FEMS Microbiol Lett, 1990 Jun 1, 57(3), 311 - 6 Comparative evaluation of three tests for the detection of Escherichia coli cytotoxic necrotizing factors (CNF1 and CNF2) using filtrates of cultures treated with mitomycin C; Blanco J et al.; Necrotizing Escherichia coli (NTEC) strains grown in the presence of mitomycin C released cell associated necrotizing factors CNF1 and CNF2 to culture medium . Using culture filtrates from 96 mitomycin C treated E . coli strains, we have found that a modified HeLa cell assay was a more sensitive and specific method for the detection of CNF1 and CNF2 than the Vero cell assay and the rabbit skin test. J Gen Microbiol, 1990 Jun, 136 ( Pt 6), 1017 - 23 A relationship between L-serine degradation and methionine biosynthesis in Escherichia coli K12; Brown EA et al.; While wild-type Escherichia coli K12 cannot grow with L-serine as carbon source, two types of mutants with altered methionine metabolism can . The first type, metJ mutants, in which the methionine biosynthetic enzymes are expressed constitutively, are able to grow with L-serine as carbon source . Furthermore, a plasmid carrying the metC gene confers ability to grow on L-serine . These observations suggest that in these mutants, L-serine deamination may be a result of a side-reaction of the metC gene product, cystathionine beta-lyase . The second type is exemplified by two newly isolated strains carrying mutations mapping between 89.6 and 90 min . These mutants use L-serine as carbon source, and also require methionine for growth with glucose at 37 degrees C and above . The phenotypes of the new mutants resemble those of both met and his constitutive mutants in some respects, but have been differentiated from both of them. Anal Biochem, 1990 Jun, 187(2), 328 - 36 Characterization of indo-1 and quin-2 as spectroscopic probes for Zn2(+)-protein interactions; Jefferson JR et al.; 1-{2-Amino-5-(6-carboxyindol-2-yl)phenoxyl}-2-(2'- amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (indo-1) and 2-{2-(bis(carboxymethyl)amino-5-methylphenoxy) methyl}-6- methyl-8-{bis-(carboxymethyl)amino}quinoline (quin-2) are sensitive, spectral indicators for Zn2+ . Additions of subsaturating Zn2+ to 10-80 microM indo-1 or quin-2 at pH 7.0 produce uv difference spectra with isosbestic wavelengths at 342 and 282 nm or at 342, 317, and 252 nm, respectively . Formation of 1:1 Zn2+:indicator complexes at pH 7.0 and 20 degrees C in the absence (presence) of 100 mM KCl gives delta epsilon max = -2.4 +/- 0.2 X 10(4) M-1 cm-1 at 367 nm (-2.1 +/- 0.2 X 10(4) M-1 cm-1 at 365 nm) for indo-1 and delta epsilon max = -2.7 +/- 0.1 X 10(4) M-1 cm-1 at 266 nm (-2.6 +/- 0.1 X 10(4) M-1 cm-1 at 265 nm) for quin-2 . Competition experiments at pH 7.0 and 20 degrees C with indo-1 and quin-2 and also 4-(2-pyridylazo)resorcinol (PAR) as the second chelator in the absence (presence) of 100 mM KCl yield apparent affinity constants: K'A = 2.5 +/- 1.0 X 10(10) M-1 (6.2 +/- 0.5 X 10(9) M-1) for indo-1 binding Zn2+ and K'A = 9.4 +/- 3.3 X 10(11) M-1 (2.7 +/- 0.1 X 10(11) M-1) for quin-2 binding Zn2+ . The above constants provide the basis for rapid steady-state spectrophotometric determinations of the affinity of a protein for Zn2+ with K'A approximately 10(10) - 10(13) M-1.(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Immunol, 1990 Jun, 20(6), 1311 - 6 Partial tolerance in beta-galactosidase-transgenic mice; Theopold U et al.; A transgenic mouse line was produced which allowed the expression of E . coli beta-galactosidase (beta-Gal) under the regulatory elements of the immunoglobulin heavy chain locus . Expression of the transgene is found in spleen and bone marrow . Upon immunization of the transgenic mice with beta-Gal, a reduced but clearly detectable antibody response was obtained . Affinity purification with sera from immunized transgenic mice suggests that they contain lower affinity antibodies as compared to normal littermates . Transgenic and nontransgenic mice immunized with bovine serum albumin (BSA) alone or as a mixture with beta-Gal gave comparable anti-BSA responses . Immunization with a chemically cross-linked (Gal-BSA)-protein, however, showed a 10- to 30-fold difference in the anti-BSA response . Partial unresponsiveness to beta-Gal in the transgenic mice is best explained by a dominant, peripheral suppression mechanism linked to the antigen-presenting potential of B cells. Biochem J, 1990 Jun 1, 268(2), 317 - 23 A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) beta-galactosidase; Selwood T et al.; 1 . Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside and 3,4-dinitrophenyl beta-D-galactopyranoside Escherichia coli (lacZ) beta-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O . 2 . For hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal {Sinnott, Withers & Viratelle (1978) Biochem . J . 175, 539-546} that glycone-aglycone fission without acid catalysis governs both V and V/Km . 3 . V for hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons {Tenu, Viratelle, Garnier & Yon (1971) Eur . J . Biochem . 20, 363-370}, and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08 . There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton . 4 . The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively . There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl beta-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V . 5 . Identification of the 'conformation change' in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard {(1973) Biochem . J . 133, 89-98} with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the conflict between the proposals and the 18O kinetic-isotope-effect data reported by Rosenberg & Kirsch {(1981) Biochemistry 20, 3189-3196} . It should be noted that the actual Km values were determined to higher precision than can be estimated from the Figures in this paper.(ABSTRACT TRUNCATED AT 400 WORDS) Mol Cell Biol, 1990 Jun, 10(6), 2840 - 7 Oncogenic transformation by vrel requires an amino-terminal activation domain; Kamens J et al.; The mechanism by which the products of the v-rel oncogene, the corresponding c-rel proto-oncogene, and the related dorsal gene of Drosophila melanogaster exert their effects is not clear . Here we show that the v-rel, chicken c-rel, and dorsal proteins activated gene expression when fused to LexA sequences and bound to DNA upstream of target genes in Saccharomyces cerevisiae . We have defined two distinct activation regions in the c-rel protein . Region I, located in the amino-terminal half of rel and dorsal proteins, contains no stretches of glutamines, prolines, or acidic amino acids and therefore may be a novel activation domain . Lesions in the v-rel protein that diminished or abolished oncogenic transformation of avian spleen cells correspondingly affected transcription activation by region I . Region II, located in the carboxy terminus of the c-rel protein, is highly acidic . Region II is not present in the v-rel protein or in a transforming mutant derivative of the c-rel protein . Our results show that the oncogenicity of Rel proteins requires activation region I and suggest that the biological function of rel and dorsal proteins depends on transcription activation by this region. Plant Mol Biol, 1990 Jun, 14(6), 899 - 908 Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization; Bates GW et al.; The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns . One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration . This paper provides evidence for the ligation of plasmid fragments by plant cells . Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24 h later . Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression . Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40-50% . Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts . CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression . Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kb Hind III fragment of pCaMVCATM . DNA isolated from nuclei of the protoplasts was used to transform competent cells of Escherichia coli, and colonies were recovered that carried pGEM with Hind III-CaMVCAT inserts . Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transform E . coli yielded an estimate of the frequency of plasmid ligation . A maximum of only 4% of the input linear DNA was recovered as circular molecules . This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms . Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning into E . coli. Biomed Environ Sci, 1990 Jun, 3(2), 146 - 55 Analysis of the phenotype and the restriction enzyme mapping level of mutations induced by the new mutagen glycidyl methacrylate; Xie DY et al.; Glycidyl methacrylate (GMA) is a recently recognized chemical mutagen . In order to explore the mutagenicity and mutagenic process of GMA, plasmid pBR322 was used for in vitro binding, mutant screening, and restriction enzyme mapping . The binding between GMA and DNA in vitro has been verified by means of a spectrophotometric method . When pBR322 and GMA-bound pBR322 were used to transform Escherichia coli HB101, the following results were obtained: (1) The transformation efficiency of GMA-bound pBR322 was much lower than that of pBR322 alone . (2) GMA-bound pBR322 induced phenotype changes in competent cells (i.e., tetracycline-resistance inactivation or ampicillin-resistance inactivation) . There were two mutants of pBR322, ApRTCS and ApSTcR, in the transformants and a deductive mutant ApsTcs in the nontransformants . (3) All of the selected mutants were stable and heritable . (4) When restriction enzyme maps were used to analyze the mutant ApRTcS, four of seven maps were changed, some sites were shifted to other resistant gene regions, for example, sites of Bg/I, EcoRI, HindIII, HincII, etc., and there was a new recognition site for HincII (252) . We did not observe any DNA fragment insertion or deletion on any maps . Our results suggest that when GMA is covalently linked to the plasmid DNA, it gives rise to a premutagenic lesion of DNA that is converted in vivo into a point mutation. New Biol, 1990 Jun, 2(6), 574 - 82 Regulation of expression of the dnaA gene in Escherichia coli: role of the two promoters and the DnaA box; Polaczek P et al.; The dnaA gene of Escherichia coli specifies a product that is a key element in the initiation of DNA replication . Expression of dnaA occurs from two promoters, 1P and 2P, which flank a DnaA protein binding site (DnaA box) . In this paper we describe the effects of DnaA box mutations on transcription from the two promoters, measured with the use of dnaA-galK gene fusions . All of the mutations examined led to a five-fold increase in expression from the upstream promoter, 1P, suggesting that expression from this promoter is negatively autoregulated by binding of DnaA protein to the box . The same mutations led to a decrease in expression from promoter 2P, suggesting that the DnaA box plays a role in activating promoter 2P under normal conditions . Overproduction of DnaA protein, above normal physiological levels, led to decreased transcription from both promoters . We present evidence from analysis of both dnaA mRNA and DnaA protein that promoter 2P is subject to growth rate-dependent control and that this promoter, but not promoter 1P, is completely shut off in the stationary phase of growth. Aliment Pharmacol Ther, 1990 Jun, 4(3), 255 - 63 Immune complex induced experimental colitis: beneficial effect of sulphasalazine in rabbits; Axelsson LG et al.; Experimental colitis was induced in rabbits by exposing the colon mucosa to 1% formalin followed by i.v . injections of soluble immune complexes made with antigen in excess . The animals were preimmunized with Escherichia coli O14:K7:H--inducing antibodies cross-reactive to intestinal epithelium . Animals with this colitis were divided in two groups . One group was treated with sulphasalazine and the other was given vehicle only . Sulphasalazine was administered daily at 125.5 mumol (50 mg) per kg body weight . The administration was started at the same day as the colitis was initiated . At Day 6, 13 and 30 following induction of colitis, biopsies were sampled and histologically evaluated . Inflammation was assessed by scores for inflammatory cells, crypt distortion, decreased crypt number and presence of crypt abscesses, thus corresponding to the picture seen in humans . A statistically significant lower score of inflammation was seen on Day 6 and 13 (P less than 0.01) and on Day 30 (P less than 0.05) following induction of colitis in animals treated with sulphasalazine. Jpn J Exp Med, 1990 Jun, 60(3), 93 - 6 Hepatitis B core antigen specific CD4 response in peripheral blood; Shirai M et al.; The proliferative response of peripheral blood CD4+ T cells to recombinant hepatitis B core antigen (rHBcAg) has been studied in patients with chronic active hepatitis (CAH) type B (CAH-B), CAH-nonA nonB, and normal volunteers . CD4+ T cells from patients with CAH-B indicated a significant proliferative response to rHBcAg in the presence of non-T antigen presenting cells . In contrast, there was no apparent T cell reaction to rHBcAg in patients with CAH-nonA nonB and healthy volunteers . We suggested the possibility of CD4-mediated HBcAg specific response even in the peripheral blood compartments of HBcAg-responsive CAH-B patients. Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4712 - 6 Targeted replacement of the homeobox gene Hox-3.1 by the Escherichia coli lacZ in mouse chimeric embryos; Le Mouellic H et al.; Through gene targeting based upon homologous recombination in embryonic stem cells, a chosen gene can be inactivated and eventually a strain of mutant mice created . We have devised a procedure to specifically replace a targeted gene by another gene . A murine homeobox gene was disrupted at high frequency in embryonic stem cells by its replacement with Escherichia coli lacZ . Injection of such stem cells into blastocysts yielded chimeric embryos in which beta-galactosidase activity was driven by the Hox-3.1 promoter . This technique will allow the visual assessment at the cellular level of gene inactivation effects in transgenic mice. J Bacteriol, 1990 Jun, 172(6), 3214 - 20 Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12; Wallace B et al.; A gene encoding a carrier protein for glutamate and aspartate was cloned into Escherichia coli K-12 strain BK9MDG by using the high-copy-number plasmid pBR322 . The gene (designated gltP) is probably identical to a gene recently cloned from E . coli B (Y . Deguchi, I . Yamato, and Y . Anraku, J . Bacteriol . 171:1314-1319) . A 1.6-kilobase DNA fragment containing gltP was subcloned into the expression plasmids pT7-5 and pT7-6, and its product was identified by a phage T7 RNA polymerase-T7 promoter coupled system (S . Tabor and C . C . Richardson, Proc . Natl . Acad . Sci . USA 82:1074-1078) as a polypeptide with an apparent mass of 38 kilodaltons . A portion of the gltP polypeptide was associated with the cytoplasmic membrane . The nucleotide sequence of the 1.6-kilobase fragment was determined . It contained an open reading frame capable of encoding a highly hydrophobic polypeptide of 395 amino acids, containing four possible transmembrane segments . Uptake of glutamate and aspartate was increased 5.5- and 4.5-fold, respectively, in strains containing gltP plasmids . Glutamate uptake was insensitive to the concentration of Na+ and was inhibited by L-cysteate and beta-hydroxyaspartate . These results suggest that gltP is a structural gene for a carrier protein of the Na(+)-independent, binding-protein-independent glutamate-aspartate transport system. J Bacteriol, 1990 Jun, 172(6), 3201 - 7 Regulation of Escherichia coli pyrC by the purine regulon repressor protein; Choi KY et al.; The purine regulon repressor, PurR, was identified as a component of the Escherichia coli regulatory system for pyrC, the gene that encodes dihydroorotase, an enzyme in de novo pyrimidine nucleotide synthesis . PurR binds to a pyrC control site that resembles a pur regulon operator and represses expression by twofold . Mutations that increase binding of PurR to the control site in vitro concomitantly increase in vivo regulation . There are completely independent mechanisms for regulation of pyrC by purine and pyrimidine nucleotides . Cross pathway regulation of pyrC by PurR may provide one mechanism to coordinate synthesis of purine and pyrimidine nucleotides. J Bacteriol, 1990 Jun, 172(6), 2862 - 70 Isolation and characterization of a Treponema pallidum major 60-kilodalton protein resembling the groEL protein of Escherichia coli; Houston LS et al.; A native structure containing the major 60-kilodalton common antigen polypeptide (designated TpN60) was isolated from Treponema pallidum subsp . pallidum (Nichols strain) through a combination of differential centrifugation and sucrose density gradient sedimentation . Gel filtration chromatography indicated that this structure is a high-molecular-weight homo-oligomer of TpN60 . Antisera to TpN60 reacted with the groEL polypeptide of Escherichia coli, as determined by immunoperoxidase staining of two-dimensional electroblots . Electron microscopy of the isolated complex revealed a ringlike structure with a diameter of approximately 16 nm which was very similar in appearance to the groEL protein . Comparison of the N-terminal amino acid sequence of TpN60 with the deduced sequences of the E . coli groEL protein, related chaperonin proteins from mycobacteria and Coxiella burnetti, the hsp60 protein of Saccharomyces cerevisiae, the wheat ribulose bisphosphate carboxylase-oxygenase-subunit-binding protein (alpha subunit), and the human P1 mitochondrial protein indicated sequence identity at 8 of 22 to 10 of 22 residues (36 to 45% identity) . We conclude that the oligomer of TpN60 is homologous to the groEL protein and related chaperonins found in a wide variety of procaryotes and eucaryotes and thus may represent a heat shock protein involved in protein folding and assembly. Infect Immun, 1990 Jun, 58(6), 1995 - 8 Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type 1 fimbriae; Krogfelt KA et al.; Type 1 fimbriae of Escherichia coli are surface organelles which mediate binding to D-mannose-containing structures . By direct binding of FimH to D-mannose attached to a carrier protein, we demonstrated that this protein was uniquely responsible for the receptor specificity . Furthermore, we show by receptor immunoelectron microscopy that the FimH protein is located laterally in the structure of the type 1 fimbriae. Infect Immun, 1990 Jun, 58(6), 1591 - 9 Signal transduction in human platelets and inflammatory mediator release induced by genetically cloned hemolysin-positive and -negative Escherichia coli strains; Konig B et al.; Incubation of human platelets with the hemolysin-producing Escherichia coli strain K-12 (pANN5211) induced the activation of protein kinase C, aggregation of platelets, calcium influx, low amounts of 12-hydroxyeicosatetraenoic acid (12-HETE), and release of serotonin from dense granules . Nonhemolytic isogenic strains of E . coli 536/21 which differed only in their types of adhesins (MSH+ MS-Fim+; S-MRH+ S-Fim+; P-MRH+ P-Fim+) released neither serotonin nor 12-HETE from human platelets nor induced platelet aggregation . All hemolysin-negative bacteria except E . coli 536/21, without any adhesins, were able to activate protein kinase C reversibly but did not induce calcium influx . Activation of platelets with fluoride, an activator of the GTP-binding protein, was associated with protein kinase C activation, calcium influx, platelet aggregation, serotonin release, and 12-HETE formation . The simultaneous stimulation of platelets with NaF and the nonhemolytic E . coli strains suppressed several of the NaF-induced platelet responses . Membrane preparations isolated from stimulated platelets with hemolysin-negative and hemolysin-positive E . coli showed increased binding of guanylylimidodiphosphate, a nonhydrolyzable GTP analog, and enhanced GTPase activity. Infect Immun, 1990 Jun, 58(6), 1500 - 8 Effects of adhesins from mannose-resistant Escherichia coli on mediator release from human lymphocytes, monocytes, and basophils and from polymorphonuclear granulocytes; Ventur Y et al.; We investigated the role of Escherichia coli expressing mannose-resistant hemagglutination and adhesins with regard to the induction of leukotrienes from a suspension of human lymphocytes, monocytes, and basophils (LMBs) compared with human polymorphonuclear granulocytes (PMNs) . Genetically cloned E . coli strains expressing various types of mannose-resistant hemagglutination (MRH+) were phagocytosed to a higher degree by monocytes than the nonadherent E . coli strain . The various strains differed in their capacity to induce a chemiluminescence response, which showed the same pattern for LMBs and PMNs . Stimulation of LMBs with bacteria alone, unlike granulocytes, did not activate the cells for the release of leukotrienes . However, preincubation of LMBs with bacteria decreased subsequent leukotriene formation when the cells were stimulated with calcium ionophore . The inhibitory effect was dependent on the concentration of bacteria used for preincubation as well as on the preincubation temperature . The various bacterial strains differed in inhibitory potency for mediator release . Preincubation of LMBs with zymosan, opsonized zymosan, the bacterial peptide FMLP, and peptidoglycan had no inhibitory effect or even increased subsequent leukotriene formation . Opsonized bacteria were far less inhibitory than nonopsonized bacteria . In contrast to human LMBs, preincubation of human PMNs with mannose-resistant bacteria led to increased leukotriene B4 generation and reduced w-oxidation of leukotriene B4 . Our data suggest that phagocytes (neutrophils, monocytes) respond in a different way for leukotriene formation after interaction with mannose-resistant E . coli. J Clin Lab Immunol, 1990 Jun, 32(2), 49 - 54 Recombinant plasmid expressing the entire coding region of the Bmyc putative protein; Steinitz M et al.; An expression vector with the entire coding region of Bmyc oncogene was constructed . The longest predicted open reading frame of Bmyc (178 amino acid residues) was expressed as a fusion protein with the trpE protein (308 amino acid residues) in transformed bacteria . The newly synthesized 58 Kd protein reacted with an anti pan-myc serum . The fusion protein isolated from a preparative Western blot was used as immunogen to generate rabbit anti myc specific immune sera. Mol Gen Genet, 1990 Jun, 222(1), 41 - 8 Introduction of hygromycin B resistance into Schizophyllum commune: preferential methylation of donor DNA; Mooibroek H et al.; Cotransformation of a trp1 strain of Schizophyllum commune with the homologous TRP gene and the Escherichia coli HPT gene was used to study the feasibility of transformation of S . commune to hygromycin B resistance . Southern blot analysis showed that 75% of the TRP transformants contained multiple integrated copies of the HPT gene . However only 7% of the transformants were resistant to 25 micrograms/ml hygromycin B and direct selection for hygromycin B resistance was hampered by the high incidence of spontaneously arising resistant colonies . Rescue of the HPT gene was possible with E . coli JA221 (mcr-) but not with JM83, suggesting methylation of the integrated donor DNA . Isoschizomer analyses confirmed heavy methylation in the HPT gene and flanking vector sequences but not in the homologous donor TRP gene and its flanking vector sequences . Also cotransforming S . commune Sc4 gene and flanking vector sequences were not methylated . A fusion between the S . commune TRP1 and the E . coli HPT genes resulted in only slight or no methylation of both vector and HPT sequences and in a higher hygromycin B resistance level . This suggests that transformation with DNA exclusively containing foreign sequences results in integration into regions where methylation occurs, possibly entailing poor transcription . Methylation of the HPT gene was also indicated by the stimulation of growth by 5-azacytidine of transformants on hygromycin B containing medium. Virus Genes, 1990 Jun, 4(1), 41 - 52 In-vitro translation of cucumoviral satellites . III . Translational efficiencies of cucumber mosaic virus-associated RNA 5 sequence variants can be related to the predicted secondary structures of their first 55 nucleotides; Steen MT et al.; The cucumber mosaic virus (CMV) satellites D- and S-CARNA 5 (CARNA 5 = Cucumber mosaic virus-Associated RNA 5), their full-length cDNA clone transcripts, and DNA clone transcripts of their open reading frames (ORFs) were used as mRNAs in the wheat-germ in-vitro translation system . Natural D-CARNA 5 yielded an anomalously large polypeptide, while transcripts made from cDNA clones of D-CARNA 5 or its first ORF had no mRNA activity . Transcripts made from the second major ORF in D-CARNA 5 yielded a smaller product, consistent with its size . Natural S-CARNA 5 and its cDNA clone transcripts both yielded the two polypeptides previously reported, while transcripts of its only major ORF yielded exclusively the smaller of the two products . The potential for an alternate initiation codon, 36 nucleotides upstream, being the source of the larger of the two polypeptides was tested . The differences in the translational properties of D- and S-CARNA 5 were related to the predicted secondary structures of the first 55 nucleotides in these CARNA 5 sequence variants . The calculated free energies of the predicted hairpins correlated inversely with their in-vitro translational activities. Scand J Clin Lab Invest, 1990 Jun, 50(4), 421 - 7 The production of tumour necrosis factor, tissue thromboplastin, lactoferrin and cathepsin C during lipopolysaccharide stimulation in whole blood; Gutteberg TJ et al.; The release of tumour necrosis factor (TNF), lactoferrin (LF) and cathepsin C (CC) into plasma and production of thromboplastin (TPL) in monocytes were studied in lipopolysaccharide (LPS) stimulated heparinized whole blood from 10 healthy donors . The influence of dextran 70, haemaccel and methylprednisolone on levels of these parameters were examined . TNF concentration in plasma 5 min after the addition of LPS (0 h) was 250 pg/ml (median), 520 pg/ml after 1 h and 1300 pg/ml after 3 h . The addition of dextran 70 to the blood in addition to LPS at the same intervals gave significantly higher values of 740 pg/ml and 1800 pg/ml after 1 h and 3 h respectively . Unstimulated cells had no TPL but after 1 h with LPS, the TPL activity in incubated cells was 2.3 mU/10(6) monocytes and after 3 h, 2.7 mU/10(6) monocytes . LPS induced the secretion of LF from granulocytes (PMN) and the levels 5 min after the addition of LPS (0 h) were 2.1 mg/l (control 0.2 mg/l) and after 1 h, 5.3 mg/l (control 1.3 mg/l) in plasma after LPS stimulation . Haemaccel enhanced the LPS-induced generation of TPL in monocytes and production of CC . The LPS-induced secretion of LF was, to a small extent, influenced by the three reagents tested . Methylprednisolone (1 mmol/l) reduced the production and appearance of TNF in plasma and the generation of TPL activity in monocytes . This model for stimulating heparinized whole blood is suitable for examination of the production and appearance of cellular factors and the influence of drugs on this production. Am J Trop Med Hyg, 1990 Jun, 42(6), 527 - 31 Safety and immunogenicity of a Plasmodium vivax sporozoite vaccine; Gordon DM et al.; A recombinant DNA Plasmodium vivax sporozoite vaccine containing the repeating region of the Salvador I strain circumsporozoite (CS) protein was produced in Escherichia coli . This vaccine was tested in 13 naive volunteers at doses of 10-1,000 micrograms . No serious adverse reactions were noted . None of 4 volunteers receiving the 10 micrograms dose developed antibodies measurable by ELISA . Six of 9 volunteers in the other dose groups developed measurable antibodies: 5 of 5 volunteers receiving 100 micrograms and 1 of 4 receiving 1,000 micrograms . Antibody responses measured by immunofluorescence assays paralleled those seen by ELISA . None of the volunteers developed antisera that inhibited sporozoite invasion of human hepatoma cells in vitro . Lack of a classical anamnestic response and lack of a typical dose response to increasing amounts of antigen suggests the possible presence of an immunosuppressive epitope in the repetitive region of the CS protein. Microbiol Rev, 1990 Jun, 54(2), 89 - 100 Metabolic growth rate control in Escherichia coli may be a consequence of subsaturation of the macromolecular biosynthetic apparatus with substrates and catalytic components; Jensen KF et al.; In this paper, the Escherichia coli cell is considered as a system designed for rapid growth, but limited by the medium . We propose that this very design causes the cell to become subsaturated with precursors and catalytic components at all levels of macromolecular biosynthesis and leads to a molecular sharing economy at a high level of competition inside the cell . Thus, the promoters compete with each other in the binding of a limited amount of free RNA polymerase and the ribosome binding sites on the mRNA chains compete with each other for the free ribosomes . The macromolecular chain elongation reactions sequester a considerable proportion of the total amount of RNA polymerase and ribosomes in the cells . We propose that the degree of subsaturation of the macromolecular biosynthetic apparatus renders a variable fraction of RNA polymerase and ribosomes unavailable for the initiation of new chain synthesis and that this, at least in part, determines the composition of the cell as a function of the growth rate . Thus, at rapid growth, the high speed of the elongation reactions enables the cell to increase the concentrations of free RNA polymerase and ribosomes for initiation purposes . Furthermore, it is proposed that the speed of RNA polymerase movement is adjusted to the performance speed of the ribosomes . Mechanistically, this adjustment of the coupling between transcription and translation involves transcriptional pause sites along the RNA chains, the adjustment of the saturation level of RNA polymerase with the nucleoside triphosphate substrates, and the concentration of ppGpp, which is known to inhibit RNA chain elongation . This model is able to explain the stringent response and the control of stable RNA and of ribosome synthesis in steady states and in shifts, as well as the rate of overall protein synthesis as a function of the growth rate. EMBO J, 1990 Jun, 9(6), 2001 - 10 Host-specificity of uropathogenic Escherichia coli depends on differences in binding specificity to Gal alpha 1-4Gal-containing isoreceptors; Stromberg N et al.; Four G adhesins, cloned from uropathogenic Escherichia coli strains, were examined for binding to glycolipids and various eukaryotic cells . PapGAD110 and PapGIA2 showed virtually identical binding patterns to Gal alpha 1-4Gal-containing glycolipids, while PapGJ96 differed slightly and PrsGJ96 markedly with respect to the effect of neighbouring groups on the binding . Their hemagglutination patterns confirmed the existence of three receptor-binding specificities . While the PapG adhesins bound to uroepithelial cells from man (T24) but not to those from the dog (MDCK II), the reverse was true of PrsG . These binding patterns were largely explained by the absence or presence of appropriate glycolipid isoreceptors, although the inability of the PapG adhesins to bind MDCK II cells was attributed to an inappropriate presentation of their receptor epitopes . The high prevalence of PrsG-like specificities observed among wild-type dog uropathogenic E . coli isolates, together with the determined isoreceptor composition of human and dog kidney target tissues, suggest variation in receptor specificity as a mechanism for shifting host specificity, and that this variation has evolved in response to the topography of the host cellular receptors . The receptor-binding half proposed for the predicted amino acid sequences of the four G adhesins and the corresponding adhesin of one of the dog E . coli isolates varied considerably among the three receptor-binding groups of adhesins, but only little within each group. Infect Immun, 1990 Jun, 58(6), 1937 - 42 Identification, sequencing, and expression of Mycobacterium leprae superoxide dismutase, a major antigen; Thangaraj HS et al.; The gene encoding a major 28-kilodalton antigen of Mycobacterium leprae has now been sequenced and identified as the enzyme superoxide dismutase (SOD) on the basis of the high degree of homology with known SOD sequences . The deduced amino acid sequence shows 67% homology with a human manganese-utilizing SOD and 55% homology with the Escherichia coli manganese-utilizing enzyme . The gene is not expressed from its own promoter in E . coli but is expressed from its own promoter in Mycobacterium smegmatis . The amino acid sequences of epitopes recognized by monoclonal antibodies against the 28-kilodalton antigen have been determined. Infect Immun, 1990 Jun, 58(6), 1870 - 8 Epitope analysis of the F4 (K88) fimbrial antigen complex of enterotoxigenic Escherichia coli by using monoclonal antibodies; van Zijderveld FG et al.; So far, three subtypes of the F4 (K88) fimbrial antigen of porcine enterotoxigenic Escherichia coli, F4ab, F4ac, and F4ad, have been distinguished by using polyclonal antisera in agglutination and precipitation tests . The a factor represents one or more common epitopes, whereas each of the b, c, and d factors represents one or more subtype-specific epitopes . We further characterized the F4 antigen complex by using a panel of 40 F4-specific monoclonal antibodies (MAbs) . The specificity of all MAbs was proven by enzyme-linked immunosorbent assays, agglutination and radioimmunoprecipitation tests, and immunoelectron microscopy . The MAbs either reacted with all F4 subtypes, reacted with two subtypes, or were subtype specific . Epitope analysis by competition enzyme-linked immunosorbent assays revealed at least 11 epitope clusters on the F4 antigen complex, designated a1 to a7, b1, b2, c, and d . The following antigenic formulas were found for the F4 subtypes: F4ab, a1a2a3a4a5a6b1b2; F4ac, a1a2a3(a4)a5a6a7c; and F4ad, a1a2a3a4a7d . All MAbs were directed against conformational epitopes located on the 27,500-dalton major fimbrial subunits . Consequences for the replacement of polyclonal antisera by MAbs in diagnostic tests are discussed. J Virol, 1990 Jun, 64(6), 2802 - 9 Mapping of B-cell epitopes of the human hepatitis B virus X protein; Stemler M et al.; The immune response to the X protein of human hepatitis B virus (HBV) was studied by epitope mapping by using a set of MS2-HBx fusion proteins and synthetic peptides . Antibodies in sera of patients with acute and chronic HBV infection showed a multispecific immune response . Each serum contained antibodies to a different set of epitopes, which taken together cover most of the HBx sequence . Some of the epitopes were detectable only by immunoblotting with fusion proteins; others were detectable only by an enzyme-linked immunosorbent assay (ELISA) with synthetic peptides . The carboxy-terminal half of the HBx protein was preferentially recognized by antibodies from patients with chronic hepatitis and contained a short immunodominant antigenic region with at least two major nonoverlapping epitopes . Anti-HBx antibody titers as revealed by peptide ELISAs were highest and most frequent in patients with chronic hepatitis and usually low in acutely infected patients and asymptomatic carriers . The data demonstrate a remarkable qualitative and quantitative heterogeneity of the humoral HBx immune response which can be monitored by HBx-specific peptide ELISAs . Such tests may become useful diagnostic tools. J Ind Microbiol, 1990 Jun, 5(4), 215 - 27 Cytoplasmic and periplasmic expression of a highly basic protein, human interleukin 4, in Escherichia coli; Lundell D et al.; Human IL-4 (hIL-4) has been cloned from a human T cell line based on its homology to the murine IL-4 cDNA sequence . We have compared cytoplasmic and extra-cytoplasmic expression of this basic protein in Escherichia coli using various combinations of promoters, replicons and host strains . Strains producing a cytoplasmic product were most successful at heterologous protein expression, producing up to 500 mg/l of an inactive aggregated form of the protein . The biological activity of the protein could be restored by refolding the protein with guanidine hydrochloride and glutathione giving a specific activity identical to that of IL-4 derived from CHO cell lines stably transformed with an hIL-4 expression plasmid . Strains designed to secrete human IL-4 into the periplasmic space produced far less protein (approximately 5 mg/l) . However, a significant fraction of this protein was detected in the culture medium . This fraction appeared to be soluble after ultracentrifugation, and demonstrated high specific activity without refolding . Leakage of heterologous protein into the culture medium may be a viable way to recover biologically active products without relying on the denaturation and refolding in vitro that can, at times, yield incorrectly folded gene product. J Ind Microbiol, 1990 Jun, 5(4), 197 - 206 Production of Streptomyces clavuligerus isopenicillin N synthase in Escherichia coli using two-cistron expression systems; Doran JL et al.; Streptomyces clavuligerus isopenicillin N synthase (IPNS) gene expression was achieved in Escherichia coli by the construction of two-cistron expression systems formed in the high copy number plasmid vector pUC119 . These two-cistron constructions were composed of the IPNS gene and its flanking sequences which encoded an upstream open reading frame (ORF), the IPNS ribosome binding site and a putative transcription terminator . No E . coli- like Streptomyces promoter motif was present upstream of the IPNS gene therefore transcriptional regulation of the two-cistron system was provided by the lac promoter of pUC119 . Enzymatically active IPNS was detected in E . coli cells harboring the recombinant plasmids thereby providing evidence for the activity of the IPNS ORF and for the feasibility of production of S . clavuligerus IPNS in E . coli . These results indicate that simple two-cistron constructions involving foreign gene flanking sequences may be used to express foreign proteins in E . coli. Gene, 1990 May 31, 90(1), 61 - 7 Cloning, characterization and sequence of a novel 59-kDa protein of Chlamydia trachomatis; Kahane S et al.; Chlamydia trachomatis (Ct) serovar L2 DNA was partially digested with BamHI, ligated with plasmid vector pBR325 and used to transform Escherichia coli JMB83 . Recombinant colonies were screened for their ability to synthesize chlamydial (chl) proteins by dot immunoblot and by in vitro transcription translation assays . A clone, B1, expressing a 59-kDa protein was further characterized, and the encoding gene was subcloned in the expression vector, pKK223-3, containing the tac promoter . Elevated levels of the 59-kDa protein were produced in E . coli in the presence of the lac inducer, IPTG . Sequencing identified one long open reading frame encoding a polypeptide of 59,075 Da (59 kDa) . The partially purified 59-kDa protein was recognized by sera from patients with chl infections as shown in immunoblotting . In addition, the 59-kDa protein was located in the sarcosyl-soluble fraction of chl lysates . When used as a DNA probe in dot hybridization assays, the clone encoding the 59-kDa protein showed high homology to all serovars of Ct and four strains of Chlamydia psittaci . The cloned 59-kDa protein is neither related to the 60-kDa heat-shock protein found in many strains of bacteria, nor to the Cys-rich sarcosylinsoluble protein described in other studies of chlamydia. Gene, 1990 May 31, 90(1), 31 - 41 Cloning and characterization of the histidine biosynthetic gene cluster of Streptomyces coelicolor A3(2); Limauro D et al.; Biochemical and genetic data indicate that in Streptomyces coelicolor A3(2) the majority of the genes involved in the biosynthesis of histidine are clustered in a small region of the chromosome {Carere et al., Mol . Gen . Genet . 123 (1973) 219-224; Russi et al., Mol . Gen . Genet . 123 (1973) 225-232} . To investigate the structural organization and the regulation of these genes, we have constructed genomic libraries from S . coelicolor A3(2) in pUC vectors . Recombinant clones were isolated by complementation of an Escherichia coli hisBd auxotroph . A recombinant plasmid containing a 3.4-kb fragment of genomic DNA was further characterized . When cloned in the plasmid vector, pIJ699, this fragment was able to complement S . coelicolor A3(2) hisB mutants . Overlapping clones spanning a 15-kb genomic region were isolated by screening other libraries with labeled DNA fragments obtained from the first clone . Derivative clones were able to complement mutations in four different cistrons of the his cluster of S . coelicolor A3(2) . Nucleotide sequence analysis of a 4-kb region allowed the identification of five ORFs which showed significant homology with the his gene products of E . coli . The order of the genes in S . coelicolor A3(2) (5'--hisD-hisC-hisBd-hisH-hisA-3') is the same as in the his operon of E . coli. Gene, 1990 May 31, 90(1), 141 - 4 Construction of Escherichia coli vectors for expression and mutagenesis: synthesis of human c-Myc protein that is initiated at a non-AUG codon in exon 1; Date T et al.; Three types of Escherichia coli vector for both gene expression and mutagenesis were constructed from a plasmid/phage chimera vector pUC118 . Each vector contains the lac (pTD-lac), tac (pTD-tac), or T7 promoter (pTD-T7) . Downstream from the promoter, these vectors have sequences in common, including a Shine-Dalgarno (SD), multiple cloning sequence, sequence-primer binding site, transcription termination signal, and M13 origin of replication . Using single-stranded circular DNA obtained by infection with helper phage, oligodeoxyribonucleotide (oligo)-directed mutagenesis allows the appropriate fusion between the vector SD sequence and the start codon in the inserted fragment . Since a complementary oligo representing a large deletion is generally used for this construction, the extra nucleotides in the opposing strand form a loop structure . Thus, we have designated this mutagenesis as 'loop-out mutagenesis' . Expression plasmid encoding the larger human c-Myc protein that is initiated at a non-AUG codon in exon 1 and its derivatives were constructed using a pTD-T7 vector . Expression experiments indicated that the wild-type (wt) protein was synthesized poorly after induction with isopropyl-beta-D-thiogalactopyranoside, while one of the derivatives, p62M1T, in which a threonine residue was added at the N terminus of the wt protein, was produced in a large quantity in E . coli. Biochem Biophys Res Commun, 1990 May 31, 169(1), 64 - 9 Influence of DNA adenine methylase on the sensitivity of Escherichia coli to near-ultraviolet radiation and hydrogen peroxide; Yallaly P et al.; Near-ultraviolet (NUV) radiation and hydrogen peroxide (H2O2) inactivation studies were performed on Escherichia coli K-12 DNA adenine methylation (dam) mutants and on cells that carry plasmids which overexpress Dam methylase . Lack of methylation resulted in increased sensitivity to NUV and H2O2 (a photoproduct of NUV) . In a dam mutant carrying a dam plasmid, the levels of Dam enzyme and resistance to NUV and H2O2 were restored . However, using a multicopy dam+ plasmid strain, increasing the methylase above wildtype levels resulted in an increase in sensitivity of the cells rather than resistance. Gene, 1990 May 31, 90(1), 135 - 40 Vectors for constructing kan gene fusions: direct selection of mutations affecting IS10 gene expression; Sussman JK et al.; We describe several vectors for constructing translational fusions to the kan gene of Tn5 . Fusions are constructed in vitro using multi-copy vectors containing unique cloning sites situated between upstream transcriptional terminators and a downstream kan gene lacking transcriptional and translational start signals . Multi-copy fusions can be converted to single-copy chromosomal fusions by in vivo recombination with specific phage lambda vectors and vice versa . We find that kan fusions are often more suitable than lacZ fusions for the direct selection of mutations that increase fusion expression . These vectors were developed for isolating mutations that increase IS10 transposase expression; we describe strategies used to isolate such mutations, which map to IS10 or the Escherichia coli himA, himD(hip), dam or infC genes. Eur J Biochem, 1990 May 31, 190(1), 85 - 92 The E2 protein of human papillomavirus type 16 . Over-expression and purification of an active transcriptional regulator; Lees EM et al.; The E2 open reading frame of human papillomavirus type 16 was inserted into the Escherichia coli vector pKK223-3, and expressed to greater than 15% of total cellular protein when induced with isopropyl beta-D-thiogalactopyranoside . The highest expressing clone was grown in bulk and the E2 protein purified to homogeneity by the following procedure: (a) isolation of the insoluble protein fraction; (b) extraction with urea; (c) quaternary amino-ethyl-Sepharose ion-exchange chromatography and (d) renaturation and chromatography on dextran sulphate . That the purified protein was fully functionally active was confirmed by its specific DNA-binding properties and its ability to activate gene transcription by over two orders of magnitude in an in vivo assay. Biochem Biophys Res Commun, 1990 May 31, 169(1), 39 - 45 Alkaline low spin form of sulfite reductase hemeprotein subunit; Young LJ et al.; The reversible reduction and reoxidation of Escherichia coli sulfite reductase hemeprotein subunit at pH 9.9 produces high and low spin ferric species, the latter with properties distinct from any alkaline low spin yet reported . With virtually no effect on the 298 degrees K optical spectrum, chloride drastically reduces the low spin EPR intensity and produces a high spin conformer pattern like that seen at pH 11 . The distribution of g = 5 and g = 2.29 species in the doubly-reduced enzyme is also pH-sensitive. Gene, 1990 May 31, 90(1), 43 - 9 Construction of lacZ promoter probe vectors for use in Synechococcus: application to the identification of CO2-regulated promoters; Scanlan DJ et al.; It was shown that the Escherichia coli lacZ gene could be expressed in the cyanobacterium Synechococcus R2 PCC7942 both as a plasmid-borne form and also integrated into the chromosome . A promoterless form of the lacZ gene was constructed and used as a reporter gene to make transcriptional fusions with cyanobacterial promoters using a shuttle vector system and also via a process of integration by homologous recombination . Synechococcus R2 promoter-lacZ gene fusions were then used to identify CO2-regulated promoters, by quantitatively assessing beta-galactosidase activity under high and low CO2 conditions using a fluorescence assay . Several promoters induced under low CO2 conditions were detected. Gene, 1990 May 31, 90(1), 125 - 8 A mutation that converts serine340 of the HsdSK polypeptide to phenylalanine and its effects on restriction and modification in Escherichia coli K-12; Zinkevich VE et al.; A hybrid hsdS gene, encoding the HsdSts + d polypeptide, was constructed by joining the proximal region of the wild-type (wt) hsdS sequence with the distal region of the hsdSts + d sequence, at the hsdS BglII site . The hybrid hsdS-Sts + d gene exerts a trans-dominant effect on restriction and modification, which points to the location of the temperature-sensitive (ts) trans-dominant (+ d) mutation in the gene hsdSts + d distal region . Sequencing of the region downstream from the HindIII target in the Escherichia coli K-12 hsdSts + d mutant was carried out . It is identical to the wt hsdS sequence (GenBank/EMBL accession number ECHSDK LV00288), except for a single base-pair transition C1245----T . The results obtained support the idea that the trans-dominant effect of the ts mutation described earlier is related to the single base-pair transition in the nonhomologous region of the hsdSts + d sequence. Biochemistry, 1990 May 29, 29(21), 5119 - 26 Preparation and properties of recombinant DNA derived tobacco mosaic virus coat protein; Shire SJ et al.; Recombinant DNA derived tobacco mosaic virus (vulgare strain) coat protein (r-TMVP) was obtained by cloning and expression in Escherichia coli and was purified by column chromatography, self-assembly polymerization, and precipitation . SDS-PAGE, amino terminal sequencing, and immunoblotting with polyclonal antibodies raised against TMVP confirmed the identify and purity of the recombinant protein . Isoelectric focusing in 8 M urea and fast atom bombardment mass spectrometry demonstrated that the r-TMVP is not acetylated at the amino terminus, unlike the wild-type protein isolated from the tobacco plant derived virus . The characterization of r-TMVP with regard to its self-assembly properties revealed reversible endothermic polymerization as studied by analytical ultracentrifugation, circular dichroism, and electron microscopy . However, the details of the assembly process differed from those of the wild-type protein . At neutral pH, low ionic strength, and 20 degrees C, TMVP forms a 20S two-turn helical rod that acts as a nucleus for further assembly with RNA and additional TMVP to form TMV . Under more acidic conditions, this 20S structure also acts as a nucleus for protein self-assembly to form viruslike RNA-free rods . The r-TMVP that is not acetylated carries an extra positive charge at the amino terminus and does not appear to form the 20S nucleus . Instead, it forms a 28S four-layer structure, which resembles in size and structure the dimer of the bilayer disk formed by the wild-type protein at pH 8.0, high ionic strength, and 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 May 29, 29(21), 5027 - 34 Site-directed mutagenesis of recombinant rat DNA polymerase beta: involvement of arginine-183 in primer recognition; Date T et al.; By site-directed mutagenesis using synthetic oligonucleotides, amino acid residues 181Phe-Arg-Arg183 of recombinant rat DNA polymerase beta were replaced by other amino acids to clarify the roles of these residues in the DNA synthesizing reaction . Replacement of Phe-181 by alanine reduced the enzyme activity only 30% . Replacement of Arg-182 by alanine and glutamine resulted in reduction of the activity by about 67% and 95%, respectively . The Arg-182----Gln replacement increased the binding strength to single-stranded DNA but did not significantly change the Km's for the primer and dTTP, suggesting that Arg-182 is involved in modulation of binding to the template rather than to the primer or deoxyribonucleoside triphosphate . Replacement of Arg-183 by Gln resulted in reduction of the activity by about 95%, and this change, although causing little change in binding strength to single-stranded DNA, resulted in a 3-4-fold increase in the Km's for the primer and deoxyribonucleoside triphosphate . A more dramatic change was observed when Arg-183 was replaced by Ala, which resulted in a 99.98% reduction of enzyme activity . Although the Km for deoxyribonucleoside triphosphate of this mutant enzyme was hardly changed, that for the primer increased 159-fold . Therefore, it is concluded that Arg-183 occupies an important part of the primer recognition site of DNA polymerase beta. Biochemistry, 1990 May 29, 29(21), 5195 - 202 The kinetic mechanism of wild-type and mutant mouse dihydrofolate reductases; Thillet J et al.; A kinetic mechanism is presented for mouse dihydrofolate reductase that predicts all the steady-state parameters and full time-course kinetics . This mechanism was derived from association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance measurements . The major features of this kinetic mechanism are as follows: (1) the two native enzyme conformers, E1 and E2, bind ligands with varying affinities although only one conformer, E1, can support catalysis in the forward direction, (2) tetrahydrofolate dissociation is the rate-limiting step under steady-state turnover at low pH, and (3) the pH-independent rate of hydride transfer from NADPH to dihydrofolate is fast (khyd = 9000 s-1) and favorable (Keq = 100) . The overall mechanism is similar in form to the Escherichia coli kinetic scheme (Fierke et al., 1987), although several differences are observed: (1) substrates and products predominantly bind the same form of the E . coli enzyme, and (2) the hydride transfer rate from NADPH to either folate or dihydrofolate is considerably faster for the mouse enzyme . The role of Glu-30 (Asp-27 in E . coli) in mouse DHFR has also been examined by using site-directed mutagenesis as a potential source of these differences . While aspartic acid is strictly conserved in all bacterial DHFRs, glutamic acid is conserved in all known eucaryotes . The two major effects of substituting Asp for Glu-30 in the mouse enzyme are (1) a decreased rate of folate reduction and (2) an increased rate of hydride transfer from NADPH to dihydrofolate.(ABSTRACT TRUNCATED AT 250 WORDS) J Immunol, 1990 May 15, 144(10), 3868 - 76 The fine specificity of anti-La antibodies induced in mice by immunization with recombinant human La autoantigen; St Clair EW et al.; Because of increasing evidence suggesting that anti-La autoantibodies are induced in humans by an Ag-specific mechanism, we investigated the antibody response of animals immunized with the human La Ag and studied its relationship to the anti-La response of autoimmune patients . Anti-La antibodies were raised in 6- to 8-wk-old male MRL(-)+/+, C57BL/6J, BALB/c, and A/J mice by immunizing with authentic human La protein obtained by recombinant expression in Escherichia coli . As we have shown previously for human autoantibodies, induced mouse anti-La antibodies reacted with recombinant fusion proteins containing nonoverlapping sequences from different portions of the La molecule . The epitope specificity of antibodies to the middle region of the La Ag was further evaluated using six synthetic La peptides predicted to be antigenic based on their hydrophilic properties . Although the induced mouse anti-La antibodies bound to five of the six synthetic La peptides, human anti-La autoantibodies failed to recognize any of the peptide homologs . These results suggest that mice respond to immunization with human La protein differently than humans who develop autoimmunity to this self Ag. Nucleic Acids Res, 1990 May 25, 18(10), 3021 - 5 Oligonucleotide correlations between infector and host genomes hint at evolutionary relationships; Barrai I et al.; The frequencies of oligonucleotides of length 3-6 were studied in 211 sequences of human DNA (659 kilobases), 22 sequences of DNA of human viruses (120 kbs), in 181 sequences of E . coli (442 kbs), and in 42 sequences of phages of E . coli (137 kbs) . The sequences were obtained from Genbank(R) 48 . The observed frequencies (O) were compared to the expected frequencies (E) obtained in two ways: 1) according to nucleotide composition for each series, and 2) according to first order Markow chains for triplets, second order for quadruplets, and third order for quintuplets and sextuplets . The ratio O/E was obtained for each oligonucleotide . Then, the correlation between the ratio O/E in a pair of series was calculated . Strong correlations were observed for sequences of man and human viruses, and for E . coli and its phages . Other correlations were small . For higher order Markov chains, there is indication of some correlation also between viruses and phages . It was concluded that through analysis of parallel oligonucleotide series it may be possible to infer some of the complex evolutionary relationships existing between cells and their infectors beyond the level of codon usage. Nucleic Acids Res, 1990 May 25, 18(10), 2869 - 73 The effect of context on synonymous codon usage in genes with low codon usage bias; Bulmer M; The effect of neighbouring bases on the usage of synonymous codons in genes with low codon usage bias in yeast and E . coli is examined . The codon adaptation index is employed to identify a group of genes in each organism with low codon usage bias, which are likely to be weakly expressed . A similar pattern is found in complementary sequences with respect to synonymous usage of A vs G or of U vs C . It is suggested that this may reflect an effect of context on mutation rates in weakly expressed genes. Nucleic Acids Res, 1990 May 25, 18(10), 2875 - 80 Quantitative analysis of Tn10 Tet repressor binding to a complete set of tet operator mutants; Sizemore C et al.; A saturating oligonucleotide-directed mutagenesis of both tet operators in the tet regulatory sequence was performed yielding mutants with four identical base pair exchanges at equivalent positions in the four tet operator half sides . The mutants were cloned between bipolar lacZ and galK indicator genes on a multicopy plasmid allowing the quantitative analysis of their effects in vivo . In the absence of Tet repressor the mutations lead to considerably different expression levels of both genes . They are discussed with respect to the promoter consensus sequences . In particular, the -10 region of the in vivo active tetPR2 promoter is unambiguously defined by these results . In the presence of Tet repressor most of the mutants exhibit a lower affinity for that protein as determined quantitatively by their reduced expression levels . In general, tet operator recognition is most strongly affected by alterations of base pairs near the center of the palindromic sequence . The most important position is the third base pair, followed by base pairs two, four, five and six, the latter showing similar effects as base pair one . At each position, the four possible base pairs show different affinities for Tet repressor . They are discussed according to their exposure of H-bond donors and -acceptors in the major and minor grooves of the B-DNA . The results are in agreement with major groove contacts at positions two, three and five . At position four a low potential correlation of efficiencies with the H-bonding in the minor groove is found, while mutations at position six seem to influence repressor binding by other mechanisms. J Biol Chem, 1990 May 25, 265(15), 8760 - 5 Complementation of two overlapping fragments of SecA, a protein translocation ATPase of Escherichia coli, allows ATP binding to its amino-terminal region; Matsuyama S et al.; SecA is a protein translocation ATPase . The secA gene was engineered so as to code for SecA fragments of different sizes, either from the amino terminus or the carboxyl terminus . These SecA fragments, most of which formed aggregates in the cytosol, were overproduced and then purified in the presence of 6 M guanidine hydrochloride . The fragments were renatured by means of dilution and dialysis, and then examined as to their ability to interact with ATP by means of photoaffinity cross-linking with {alpha-32P}ATP . Individual SecA fragments thus renatured were inactive as to ATP binding . However, when two fragments (amino- and carboxyl-terminal ones), which structurally complemented each other and which had an overlapping region, were mixed, cross-linking was observed at the amino-terminal segments . The cross-linking was appreciably enhanced when two such fragments were first mixed together in 6 M guanidine hydrochloride and then renatured . It is concluded that SecA has an ATP-binding domain near its amino-terminal region and that the binding requires a carboxyl-terminal fragment that is large enough to cover the region deleted from the amino-terminal fragment . An amino-terminal fragment, which constituted about 92% of the entire SecA molecule, was active in not only ATP binding but also protein translocation . Based on these findings, the structure-function relationship of SecA is discussed. J Biol Chem, 1990 May 25, 265(15), 8354 - 7 A uniform isopeptide-linked multiubiquitin chain is sufficient to target substrate for degradation in ubiquitin-mediated proteolysis; Gregori L et al.; The proteolytic targeting function of ubiquitin was investigated by a combination of site-specific mutagenesis and covalent modification . Lys48 was replaced by a cysteine via mutagenesis of a synthetic ubiquitin gene to generate the mutant Ub-C48 . The single cysteine residue in Ub-C48 can be converted into a lysine analog by modification with the sulfhydryl-specific reagent, aminoethyl-8 (N-(iodoethyl)trifluoroacetamide) . The resulting protein, Ub-(S-aminoethyl)C48, is equivalent to a wild type ubiquitin except for the substitution of a sulfur atom at the gamma carbon of Lys48 . We have tested the ability of these two modified ubiquitins to target the degradation of an engineered beta-galactosidase substrate protein in ubiquitin-depleted reticulocyte lysates . Ub-C48 was unable to stimulate the degradation of this protein substrate although a monoubiquitinated beta-galactosidase was formed . In contrast, Ub-(S-aminoethyl)C48 appears to be as effective as wild type ubiquitin in targeting this substrate protein's degradation as well as the formation of multiply ubiquitinated beta-galactosidase intermediates . In conjunction with the cysteine substitution and modification, we have also examined the effects of blocking the amino groups in ubiquitin with reductive methylation . The methylation of either Lys48 in ubiquitin or its S-aminoethylcysteine counterpart abolished its proteolytic function while the blockage of the remaining six lysines in Ub-(S-aminoethyl)C48 did not alter its competence . Thus, of the seven lysine residues in ubiquitin, only Lys48 is essential . These results established unambiguously that a uniform multiubiquitin chain with ubiquitin-ubiquitin linkage solely at Lys48 is sufficient to target the degradation of a substrate protein in ubiquitin-mediated proteolysis. J Biol Chem, 1990 May 25, 265(15), 8957 - 65 Probing the activation stages of the RecA protein by monoclonal IgGs during the pairing of homologous DNA molecules; Ikeda M et al.; The biochemical properties of the RecA protein change in a stepwise manner with the binding of ATP and/or DNA during the course of the ATP-dependent formation of homologous joint molecules, which is assumed to be due to transition in the higher order structure . This transition was supposed to be detectable as changes in the affinity of the protein for monoclonal IgGs . In this study, we introduced an enzyme-linked immunosorbent assay, which enabled us to assay unbound IgG sensitively and quantitatively under the conditions for the RecA protein-mediated joint molecule formation . Through the use of this method, we studied the affinity of three anti-RecA protein monoclonal IgGs toward the RecA protein at the various stages of its activation during the formation of homologous joints . We found that the affinity of the RecA protein changed for each anti-RecA protein-IgG in a specific manner and the change in the cross-reactivity of the RecA protein correlated with its multiple activation stages . This result suggests that the RecA protein changes its higher order structure which is specific to each activation stage during the formation of homologous joint molecules. Nucleic Acids Res, 1990 May 25, 18(10), 3007 - 13 Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice; Kohler SW et al.; Transgenic mice suitable for the in vivo assay of suspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector . The shuttle vector, which contains a beta-galactosidase (beta-gal) target gene, can be rescued from genomic DNA with in vitro packaging extracts . Mutations in the target gene are detected by a change in lambda phage plaque color on indicator agar plates . Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100 micrograms of genomic DNA were too low for mutation analysis . We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals . The rescue efficiency of 5-azacytidine-treated cells increased 50-fold over non-treated controls indicating that methylation was inhibiting rescue . The inhibitory role of methylation was supported by the observation that mcr deficient E . coli plating strains and mcr deficient lambda packaging extracts further improved lambda rescue efficiency . Present rescue efficiencies of greater than 2000 pfu/copy/micrograms of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis . The background mutagenesis rate was estimated at 1 x 10(-5) in two separate lineages . Following treatment with the mutagen N-ethyl-N-nitrosourea (EtNU), a dose dependent increase in the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed in mouse testes DNA. Science, 1990 May 25, 248(4958), 1006 - 9 Blocking of the initiation-to-elongation transition by a transdominant RNA polymerase mutation; Kashlev M et al.; RNA polymerase, the principal enzyme of gene expression, possesses structural features conserved in evolution . A substitution of an evolutionarily invariant amino acid (Lys1065----Arg) in the beta subunit of Escherichia coli RNA polymerase apparently disrupts its catalytic center . The mutant protein inhibited cell growth when expressed from an inducible promoter . The assembled holoenzyme carrying the mutant subunit formed stable promoter complexes that continuously synthesized promoter-specific dinucleotides but that did not enter the elongation step . The mutant polymerase inhibited transcription by blocking the access of the wild-type enzyme to promoters. J Biol Chem, 1990 May 25, 265(15), 8948 - 56 Epitopes and active sites of the RecA protein; Ikeda M et al.; The RecA protein is indispensable for homologous genetic recombination in Escherichia coli . This protein alone promotes the ATP-dependent formation of homologous joint molecules and their processing in vitro . Through the use of a set of anti-RecA protein mouse monoclonal IgGs, we have been attempting to divide the whole process into elementary steps to determine the basic functions of the protein . In order to correlate the basic functions with the active sites on the recA polypeptide, we located the epitopes for the anti-RecA protein-IgGs on the recA polypeptide by means of immunoblotting experiments and an enzyme-linked immunosorbent assay involving isolated proteolytic polypeptides or synthetic ones derived from various regions of the recA polypeptide . The epitopes for anti-RecA protein-IgGs ARM321 and ARM414, both of which are shown to inhibit the DNA-dependent ATP hydrolysis and the formation of homologous joints by the RecA protein, were found to be located between Thr89 and Glu127 and between Glu233 and Lys256, respectively, on the RecA polypeptide . IgG ARM193 had been shown to interfere with the protein-protein interaction between two RecA protein molecules, and ARM191 had been suggested to inhibit the binding of double-stranded DNA to the RecA protein . The epitopes for ARM193 and ARM191 were found to be located in a approximately 90-amino acid region at the C terminus . These results suggest the locations of the active sites and a functional core on the RecA polypeptide. J Biol Chem, 1990 May 25, 265(15), 8490 - 6 Reverse transcriptase from Escherichia coli exists as a complex with msDNA and is able to synthesize double-stranded DNA; Lampson BC et al.; Reverse transcriptase required for the synthesis of msDNA.Ec67 in an Escherichia coli strain was purified as a large molecular weight complex with msDNA . The complex sedimented in a glycerol gradient at an s value greater than 19 . The predominant protein species co-purifying with reverse transcriptase activity in the complex had a molecular weight estimated at 65,000 which is close to the expected size of 67,227 for the Ec67-reverse transcriptase . In addition, the large complex also contained msDNA.Ec67 . The purified complex was able to synthesize cDNA using 5 S rRNA as a template (annealed to a synthetic DNA primer), and a double-stranded DNA using a synthetic DNA template (annealed to a synthetic DNA primer) . When msDNA.Ec67 was used as a natural template:primer, the purified complex produced two major products: a 103-base single-stranded DNA by extending the 3' end of msDNA using msdRNA as a template, and a 60-base double-stranded DNA product resulting from the converse reaction in which the 3' end of msdRNA is extended using msDNA as a template . The results suggest that bacterial reverse transcriptase is capable of producing single-stranded cDNA and possibly double-stranded DNA as well . Possible implications of these findings on the biology of the msDNA-retron system are discussed. Biochim Biophys Acta, 1990 May 24, 1049(1), 93 - 5 The primary structure of rat ribosomal protein S20; Chan YL et al.; The amino acid sequence of the rat 40 S ribosomal subunit protein S20 was deduced from the sequence of nucleotides in two recombinant cDNAs . Ribosomal protein S20 has 119 amino acids and has a molecular weight of 13,364 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 16-19 copies of the S20 gene . The mRNA for the protein is about 600 nucleotides in length . Rat S20 is homologous to Xenopus laevis S22 and is related to Mycoplasma capricolum S10 and to Escherichia coli S10 . The protein contains a possible internal duplication of seven residues. Nature, 1990 May 24, 345(6273), 359 - 61 How different eukaryotic transcriptional activators can cooperate promiscuously; Lin YS et al.; A striking characteristic of many different eukaryotic transcriptional activators is their ability to activate gene expression synergistically . Thus, for example, the rat glucocorticoid receptor and the yeast activator GAL4 cooperatively activate transcription of a mammalian gene bearing binding sites for each of the proteins: activation by both activators is greater than the sum of the effects of each working alone . It would seem unlikely that these two proteins from such different organisms directly interact; rather, the idea has been suggested that these and at least some other eukaryotic activators can work synergistically by simultaneously touching some part of the transcriptional machinery . An important prediction of this idea is that synergy between two such activators would be seen under conditions where each is present at concentrations sufficiently high to saturate its site on DNA . In this paper we use transcription in vitro to confirm that prediction using a derivative of the yeast activator GAL4 and the mammalian transcription factor ATF . The accompanying paper describes a similar conclusion comparing the effects of singly and multiply bound GAL4 molecules. Biochemistry, 1990 May 22, 29(20), 4959 - 66 Macromolecular binding equilibria in the lac repressor system: studies using high-pressure fluorescence spectroscopy; Royer CA et al.; High hydrostatic pressure coupled with fluorescence polarization has been used to investigate protein subunit interactions and protein-operator association in lac repressor labeled with a long-lived fluorescent probe . On the basis of observation of a concentration-dependent sigmoidal decrease in the dansyl fluorescence polarization, we conclude that application of high hydrostatic pressure results in dissociation of the lac repressor tetramer . The 2-fold decrease in the rotational relaxation time and the high-pressure plateau are consistent with a tetramer to dimer transition . The volume change for tetramer dissociation to dimer is -82 +/- 5 mL/mol . The dissociation constant calculated from the data taken at 4.5 degrees C is 4.3 +/- 1.3 nM . The tetramer dissociation constant increases by a factor of 3 when the temperature is raised from 4.5 to 21 degrees C . A very small effect of inducer binding on the subunit dissociation is observed at 4.5 degrees C; the Kd increases from 4.5 to 7.1 nM . At 21 degrees C, however, inducer binding stabilizes the tetramer by approximately 0.8 kcal/mol . Pressure-induced monomer formation is indicated by the curves obtained upon raising the pH to 9.2 . The addition of IPTG shifts the pressure transition to only slightly higher pressures at this pH, indicating that the stabilization of the tetramer by inducer is not as marked as that observed at pH 7.1 . From the decrease in the polarization of the dansyl repressor-operator complexes, we also conclude that the application of pressure results their dissociation and that the volume change is large in absolute value (approximately 200 mL/mol) . The lac repressor-operator complex is more readily dissociated upon the application of pressure than the tetramer alone, indicating that operator binding destabilizes the lac repressor tetramer. Biochemistry, 1990 May 22, 29(20), 4880 - 5 Slow-binding inhibition of the Escherichia coli pyruvate dehydrogenase multienzyme complex by acetylphosphinate; Schonbrunn-Hanebeck E et al.; The pyruvate analogue acetylphosphinate (CH3-CO-PO2H2) inhibits the pyruvate dehydrogenase component (E1) of the Escherichia coli pyruvate dehydrogenase multienzyme complex in a time-dependent process with biphasic reaction kinetics . The formation of an initial, rapidly reversible enzyme-inhibitor complex (EI) with an apparent Ki of 0.12 +/- 0.025 microM is followed by the conversion to a tighter complex (EI) at a maximal rate of k3 = 0.87 +/- 0.34 min-1 . The inhibition is reversible (dissociation rate constant k4 = 0.038 +/- 0.002 min-1), requires the presence of the cofactors thiamin pyrophosphate and Mg2+, and is competitive with regard to pyruvate . The microscopic rate constants give a value of 5 nM for the overall dissociation constant {Ki = {E} {I}/{( EI} + {EI}) = Kik4/(k3 + k4)} compared with values of 10 and 3.5 nM obtained by steady-state methods . Thus acetylphosphinate binds by 5 orders of magnitude more tightly to pyruvate dehydrogenase than does pyruvate (Km = 0.35 mM) . Acetylphosphinate also affects the pyruvate dehydrogenase complex fluorescence when excited at 290 nm in a time-dependent manner with a maximal rate constant of 0.99 min-1, suggesting a conformational change in the enzyme complex as the slow step in conversion of EI to EI (k3) . All these features taken together suggest that the interaction of the pyruvate dehydrogenase with acetylphosphinate involves the formation of a thiamin pyrophosphate-acetylphosphinate adduct that resembles the normal reaction intermediate, 2-(1-carboxy-1-hydroxyethyl)thiamin pyrophosphate (alpha-lactylthiamin pyrophosphate). Biochemistry, 1990 May 22, 29(20), 4758 - 66 Targeting of cloned firefly luciferase to yeast mitochondria; Aflalo C; The firefly luciferase gene (luc) was fused to a 5' fragment of the 70-kDa protein gene (70K) from yeast . The fragment codes for the N-terminal putative signal sequence which targets and anchors the 70-kDa protein to the cytoplasmic side of the outer membrane in mitochondria . Two versions of the fusion gene, 70K{232}::luc and 70K{93}::luc (containing 292 and 93 5' codons from 70K, respectively), were constructed in a bacterial expression plasmid . Both the genes were expressed in Escherichia coli, and in both cases, bioluminescence activity was associated with the expression . The 70K{93}::luc gene was transferred to a yeast-bacteria shuttle vector used to transform Saccharomyces cerevisiae cells . As a control, the same strain was transformed with a plasmid including the original luc . With both transformants, bioluminescence activity was detected in intact cells and crude extracts . Upon growth on a nonfermentable carbon source and fractionation, the product of the fusion gene was associated mostly with mitochondria . In the control transformant, the product of luc was more delocalized . However, a significant amount remained associated with isolated mitochondria . No such spontaneous association of purified luciferase with wild-type mitochondria was observed in vitro . Trypsin treatment of mitochondria isolated from both transformed strains indicated that the fusion protein is anchored to the outer membrane and exposed to the medium while the unfused luciferase retained with the mitochondria is occluded in a compartment unaccessible to trypsin and released in the presence of detergent . The fusion protein retained the major catalytic properties of the parent firefly luciferase, as determined in solution.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1990 May 21, 264(2), 215 - 7 Molecular weight analysis of isopenicillin N synthase by electrospray mass spectrometry; Aplin RT et al.; The use of electrospray mass spectrometry as a tool in analytical biochemistry was illustrated by determination of the molecular weights of wildtype and recombinant isopenicillin N synthase (IPNS) . The molecular weight of recombinant IPNS produced using an expression system which generated soluble protein was found to be between 38,364 and 38,376 Da, ca 60 mass units higher than that of the wildtype material, consistent with the presence of an additional N-terminal glycine in the former . Observed molecular weights were all ca 70 Da higher than that calculated from sequence information, consistent with the complexion of a partially hydrated iron atom to the enzyme during analysis. Eur J Biochem, 1990 May 20, 189(3), 667 - 73 Assignment of tyrosine resonances in the 1H-NMR spectrum of tryptophan synthase alpha-subunit . Monitoring conformational changes due to substitutions at position 49; Sawada S et al.; In order to monitor the conformational changes of tryptophan synthase alpha-subunit from Escherichia coli in solution resulting from amino acid substitutions, we have assigned the Tyr resonances in the aromatic region of the 1H-NMR spectrum to specific residues . In the spectrum of the alpha-subunit deuterated with {2,3,4,5,6-2H5}Phe and {3,5-2H2}Tyr, the C2 and C6 protons of Tyr gave completely isolated signals at acidic p2H . Some of the C3 and C5 proton resonances overlapped with each other at acidic p2H . By using a series of mutant alpha-subunits in which each Tyr was singly substituted with His or Phe, we can now assign each of seven Tyr resonances in the aromatic region to a specific residue . We have previously studied the conformational stability of a series of variant alpha-subunits at position 49 {Yutani et al . (1987) Proc . Natl Acad . Sci . USA 84, 4441-4444} . We now compare the 1H-NMR spectra in the aromatic region of the wild-type alpha-subunit and mutant alpha-subunits substituted with Phe or Gly in place of Glu-49 . The results suggest that the major conformational effects of substitutions at position 49 are localized close to the position of substitution. J Mol Biol, 1990 May 20, 213(2), 239 - 46 DNA sequence analysis of mutations induced by N-2-acetylamino-7-iodofluorene in plasmid pBR322 in Escherichia coli; Hoffmann GR et al.; The spectrum of mutations induced by N-2-acetylamino-7-iodofluorene (AAIF) was analyzed in a forward mutation system based on mutagenesis directed to a small restriction fragment in the tetracycline resistance gene of plasmid pBR322 . AAIF was found to induce frameshift mutations and base-pair substitutions at approximately equal frequencies . The frameshift mutations were mostly deletions of single base-pairs, but -2 frameshifts and +1 frameshifts were also detected . With one exception, the substitutions were transversions initiated at a G.C base-pair . Both frameshift mutations and transversions occurred preferentially at sites of repetitive guanine residues . Although AAIF and the related aromatic amines N-2-acetylaminofluorene (AAF) and N-2-aminofluorene (AF) all bind to the C-8 position of guanine, they have different effects on DNA conformation, and these differences are reflected in their mutation spectra . Previous studies have provided evidence that AAF adducts can trigger a B to Z conformational change in alternating GC sequences or displacement of the guanine by the fluorene ring in other sequences; the principal result is two classes of frameshift mutations . AF, whose DNA interaction involves outside binding rather than insertion and denaturation, primarily induces base-pair substitutions . AAIF adducts are chemically similar to AAF adducts, but the iodo group apparently hinders insertion of the fluorene ring into DNA . Consistent with this model, the mutation spectrum of AAIF combines properties of the mutation spectra of both AAF and AF. J Mol Biol, 1990 May 20, 213(2), 219 - 20 Crystallization and preliminary X-ray diffraction analysis of catalase HPII from Escherichia coli; Tormo J et al.; Green crystals of the hexameric catalase HPII from Escherichia coli have been obtained by the hanging-drop method . The crystals belong to the monoclinic space group P2 with a = 123 A, b = 132 A, c = 93 A, beta = 112.5 degrees . There are three subunits in the asymmetric unit . The crystals diffract at least to 3.2 A resolution and are suitable for further X-ray diffraction studies. Eur J Biochem, 1990 May 20, 189(3), 559 - 65 Divalent cation binding to reduced and octanoyl acyl-carrier protein; Tener DM et al.; Mn(II) EPR binding studies with reduced acyl-carrier protein (ACP-SH) strongly suggest the presence of two relatively high-affinity manganese-binding sites (average Kd/site approximately 80 microM) at physiological pH . Lowering the pH or titrating with sodium chloride reduces the average number of bound divalent cations and decreases the binding affinity . This is consistent with the idea that anionic ligand(s), e.g . the carboxylate of glutamic or aspartic acid, on the protein are involved in manganese ion coordination . At pH values above 8.0, binding affinity is also reduced, whereas the average number of bound metal ions increases to about five at pH 8.5 . By interacting weakly with divalent cations (average Kd/site approximately 1 mM), octanoyl acyl-carrier protein (OcoACP) exhibits dramatically different metal-ion-binding properties compared to ACP-SH . Calcium and magnesium can compete in either ACP species for manganese binding . Photochemically-induced dynamic nuclear polarisation 1H-NMR experiments strongly suggest that ACP-SH and OcoACP undergo at pH-induced conformational change between pH 5.5 and pH 7.0, and that divalent cations stabilize the protein against such pH-induced structural perturbations. J Mol Biol, 1990 May 20, 213(2), 229 - 37 Mapping of insertion element IS5 in the Escherichia coli K-12 chromosome . Chromosomal rearrangements mediated by IS5; Umeda M et al.; We identified phage clones containing insertion element IS5 in a set of 476 lambda phage clones carrying chromosomal segments that cover almost the entire chromosome of Escherichia coli K-12 W3110 . Precise locations and orientations of IS5 were then determined by cleavage analysis of phage DNAs containing them . We mapped 23 copies of IS5 (named is5A to is5W) on the W3110 chromosome . Among them, ten were identified as the common elements present at the same locations in both chromosomes of W3110 and another E . coli K-12 strain, JE5519 . While most of the mapped IS5 elements were scattered over the W3110 chromosome, four copies of IS5 (designated is5L, is5M, is5N and is5O) were in a region representing tandem duplication of a DNA segment flanked by two copies of IS5 . Interestingly, one unit of this DNA segment as well as a portion of it was seen also in a tandem array in a different region where two copies of IS5 (designated is5P and is5Q) were present . In particular two pairs of the mapped IS5 elements may have been involved in inversion of the chromosomal segments in two of the E . coli K-12 derivatives. Eur J Biochem, 1990 May 20, 189(3), 487 - 92 Role of the C-terminal region in the allosteric properties of Escherichia coli phosphofructokinase-1; Serre MC et al.; In order to investigate the role of the carboxy-terminal segment in the catalytic, regulatory, and structural properties of the major allosteric phosphofructokinase (ATP:D-fructose-6-phosphate-1-phosphotransferase: EC 2.7.1.11) from Escherichia coli, the corresponding gene has been modified at either of two sites using oligonucleotide-directed mutagenesis: the codon at position 279 was changed from TAC (Tyr) into TAA (Ochre), and the codon at position 311 from TGG (Trp) into TAG (Amber) . The gene mutated at position 279 is not expressed as an active enzyme, probably because a polypeptide chain lacking 41 C-terminal residues cannot fold and/or assemble under the intracellular conditions . The gene mutated at position 311 is expressed as an active enzyme which has been purified to homogeneity . The fluorescence of this protein shows that it has no tryptophan, which confirms that the last nine residues at the carboxy terminal are missing . This derivative has almost the same specific activity and affinities for the two substrates (fructose-6-phosphate and ATP) as intact phosphofructokinase; the saturation by fructose 6-phosphate is also very cooperative . The last nine residues are thus not important for substrate binding, homotropic cooperativity, and catalytic efficiency . The activity of the mutant enzyme is still sensitive to activation by GDP or inhibition by phosphoenolpyruvate, but its affinity for the allosteric effectors is reduced . The carboxy-terminal segment also appears to contribute to the stability of the interactions between subunits: the mutant protein is less stable than the wild type towards denaturation by heat or guanidinium hydrochloride. Science, 1990 May 18, 248(4957), 860 - 3 No specific recognition of leader peptide by SecB, a chaperone involved in protein export; Randall LL et al.; Most proteins destined for export from Escherichia coli are made as precursors containing amino-terminal leader sequences that are essential for export and that are removed during the process . The initial step in export of a subset of proteins, which includes maltose-binding protein, is binding of the precursor by the molecular chaperone SecB . This work shows directly that SecB binds with high affinity to unfolded maltose-binding protein but does not specifically recognize and bind the leader . Rather, the leader modulates folding to expose elements in the remainder of the polypeptide that are recognized by SecB. Science, 1990 May 18, 248(4957), 863 - 6 Inhibition of FKBP rotamase activity by immunosuppressant FK506: twisted amide surrogate; Rosen MK et al.; The immunosuppressive agents cyclosporin A and FK506 inhibit the transcription of early T cell activation genes . The binding proteins for cyclosporin A and FK506, cyclophilin and FKBP, respectively, are peptidyl-prolyl-cis-trans isomerases, or rotamases . One proposed mechanism for rotamase catalysis by cyclophilin involves a tetrahedral adduct of an amide carbonyl and an enzyme-bound nucleophile . The potent FKBP rotamase inhibitor FK506 has a highly electrophilic carbonyl that is adjacent to an acyl-pipicolinyl (homoprolyl) amide bond . Such a functional group would be expected to form a stabilized, enzyme-bound tetrahedral adduct . Spectroscopic and chemical evidence reveals that the drug interacts noncovalently with its receptor, suggesting that the alpha-keto amid of FK506 serves as a surrogate for the twisted amide of a bound peptide substrate. Biochem Biophys Res Commun, 1990 May 16, 168(3), 1285 - 91 High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH:nitrate reductase and its comparison to human NADH:cytochrome B5 reductase; Hyde GE et al.; Higher plant nitrate reductase can be divided into three functional domains representing its prosthetic groups: 1) flavin; 2) cytochrome b; and 3) Mo-pterin . The flavin domain has been synthesized by heterologous expression in Escherichia coli using a fragment of a corn leaf NADH:nitrate reductase cDNA clone, Zmnr1, which we had previously isolated and sequenced . A Xho2-BamH1 fragment was cut from Zmnr1, containing the sequence for the flavin domain, and ligated in the BamH1 site of expression vector pET3c . When this construct was expressed in E . coli, a 30 kD polypeptide was found to be newly synthesized . The flavin domain was purified to homogeneity using blue Sepharose and shown to have a molecular weight of 30 kD . The recombinant flavin domain has a ferricyanide reductase specific activity of 1000 mumols NADH oxidized/min/mg protein and a visible spectrum virtually identical to that of human NADH:cytochrome b5 reductase. Biochem Biophys Res Commun, 1990 May 16, 168(3), 1095 - 102 Evidence for nonintercalative complexes formed from the reversible binding of benzo{a}pyrene metabolites to closed-circular, single-stranded M13mp19 DNA; Price HL et al.; The fluorescence excitation spectrum of complexes formed from the reversible binding of the proximate carcinogen, trans-7,8-dihydroxy-7,8-dihydro-benzo{a}pyrene (BP78D) to closed-circular, single-stranded, viral M13mp19 DNA (SS M13 DNA) exhibits a red-shift of 5 nm compared to the spectrum of BP78D measured without DNA or with native, calf thymus DNA . In SS M13 DNA which is 0.10 mM in PO4-, the fluorescence intensity of BP78D is 2.3 times smaller than the intensity measured without DNA; however, the fluorescence lifetime (42.7 nsec) of BP78D with SS M13 DNA is 1.7-1.8 times larger than the lifetimes of BP78D measured without DNA or with calf thymus DNA . These results are consistent with the conclusion that, in addition to binding sites which cause fluorescence quenching, SS M13 DNA contains sites which permit formation of BP78D inclusion complexes that have weaker interactions with nucleotide bases than those occurring in intercalated complexes . The association constant (1.45 +/- 0.01 x 10(5) M-1) for the binding of BP78D to SS M13 DNA is more than 9.0 times larger than that for binding to calf thymus DNA . It is 7.1 times larger than that for the binding of the less genotoxic metabolite, trans-4,5-dihydroxy-4,5-dihydrobenzo{a}pyrene (BP45D) to SS M13 DNA . UV Photoelectron data and results from ab initio molecular orbital calculations suggest that a difference in polarizability contributes to the greater SS M13 DNA binding of BP78D compared to that of BP45D. Biochem Biophys Res Commun, 1990 May 16, 168(3), 1211 - 6 Target of serine inhibition in Escherichia coli; Hama H et al.; L-serine has long been known to inhibit growth of Escherichia coli cells cultured in minimal medium supplemented with glucose, lactate, or another carbohydrate as the sole source of carbon . However, the target of serine inhibition was not known . The growth inhibition was released by adding isoleucine, 2-ketobutyric acid, threonine or homoserine, but not by aspartate . Thus the inhibition site must be between aspartate and homoserine in the isoleucine biosynthetic pathway . We found that homoserine dehydrogenase I was strongly inhibited by serine . We isolated serine-resistant mutants, and found that in these mutants homoserine dehydrogenase I was resistant to serine . Thus, we conclude that the target of serine inhibition in Escherichia coli is homoserine dehydrogenase I. Biochemistry, 1990 May 15, 29(19), 4704 - 13 Multiple non-B-DNA conformations of polypurine.polypyrimidine sequences in plasmids; Shimizu M et al.; A polypurine.polypyrimidine (Pur.Pyr) sequence with a central interruption in a plasmid can adopt multiple non-B-DNA conformations depending on the conditions as revealed by specific chemical probes (OsO4, diethyl pyrocarbonate, and dimethyl sulfate) and two-dimensional electrophoresis . The relatively long mirror repeat Pur.Pyr sequences (GAA)9TTC(GAA)8 and (GGA)9TCC(GGA)8 form single canonical intramolecular triplexes at pH 7.0-6.0 in negatively supercoiled plasmids as isolated from Escherichia coli . With a lowering of the pH and/or an increase in the degree of negative supercoiling, these sequences undergo a novel conformational change as revealed by diethyl pyrocarbonate hypermodification of adenines in the middle of the polypurine strand and OsO4 reaction with thymines in the center and the quarter points of the polypyrimidine strand . To evaluate this structure, a family of related Pur.Pyr sequences were cloned and studied . The non mirror repeat sequence (GGA)9TCC(GAA)8 forms a non-B conformation only under acidic pH conditions, but the structural properties are different from those of the mirror repeat sequences . Furthermore, when the central interruptions of a mirror repeat sequence were increased from 3 to 9 bp, two canonical triplexes formed independently at pH 5.0 {at the (GAA)9 and (GAA)8 regions in the sequence (GAA)9TTAATTCGC(GAA)8} . Thus, if an interruption is sufficiently long, the two halves of the Pur.Pyr sequence do not interact with each other . Novel types of folded DNA geometries which explain these results are described. Biochem Pharmacol, 1990 May 15, 39(10), 1557 - 64 Inactivation of human synovial fluid phospholipase A2 by the marine natural product, manoalide; Jacobson PB et al.; The marine natural product, manoalide (MLD), was investigated to determine if this drug inhibited purified human synovial fluid phospholipase A2 (HSF-PLA2) . Utilizing classical Michaelis-Menten kinetics, apparent Km and Vmax values for HSF-PLA2 of 1.34 mM and 0.47 mumol {3H}palmitic acid released/min/mg protein were obtained using dipalmitoylphosphatidylcholine (DPPC) as the substrate, and 38.0 microM and 18.8 mumol {3H}arachidonic acid released/min/mg protein with Escherichia coli as a natural substrate . These kinetic parameters were utilized subsequently to evaluate the inhibitory effects of manoalide on HSF-PLA2 . Inhibition of HSF-PLA2 by MLD was concentration and time dependent with IC50 values of 0.2 and 0.02 microM for DPPC and E . coli respectively . Dialysis studies and examination of DPPC or E . coli hydrolysis versus enzyme concentration indicate that MLD is an irreversible inhibitor of HSF-PLA2 . Substrate specificity was also examined in the absence and presence of MLD using dipalmitoylphosphatidylethanolamine (DPPE) as a substrate . MLD inhibited the hydrolysis of DPPE (greater than 90% inhibition at 2 microM), and preliminary results indicate that DPPC was more readily hydrolyzed than DPPE under the substrate conditions of the assay . While the cellular source of secreted HSF-PLA2 is unknown, these studies indicate that MLD can inactivate secreted phospholipase A2 isolated from patients with inflammatory joint disease. Arch Biochem Biophys, 1990 May 15, 279(1), 138 - 45 Responsibility of tRNA(Ile) for spermine stimulation of rat liver Ile-tRNA formation; Peng Z et al.; To determine whether tRNA or aminoacyl-tRNA synthetase is responsible for spermine stimulation of rat liver Ile-tRNA formation, homologous and heterologous Ile-tRNA formations were carried out with Escherichia coli and rat liver tRNA(Ile) and their respective purified Ile-tRNA synthetases . Spermine stimulation was observed only when tRNA from the rat liver was used . Spermine bound to rat liver tRNA(Ile) but not to the purified aminoacyl-tRNA synthetase complex . Kinetic analysis of Ile-tRNA formation revealed that spermine increased the Vmax and Km values for rat liver tRNA(Ile) . The Km value for ATP and isoleucine did not change significantly in the presence of spermine . Furthermore, higher concentrations of rat liver tRNA(Ile) tended to inhibit Ile-tRNA formation if spermine was absent . Spermine restored isoleucine-dependent PPi-ATP exchange in the presence of rat liver tRNA(Ile), an inhibitor of this exchange . The nucleotide sequence of rat liver tRNA(Ile) was determined and compared with that of E . coli tRNA(Ile) . Differences in nucleotide sequences of the two tRNAs(Ile) were observed mainly in the acceptor and anticodon stems . Limited ribonuclease V1 digestion of the 3'-32P-labeled rat liver tRNA(Ile) showed that both the anticodon and acceptor stems were structurally changed by spermine, and that the structural change by spermine was different from that by Mg2+ . The influence of spermine on the ribonuclease V1 digestion of E . coli tRNA(Ile) was different from that of rat liver tRNA(Ile) . The results suggest that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation. Cancer Res, 1990 May 15, 50(10), 2885 - 90 Interleukin 6 perfusion stimulates reconstitution of the immune and hematopoietic systems after 5-fluorouracil treatment; Takatsuki F et al.; Effects of interleukin 6 (IL-6) on the functional capacity of the immune and hematopoietic systems in 5-fluorouracil (5-FU)-treated mice were determined . IL-6 (5 x 10(4) units/mouse/day) was administered s.c . for 7 days by implantation of an osmotic pump, since it was demonstrated that a much higher increase in the primary response to sheep RBC was observed by administration of slowly released rather than daily s.c . injection of IL-6 . IL-6 perfusion significantly augmented anti-sheep RBC antibody responses depressed by 5-FU (150 mg/kg) treatment . IL-6 also was shown to stimulate hematological recovery in mice treated with 5-FU . Namely, IL-6 perfusion accelerated the recovery of the number of hematopoietic stem cells, granulocyte-macrophage progenitors, and mature neutrophils in the spleen, although IL-6 did not stimulate the recovery of the neutrophil count in blood . Recovery of the platelet count in blood was stimulated by IL-6 . Furthermore, it was found that the endogenous IL-6 level in serum increased after 5-FU treatment, which suggests that IL-6 may play some role in the recovery of the immune and hematopoietic systems . Finally, we examined the effect of IL-6 on the survival of mice treated with a higher dosage of 5-FU (300 mg/kg) . IL-6 perfusion produced a distinct increase in survival rate at Day 30 (74% versus 28%) . It is of note that the number of bacteria (identified as Escherichia coli) cultured from the spleen and the liver decreased in IL-6-perfused mice . This IL-6-induced effect was accompanied by enhancement of an oxidative burst response . Moreover, the anti-E . coli antibody titer in serum was higher in IL-6-perfused mice than in control mice . These results suggest the possible use of IL-6 for stimulating the reconstitution of the immune and hematopoietic systems after chemotherapy treatment. Biochem J, 1990 May 15, 268(1), 69 - 75 Inhibition of pyruvate:ferredoxin oxidoreductase from Trichomonas vaginalis by pyruvate and its analogues . Comparison with the pyruvate decarboxylase component of the pyruvate dehydrogenase complex; Williams KP et al.; Pyruvate:ferredoxin oxidoreductase and the pyruvate dehydrogenase multi-enzyme complex both catalyse the CoA-dependent oxidative decarboxylation of pyruvate but differ in size, subunit composition and mechanism . Comparison of the pyruvate:ferredoxin oxidoreductase from the protozoon Trichomonas vaginalis and the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex shows that both are inactivated by incubation with pyruvate under aerobic conditions in the absence of co-substrates . However, only the former is irreversibly inhibited by incubation with hydroxypyruvate, and only the latter by incubation with bromopyruvate . Pyruvate:ferredoxin oxidoreductase activity is potently, but reversibly, inhibited by addition of bromopyruvate in the presence of CoA, and it is suggested that the mechanism involves formation of an adduct between CoA and bromopyruvate in the active site of the enzyme . It is proposed that both enzymes are inactivated by pyruvate through a mechanism involving oxidation of an enzyme-bound thiamin pyrophosphate/substrate adduct to form a tightly bound inhibitory species, possibly thiamin thiazolone pyrophosphate as hypothesized by Sumegi & Alkonyi. J Biol Chem, 1990 May 15, 265(14), 8027 - 32 The importance of loop region residues 40-46 in human dihydrofolate reductase as revealed by site-directed mutagenesis; Tan XH et al.; Site-directed mutagenesis has been used to delete 2 residues (Gly45-Lys46) from a flexible "loop" region between residues 40 and 46 of human dihydrolate reductase . Steady-state kinetic studies show that the Km values for the deletion mutant enzyme for both dihydrofolate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) as well as the pH rate profile are virtually identical to that of the wild type . In contrast, the Vmax value of the mutant enzyme is decreased 2.5-fold . The results suggest that the loop region may play a role in the catalytic efficiency but not necessarily in the binding of substrates . Agents such as KCl, urea, and organomercurials at concentrations which show activating effects on the wild-type human dihydrofolate reductase have little or no effect on the deletion mutant . Competitive enzyme-linked immunosorbent assay experiments using peptide-specific antibodies against cyanogen bromide fragments generated from human dihydrofolate reductase show that the binding of folate, NADPH, and methotrexate, either in binary or in ternary complexes with the wild-type enzyme, causes a striking reduction in the binding of the antibodies . Compared with wild type, the binding of these ligands with the deletion mutant enzyme causes much less inhibition (2-16-fold less) in the binding of all three antibodies . The altered properties of the mutant enzyme can be explained on the basis of a need for the flexible loop 40-46 for reversible protein unfolding during activation and also for conformational changes induced by ligand binding, thus "communicating" the effects of ligand binding. J Biol Chem, 1990 May 15, 265(14), 7886 - 93 The ATP-dependent Clp protease of Escherichia coli . Sequence of clpA and identification of a Clp-specific substrate; Gottesman S et al.; The clpA gene, which codes for the ATP-binding subunit of the ATP-dependent Clp protease of Escherichia coli, has been sequenced . The coding region contains a single open reading frame for a protein of 758 amino acids; within the amino acid sequence are two consensus sequences for ATP-binding sites . The sequence of ClpA does not resemble that of other previously described ATPases or Lon, the other sequenced ATP-dependent protease of E . coli, except in the ATP-binding site consensus region . The clpA gene is expressed as a monocistronic message . Primer extension experiments define a major start point of transcription at -183 relative to the start of translation . A rho-independent terminator is located 23 bases beyond the end of the coding region . The ClpA protein is degraded in vivo in a Clp-dependent fashion (t1/2 approximately 60 min) . A fusion protein containing the first 40 amino acids of ClpA fused in frame to beta-galactosidase is degraded very rapidly in a clpA+ host (t1/2 approximately 3 min) but not in a clpA- host . This fusion protein is the first Clp-specific substrate described. J Biol Chem, 1990 May 15, 265(14), 7808 - 13 Isolation and characterization of the Drosophila nuclear envelope otefin cDNA; Padan R et al.; We have recently identified and characterized a 53-kDa inner nuclear membrane-associated protein in Drosophila and termed it otefin . Here we report the isolation and characterization of cDNA and genomic clones of the otefin gene . Based on sequence analysis, we deduced that the primary translation product has a calculated mass of 45 kDa, contains many serine and threonine residues, and is mostly hydrophilic . However, in the carboxyl terminus, there is a hydrophobic region which may serve as a membrane anchoring domain . RNA blot analysis indicated that the otefin gene codes for a single poly(A+) transcript of 1.6 kilobases and that relatively large amounts of this transcript are present during developmental stages in which many nuclear divisions occur . Polyclonal antibodies raised against the cDNA translation product react with a 58-kDa mammalian nuclear envelope protein, demonstrating evolutionary conservation. J Biol Chem, 1990 May 15, 265(14), 7742 - 7 Dissection of the functional domains of Escherichia coli carbamoyl phosphate synthetase by site-directed mutagenesis; Post LE et al.; The catalytic functions of the amino-terminal and carboxyl-terminal halves of the large subunit of carbamoyl phosphate synthetase from Escherichia coli have been identified using site-directed mutagenesis . Glycine residues at positions 176, 180, and 722 within the putative mononucleotide-binding site were replaced with isoleucine residues . Each of these mutations resulted in at least a 1 order of magnitude reduction in the Vmax for carbamoyl phosphate synthesis . The mutations on the amino-terminal half, G176I and G180I, caused slight reduction in the rate of synthesis of ATP from ADP and carbamoyl phosphate (the partial ATP synthesis reaction) but the bicarbonate-dependent ATPase reaction velocity was reduced to less than 10% of the wild-type rate . The mutant G722I, which is on the carboxy-terminal half, caused the partial ATP synthesis reaction to be reduced by 1 order of magnitude but the bicarbonate-dependent ATPase reaction was reduced only slightly . All three mutations are within regions which show homology to the putative glycine-rich loops of many ATP-binding proteins . These results have been interpreted to suggest that the two homologous halves of the large subunit of carbamoyl phosphate synthetase each contain a binding site for ATP . The NH2-terminal domain contains the portion of the large subunit that is primarily involved with the phosphorylation of bicarbonate to carboxy phosphate while the COOH-terminal domain contains the region of the enzyme that catalyzes the phosphorylation of carbamate to carbamoyl phosphate. Arch Biochem Biophys, 1990 May 15, 279(1), 151 - 7 Amino acid sequence of spinach chloroplast fructose-1,6-bisphosphatase; Marcus F et al.; The amino acid sequence of the spinach chloroplast fructose-1,6-bisphosphatase (FBPase) subunit has been determined . Placement of the 358 residues in the polypeptide chain was based on automated Edman degradation of the intact protein and of peptides obtained by enzymatic or chemical cleavage . The sequence of spinach chloroplast FBPase shows clear homology (ca . 40%) to gluconeogenic (mammalian, yeast, and Escherichia coli) fructose-1,6-bisphosphatases and 80% homology with the wheat chloroplast enzyme . The two chloroplast enzymes show near the middle of the structure a unique sequence insert probably involved in light-dependent regulation of the chloroplast FBPase enzyme activity . This sequence insert contains two cysteines separated by only 4 amino acid residues, a characteristic feature of some enzymes containing redox-active cysteines . The recent X-ray crystallographic resolution of pig kidney FBPase (H . Ke, C . M . Thorpe, B . A . Seaton, F . Marcus, and W . N . Lipscomb, 1989, Proc . Natl . Acad . Sci . USA 86, 1475-1479) has allowed the discussion of the amino acid sequence of spinach chloroplast FBPase in structural terms . It is to be noted that most of pig kidney FBPase residues shown to be either at (or close to) the sugar bisphosphate binding site or located at the negatively charged metal binding pocket are conserved in the chloroplast enzyme . The unique chloroplast FBPase insert presumably involved in light-dependent activation of the enzyme via a thioredoxin-linked mechanism can be accommodated in the surface of the FBPase molecule. J Biol Chem, 1990 May 15, 265(14), 8243 - 51 Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli; Linder ME et al.; Complementary DNAs for the G protein alpha subunits Gi alpha 1, Gi alpha 2, Gi alpha 3, and Go alpha were expressed in Escherichia coli, and the four proteins were purified to homogeneity . The recombinant proteins exchange and hydrolyze guanine nucleotide, are ADP-ribosylated by pertussis toxin, and interact with beta gamma subunits . The rates of dissociation of GDP from Gi alpha 1 and Gi alpha 3 (0.03 min-1) are an order of magnitude slower than that from rGo alpha; release of GDP from Gi alpha 2 is also relatively slow (0.07 min-1) . However, the values of kcat for the hydrolysis of GTP by rGo alpha and the three rGi alpha proteins are approximately the same, about 2 min-1 at 20 degrees C . The recombinant proteins restore inhibition of Ca2+ currents in pertussis toxin-treated dorsal root ganglion neurons in response to neuropeptide Y and bradykinin, indicating that the proteins can interact functionally with all necessary components of at least one signal transduction system . The two different receptors function with different arrays of G proteins to mediate their responses, since all four G proteins restored responses to bradykinin, while Gi alpha 2 was inactive with neuropeptide Y . Despite these results, high concentrations of activated Gi alpha proteins are without effect on adenylyl cyclase activity, either in the presence or absence of forskolin or Gs alpha, the G protein that activates adenylyl cyclase . These results are consistent with the hypothesis that G protein beta gamma subunits are primarily responsible for inhibition of adenylyl cyclase activity. J Biol Chem, 1990 May 15, 265(14), 8164 - 9 SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli; Akita M et al.; SecA is an acidic, peripheral membrane protein involved in the translocation of secretory proteins across the cytoplasmic membrane . The direct interaction of SecA with secretory proteins was demonstrated by means of chemical cross-linking with 1-ethyl-3-(3-dimethylaminoprophyl)carbodiimide . OmpF-Lpp, a model secretory protein, carries either an uncleavable or cleavable signal peptide, and mutant secretory proteins derived from uncleavable OmpF-Lpp were used as translocation substrates . The interaction was SecA-specific . None of the control proteins, which are as acidic as SecA, was cross-linked with uncleavable OmpF-Lpp . The interaction was signal peptide-dependent . The interaction was increasingly enhanced as the number of positively charged amino acid residues at the amino-terminal region of the signal peptide was increased, irrespective of the species of amino acid residues donating the charge . Finally, parallelism was observed between the efficiency of interaction and that of translocation among mutant secretory proteins . It is suggested that precursors of