protein kinase C gamma (PKC gamma); 15G4, 5A2 and 36G9, were shown to display distinct properties with respect to PKC gamma kinase activity {Cazaubon, S., Marais, R., Parker, P . & Strosberg, A.D . (1989) Eur . J . Biochem . 182, 401-406} . The mAb 5A2 and 36G9, which act as potent inhibitors of the cofactor-dependent kinase activity, can no longer bind PKC gamma in the presence of phosphatidylserine and phosphatidylserine/phorbol ester, respectively; 15G4 binding is not influenced by effectors . Due to this functional relationship between the inhibitory mAb- and cofactor-binding sites, we sought to localize the mAb epitopes with respect to the functional sites of PKC gamma . For this purpose, several deletions were introduced at the 5' end of the PKC gamma cDNA and the mutant proteins were expressed in Escherichia coli . The determination of the immunoreactivity of the deleted PKC gamma proteins shows that the amino acid residues essential to the binding of 5A2 and 36G9 are directly adjacent to the second cysteine-rich motif: these are contained in the sequences at positions 151-163 and 164-197, respectively . In addition, various deletions around the C1 region of the regulatory domain allowed the identification of the second cysteine-rich motif as a functional binding site for phorbol dibutyrate . These deletion studies thus demonstrate that the epitopes recognized by the inhibitory mAbs 5A2 and 36G9 are distinct from the cofactor-binding sites . This suggests that the binding of phosphatidylserine and phorbol ester induce conformational changes in the regulatory domain of PKC, which are thus responsible for the loss of the 5A2 and 36G9 immunoreactivity of the native protein . In this conformational state, PKC gamma conserves its ability to interact with the non-inhibitory mAb 15G4 . By using synthetic peptides, the 15G4 epitope was localized to the sequence 297-310 in the V3 variable region . This indicates that the flexibility of the V3 region, which delimits the C-terminus of the regulatory domain, may not be necessary for the allosteric activation of PKC . In view of these results, we propose that PKC activation by its cofactors results in intramolecular changes which allow the enzyme to bind exogenous substrates. Biochemistry, 1990 Dec 25, 29(51), 11266 - 73 Affinity labeling of aminoacyl-tRNA synthetases with adenosine triphosphopyridoxal: probing the Lys-Met-Ser-Lys-Ser signature sequence as the ATP-binding site in Escherichia coli methionyl-and valyl-tRNA synthetases; Hountondji C et al.; Pyridoxal 5'-triphospho-5'-adenosine (AP3-PL), the affinity labeling reagent specific for lysine residues in the nucleotide-binding site of several enzymes {Tagaya, M., & Fukui, T . (1986) Biochemistry 25, 2958-2964; Yagami, T., Tagaya, M., & Fukui, T . (1988) FEBS Lett . 229, 261-264}, was used to identify the ATP-binding site of Escherichia coli methionyl-tRNA synthetase (MetRS) . Incubation of this enzyme with AP3-PL followed by reduction with sodium borohydride resulted in a rapid inactivation of both the tRNA(Met) aminoacylation and the methionine-dependent ATP-PPi exchange activities . Complete inactivation corresponded to the incorporation of 0.98 mol of AP3-PL/mol of monomeric trypsin-modified MetRS . ATP or MgATP protected the enzyme from inactivation . The labeling with AP3-PL was also applied to E . coli valyl-tRNA synthetase (ValRS) . Both the tRNA(Val) aminoacylation and the valine-dependent ATP-PPi exchange activities were abolished by the incorporation of 0.91 mol of AP3-PL/mol of monomeric ValRS . AP3-PL was found attached to lysine residues 335, 402, and 528 in the primary structure of MetRS . In the case of ValRS, the AP3-PL-labeled residues corresponded to lysines 557, 593, and 909 . We therefore conclude that these lysines of MetRS and ValRS are directed toward the ATP-binding site of these synthetases, more specifically at or close to the subsite for the gamma-phosphate of ATP . AP3-PL-labeled Lys-335 of MetRS and Lys-557 of ValRS belong to the consensus tRNA CCA-binding Lys-Met-Ser-Lys-Ser sequence {Hountondji, C., Dessen, P., & Blanquet, S . (1986) Biochimie 68, 1071-1078}.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Dec 25, 29(51), 11180 - 8 Binding of p-nitrophenyl alpha-D-galactopyranoside to lac permease of Escherichia coli; Lolkema JS et al.; Binding of the substrate analogue p-nitrophenyl alpha-D-galactopyranoside (NPG) to lac permease of Escherichia coli in different membrane preparations was investigated . Binding was assayed with an improved version of the centrifugation technique introduced by Kennedy et al . {Kennedy, E.P., Rumley, M.V., Armstrong, J.B . (1974) J . Biol . Chem . 249, 33-37} . Two binding sites for NPG were found with dissociation constants of about 16 microM and 1.6 mM at pH 7.5 and room temperature . With purified lac permease reconstituted into proteoliposomes, it could be shown that one permease molecule binds two substrate molecules . Oxidation of lac permease with the lipophilic quinone plumbagin or alkylation with the sulfhydryl reagent N-ethylmaleimide caused a 12-fold increase in the first dissociation constant . The second dissociation constant seemed to be increased as well, but its value could not reliably be estimated . Ethoxyformylation of lac permease with diethyl pyrocarbonate totally abolished NPG binding . The implications of these results for the catalytic performance of the enzyme are discussed. J Biol Chem, 1990 Dec 25, 265(36), 22520 - 5 A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity; Tsuji T et al.; A plasmid encoding a mutant gene of heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, was induced by treatment of plasmid EWD 299 with hydroxylamine . A mutant strain of E . coli HB 101 carrying the mutant plasmid pTUH 6A produced a low toxic LT analogue (mutant LT), which was cross-reactive with anti-LT antibody . The mutant LT activity was less than 0.15 and 0.006% of the normal LT in the rabbit ileal loop test and in the rabbit skin permeability test, respectively . The amino acid composition of the mutant LT-B subunit was the same as that of the normal B subunit . Though the A2 fragment of the mutant LT was identical to normal LT by DNA analysis, the A1 fragment of the mutant LT differed from the normal A1 fragment in one amino acid at position 112; namely it had lysine instead of glutamic acid from the N terminus . These data suggest that glutamic acid at position 112 from the N terminus of the A1 fragment is important for the A subunit to express its biological activity. J Biol Chem, 1990 Dec 25, 265(36), 22506 - 12 Construction and processing of transfer RNA precursor models; Surratt CK et al.; Several "dimeric" tRNA molecules were constructed as potential substrates for ribonuclease P (RNase P) and for M1 RNA, the catalytic subunit of RNase P . Construction was affected by the T4 RNA ligase-mediated coupling of a mature Escherichia coli tRNA (acceptor substrate) and nucleotides 1-36 of yeast tRNAPhe (donor substrate), followed by annealing of the 3'-half of yeast tRNAPhe (nucleotides 38-76) . E . coli RNase P and M1 RNA were both found to cleave the dimeric tRNA precursor model constructed from E . coli tRNAPhe (5'-tRNA) and yeast tRNAPhe (3'-tRNA) in a reaction that was dependent on the presence of the annealed 3'-half molecule derived from yeast tRNAPhe, or on some conformation imposed by the presence of this species; the product had the same mobility as authentic E . coli tRNAPhe on a polyacrylamide gel . By utilizing tRNA precursor models radiolabeled at phosphodiesters immediately preceding or following the putative site of processing, cleavage of the substrate by both M1 RNA and the holoenzyme was demonstrated to occur at the expected phosphate ester linkage . The results obtained here suggest that the endonucleolytic separation of two tRNAs by RNase P is dependent on one or more structural features in the 3'-half of the 3'-tRNA, and thus are consistent with the report of McClain et al . (McClain, W . H., Guerrier-Takada, C., and Altman, S . (1987) Science 238, 527-530) that identifies the T stem and loop as a possible recognition site. J Biol Chem, 1990 Dec 25, 265(36), 22402 - 8 Cryoelectron microscopy of frozen-hydrated alpha-ketoacid dehydrogenase complexes from Escherichia coli; Wagenknecht T et al.; The native architectures of the pyruvate and 2-oxoglutarate dehydrogenase complexes have been investigated by cryoelectron microscopy of unstained, frozen-hydrated specimens . In pyruvate dehydrogenase complex and 2-oxoglutarate dehydrogenase complex the transacylase (E2) components exist as 24-subunit, cube-shaped assemblies that form the structural cores of the complexes . Multiple copies (12-24) of the alpha-ketoacid dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) components bind to the surface of the cores . Images of the frozen-hydrated enzyme complexes do not appear consistent with a symmetric arrangement of the E1 and E3 subunits about the octahedrally symmetric E2 core . Often the E1 or E3 subunits appear separated from the surface of the E2 core by 3-5 nm, and sometimes thin bridges of density appear in the gap between the E2 core and the bound subunits; studies of subcomplexes consisting of the E2 core from 2-oxoglutarate dehydrogenase complex and E1 or E3 show that both E1 and E3 are bound in this manner . Images of the E2 cores isolated from pyruvate dehydrogenase complex appear surrounded by a faint fuzz that extends approximately 10 nm from the surface of the core and likely corresponds to the lipoyl domains of the E2. J Biol Chem, 1990 Dec 25, 265(36), 22300 - 5 Interaction of the Mnt repressor with 37-base pair synthetic operator DNA fragments . Importance of symmetric GC pairs; Rajendrakumar GV et al.; Mnt repressor is indirectly responsible for the maintenance of lysogeny of the phage P22 . This repressor interacts with a 21-base pair operator DNA constituting within it a 17-base pair perfect 2-fold symmetric sequence whose bases make a direct contact with the protein . We have synthesized six 37-base pair DNAs consisting of 21 base pair natural operator and its modifications in which certain symmetrically situated GC base pairs were replaced systematically with ATs to understand their importance . The binding interaction studies of Mnt repressor to such natural and modified operator DNAs reported here indicate that the GCs close to the center of symmetry make major contacts with the protein whereas, GCs nearer to the periphery form weak contacts . Methylation protection experiments indicated that when the GCs near the center of symmetry were replaced with AT, the central GC became more accessible for dimethyl sulfate methylation with possible conformational change in DNA . The circular dichroism studies indicated that upon repressor binding conformational changes in DNA takes place with a possible increase in helicity of the repressor protein. J Biol Chem, 1990 Dec 25, 265(36), 22174 - 80 Structural and functional properties of the 14-kDa envelope protein of vaccinia virus synthesized in Escherichia coli; Lai CF et al.; Vaccinia virus is a highly cytocidal virus, but the steps that lead to virus penetration into cells, the first event in virus pathogenesis, have not been elucidated . We have shown that a 14-kDa envelope protein of vaccinia virus might play a major role in virus-penetration acting at the level of cell fusion (Rodriguez, J . F., Paez, E., and Esteban, M . (1987) J . Virol . 61, 395-404; Gong, S., Lai, C., and Esteban, M . (1990) Virology 178, 81-91) . To carry out structural and functional studies on the vaccinia 14-kDa protein, it would be desirable to have a high level expression system, since the amount of protein that can be obtained from purified virus or from infected cells is very limited . In this investigation we demonstrate that the 14-kDa envelope protein of vaccinia virus is expressed in Escherichia coli in soluble form and at high levels . We establish, by several criteria, that the 14-kDa vaccinia virus protein expressed in E . coli is similar to the protein found in the virus particle based on apparent molecular mass, occurrence of disulfide-linked oligomers, reactivity against specific monoclonal antibody, and identity in amino-terminal sequence with the predicted DNA sequence of the gene . We define several structural and functional properties concerning the 14-kDa envelope protein of vaccinia virus . 1) 14 kDa is a trimer of identical subunits . 2) A monomer binds to itself more strongly than to a dimer or a trimer . 3) Oligomerization does not require cellular factors . 4) Trimers induce high titer neutralizing antibodies in animals which correlate with overall immunogenicity . 5) 14-kDa binds with specificity to the cell surface of cultured cells. Nucleic Acids Res, 1990 Dec 25, 18(24), 7367 - 72 Promoter selectivity of Escherichia coli RNA polymerase: effect of base substitutions in the promoter -35 region on promoter strength; Kobayashi M et al.; A set of 18 variant lac UV5 promoters was constructed, each carrying a single base substitution within the -35 region (nucleotide positions from -36 to -31 relative to the transcription start site) . Using truncated DNA fragments carrying these variant promoters and purified Escherichia coli RNA polymerase holoenzyme, in vitro mixed transcription assays were performed to determine two parameters governing promoter strength: i.e., the binding affinity to RNA polymerase (parameter I) and the rate of open complex formation (parameter II) . The following conclusions were drawn from the data presented: (1) Alteration in the promoter strength of variant promoters is dependent on both the position and base species of substitutions; (2) the consensus sequence (TTGACA) exhibits the highest values for both parameters; (3) base substitutions at nucleotide position -34 cause marked effect on both parameters; (4) cytosine at nucleotide position -32 can not be replaced with other nucleotides without significant reduction of the promoter strength; and (5) base substitution at nucleotide position -31 exerts only a little effect on parameter I . All these findings were confirmed by abortive initiation assays. Nucleic Acids Res, 1990 Dec 25, 18(24), 7243 - 50 Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription; Bell A et al.; The effects of a number of mutations in the E . coli cyclic AMP receptor protein (CRP) have been determined by monitoring the in vivo expression and in vitro open complex formation at two semi-synthetic promoters that are totally CRP-dependent . At one promoter the CRP-binding site is centered around 41.5 base pairs upstream from the transcription start whilst at the other promoter it is 61.5 base pairs upstream . The CRP mutation E171K reduces expression from both promoters whilst H159L renders CRP totally inactive: neither mutation stops CRP binding at either promoter . The mutations K52N and K52Q reverse the effect of H159L and 'reeducate' CRP to activate transcription . CRP carrying both H159L and K52N activates transcription from the promoter with the CRP site at -41.5 better than wild type CRP . In sharp contrast, this doubly changed CRP is totally inactive with respect to the activation of transcription from the promoter carrying the CRP site at -61.5 . Our results suggest that CRP can use different contacts and/or conformations during transcription activation at promoters with different architectures. J Biol Chem, 1990 Dec 25, 265(36), 22499 - 505 Induction of an interleukin-1 receptor (IL-1R) on monocytic cells . Evidence that the receptor is not encoded by a T cell-type IL-1R mRNA; Spriggs MK et al.; Primary human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-1 alpha (IL-1 alpha) and IL-1 beta . Treatment of primary monocytes with dexamethasone resulted in a 10-fold increase in receptor number over untreated cells, to approximately 2,000 receptors/cell . Treatment of THP-1 cells with phorbol ester followed by prostaglandin E2 and dexamethasone resulted in the expression of approximately 30,000 receptors/cell . Competitive binding assays on THP-1 cells showed that both IL-1 alpha and IL-1 beta bind to the same receptor . The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct . However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of approximately 5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R . Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region . A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger . Data are presented which suggest that neither form of this message encodes the 63-kDa IL-1R, but rather that this protein is the product of a separate nonhomologous mRNA. Proc R Soc Lond B Biol Sci, 1990 Dec 22, 242(1305), 217 - 24 Active site complementation in engineered heterodimers of Escherichia coli glutathione reductase created in vivo; Scrutton NS et al.; By directed mutagenesis of the cloned Escherichia coli gor gene encoding the dimeric flavoprotein glutathione reductase, Cys-47 (a cysteine residue forming an essential charge-transfer complex with enzyme-bound FAD) was converted to serine (C47S) and His-439 (required to facilitate protonation of the reduced glutathione) was converted to glutamine (H439Q) . Both mutant genes were placed in the same plasmid, pHD, where each of them came under the control of a strong tac promoter . This was designed to achieve equal over-expression of both genes in the same E . coli cell . The parental homo-dimers show no (C47S) or very little (H439Q) activity as glutathione reductases . The formation in vivo of heterodimers, carrying one crippled and one fully functional active site, was detected by absorbance spectroscopy and fluorescence emission spectrometry of enzyme-bound FAD and by active site complementation . The fractional distribution of homo- and hetero-dimers was in accord with that expected for a random association of enzyme subunits . In a homo-dimer, the H439Q mutation leads to a big fall in the value of Km for NADPH which binds some 1.8 nm from the point of mutation (Berry, A., Scrutton, N.S . & Perham, R . N . Biochemistry 28, 1264-1269 (1989)) . However, the one active site in the H439Q/C47S hetero-dimer exhibited kinetic parameters similar to those of the wild-type enzyme . Thus, the effect of the H439Q mutation must be retained within the active site that accommodates it and is not transmitted through the protein to the second active site across the subunit interface.(ABSTRACT TRUNCATED AT 250 WORDS) Cell, 1990 Dec 21, 63(6), 1159 - 65 The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor; McDonald HB et al.; The Drosophila ncd gene is required for chromosome segregation during female meiosis . Previous analyses suggested that the ncd gene encoded a protein with sequence similarity to the kinesin motor domain, which suggested that, like kinesin, the ncd protein might be a plus end-directed microtubule motor . Here we describe the expression of ncd protein in E . coli and the initial characterization of the ncd protein's motor properties . The ncd protein is indeed a microtubule motor, but the polarity of movement is minus end directed . The ncd protein also has microtubule bundling activity . These findings limit possible models for the in vivo functions of the ncd protein and suggest that motor proteins with similar sequence can generate movement in opposite directions along a microtubule. Science, 1990 Dec 21, 250(4988), 1712 - 5 Stable, monomeric variants of lambda Cro obtained by insertion of a designed beta-hairpin sequence; Mossing MC et al.; lambda Cro is a dimeric DNA binding protein . Random mutagenesis and a selection for Cro activity have been used to identify the contacts between Cro subunits that are crucial for maintenance of a stably folded structure . To obtain equivalent contacts in a monomeric system, a Cro variant was designed and constructed in which the antiparallel beta-ribbon that forms the dimer interface was replaced by a beta-hairpin . The engineered monomer has a folded structure similar to wild type, is significantly more stable than wild type, and exhibits novel half-operator binding activity. J Mol Biol, 1990 Dec 20, 216(4), 949 - 64 RecA protein self-assembly . II . Analytical equilibrium ultracentrifugation studies of the entropy-driven self-association of RecA; Brenner SL et al.; We have investigated the self-association of RecA protein from Escherichia coli by equilibrium ultracentrifugation . Monomeric RecA (Mr = 37,842) was observed in reversible equilibrium with trimers, hexamers and dodecamers in the presence of 1.5 M-KCl, 5 mM-Hepes, 1 mM-EDTA, 2 mM-ATP (pH 7.0) at 1 degrees C . The equilibrium was strongly temperature-dependent, with polymerization being favored as the temperature was raised from 1 degrees C 21 degrees C, and was reversible with respect to temperature . The values of both the standard enthalpy and entropy of self-association were positive, indicating that it is an entropy-driven process under these conditions . In the absence of KCl, in 50 mM-citrate, 5 mM-ATP, 5% (v/v) glycerol (pH 6.0) at 4 degrees C, only small amounts of RecA monomer could be detected, while in 10 mM-Tris-acetate, 10% glycerol (pH 7.5) at 4 degrees C, the smallest species present in significant concentration appeared to be the trimer . The majority of the species observed had molecular weights between 228,000 and 456,000, suggesting dominant stoichiometries of six to 12 monomers per oligomer . At pH 6.0, in the absence of ATP, much larger oligomers containing at least 24 monomers also appeared to be present . The data are consistent with an equilibrium mixture of monomers, trimers, hexamers, dodecamers, 24-mers and higher oligomers, with the distribution of oligomers being dependent on solution conditions . Thermodynamic analysis indicates that these oligomeric species are in reversible equilibrium with each other . It is not certain whether trimers assemble directly into hexamers, or whether disassembly into monomers is a prerequisite for the formation of higher oligomers . The possible role of higher-order RecA oligomers in the formation of RecA nucleoprotein filaments is discussed. J Mol Biol, 1990 Dec 20, 216(4), 803 - 7 Error-prone SOS repair can be error-free; Liu SK et al.; Most of the mutagenesis that accompanies the SOS repair of ultraviolet light-induced lesions in the single-stranded DNA of phage S13 is eliminated when the groES or the groEL gene of Escherichia coli is defective . Therefore, this SOS mutagenesis is not a necessary consequence of what is commonly called error-prone repair, but is additionally imposed on the repair system by the GroE heat shock proteins, which are responsible for the assembly of polypeptides into multimeric structures. Biochemistry, 1990 Dec 18, 29(50), 11101 - 9 Participation of cob(I) alamin in the reaction catalyzed by methionine synthase from Escherichia coli: a steady-state and rapid reaction kinetic analysis; Banerjee RV et al.; The kinetic mechanism of the reaction catalyzed by cobalamin-dependent methionine synthase from Escherichia coli K12 has been investigated by both steady-state and pre-steady-state kinetic analyses . The reaction catalyzed by methionine synthase involves the transfer of a methyl group from methyltetrahydrofolate to homocysteine to generate tetrahydrofolate and methionine . The postulated reaction mechanism invokes an initial transfer of the methyl group to the enzyme to generate enzyme-bound methylcobalamin and tetrahydrofolate . Enzyme-bound methylcobalamin then donates its methyl group to homocysteine to generate methionine and cob(I)alamin . The key questions that were addressed in this study were the following: (1) Does the reaction involve a sequential or ping-pong mechanism? (2) Is enzyme-bound cob(I)alamin a kinetically competent intermediate? (3) If the reaction does involve a sequential mechanism, what is the nature of the "free" enzyme to which the substrates bind; i.e., is the prosthetic group in the cob(I)alamin or methylcobalamin state? Both the steady-state and rapid reaction studies were conducted at 25 degrees C under anaerobic conditions . Initial velocity analysis under steady-state conditions revealed a family of parallel lines suggesting either a ping-pong mechanism or an ordered sequential mechanism . Steady-state product inhibition studies provided evidence for an ordered sequential mechanism in which the first substrate to bind is methyltetrahydrofolate and the last product to be released is tetrahydrofolate . Pre-steady-state kinetic studies were then conducted to determine the rate constants for the various reactions . Enzyme-bound cob(I)alamin was shown to react very rapidly with methyltetrahydrofolate (with an observed rate constant of 250 s-1 versus a turnover number under maximal velocity conditions of 19 s-1).(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Dec 18, 29(50), 11029 - 32 Ribosome subunit to polysome ratios affect the synthesis of rRNA in Drosophila cells; Yamamoto KK et al.; Many investigations have revealed that ribosome numbers increase in parallel with the growth rate of cells . Here we show that the absolute level of protein synthesis may not be the only factor influencing rRNA synthesis in a nondividing eukaryotic cell . Under conditions of complete (greater than 99%) inhibition of protein synthesis by four different antibiotics, there is a corresponding inhibition of rRNA synthesis . At lower levels of inhibition of protein synthesis (70%), a different effect of individual antibiotics on rRNA synthesis is observed . Cycloheximide and anisomycin, which cause a decrease in the free subunit pool due to a buildup of polysomes, stimulate rRNA synthesis, whereas puromycin and pactamycin, which cause an increase in the free subunit pool, cause a decrease in rRNA synthesis . These effects on rRNA synthesis are not solely due to a low level of completed proteins . Pactamycin treatment allows completed proteins to be made yet lowers rRNA labeling, while anisomycin treatment does not show synthesis of complete proteins yet increases rRNA labeling . The result suggest that eukaryotic cells may regulate ribosome synthesis in response to the number of free versus translating (polysomal) ribosomes as do Escherichia coli cells. Biochemistry, 1990 Dec 18, 29(50), 11051 - 7 Cooperative binding of R17 coat protein to RNA; Witherell GW et al.; The binding of the R17 coat protein to synthetic RNAs containing one or two coat protein binding sites was characterized by using nitrocellulose filter and gel-retention assays . RNAs with two available sites bound coat protein in a cooperative manner, resulting in a higher affinity and reduced sensitivity to pH, ionic strength, and temperature when compared with RNAs containing only a single site . The cooperativity can contribute up to -5 kcal/mol to the overall binding affinity with the greatest cooperativity found at low pH, high ionic strength, and high temperatures . Similar solution properties for the encapsidation of the related fr and f2 phage suggest that the cooperativity is due to favorable interactions between the two coat proteins bound to the RNA . This system therefore resembles an intermediate state of phage assembly . No cooperative binding was observed for RNAs containing a single site and a 5' or 3' extension of nonspecific sequence, indicating that R17 coat protein has a very low nonspecific binding affinity . Unexpectedly weak binding was observed for several RNAs due to the presence of alternative conformational states of the RNA. FEBS Lett, 1990 Dec 17, 277(1-2), 105 - 8 Amino-terminal amino acid sequence of beef heart mitochondrial coupling factor B; Kantham L et al.; Bovine heart mitochondrial coupling factor B was isolated and purified to homogeneity in its active form . The amino-terminal amino acid sequence of the alkylated protein was determined . Two chains with exactly the same sequence except for the presence of an additional Phe at the amino-terminus on one of them were obtained . The 55 amino acid sequence appears to be largely hydrophilic with several charged amino acid residues . This sequence showed no homology with the E . coli unc operon, oligomycin sensitivity conferring protein, or coupling factor 6 or any protein in the data base. FEBS Lett, 1990 Dec 17, 277(1-2), 267 - 71 The serine acetyltransferase from Escherichia coli . Over-expression, purification and preliminary crystallographic analysis; Wigley DB et al.; An expression vector has been constructed which increases the expression of serine acetyltransferase (SAT) from E . coli to 17% of the soluble cell protein . A novel purification procedure, using dye-affinity chromatography, allows purification of SAT to homogeneity . The enzyme has been crystallised from polyethylene glycol, in the presence of L-cysteine (an inhibitor of SAT) . The crystals which diffract to beyond 3.0 A resolution are of the tetragonal spacegroup P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions a = b = 123 A, c = 79 A . Since ultracentrifugation and gel-filtration experiment indicate that purified SAT is a tetramer, there appears to be one-half tetramer in the asymmetric unit (Vm = 2.55 A3/Da). Gene, 1990 Dec 15, 96(2), 305 - 9 High-level synthesis of biologically active human plasminogen activator inhibitor type 1 (PAI-1) in Escherichia coli; Sisk WP et al.; Segments of a cDNA encoding human plasminogen activator inhibitor type 1 (PAI-1) were subcloned into a highly regulated and inducible Escherichia coli expression system . A plasmid encoding the mature form of human endothelial PAI-1 produced a functional recombinant molecule, as indicated by its ability to inhibit tissue plasminogen activator's enzymatic activity . In contrast to PAI-1 isolated from human fibrosarcoma cells, the biological activity of the recombinant PAI-1 was not dependent on pretreatment with denaturing agents . A construct encoding a polypeptide lacking the first 80 amino acids of PAI-1 also produced elevated levels of the truncated recombinant protein . However, this truncated product was functionally inactive, indicating that an intact N terminus is required for activity. Gene, 1990 Dec 15, 96(2), 271 - 6 Random silent mutagenesis in the initial triplets of the coding region: a technique for adapting human glutathione reductase-encoding cDNA to expression in Escherichia coli; Bucheler US et al.; The introduction of random silent mutations into the 5'-coding region of a human cDNA as the basis for successful expression in Escherichia coli is demonstrated in four steps . (1) Plasmid pUB200 containing the pRpL promoters of phage lambda was found not to serve as an expression vector for a unchanged human glutathione reductase (hGR)-encoding cDNA . (2) When this cDNA was expressed in a two-cistron context using high-copy-number plasmids, recombinant protein was detected in low yield (0.03% of the total cell protein) . (3) Silent mutations were introduced into the triplets coding for the N-terminal amino acids . When screening E . coli colonies transformed with expression plasmids containing cDNA mutants, we identified adapted clones that produced hGR in up to 70-fold higher yield than the clone containing the unchanged cDNA . Sequence analyses of adapted cDNA species revealed lower G + C contents in the modified regions, suggesting altered mRNA structures . (4) When the adapted cDNA sequences were recloned in the vector which had failed to express unchanged hGR cDNA in step 1, synthesis of recombinant protein was as high as in step 3 . This means that the yield of expression for adapted cDNA was at least 1000-fold higher than for unchanged cDNA . In conclusion, random silent mutations introduced into the translation initiation region of cDNA might be a useful technique for designing sequence features which favour gene expression. Gene, 1990 Dec 15, 96(2), 241 - 7 A tissue-specific puff (Balbiani ring a) in Chironomus thummi may contain a gene encoding a 67-kDa protein which exhibits non-tissue-specific expression; Bogachev SS et al.; A 2.3-kb genomic clone has been isolated from the region where the tissue-specific puff, Balbiani ring a (BRa), is found on chromosome IV of the special lobe of Chironomus thummi salivary gland cells . The clone was characterized by nucleotide sequence analysis . Two clusters of direct tandem repeats were identified, as well as large and small open reading frames (ORFs) . The large ORF was fused to an Escherichia coli lacZ gene . Antibodies against the beta-galactosidase/ORF fusion protein reacted selectively on Western blots with a 67-kDa protein . Western-blot analysis and immunoelectron microscopy showed that this protein was distributed in the cells of all larval tissues examined . We concluded that BRa, a tissue-specific puff, whose activity correlates with the synthesis of 160-kDa secretory protein {Kolesnikov et al., Chromosoma 83 (1981) 661-677}, may also contain a gene which is not expressed in a tissue-specific manner. Biochem J, 1990 Dec 15, 272(3), 805 - 11 Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron; Ramotar K et al.; The cistron encoding the B subunit of Escherichia coli Shiga-like toxin I (SLT-I) was cloned under control of the tac promoter in the expression vector pKK223-3 and the SLT-I B subunit was expressed constitutively in a wild-type background and inducibly in a lacIq background . The B subunit was located in the periplasmic space, and less than 10% was found in the culture medium after 24 h incubation . Polymyxin B extracts contained as much as 160 micrograms of B subunit/ml of culture . B subunit was purified to homogeneity by ion-exchange chromatography followed by chromatofocusing . Cross-linking analysis of purified native B subunit showed that it exists as a pentamer . In gels containing 0.1% SDS the native protein dissociated into monomers . B subunit was found to have the same glycolipid-receptor-specificity as SLT-I holotoxin . Competitive binding studies showed that B subunit and holotoxin had the same affinity for the globotriosylceramide receptor . We conclude that this recombinant plasmid is a convenient source of large amounts of purified SLT-I B subunit, which could be used for biophysical and structural studies or as a natural toxoid. Biochem J, 1990 Dec 15, 272(3), 767 - 73 Human brain n-chimaerin cDNA encodes a novel phorbol ester receptor; Ahmed S et al.; A human brain-specific cDNA encoding n-chimaerin, a protein of predicted molecular mass 34 kDa, has sequence identity with two different proteins: protein kinase C (PKC) at the N-terminus and BCR protein {product of the breakpoint-cluster-region (BCR) gene, involved in Philadelphia chromosome translocation} at the C-terminus {Hall, Monfries, Smith, Lim, Kozma, Ahmed, Vannaisungham, Leung & Lim (1990) J . Mol . Biol . 211, 11-16} . The sequence identity of n-chimaerin with PKC includes the cysteine-rich motif CX2CX13CX2CX7CX7C, and amino acids upstream of the first cysteine residue, but not the kinase domain . This region of PKC has been implicated in the binding of diacylglycerol and phorbol esters in a phospholipid-dependent fashion . Part of this cysteine-rich motif (CX2CX13CX2C) has the potential of forming a 'Zn-finger' structure . Phorbol esters cause a variety of physiological changes and are among the most potent tumour promoters that have been described . PKC is the only known protein target for these compounds . We now report that n-chimaerin cDNA encodes a novel phospholipid-dependent phorbol ester receptor, with the cysteine-rich region being responsible for this activity . This finding has wide implications for previous studies equating phorbol ester binding with the presence of PKC in the brain. Biochem J, 1990 Dec 15, 272(3), 659 - 64 Translation of preprochymosin in vitro . Evidence for folding of prochymosin to the native conformation; Sheikh A et al.; 1 . The cDNA coding for preprochymosin has been sub-cloned into the transcription/translation vector pGEM-3Z, the T7 promoter used to transcribe the gene and the product expressed in an 'in vitro' cell-free system comprising rabbit reticulocyte lysate and dog pancreatic microsomes . 2 . Translations in various conditions, and analyses of the translation product in reducing and non-reducing conditions, indicate that oxidizing translation conditions and the cleavage of the N-terminal 'pre-' sequence are essential for generation of a disulphide-bonded translation product . 3 . The disulphide-bonded translation product was resistant to proteinases, as expected for a translation product segregated within microsomal vesicles; in the presence of detergent to solubilize the membranes, the product was not readily susceptible to proteolysis, and was converted to a proteinase-resistant core fragment . 4 . Segregated prochymosin, synthesized in reducing conditions, was completely degraded by proteinases under similar conditions . 5 . Proteinase treatment of purified recombinant prochymosin gave rise to a proteinase-resistant fragment of similar Mr, suggesting that the disulphide-bonded product of translation in vitro was correctly folded . 6 . The translocated, disulphide-bonded and folded prochymosin could be converted into pseudochymosin at pH 2.0, and addition of chymosin to the activation mixture resulted in increased pseudochymosin production. J Immunol, 1990 Dec 15, 145(12), 4322 - 5 Identification and analysis of cDNA clones encoding CD53 . A pan-leukocyte antigen related to membrane transport proteins; Amiot M; CD53 is a human cell-surface Ag expressed exclusively by nucleated cells of hemopoietic origin . In this work a cDNA clone encoding the CD53 Ag was isolated from a COS cell-expression library . The sequence of the cDNA predicts a protein of 219 residues bearing four putative membrane-spanning hydrophobic domains . Sequence analysis shows that CD53 is related to three other recently described molecules: a melanoma Ag, ME491; a B cell Ag, CD37; and the broadly distributed hemopoietic cell Ag S5.7 . Comparison of NH2-terminal protein sequence of OX44 rat Ag and CD53 suggest that CD53 is the human homologue of OX44 . In addition, CD53 is distantly related to Escherichia coli lac Y permease, a type III integral membrane protein that ferries lactose into the bacterial cell . CD53 transcripts increase in prevalence after mitogenic stimulation, suggesting that the protein may be involved in the transport of factors essential for cell proliferation. J Biol Chem, 1990 Dec 15, 265(35), 22004 - 10 The regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase requires a short-lived protein and occurs in the endoplasmic reticulum; Chun KT et al.; A chimeric gene consisting of the coding sequence for the membrane domain of the endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, fused to the coding sequence for the soluble enzyme, beta-galactosidase of Escherichia coli, has been previously constructed . This fusion protein, HMGal, has been localized to the membrane of the endoplasmic reticulum of Chinese hamster ovary cells transfected with this chimeric gene, and its beta-galactosidase activity has declined in the presence of low density lipoprotein (Skalnik, D . G., Narita, H., Kent, C., and Simoni, R . D . (1988) J . Biol . Chem . 263, 6836-6841) . In this report, we demonstrate that the loss of beta-galactosidase activity results from the accelerated degradation of the HMGal protein . Taking advantage of a fluorescence-activated cell sorter technique, we have selected transfected cells which express sufficient levels of HMGal to improve its immunodetection . Based on pulse-chase experiments, the half-life of HMGal is 6.0 h, and, in the presence of 20 mM mevalonate, the half-life declines 1.7-fold . Under these conditions, mevalonate accelerates the degradation of HMG-CoA reductase in these cells 1.6-fold, from 8.4 h to 5.3 h, most probably by the same mechanism . This mevalonate-regulated degradation of HMGal is not due to a heteromeric association of HMGal with reductase, since the same effect has been observed in cells lacking the reductase protein . In addition, we demonstrate that inhibition of protein synthesis with cycloheximide abolishes the mevalonate-dependent accelerated degradation of HMGal, in agreement with previous studies which have presented indirect evidence that a short-lived protein is essential for mediating the loss of HMG-CoA reductase activity . Finally, using brefeldin A, we show that the mevalonate-dependent accelerated degradation of HMGal may occur in the endoplasmic reticulum. J Biol Chem, 1990 Dec 15, 265(35), 21966 - 70 Anaerobic biosynthesis of the manganese-containing superoxide dismutase in Escherichia coli . Effects of diazenedicarboxylic acid bis(N,N'-dimethylamide) (diamide); Privalle CT et al.; Anaerobically grown Escherichia coli accumulate active manganese-containing superoxide dismutase (MnSOD) upon exposure to diamide . This induction requires de novo biosynthesis of MnSOD . Catalase, glutathione disulfide reductase, and glucose-6-phosphate dehydrogenase were also induced by diamide in anaerobic E . coli . A GSH-negative strain of E . coli did not produce MnSOD under anaerobic conditions and was as responsive to diamide as was the wild type strain . Diamide which had been prereduced, by incubation with GSH, was ineffective . NO3- plus paraquat, which elicits increased anaerobic biosynthesis of the MnSOD polypeptide, but not of active MnSOD, synergized with diamide in the induction of active MnSOD . A similar increase in the ability of diamide to cause anaerobic biosynthesis of active MnSOD was seen when the production of the MnSOD polypeptide was increased by isopropyl-beta-D-thiogalactopyranoside, in a strain bearing the MnSOD gene under the control of the tac promoter . These results are explained in terms of a dual action of diamide, i.e . at both the transcriptional and the maturational levels of biosynthesis of MnSOD . Oxidative inactivation of an Fe(II)-containing repressor and oxidative facilitation of insertion of manganese, in place of iron, into the nascent MnSOD polypeptide, are the postulated bases of this dual action. J Biol Chem, 1990 Dec 15, 265(35), 21889 - 95 The chemical synthesis of a gene coding for bovine pancreatic DNase I and its cloning and expression in Escherichia coli; Worrall AF et al.; A gene coding for bovine pancreatic DNase I has been constructed from synthetic oligonucleotides . This gene has been cloned into a plasmid vector pDOC55 designed to allow very tight control of expression of potentially lethal proteins . Induction of protein synthesis from the gene yielded a peptide of molecular weight of approximately 31,000, consistent with DNase I . The yield of this protein from the pDOC55 construct (pAW5) was approximately 150 micrograms/liter of cell culture . Attempts to clone the gene into a less tightly controlled expression vector based on the tac-promoter (pKK223-3) were unsuccessful, presumably due to the expected lethality of the product . Mutagenesis of the gene to replace the active site histidine (His-134) in the protein with glutamine yielded a gene readily clonable into both expression systems . Yields of the mutagenized protein were approximately 6 micrograms/liter from a pDOC55 system and 20 mg/liter from a pKK223-3 system . The activity of the proteins were assayed using the Kunitz procedure and their cleavage selectivities by digestion of the Escherichia coli tyr T promoter . The recombinant native enzyme had both the same specific activity and DNA cleavage selectivity as the protein isolated from bovine pancreas using these two assays . The H134Q mutant had a specific activity of about 0.001% of the native protein but had an unaltered DNA cleavage selectivity. J Biol Chem, 1990 Dec 15, 265(35), 21852 - 8 Dephosphorylation-induced interactions of neurofilaments with microtubules; Hisanaga S et al.; Effects of dephosphorylation on interactions of neurofilaments (NFs) with microtubules (MTs) were studied by the cosedimentation method . Centrifugation conditions were chosen so that MTs pelleted but NFs did not . While NFs isolated from bovine spinal cords did not cosediment with MTs polymerized in the presence of taxol, NFs dephosphorylated with Escherichia coli alkaline phosphatase began to coprecipitate with MTs . The dephosphorylated NFs bound to MTs but not to the unpolymerized tubulin dimer . The binding was not observed in the presence of high salt or with MTs containing microtubule-associated proteins . The cosedimentation experiments using purified NF subunit proteins showed that the dephosphorylation-induced binding of NFs to MTs was mediated by the largest subunit of NF (NF-H) . Negative staining electron microscopy confirmed bindings of the dephosphorylated NFs and NF-H to MTs . Densitometric measurement of the bound and unbound NF-H after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the binding of the dephosphorylated NF-H to MT was saturable and gave the following binding parameters . Approximately 1 mol of NF-H bound per 10 mol of tubulin dimer with a high affinity site (Kd = 3.8 x 10(-8) M) and per 16 mol of tubulin dimer with a low affinity site (Kd = 1.1 x 10(-7) M). J Biol Chem, 1990 Dec 15, 265(35), 21804 - 10 Identification of the MAP2- and P75-binding domain in the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase . Cloning and expression of the cDNA for bovine brain RII beta; Luo Z et al.; cDNA clones coding for the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a bovine brain cDNA expression library in lambda gt11 . The cDNA codes for a protein of 418 amino acids which is 98% homologous to the rat and human RII beta proteins . A series of expression vectors coding for truncated RII beta proteins were constructed in pATH plasmids and fusion proteins were expressed in Escherichia coli . Polyclonal and monoclonal antibodies made against purified bovine brain RII were immunoreactive with the fusion proteins on Western blots . The expressed RII beta-fusion proteins were used in overlay assays to identify the region in RII beta which binds to microtubule-associated protein 2 (MAP2) and to the 75,000-dalton calmodulin-binding protein (P75) (Sarkar, D., Erlichman, J., and Rubin, C.S . (1984) J . Biol . Chem . 259, 9844-9846) in bovine brain . Fusion protein containing amino acids 1-50 of the RII beta NH2 terminus (RII beta(1-50)} bound to both MAP2 and P75 immobilized on nitrocellulose filters . A pATH11-directed fusion protein containing the 31 amino acid RII-binding site of the human MAP2 protein (MAP2(31)) (Rubino, H.M., Dammerman, M., Shafit-Zagardo, B., and Erlichman, J . (1989) Neuron 3, 631-638) also bound RII beta-fusion proteins containing RII beta amino acids 1-50 . Three fusion proteins, RII beta(1-25), RII beta(25-96), and RII beta(1-265,25-96 deleted) did not bind to MAP2(31) nor P75 . The results showed that the binding domain for MAP2 and P75 was located within the NH2-terminal 50 amino acids of RII beta . Preincubation of bovine heart protein kinase II alpha and RII beta(1-50) with MAP2(31) prevented their binding to both P75 and MAP2(31) that were immobilized on nitrocellulose, suggesting that the binding sites for MAP2 and P75 are located near each other or that the same site on RII was binding to both proteins. J Biol Chem, 1990 Dec 15, 265(35), 21704 - 8 Nucleotide sequence of gltS, the Na+/glutamate symport carrier gene of Escherichia coli B; Deguchi Y et al.; The nucleotide sequence of the gltS gene coding for an Na+/glutamate symport carrier of Escherichia coli B has been determined, and the amino acid sequence of the carrier protein was deduced . The predicted glutamate carrier consists of 401 amino acids with a molecular weight of 42,455 . A Shine-Dalgarno sequence and putative promoter sequences were found in the 5'-flanking region of the putative gltS gene . The predicted protein is very hydrophobic (73% nonpolar amino acids), and judging from its hydropathy profile, the protein is composed of 12 hydrophobic membrane-spanning segments with a mean length of 21.6 residues/segment . A typical rho-independent transcription termination signal was found downstream of the gltS gene . We found a conserved alignment of 5 amino acid residues (Gly42--Ala82-X-X-X-X-Leu87-X-X-X-Gly91-Arg92 ), which commonly exists in four Na+ symport carrier proteins, the glutamate carrier, and the proline carrier of E . coli, and the Na+/glucose co-transporters of rabbit and human intestines . We propose that this consensus sequence (or motif) may play an important role in cation recognition or binding in the Na+/solute symport reaction. J Biol Chem, 1990 Dec 15, 265(35), 21573 - 9 Structure of chicken 16-kDa beta-galactoside-binding lectin . Complete amino acid sequence, cloning of cDNA, and production of recombinant lectin; Sakakura Y et al.; The complete primary structure of chicken 16-kDa beta-galactoside-binding lectin (C-16) was determined . It was composed of 134 amino acid residues and has an acetylated NH2 terminus . A cDNA was also cloned, but no signal sequence was found in the initiator region . The initiator methionine remained as the NH2 terminus of the mature lectin . Although C-16 is distinct from chicken 14-kDa beta-galactoside-binding lectin (C-14), it proved to be a member of the vertebrate 14-kDa-type lectin family . Comparison of the primary structures between the vertebrate 14-kDa-type lectins suggests that C-14 and C-16 were produced by gene duplication of an ancestral lectin gene at a time close to the divergence of birds and mammals . Northern and Southern blot analysis indicated that these isolectins are encoded by individual genes which are differently regulated during the development of the embryo . A recombinant C-16 lectin was produced in Escherichia coli . The product was indistinguishable from the authentic C-16 lectin except that the NH2 terminus of the former was found to begin with free methionine. J Biol Chem, 1990 Dec 15, 265(35), 21532 - 5 pheAo mutants of Escherichia coli have a defective pheA attenuator; Gavini N et al.; Two classes of mutants affecting the regulation of pheA expression in Escherichia coli have been reported previously: trans-acting mutants involving the locus pheR, and cis-acting mutants involving the locus pheAo . The effects of these mutants have been found to be mediated through one regulatory mechanism . The gene pheR has been shown to encode tRNA(Phe) (Gavini, N., and Davidson, B . E . (1990) J . Biol . Chem . 265, 21527-21531) . In this paper we report the cloning and nucleotide sequencing of the promoter-attenuator regions from two of the cis-acting mutants pheAo351 and pheAo352 . Both pheAo351 and pheAo352 contained a G:C to A:T base pair transition, at different positions in the 3:4 stem of the pheA attenuator terminator . Since these changes would destabilize the G:C stem of the attenuator terminator we propose that the enhanced expression of pheA observed in the pheAo mutants is due to increased transcription readthrough at the defective attenuator terminator. J Biol Chem, 1990 Dec 15, 265(35), 21520 - 6 Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements; Hager PW et al.; The x-ray structure of the EcoRI endonuclease-DNA complex (3) suggests that hydrogen bonds between amino acids, glutamic acid 144, arginine 145, and arginine 200, and major groove base moieties are the molecular determinants of specificity . We have investigated residue 144 using aspartate and glutamine substitutions introduced by site-directed mutagenesis . Substitution with glutamine results in a null phenotype (at least a 2000-fold reduction in activity) . On the other hand, the aspartic acid mutant (ED144) retained in vivo activity . Substrate binding and catalytic studies were done with purified ED144 enzyme . The affinity of the ED144 enzyme for the canonical sequence 5'-GAATTC-3' is about 340-fold less than the wild-type (WT) enzyme, while its affinity for nonspecific DNA is about 50 times greater . The ED144 enzyme cleaves one strand in the EcoRI site in plasmid pBR322 with a kcat/Km similar to WT . In contrast to the WT enzyme, the ED144 enzyme dissociates after the first strand cleavage . Partitioning between cleavage and dissociation at the first and second cleavage steps for the ED144 enzyme is extremely salt-sensitive . The altered partitioning results largely from a destabilization of the enzyme-DNA complex, particularly the enzyme-nicked DNA complex, with only small changes in the respective cleavage rates . The hydrogen bonds of Glu-144 are critical, they appear to act cooperatively with other specificity contacts to stabilize the enzyme-DNA complex. J Biol Chem, 1990 Dec 15, 265(35), 22016 - 22 Expression and characterization of an active human estrogen receptor as a ubiquitin fusion protein from Escherichia coli; Wittliff JL et al.; The gene coding for the human estrogen receptor protein was expressed as a ubiquitin fusion under the control of the lambda PL promoter in Escherichia coli . Analysis of extracts by Western blot showed that intact receptor protein was produced only when PL promoter was depressed by nalidixic acid at 30 degrees C . To ascertain the intactness of the expressed protein, estrogen receptor in bacterial extracts was titrated with either 17 beta-{3H}estradiol or 17 beta-{125I}iodoestradiol, and the mass was quantified by enzyme immunoassay . Cell extracts contained 484 fmol of estrogen receptor per mg of soluble protein by titration with a Kd of 3.2 x 10(-10) M . The simultaneous analysis by enzyme immunoassay resulted in 487 fmol/mg of extract protein indicating that most of the expressed protein bound ligand with wild type properties . Ligand competition analysis demonstrated that expressed receptor contained a binding domain preferentially recognizing estrogenic substances identical with that of wild-type estrogen receptor . The E . coli-expressed estrogen receptor also associated specifically with estrogen response DNA elements . Full length receptor protein was detected by ligand binding and immunorecognition using size exclusion chromatography . Hydrophobic interaction chromatography showed a major isoform which exhibited both ligand binding and immuno-recognition identical with the estrogen receptor from human breast tumor tissues . These data indicate that estrogen receptor expressed in E . coli retained characteristics of the native protein found in human tissues. J Biol Chem, 1990 Dec 15, 265(35), 21859 - 66 Human alpha-N-acetylgalactosaminidase-molecular cloning, nucleotide sequence, and expression of a full-length cDNA . Homology with human alpha-galactosidase A suggests evolution from a common ancestral gene; Wang AM et al.; Human alpha-N-acetylgalactosaminidase (alpha-GalNAc, E.C . 3.2.1.49), the lysosomal glycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties from glycoconjugates, is encoded by a gene localized to chromosome 22q13----qter . The deficient activity of alpha-GalNAc is the enzymatic defect in Schindler disease, an inherited neuroaxonal dystrophy . To isolate a full-length cDNA, the enzyme from human lung was purified to homogeneity, 129 non-overlapping amino acids were determined by microsequencing the N terminus and seven tryptic peptides, and four synthetic oligonucleotide mixtures were used to screen a human fibroblast cDNA library . A full-length cDNA, pAGB-3, isolated from a placental lambda gt11 cDNA library, had a 2158-base pair (bp) insert with an open reading frame which predicted an amino acid sequence that was colinear with all 129 microsequenced residues of the purified enzyme . The pAGB-3 insert had a 344-bp 5'-untranslated region, a 1236-bp open reading frame encoding 411 amino acids, a 514-bp 3'-untranslated region, and a 64-bp poly(A) tract . A signal peptide sequence of 17 amino acids as well as six N-glycosylation sites were predicted . The pAGB-3 cDNA was subcloned into the p91023(B) mammalian expression vector and human alpha-GalNAc activity was transiently expressed in COS-1 cells, demonstrating the functional integrity of the full-length cDNA . Northern hybridization analysis of mRNA revealed two transcripts of about 3.6 and 2.2 kilobases (kb), and primer extension studies indicated a cap site at nucleotide -347 for the 2.2-kb transcript . The 3.6-kb cDNA (pAGB-35) was isolated; the 3598-bp pAGB-35 insert was identical to that of the 2.2-kb insert but had additional 5'- and 3'-untranslated sequences including a second downstream polyadenylation signal at nucleotide 3100-3105 . Isolation of a genomic clone, gAGB-1, and sequencing the 2048-bp region including pAGB-3 revealed a 1754-bp intron between codons 319 and 320, which also was the site of a 70-bp insertion and a45-bp deletion in other cDNA clones . Notably, the alpha-GalNAc cDNA had remarkable amino acid homology with the human alpha-galactosidase A (alpha-Gal A) cDNA suggesting the evolutionary relatedness of these genes . The alpha-GalNAc cDNA had 46.9-64.7% amino acid identity in sequences (codons 1-319) corresponding to alpha-Gal A exons 1 through 6, while the comparable exon 7 sequence (pAGB-3 codons 320-411) had only 15.8% homology with numerous gaps.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1990 Dec 15, 265(35), 21527 - 31 The pheR gene of Escherichia coli encodes tRNA(Phe), not a repressor protein; Gavini N et al.; Nucleotide sequence analysis and transposon 5 (Tn5) insertional mutagenesis indicate that the Escherichia coli gene pheR encodes tRNA(Phe) and not a repressor protein as previously reported . The coding region of pheR is identical to that of three other cloned tRNA(Phe) genes, pheU, pheV, and pheW . Multicopy plasmids carrying pheR, like those carrying pheU, pheV, or pheW, complement a temperature-sensitive lesion in the gene for the alpha-subunit of phenylalanyl-tRNA synthetase (pheS) . The nucleotide sequences of the 5'-flanking DNA of pheR, pheU, and pheW are almost identical but are quite different from the same region of pheV . By comparison with pheV, which has two tandem promoters, pheR was found to have a single promoter . The expression of pheA (encoding chorismate mutase/prephenate dehydratase) in strains carrying the pheR374 allele was decreased to similar extents by multicopy plasmids containing either pheR or pheV . It is proposed that this decrease in pheA expression and the increase in expression of pheA previously reported for chromosomal pheR mutants are both mediated through the attenuation control mechanism that regulates pheA. Cancer Res, 1990 Dec 15, 50(24), 7754 - 7 Suramin affects DNA synthesis in HeLa cells by inhibition of DNA polymerases; Jindal HK et al.; Suramin, a polysulfonated naphthylurea widely used in the treatment of trypanosomiasis and onchocerciasis, is currently being investigated as an antitumor agent for the treatment of advanced cancer . Suramin exerts a wide variety of biological effects . We have shown that suramin inhibits cell proliferation and DNA synthesis in cultured HeLa cells . The replication in vitro of SV40 DNA is completely abolished by 40 microM suramin . The inhibition of DNA replication is due to inhibition of DNA polymerases alpha and delta, the replicative enzymes in eukaryotic cells . DNA polymerase alpha is sensitive to lower concentrations of suramin {concentration to achieve 50% inhibition (IC50) of 8 microM} than is DNA polymerase delta (IC50 36 microM), whereas DNA polymerase beta is relatively insensitive to the drug (IC50 of 90 microM) . Suramin inhibits other replicative DNA polymerases such as Escherichia coli polymerase I (Klenow fragment) and Thermus aquaticus polymerase . Suramin is noncompetitive with both substrate deoxyribonucleotides and template-primers with respect to DNA polymerase inhibition . Much lower concentrations (8-30 microM) of the drug are required for 50% inhibition of DNA polymerases than for 50% inhibition of other enzymes such as protein kinase C and reverse transcriptase . These results show an important biological effect of this drug and indicate the need for more studies before its clinical use as an antitumor agent. Gene, 1990 Dec 15, 96(2), 205 - 11 Prophage lambda libraries for isolating cDNA clones by functional screening; Mutzel R et al.; Isolation of cDNA clones from lambda gt11 phage libraries by functional screening is limited by the low amount of lacZ-cDNA-encoded fusion protein synthesized in an isolated phage plaque . The amount of specific cDNA-encoded protein can be significantly enhanced by expression in bacterial colonies rather than phage plaques . Escherichia coli was lysogenized with a lambda gt11 cDNA expression library from Dictyostelium discoideum . Bacteria were selected for the presence of the lambda gt11 prophage by elimination of nonlysogenic parental cells with a lambda cI phage . The usefulness of the lysogen library was demonstrated by immuno-screening and functional screening with two different radiolabeled ligands . cDNA clones encoding a well-characterized D . discoideum protein, the regulatory subunit of the cAMP-dependent protein kinase, were isolated by screening the lysogen library with antibodies . Clones encoding this protein could also be identified by functional screening with {3H}cAMP, demonstrating that the limit of detection of positive clones by ligand screening is at least an order of magnitude lower for the lysogen library than for the corresponding phage library . We have subsequently used the lysogen library to isolate cDNA clones encoding calmodulin-binding protein(s) from D . discoideum by functional screening with {125I}calmodulin . For these clones, screening of the corresponding phage library had previously been found unsuccessful. J Biol Chem, 1990 Dec 15, 265(35), 21567 - 72 Suppression mutations in the defective beta subunit of F1-ATPase from Escherichia coli; Miki J et al.; The Escherichia coli mutant of the proton-translocating ATPase KF11 (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., and Futai, M . (1980) J . Biochem . (Tokyo) 88, 695-703) has a defective beta subunit with serine being replaced by phenylalanine at codon 174 . Four suppression mutants (RE10, RE17, RE18, and RE20) from this strain capable of growth on minimal plate agar supplemented by succinate were isolated . The original point mutation at codon 174 was intact in these strains . Additional point mutations, Ala-295 to Thr, Gly-149 to Ser, Leu-400 to Gln, Ala-295 to Pro, for RE10, RE17, RE18, and RE20, respectively, were identified by the polymerase chain reaction and sequencing . These mutations, except for RE10, were confirmed as a single mutation conferring a suppressive phenotype by genetic suppression assay using KF11 as the host cells . The results indicated that Ser-174 has functional interaction with Gly-149, Ala-295, and Leu-400 . The residues are located within the previously estimated catalytic domain of the beta subunit, indicating that this domain is indeed folded for the active site of catalytic function . Growth rates of the revertants in the minimal medium with succinate increased compared with that of KF11, showing that ATP synthesis recovered to some extent . The ATP hydrolytic activity in the revertant membranes increased in RE17 and RE20 but did not in RE10 and RE18, suggesting that synthesis and hydrolysis are not necessarily reversible in the proton-translocating ATPase (F1F0). J Immunol, 1990 Dec 15, 145(12), 4185 - 91 Anti-IL-6 monoclonal antibodies protect against lethal Escherichia coli infection and lethal tumor necrosis factor-alpha challenge in mice; Starnes HF Jr et al.; Potentially fatal physiologic and metabolic derangements can occur in response to bacterial infection in animals and man . Recently it has been shown that alterations in the levels of circulating cytokines such as IL-6 and TNF-alpha occur shortly after bacterial challenge . To understand better the role of IL-6 in inflammation, we investigated the effects of in vivo anti-mouse IL-6 antibody treatment in a mouse model of septic shock . Rat anti-mouse IL-6 neutralizing mAb was produced from splenocytes of an animal immunized with mouse rIL-6 . This mAb, MP5-20F3, was a very potent and specific antagonist of mouse IL-6 in vitro bioactivity, demonstrated using the NFS60 myelomonocytic and KD83 plasmacytoma target cell lines, and also immunoprecipitated radiolabeled IL-6 . Anti-IL-6 mAb pretreatment of mice subsequently challenged with lethal doses of i.p . Escherichia coli or i.v . TNF-alpha protected mice from death caused by these treatments . Pretreatment of E . coli-challenged mice with anti-IL-6 led to an increase in serum TNF bioactivity, in comparison to isotype control antibody, implicating IL-6 as a negative modulator of TNF in vivo . Anti-TNF-alph