Escherichia coli

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 secretory proteins interact with SecA to initiate the translocation reaction.

J Biol Chem, 1990 May 15, 265(14), 7976 - 81
Location of the protease-inhibitory region of secretory leukocyte protease inhibitor; Eisenberg SP et al.; Secretory leukocyte protease inhibitor (SLPI) is a two-domain protein that inhibits a wide range of proteases including chymotrypsin, leukocyte elastase, and trypsin . Based on its homology to other protease inhibitors and on x-ray crystallography of an SLPI-chymotrypsin complex it has been proposed that the elastase and chymotrypsin-inhibitory site is in the COOH-terminal domain and that the trypsin-inhibitory site is in the NH2-terminal domain . We have prepared muteins of SLPI by site-directed mutagenesis of a synthetic gene for the protein, followed by expression in Escherichia coli . The protease-inhibitory activities of these muteins indicate that leucine 72 in the COOH-terminal domain is at the inhibitory site for elastase and chymotrypsin . Unexpectedly, our measurements indicate that the trypsin-inhibitory site is not in the NH2-terminal domain . Instead they suggest that leucine 72 is also the inhibitory site for trypsin, even though the amino acid residues at the inhibitory sites of other trypsin inhibitors are almost always either lysine or arginine.

J Biol Chem, 1990 May 15, 265(14), 7853 - 8
Enhanced operator binding by trp superrepressors of Escherichia coli; Hurlburt BK et al.; The trp repressor of Escherichia coli binds to the operators of three operons concerned with tryptophan biosynthesis and regulates their expression . trp superrepressors can repress expression of the trp operon in vivo at lower tryptophan concentrations than those required by the wild-type repressor . The five known superrepressors have been purified and characterized using a modified filter binding assay . In four of the five superrepressors, EK13, EK18, DN46 and EK49, negatively charged wild-type residues located on the surface of the repressor that faces the operator are replaced by positively charged or neutral residues . Each of these proteins has higher affinity for the trp operator than wild-type repressor . Decreased rates of dissociation of the repressor-operator complex were found to be responsible for the higher affinities . The fifth superrepressor, AV77, has an amino acid substitution in the turn of the helix-turn-helix DNA-binding motif . This superrepressor was indistinguishable from wild-type repressor in our filter binding assay . We conclude that rapid dissociation of repressor from operator is important for trp repressor function in vivo . The negatively charged wild-type residues that are replaced in superrepressors are probably responsible for the characteristic rapid dissociation of the trp repressor from the trp operator.

Cancer Lett, 1990 May 15, 51(1), 59 - 65
Differential inhibition by an antitumoral drug 10-{gamma-diethylaminopropylamino}-6-methyl-5H-pyrido{3',4': 4,5}pyrrolo {2,3-G}isoquinoline (BD-40), a pyrido-pyrrolo-isoquinoline derivative, of in vitro DNA synthesis catalyzed by various DNA polymerases; Ono K et al.; The effects of BD-40, a pyrido-pyrrolo-isoquinoline analogue of ellipticines, and its 2-acetylated derivative (BD-84) and in vitro DNA synthesis catalyzed by purified preparations of various DNA polymerases were examined . The major conclusions are: (1) Both BD-40 and BD-84 strongly inhibit the DNA synthesis by DNA polymerase or reverse transcriptase with poly(rA).oligo(dT) as the template.primer . (2) Both compounds moderately inhibit the DNA synthesis by DNA polymerase alpha or E . coli DNA polymerase I with activated DNA . However, the DNA synthesis by DNA polymerase beta is resistant to inhibition by BD-40 and slightly sensitive to that by BD-84 . (3) BD-84 is more inhibitory than BD-40 in DNA syntheses by various DNA polymerases except in those by DNA polymerase alpha and terminal deoxyneuclotidyltransferase to which both compounds are similarly inhibitory . (4) Kinetic analyses revealed that the observed inhibitions are due to competition between the drug or the drug-bound template.primer and the free template.primer for the same binding site of the enzyme.

Blood, 1990 May 15, 75(10), 2049 - 52
Inducible production of interleukin-6 by human polymorphonuclear neutrophils: role of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha; Cicco NA et al.; The recent demonstration of the ability of human polymorphonuclear neutrophils (PMN) to secrete various cytokines in response to the granulocyte activator granulocyte-macrophage colony-stimulating factor (GM-CSF) but not to other cytokines, has led to the identification of PMN as biosynthetically active cells . In this study we have investigated the ability of PMN to secrete interleukin-6 (IL-6), a molecule known to be involved in inflammatory reactions . Using RNA blotting analysis and bioassays, we show that PMN could be induced to synthesize transcripts specific for IL-6, indistinguishable in size from IL-6 mRNA produced by activated human macrophages . Consequently, PMN released IL-6-like activity into their culture supernatants that could be neutralized by monospecific anti-IL-6 antibody . Interleukin-6 secretion by PMN, however, required previous stimulation with GM-CSF or tumor necrosis factor-alpha (TNF-alpha), whereas other cytokines, including interleukin-3 (IL-3), granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), interferon gamma (IFN-gamma), and lymphotoxin (LT), failed to induce IL-6 mRNA accumulation and protein secretion by PMN . Similar to GM-CSF and TNF-alpha, other compounds, including the inhibitor of protein synthesis cyclohexemide (CHX), endotoxin (Escherichia coli-derived lipopolysaccharide), and phorbol myristate acetate (PMA) (but not the chemoattractant N-formyl-methionyl-leucyl-phenylalanine {FMLP}), induced detectable levels of IL-6 transcripts in PMN.

J Biol Chem, 1990 May 15, 265(14), 7987 - 93
Mechanism of inactivation of the beta 2 subunit of Escherichia coli tryptophan synthase by monoclonal antibodies; Murry-Brelier A et al.; Monoclonal antibodies directed against the native form of the beta 2 subunit of Escherichia coli tryptophan synthase strongly inhibit both its tryptophan synthase and its serine deaminase activities . The mechanism of this inactivation is studied here, by monitoring quantitatively the absorption and fluorescence properties of different well-characterized successive intermediates in the catalytic cycle of tryptophan synthase . It is shown that the antibodies interfere specifically with the formation of one or the other of these intermediates . It is concluded that the antibodies either modify or block the molecular flexibility of the protein, thus preventing conformational changes that the protein has to undergo during the catalysis . At least two different stages of the catalytic process, each one sensitive to a different class of antibodies, are shown to involve molecular movements of the polypeptide chain . Indications are given on the regions of the molecule involved in these movements.

J Biol Chem, 1990 May 15, 265(14), 7832 - 6
Mutagenesis of a nucleotide-binding site of an anion-translocating ATPase; Karkaria CE et al.; The ars operon of the conjugative R-factor R773 confers resistance to arsenicals by coding for an anion pump for extrusion of arsenicals from cells of Escherichia coli . The operon encodes three structural genes arsA, arsB, and arsC . The anion pump requires only two polypeptides, the ArsA and ArsB proteins . Purified ArsA protein exhibits oxyanion-stimulated ATPase activity and was demonstrated to bind ATP by photoaffinity labeling with {alpha-32P}ATP . Analysis of the amino acid sequence deduced from the nucleotide sequence of the arsA gene suggests that the ArsA protein contains two potential nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein . A combination of site-directed and bisulfite mutagenesis was used to alter the glycine-rich region of the N-terminal putative nucleotide-binding sequence G15KGGVGKTS23 . Four mutant proteins (G18----D, G18----R, G20----S, and T22----I) were analyzed . Strains bearing the mutated plasmids were all arsenite sensitive and were unable to extrude arsenite . Each purified mutant protein lacked oxyanion-stimulated ATPase activity and ATP binding . These results suggest that the N-terminal sequence is part of a nucleotide-binding domain required for catalysis.

Gene, 1990 May 14, 89(2), 187 - 93
A general strategy for polymerization, assembly and expression of epitope-carrying peptides applied to the Plasmodium falciparum antigen Pf155/RESA; Stahl S et al.; Polymerization of DNA fragments in a head-to-tail arrangement provides a convenient way to obtain multimeric expression of a specific gene product, e.g., epitope-carrying peptides for immunological studies . A novel technique for the polymerization and assembly of peptides has been developed, involving the use of the class-IIS restriction enzyme BspMI which enables unidirectional insertion of the DNA fragments to be polymerized {Kim and Szybalski, Gene 71 (1988) 1-8} . One or several DNA fragments are polymerized in subsequent steps, using in vitro DNA polymerization, and the obtained gene constructs containing several repeats are screened and sequenced using polymerase chain reaction techniques . Using a two-step polymerization strategy a peptide, comprising two repetitive sequences from the Plasmodium falciparum malaria blood-stage antigen Pf155/RESA, was assembled and subsequently synthesized in Escherichia coli . Two different fusion proteins suitable for affinity purification were produced using a dual affinity system . Rabbits were immunized with one of the fusion proteins and the antibody response was analyzed by the enzyme-linked immunosorbent assay and immunofluorescence using the second fusion protein.

Nucleic Acids Res, 1990 May 11, 18(9), 2649 - 51
The complete AvrII restriction map of the Escherichia coli genome and comparisons of several laboratory strains; Daniels DL; The complete 13 site AvrII restriction map of the genome of E coli strain MG1655 is presented and compared with several other E . coli strains . The map was determined primarily by isolating individual AvrII fragments from pulsed-field gels, and hybridizing these large probes to a battery of mapped E . coli clones in lambda vectors . AvrII restriction patterns for eight other laboratory strains were determined and maps for seven of them deduced from the gel and comparisons between the strain genotypes, the MG1655 map, and AvrII sites in E . coli sequences taken from Genbank.

Nucleic Acids Res, 1990 May 11, 18(9), 2777 - 82
Effect of in vitro transcription on cruciform stability; Morales NM et al.; We have investigated the effect of in vitro transcription on cruciform stability . Replicative form DNA of phiX174 strain ins6240, containing a 48 bp synthetic palindrome in the J-F intercistronic region, was supercoiled in vitro to mean negative superhelical densities (sigma) ranging from 0 to 0.15 . The presence of cruciforms was probed by limited digestion with the single-strand specific nuclease Bal31 . The 48 bp palindrome was extruded at a mean sigma = -0.05, but only after heating the DNA . An in vitro transcription reaction with E . coli RNA polymerase and {alpha-32P}UTP gave identical transcripts with heated or unheated template DNA . The synthetic cruciform was stable upon binding of the RNA polymerase to the template, but it was destabilized upon movement of the transcription complex along the template . Transcription of unheated templates did not result in cruciform formation . We propose that cruciform structures in supercoiled template DNAs present no hindrance to RNA polymerase, and thus have no detectable effect on transcription elongation in vitro.

Nucleic Acids Res, 1990 May 11, 18(9), 2707 - 14
Interaction of RecA protein with pBR322 DNA modified by N-hydroxy-2-acetylaminofluorene and 4-hydroxyaminoquinoline 1-oxide; Kojima M et al.; Interaction of RecA protein of Escherichia coli with pBR322 DNA modified by N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and 4-hydroxyaminoquinoline 1-oxide (4HAQO) was investigated . RecA protein bound more efficiently to modified DNA than to unmodified DNA as judged by filter-binding and gel electrophoresis assay . The binding of RecA protein with modified DNA resulted in the stimulation of ATPase activity and the activation for RecA protein to stimulate the repressor cleavage . These abilities of RecA protein were increased proportionally to the number of adducts in the plasmid DNA (0-5 adducts) . Apurinic and alkylated DNA did not activate RecA protein . We suggest that modification of DNA by N-OH-AAF and 4HAQO provides binding sites for RecA protein and may act as an activation signal for SOS response.

J Chromatogr, 1990 May 11, 506, 319 - 26
High-performance liquid chromatography of transfer ribonucleic acids on spherical hydroxyapatite beads; Yamakawa Y et al.; High-performance liquid chromatography (HPLC) on newly developed spherical beads of hydroxyapatite was applied to the analysis of purified E . coli tRNAs (Val, Met, Tyr and Phe) . tRNAs were eluted from the column separately with appreciable differences in retention time by a 45-min gradient of phosphate buffer (pH 6.8) of concentration from 64 to 123 mM; both the retention times and peak areas of respective tRNAs were highly reproducible . Total tRNA (tRNA(Total)) preparations obtained from E . coli and B . subtilis were also analysed on the column . It is possible even to elute tRNA(Total) which may, in general, contain 60 or more tRNA species with a relatively shallow gradient such as 75-132 mM . The recovery of tRNA from the column was as high as 90% . Owing to the complicated composition, the elution profile of tRNA(Total) had a wide spread irregular shape but, with a 1-h gradient more than ten peaks were easily detected . When the amino acid-accepting activity of tRNA in the eluate of tRNA(Total) was determined, for ten specific tRNAs, each activity peak was eluted sharply from the column . In addition, several tRNA activities were eluted in different fractions . This indicates that isoacceptors were separated by the column . The results show that HPLC on hydroxyapatite beads is useful for the purification and characterization of tRNA.

Nature, 1990 May 10, 345(6271), 182 - 4
Probing the calcium-induced conformational transition of troponin C with site-directed mutants; Fujimori K et al.; The contraction of skeletal muscle is regulated by calcium binding to troponin C (TnC) . TnC consists of two spatially independent domains, each of which contains two metal ion binding sites . Calcium binding to the regulatory sites of the N-terminal domain triggers muscle contraction by a series of conformational changes . Site-directed mutagenesis offers a means of elucidating the links in this signal path between TnC and actin-myosin crossbridges . Such mapping is possible if the mutants shift the equilibrium between 'on' and 'off' states of the regulatory complex while maintaining the coupling between calcium binding and tension development . Candidate amino-acid residues for yielding this information would be in positions remote from the calcium-binding sites and from the site of development of tension . Analysis of the crystal structure of TnC and of the model of the calcium-activated molecule has enabled us to identify two such residues: Glu 57 and Glu 88 . In separate experiments we have replaced each of these residues by lysines . The resulting reduction in calcium affinity indicates that these residues have a long-range effect on calcium binding . This result may reflect the formation of a salt bridge between positions 57 and 88 that is not present in the native molecule . Moreover, the level of tension recovery when the mutants are incorporated into muscle suggests that the interaction between TnC and other muscle components has also been altered . Thus, these residues may participate in the contraction signal transmission.

Biochim Biophys Acta, 1990 May 9, 1024(1), 111 - 21
Ion channel activities in the Escherichia coli outer membrane; Buechner M et al.; The electrical properties of Escherichia coli cells were examined by the patch-clamp technique . Giant cells or giant spheroplasts were generated by five different methods . By electron micrographic and other criteria we determined that the patches are most likely from the outer membrane . We regularly observed currents through at least two types of channels in this membrane . The first current is mechanosensitive and voltage-dependent, and can be observed in single gene mutants of the known major porins (ompF, ompC, phoE, lamB); this channel may represent a minor porin or a new class of outer membrane protein . The possible identity of the second, voltage-sensitive channel with one of the known outer membrane proteins is being explored . The high-resistance seals consistently formed on these patches and the presence of gated ion channels suggest that most of the pores of the outer membrane are not statically open, as commonly held, but are closed at rest and may be openable by physiological stimuli.

Biochemistry, 1990 May 8, 29(18), 4440 - 6
Effects of an anti-alpha monoclonal antibody on interaction of Escherichia coli RNA polymerase with lac promoters; Riftina F et al.; The anti-alpha monoclonal antibody, mAb 126C6, has been used to investigate the role of the alpha subunit in transcription initiation . mAb 126C6 strongly inhibits cAMP-CRP-dependent abortive initiation with lac P+, partially inhibits abortive initiation with the lac L8UV5 promoter, and is without effect on the d(A-T)n-directed synthesis of r(A-U)n . DNase I footprinting shows that the preformed mAb 126C6-RNA polymerase complex does not bind to cAMP-CRP-lac P+; RNA polymerase specific protection is largely lost after incubation of the preformed RPo with mAb 126C6 . Kinetic analysis of open complex formation by mAb 126C6-RNA polymerase with lac L8UV5 showed that changes in both the binding and the rate of isomerization account for the observed inhibition, with the isomerization step affected to a greater extent . Binding of cAMP-CRP to lac L8UV5 is RNA polymerase dependent . DNase I footprints show that as a consequence of mAb 126C6 binding of the preformed cAMP-CRP-lac L8UV5-RNA polymerase RPo, CRP dissociates from its site on the promoter . RNA polymerase protection of the promoter upstream from -41 is also lost . DNase I footprinting of mAb 126C6-RNA polymerase complexed with cAMP-CRP-lac P+ or -lac L8UV5 suggests that interactions between CRP and RNA polymerase are affected by binding of the anti-alpha mAb 126C6 to RNA polymerase . Protection methylation studies demonstrate that the formation of the mAb 126C6-RNA polymerase-lac L8UV5 open complex occurs at a slower rate and that nonoptimal contacts are established between mAb 126C6-RNA polymerase-lac L8UV5 promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 May 8, 29(18), 4268 - 77
Fluorescence characterization of the interaction of various transfer RNA species with elongation factor Tu.GTP: evidence for a new functional role for elongation factor Tu in protein biosynthesis; Janiak F et al.; The ubiquity of elongation factor Tu (EF-Tu)-dependent conformational changes in amino-acyl-tRNA (aa-tRNA) and the origin of the binding energy associated with aa-tRNA.EF-Tu.GTP ternary complex formation have been examined spectroscopically . Fluorescein was attached covalently to the 4-thiouridine base at position 8 (s4U-8) in each of four elongator tRNAs (Ala, Met-m, Phe, and Val) . Although the probes were chemically identical, their emission intensities in the free aa-tRNAs differed by nearly 3-fold, indicating that the dyes were in different environments and hence that the aa-tRNAs had different tertiary structures near s4U-8 . Upon association with EF-Tu.GTP, the emission intensities increased by 244%, 57%, or 15% for three aa-tRNAs due to a change in tRNA conformation; the fourth aa-tRNA exhibited no fluorescence change upon binding to EF-Tu.GTP . Despite the great differences in the emission intensities of the free aa-tRNAs and in the magnitudes of their EF-Tu-dependent intensity increases, the emission intensity per aa-tRNA molecule was nearly the same (within 9% of the average) for the four aa-tRNAs when bound to EF-Tu-GTP . Thus, the binding of EF-Tu.GTP induced or selected a tRNA conformation near s4U-8 that was very similar, and possibly the same, for each aa-tRNA species . It therefore appears that EF-Tu functions, at least in part, by minimizing the conformational diversity in aa-tRNAs prior to their beginning the recognition and binding process at the single decoding site on the ribosome . Since an EF-Tu-dependent fluorescence change was also observed with fluorescein-labeled tRNA(Phe), the protein-dependent structural change is effected by direct interactions between EF-Tu and the tRNA and does not require the aminoacyl group . The Kd of the tRNA(Phe).EF-Tu.GTP ternary complex was determined, at equilibrium, to be 2.6 microM by the ability of the unacylated tRNA to compete with fluorescent Phe-tRNA for binding to the protein . Comparison of this Kd with that of the Phe-tRNA ternary complex showed that in this case the aminoacyl moiety contributed 4.3 kcal/mol toward ternary complex formation at 6 degrees C but that the bulk of the binding energy in the ternary complex was derived from direct protein-tRNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1990 May 8, 29(18), 4296 - 304
Mechanism of adenylate kinase . Are the essential lysines essential?
Tian GC, Yan HG, Jiang RT, Kishi F, Nakazawa A, Tsai MD.
Using site-specific mutagenesis, we have probed the structural and functional roles of lysine-21 and lysine-27 of adenylate kinase (AK) from chicken muscle expressed in Escherichia coli . The two residues were chosen since according to the nuclear magnetic resonance (NMR) model {Mildvan, A . S., & Fry, D . C . (1987) Adv . Enzymol . 58, 241-313}, they are located near the alpha- and the gamma-phosphates, respectively, of adenosine 5'-triphosphate (ATP) in the AK-MgATP complex . In addition, a lysine residue (Lys-21 in the case of AK) along with a glycine-rich loop is considered "essential" in the catalysis of kinases and other nucleotide binding proteins . The Lys-27 to methionine (K27M) mutant showed only slight increases in kcat and Km, but a substantial increase (1.8 kcal/mol) in the free energy of unfolding, relative to the WT AK . For proper interpretation of the steady-state kinetic data, viscosity-dependent kinetics was used to show that the chemical step is partially rate-limiting in the catalysis of AK . Computer modeling suggested that the folded form of K27M could gain stability (relative to the wild type) via hydrophobic interactions of Met-27 with Val-179 and Phe-183 and/or formation of a charge-transfer complex between Met-27 and Phe-183 . The latter was supported by an upfield shift of the methyl protons of Met-27 in 1H NMR . Other than this, the 1H NMR spectrum of K27M is very similar to that of WT, suggesting little perturbation in the global or even local conformations.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 May 8, 29(18), 4335 - 40
Activation of Escherichia coli F1-ATPase by lauryldimethylamine oxide and ethylene glycol: relationship of ATPase activity to the interaction of the epsilon and beta subunits; Dunn SD et al.; The stimulation of the ATPase activity of Escherichia coli F1-ATPase by the detergent lauryldimethylamine oxide (LDAO) and the relationship of this activation to removal of the inhibitory epsilon subunit were studied . The detergent caused a dramatic decrease in the affinity of epsilon-depleted enzyme for epsilon subunit, suggesting that release of epsilon is involved in LDAO activation . However, even in the absence of any epsilon subunit, the detergent caused a 140% increase in activity, indicating activation by effects independent of epsilon . In contrast, the addition of 30% ethylene glycol to the reaction buffer caused a modest inhibition of the ATPase activity of epsilon-depleted F1-ATPase but rendered the enzyme insensitive to inhibition by epsilon subunit . This solvent prevented the cross-linking of epsilon to beta by a water-soluble carbodiimide, although epsilon remained linkable to both beta and gamma by dithiobis(succinimidyl propionate) . Thus, epsilon was not dissociated from F1-ATPase, but its intimate interaction with the beta subunit was altered . These results suggest that the inhibitory action of epsilon is expressed through its interaction with beta . Kinetic analysis revealed that LDAO activated hydrolysis at both the high- and low-affinity promotional sites, with little change in Km values . Ethylene glycol caused a substantial increase in Km at the low-affinity promotional site and made the enzyme resistant to inhibition by aurovertin D.

Biochemistry, 1990 May 8, 29(18), 4434 - 9
Evidence for allosteric coupling between the ribosome and repressor binding sites of a translationally regulated mRNA; Tang CK et al.; Escherichia coli ribosomal protein S4 is a translational repressor regulating the expression of four ribosomal genes in the alpha operon . In vitro studies have shown that the protein specifically recognizes an unusual mRNA pseudoknot secondary structure which links sequences upstream and downstream of the ribosome binding site for rpsM (S13) {Tang, C . K., & Draper, D . E . (1989) Cell 57, 531} . We have prepared fusions of the rpsM translational initiation site and lacZ that allows us to detect repression in cells in which overproduction of S4 repressor can be induced . Twenty-five mRNA sequence variants have been introduced into the S13-lacZ fusions and the levels of translational repression measured . Sets of compensating base changes confirm the importance of the pseudoknot secondary structure for translational repression . An A residue in a looped, single-stranded sequence is also required for S4 recognition and may contact S4 directly . Comparison of translational repression levels and S4 binding constants for the set of mRNA mutations show that nine mutants are repressed much more weakly than predicted from their affinity for S4; in extreme cases no repression can be detected for variants with unchanged S4 binding . We suggest that the mRNA contains functionally distinct ribosome and repressor binding sites that are allosterically coupled . Mutations can relieve translational repression by disrupting the linkage between the two sites without altering S4 binding . This proposal assigns to the mRNA a more active role in mediating translational repression than found in other translational repression systems.

Biochemistry, 1990 May 8, 29(18), 4263 - 8
Initiation of in vivo protein synthesis with non-methionine amino acids; Chattapadhyay R et al.; Methionine is the universal amino acid for initiation of protein synthesis in all known organisms . The amino acid is coupled to a specific initiator methionine tRNA by methionyl-tRNA synthetase . In Escherichia coli, attachment of methionine to the initiator tRNA (tRNA(fMet)) has been shown to be dependent on synthetase recognition of the methionine anticodon CAU (complementary to the initiation codon AUG), {Schulman, L . H., & Pelka, H . (1983) Proc . Natl . Acad . Sci . U.S.A . 80, 6755-6759} . We show here that alteration of the anticodon of tRNA(fMet) to GAC or GAA leads to aminoacylation of the initiator tRNA with valine or phenylalanine . In addition, tRNA(fMet) carrying these amino acids initiates in vivo protein synthesis when provided with initiation codons complementary to the modified anticodons . These results indicate that the sequence of the anticodon of tRNA(fMet) dictates the identity of the amino acid attached to the initiator tRNA in vivo and that there are no subsequent steps which prevent initiation of E . coli protein synthesis by valine and phenylalanine . The methods described here also provide a convenient in vivo assay for further examination of the role of the anticodon in tRNA amino acid acceptor identity.

Biochim Biophys Acta, 1990 May 8, 1038(3), 291 - 4
Zinc binding and its trapping by allosteric transition in glucosamine-6-phosphate deaminase from Escherichia coli; Altamirano MM et al.; Glucosamine-6-phosphate isomerase deaminase from Escherichia coli, a typical allosteric enzyme, becomes less cooperative and 50% inhibited when treated with zinc . This metal cation behaving as a tight-bound and slow partial inhibitor . Modification of a pair of vicinal reactive thiols with some sulfhydryl reagents mimics this effect . On the other hand, sulfhydryl reactivity disappears in the presence of saturating concentrations of Zn2+, which does not modify the kinetics of S-methylated enzyme, a finding that indicates that vicinal thiols are an essential part of the zinc-binding site . Allosteric activation of the deaminase causes trapping of the metal, which cannot be released by dialysis against a buffer containing EDTA . Cadmium and nickel(II) cations also produce a similar effect.

Biochemistry, 1990 May 8, 29(18), 4410 - 9
Denaturant-dependent folding of bovine pancreatic trypsin inhibitor mutants with two intact disulfide bonds; Hurle MR et al.; The equilibrium and kinetic behavior of the guanidine hydrochloride (Gdn-HCl) induced unfolding/refolding of four bovine pancreatic trypsin inhibitor (BPTI) mutants was examined by using ultraviolet difference spectroscopy . In three of the mutants, we replaced the buried 30-51 disulfide bond with alanine at position 51 and valine (Val30/Ala51), alanine (Ala30/Ala51), or threonine (Thr30/Ala51) at position 30 . For the fourth mutant, the solvent-exposed 14-38 disulfide was substituted by a pair of alanines (Ala14/Ala38) . All mutants retained the 5-55 disulfide . Experiments were performed under oxidizing conditions; thus, both the unfolded and folded forms retained two native disulfide bonds . Equilibrium experiments demonstrated that all four mutants were destabilized relative to wild-type BPTI . However, the stability of the 30-51 mutants increased with the hydrophobicity of the residue substituted at position 30 . Kinetic experiments showed that all four mutants contained two minor slow refolding phases with characteristics of proline isomerization . The specific behavior of the phases depended on the location of the disulfide bonds . The major unfolding/refolding phase for each of the 30-51 mutants was more than an order of magnitude slower than for Ala14/Ala38 or for BPTI in which the 14-38 disulfide bond was specifically reduced and blocked with iodoacetamide {Jullien, M., & Baldwin, R . L . (1981) J . Mol . Biol . 145, 265-280} . Since this effect is independent of the stability of the protein, it is consistent with a model in which the proper docking of the interior residues of the protein is the rate-limiting step in the folding of these mutants.

FEBS Lett, 1990 May 7, 264(1), 121 - 4
A nucleotide sequence in the translation start signal region is involved in heat shock-induced translation arrest in Escherichia coli; Kuriki Y; In Escherichia coli synthesis of several proteins is transiently depressed upon heat shock treatment . A comparison of nucleotide sequences of the genes encoding these proteins revealed the occurrence of a consensus sequence, GAGGAA(N)3-6ATG, in their translation start signal region . To examine whether this sequence is involved in heat shock-induced depression of protein synthesis, DNA segments corresponding to this region of four of these genes, fusA, rpoB, glnS, and pheT, were synthesized, and each of them was fused in frame with the lacZ gene on the open reading frame vector pORF1 . The effect of heat shock on the synthesis of beta-galactosidase encoded by these fused genes was then studied in E . coli . It was thus found that beta-galactosidase synthesis starting from the inserted translation start signal was arrested transiently upon temperature shift-up from 30 to 42 degrees C . I conclude that the heat shock-induced depression of gene expression is an event taking place at the initiation of translation.

FEBS Lett, 1990 May 7, 264(1), 25 - 8
Modification of a glnB-like gene product by photosynthetic electron transport in the cyanobacterium Synechococcus 6301; Harrison MA et al.; Covalent modification of a 13 kDa soluble-phase protein occurs during adaptation of cells of the cyanobacterium Synechococcus 6301 (mutant AN112) to light specifically absorbed by photosystem II . This adaptation is accompanied by functional changes indicative of altered excitation energy distribution between the photosystems . The 13 kDa protein is identified by solid-phase N-terminal sequencing as a protein related to PII, the glnB gene product of E . coli . In E . coli, the PII protein undergoes uridylylation and acts as a regular of glutamine synthetase at both the post-translational and transcriptional levels . The implications of modification of a transcriptional regulator by photosynthetic electron transport are discussed.

J Mol Biol, 1990 May 5, 213(1), 27 - 36
Promoters and autogenous control of the Escherichia coli hupA and hupB genes; Kohno K et al.; Three start sites and a single start site for transcription of the hupB and hupA genes, respectively, have been identified in Escherichia coli . Preceding the RNA start sites are DNA sequences that conform to canonical promoter consensus sequences . The two most upstream promoters of the hupB gene function in vivo at comparable efficiency, while the third is not expressed significantly . Both hupB and hupA genes possess a DNA sequence with a rho-independent transcriptional terminator in their respective regions downstream from the coding regions . The hup genes are both transcribed in vivo into monocistronic mRNA molecules . Upon introduction of an HU-overproducing plasmid carrying either the hupB or the hupA gene into the wild-type and hup single deletion mutants, the intracellular levels of mRNA from the chromosomal hup genes are reduced . The HU-1 and HU-2 proteins both repress both hup genes, repression of the hupB gene being less efficient . The HU protein selectively represses mRNA synthesis starting at the hup promoters in the hupB promoter-CmR and hupA promoter-KmR fusion genes, but does not have a negative regulatory effect on mRNA synthesis from the true CmR and KmR promoters . These findings suggest that the signals for the actions of HU proteins are located in the DNA regions upstream from the sites near the 5' extremities of the coding regions of the hupB and hupA genes.

J Mol Biol, 1990 May 5, 213(1), 123 - 34
Antitermination of characterized transcriptional terminators by the Escherichia coli rrnG leader region; Albrechtsen B et al.; We have used a plasmid antitermination test system to examine the response of an Escherichia coli rRNA operon antiterminator to transcription through Rho-dependent and Rho-independent terminator-containing fragments . We also monitored transcription through multiple copies of a terminator to explore the mechanism of rrn antitermination . Four principal observations were made about antitermination and transcriptional terminators . (1) The rrn antiterminator mediated efficient transcription through Rho-dependent terminators . (2) Under the influence of the rrn antiterminator, RNA polymerase transcribed through two and three copies of the Rho-dependent 16 S----terminator with nearly the same efficiency as through one . (3) The antiterminator had less effect on fragments containing Rho-independent terminators; the rpoC t fragment and three fragments derived from the rrnB terminator region stopped antiterminated transcription . Four other Rho-independent terminator fragments were weakly antiterminated in our test system . (4) Surprisingly, the strength of these terminator fragments was not strongly related to properties such as the -delta G or number of trailing uridine residues of their canonical Rho-independent structures, but appears to be related to additional downstream terminators . We have drawn the following conclusions from these experiments . First, that ribosomal antitermination primarily reverses Rho-dependent termination by modifying the RNA polymerase elongation complex . Transcription through a 1700 nucleotide, multiple terminator sequence showed that the antiterminator caused persistent changes in the transcription process . Second, that fragments derived from the Rho-independent rrnB and rpoBC terminator regions can effectively stop antiterminated transcription . Third, that efficient in vivo termination may often involve regions with complex multiple terminators.

J Biol Chem, 1990 May 5, 265(13), 7501 - 6
Isolation, characterization, and expression in Escherichia coli of a cDNA encoding rat heme oxygenase-2; Rotenberg MO et al.; In a recent study (Cruse, I., and Maines, M.D . (1988) J . Biol . Chem . 263, 3348-3353), we reported the isolation of a small cDNA fragment encoding a portion of heme oxygenase-2 through immunological screening of a rat testis cDNA library in lambda gt11 . We have now used this 274-base pair (bp) cDNA fragment as a hybridization probe for rescreening of the same library, and have thereby recovered a number of additional positive isolates . Of these, three candidates of approximately 900, 1100, and 1300 bp, respectively, were subsequently subcloned and sequenced . Although differing in length, the sequences of these clones were found to be otherwise identical . Moreover, the length of isolate 18B, 1284 bp, corresponded well with that of the single mRNA species (approximately 1300-1350 nucleotides) detected through Northern blot hybridization analysis of rat testis total and poly(A)+RNA . This full- or near full-length cDNA encodes a 315-amino acid protein with a molecular weight of 35,757, in good agreement with the 36,000 estimated molecular weight of heme oxygenase-2 . When expressed in Escherichia coli, cDNA encodes a protein that cross-reacts with heme oxygenase-2 antiserum (as assayed by Western immunoblotting) and yields high levels of heme oxygenase activity in bacterial soluble cell extracts . Finally, computer analysis of the heme oxygenase-2 cDNA sequence indicates that the predicted amino acid sequence and hydropathy profile of the heme oxygenase-2 protein exhibit similarity with heme oxygenase-1.

J Biol Chem, 1990 May 5, 265(13), 7478 - 84
Truncations of a secretory protein define minimum lengths required for binding to signal recognition particle and translocation across the endoplasmic reticulum membrane; Okun MM et al.; Nascent preproinsulin interacts with endoplasmic reticulum membranes after approximately 70-80 residues of the 116-amino acid precursor are polymerized (Eskridge, E . M., and Shields, D . (1983) J . Biol . Chem . 258, 11487-11491) . To understand the relationship between the size of a nascent presecretory polypeptide and the efficiency of its translocation across the endoplasmic reticulum membrane, recombinant DNA molecules were generated that encoded a series of preproinsulin derivatives with the same NH2 terminus as preproinsulin and progressively shorter COOH termini . The DNA was transcribed, the in vitro transcription products were translated in the wheat germ cell-free translation system, and the interaction of the resulting truncated polypeptides with signal recognition particle (SRP) and with microsomal membranes was analyzed . Truncations composed of 78 and 64 amino acids were translocated across the endoplasmic reticulum membrane, and translocation was found to be strictly co-translational and SRP-dependent . Translocation efficiency at low membrane concentrations was reduced for these truncated molecules relative to full-length preproinsulin . Most significantly, translation of the 64-residue polypeptide was arrested by SRP after only 50 amino acids were polymerized . This suggests that the initial interaction of nascent secretory proteins with SRP occurs when only 10 residues of the signal peptide protrude from the large ribosomal subunit.

J Biol Chem, 1990 May 5, 265(13), 7351 - 9
Kinetic analysis of the pre-equilibrium steps in the self-assembly of RecA protein from Escherichia coli; Wilson DH et al.; Total intensity light scattering is employed to investigate the self-assembly kinetics of RecA protein . Reaction conditions are employed where the kinetics of self-assembly are slow enough to yield reliable scattered intensity measurements over the range of scattering angles from 40 to 130 degrees as a function of time . From these measurements the time-dependent behavior of the weight average molecular weight, Mr, and radius of gyration, RG, of the associating protein species as a function of {MgCl2}, {NaCl}, {RecA}, and pH was determined . The temperature dependence of RecA self-assembly was also investigated and allowed an evaluation of the activation thermodynamic parameters of association . Results reveal RecA self-assembly is bi-phasic under all conditions examined . The first phase, referred to as "filamentation" is second-order in {RecA} and occurs via a quasi linear condensation scheme with an Arrhenius activation energy of 88.6 kcal/mol . Filamentation assembly involves the uptake of one proton, one MgCl2, the release of five to six NaCls, and is driven by the release of approximately 70 water molecules . The evaluated activation parameters of the first kinetic phase are consistent with the proposition that linear self-assembly of RecA protein into ordered filaments is entropically driven . The second kinetic phase, referred to as "bundling" is greater than second-order in both {RecA} and {MgCl2}, is considerably slower that filamentation assembly, and is apparently initiated by 2nd order collisions of linear filaments.

J Mol Biol, 1990 May 5, 213(1), 159 - 66
Crystallization of Escherichia coli catabolite gene activator protein with its DNA binding site . The use of modular DNA; Schultz SC et al.; To obtain crystals of the Escherichia coli catabolite gene activator protein (CAP) complexed with its DNA-binding site, we have searched for crystallization conditions with 26 different DNA segments greater than or equal to 28 base-pairs in length that explore a variety of nucleotide sequences, lengths, and extended 5' or 3' termini . In addition to utilizing uninterrupted asymmetric lac site sequences, we devised a novel approach of synthesizing half-sites that allowed us to efficiently generate symmetric DNA segments with a wide variety of extended termini and lengths in the large size range (greater than or equal to 28 bp) required by this protein . We report three crystal forms that are suitable for X-ray analysis, one of which (crystal form III) gives measurable diffraction amplitudes to 3 A resolution . Additives such as calcium, n-octyl-beta-D-glucopyranoside and spermine produce modest improvements in the quality of diffraction from crystal form III . Adequate stabilization of crystal form III is unexpectedly complex, requiring a greater than tenfold reduction in the salt concentration followed by addition of 2-methyl-2,4-pentanediol and then an increase in the concentration of polyethylene glycol.

J Biol Chem, 1990 May 5, 265(13), 7604 - 9
Deletion of lysine 84 to lysine 89 enhances the cytotoxicity and the receptor binding affinity of human lymphotoxin; Wakabayashi T et al.; Human lymphotoxin (hLT) and its mutant genes have been constructed by in vitro mutagenesis and expressed in Escherichia coli . A deletion of Lys84 to Lys89 in hLT remarkably enhanced both the cytotoxicity against human WiDr cells (colon adenocarcinoma cell line) and the prostaglandin E2-inducing activity toward human synovial cells by approximately 1000- and 50-fold, respectively . The enhanced biological potency coincided with an increase of receptor binding affinity . Circular dichroism studies and the heat and folding stabilities were similar to those of the native form . We propose that the region of Lys84 to Lys89 forms a loop structure at the hLT molecular surface and plays an important role in modulating the receptor binding and biological activities of hLT.

J Biol Chem, 1990 May 5, 265(13), 7104 - 7
Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s; Hata Y et al.; ras p21 GTPase-activating protein (GAP) has been proposed to interact with the putative effector domain of ras p21s, and smg p21, a ras p21-like guanine nucleotide binding protein (G protein), has been shown to have the same amino acid sequence as ras p21s in this region . In the present studies, we examined the effects of ras p21 GAP on the GTPase activity of smg p21 purified from human platelets, of smg p21 on the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 purified from Escherichia coli, and of c-Ha-ras p21 on the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21 . ras p21 GAP stimulated the GTPase activity of c-Ha-ras p21 but not that of smg p21 . The GTP-bound form of smg p21, however, inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 in a dose-dependent manner . The half-maximum inhibition by smg p21 was obtained at 0.4 microM which was more potent than previously observed for ras p21 (2-200 microM) . The GDP-bound form also inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21, but the efficiency was 40-50% that of the GTP-bound form . smg p21 GAP1 and -2 stimulated the GTPase activity of smg p21 but not that of c-Ha-ras p21 . c-Ha-ras p21 did not inhibit the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21 . These results indicate that ras p21 GAP interacts with smg p21 without the subsequent stimulation of its GTPase activity.

J Biol Chem, 1990 May 5, 265(13), 7158 - 65
The role of Escherichia coli UvrB in nucleotide excision repair; Seeley TW et al.; The role of UvrB in determining the nucleotide dependence of Escherichia coli excision repair has been investigated . The mutation of lysine 45 in the ATPase motif of UvrB to alanine leads to an acute defect in ATP hydrolysis and failure to support incision of UV-damaged DNA . This ATP hydrolysis activity is not required for interaction of UvrB with UvrA in solution, or for formation of a damage-independent nucleoprotein complex in the presence of UvrA and nucleotide . This UvrB mutant fails, however, to support damage-specific nucleoprotein complex formation, and does not participate in a UvrA-UvrB-dependent helicase-like activity . We conclude from these results that mutation at lysine 45 in the ATPase motif of UvrB specifically inhibits a key step in nucleotide excision repair involving the UvrB ATPase-dependent translocation of nucleoprotein complexes from undamaged to damaged DNA sites.

J Biol Chem, 1990 May 5, 265(13), 7116 - 9
Characterization of wild-type and an active site mutant of human medium chain acyl-CoA dehydrogenase after expression in Escherichia coli; Bross P et al.; The cDNA of human medium chain acyl-CoA dehydrogenase (MCADH) was modified by in vitro mutagenesis, and the sequence encoding the mature form of MCADH was introduced into an inducible expression plasmid . We observed synthesis of the protein in Escherichia coli cells transformed with this plasmid with measurable MCADH enzyme activity in cell extracts . Glutamic acid 376, which has been proposed by Powell and Thorpe (Powell, P . J., and Thorpe, J . (1988) Biochemistry 27, 8022-8028) as an essential residue and the proton-abstracting base at the active site of the enzyme, was mutated to glutamine . After expression in bacteria of this plasmid, the corresponding extracts show no detectable MCADH activity, although mutant MCADH-protein production was detected by protein immunoblots . The mature enzyme and the Gln376 mutant were purified to apparent homogeneity . The wild-type enzyme is a yellow protein due to the content of stoichiometric FAD and had a specific activity which is 50% of MCADH purified from pig kidney . The Gln376 mutant is devoid of activity (less than 0.02% that of wild type, expressed enzyme) and is green because of bound CoA persulfide . Properties of the mutant enzyme suggest that the Glu376----Gln change specifically affects substrate binding . These results prove that Glu376 plays an important role in the initial step of dehydrogenation catalysis.

J Mol Biol, 1990 May 5, 213(1), 79 - 108
RNA chain elongation by Escherichia coli RNA polymerase . Factors affecting the stability of elongating ternary complexes; Arndt KM et al.; We have devised a method to follow the stability of individual ternary transcription complexes containing Escherichia coli RNA polymerase halted at many different sites along a DNA template during the transcription process . Studies of complexes formed with phage T7 DNA templates reveal at least three general classes of ternary complexes that differ dramatically in their properties . Complexes of one sort (normal complexes) are highly stable to dissociation and denaturation under a variety of solution conditions . They remain intact and active for up to 24 hours even in salt concentrations up to 1 M-K+ . This suggests that they are stabilized to a significant extent by non-ionic interactions between RNA polymerase and the nucleic acids . We consider these to be the normal complexes formed during RNA chain elongation . Complexes of a second sort (release complexes) dissociate rapidly, releasing free RNA transcripts and active RNA polymerase . The rate of dissociation is substantially enhanced by elevated concentrations of K+, hence the interaction between RNA polymerase and nucleic acids in these complexes is stabilized predominantly by ionic interactions . However, release complexes are stabilized by millimolar concentrations of Mg2+, which as been implicated in stabilization of the binding of RNA to free RNA polymerase . These complexes are formed at DNA sequences that we refer to as release sites . Analysis of DNA sequences at release sites reveals that all share a common feature, the potential to form an RNA hairpin in the region just upstream from the actual 3' end of the released RNA . Experiments incorporating IMP in the transcript and blocking potential hairpin formation with DNA oligomers support a direct role for an RNA hairpin in triggering the release reaction . Changes in the 3'-proximal DNA sequences generally have little effect on the presence or rate of the release reaction, although there are significant exceptions . The results suggest that the presence of certain RNA hairpins in the region six to ten nucleotides upstream from the transcript growing point can trigger a substantial structural transition in the ternary transcription complex, forming a "release mode" complex from which transcript dissociation is facilitated . This release, mode complex may be a central intermediate in RNA chain termination . A final class of complexes (dead-end complexes) appear to be elongating complexes that have entered a state or conformation that is stable, but is blocked in resuming the normal elongation reaction . Such complexes bear active RNA polymerase, and can be restarted after limited pyrophosphorolysis . The structural elements that determine the formation of dead-end complexes are not yet known.

J Mol Biol, 1990 May 5, 213(1), 67 - 78
Escherichia coli ribosomal protein L4 stimulates transcription termination at a specific site in the leader of the S10 operon independent of L4-mediated inhibition of translation; Zengel JM et al.; Transcription of the 11-gene S10 operon of Escherichia coli is inhibited by excess ribosomal protein L4, the product of the third gene of the operon . Previous studies suggested that L4 regulates transcription by modulating the level of readthrough at an attenuator in the S10 leader . To understand better the molecular details of the transcriptional regulation, we have determined the site of L4-induced termination of transcription using a method that allows us to map the 5' and 3' ends of newly synthesized RNA . Our results indicate that L4 stimulates termination about 140 bases from the transcription start site . Thus, the termination point is more than 30 bases upstream from the most proximal structural gene of the S10 operon, and coincides with a string of U residues on the descending side of a terminator-like hairpin structure . Since L4 is also known to inhibit translation of the S10 operon, we have analyzed the role of translation control in the protein's regulation of transcription by deleting sequences downstream from the termination site, including bases involved in translation initiation of the proximal structural gene . We find that the first 150 bases of the S10 leader contain the information sufficient for L4-mediated attenuation control and, therefore, that L4 regulates transcription by a mechanisms that is independent of the protein's inhibition of translation.

J Biol Chem, 1990 May 5, 265(13), 7662 - 8
Active site labeling of Escherichia coli transcription elongation complexes with 5-{4-azidophenacyl)thio)uridine 5'-triphosphate; Dissinger S et al.; Escherichia coli RNA polymerase transcription elongation complexes have been prepared that contain a photo-cross-linking uridine analog at only the 3' end, or one or two nucleotides removed from the 3' end, in the nascent RNA chain . Additionally, complexes have been isolated in which the analog has been substituted for every UMP residue, at positions ranging from 20 to 140 nucleotides from the 3' end . The RNA has been photochemically cross-linked to the RNA polymerase to identify the subunits that form the binding site(s) for these regions in the nascent RNA . The photo-cross-linking nucleotide analog used for these studies was 5-{4-azidophenacyl)thio)uridine-5'-triphosphate (5-APAS-UTP), which acts as a 10-15 A probe . With 5-APAS-UMP positioned only at the 3' end of the RNA, or one or two nucleotides from the 3' end, only the beta subunit appeared to be contacted . When the analog was positioned throughout the RNA, both the beta and beta' subunits were contacted . No labeling of the sigma or alpha subunits was observed with any RNA length . In addition to placing this analog at specific positions in short RNAs, we have carried out transcription studies with 5-APAS-UTP to determine the optimal UTP to analog ratio for production of full length, photoreactive transcripts . Surprisingly, we found that when transcription complexes were stalled shortly after initiation, by deletion of one ribonucleoside triphosphate to synchronize transcription, changes in transcriptional pausing occurred downstream . These results suggest that events that occur early in transcription can affect the elongation and/or termination events that occur far downstream from the promoter . This effect occurred even with UTP but was greatly enhanced by replacement of UTP with either this analog or 4-thio-UTP . By enhancing the normal transcriptional pausing event, these analogs can serve as probes of the conformational changes that may exist in paused transcription complexes.

Cell, 1990 May 4, 61(3), 497 - 504
Jun-Fos and receptors for vitamins A and D recognize a common response element in the human osteocalcin gene; Schule R et al.; We present evidence that the vitamin D response element in the human osteocalcin gene confers responsiveness to the vitamin A metabolite, retinoic acid . Retinoic acid receptor (RAR) expressed in E . coli binds to this sequence in vitro . Transfection of RAR expression vectors in cultured cells activates heterologous promoters containing this sequence in vivo . This response element contains a consensus AP-1 site TGACTCA and in vitro is bound by the Jun-Fos complex . Unexpectedly, cotransfection of Jun and Fos expression vectors suppresses basal level transcription of the osteocalcin gene and suppresses induction by both retinoic acid and vitamin D3 . Additional studies delimit an 11 nucleotide segment as a minimal hormone response element containing the AP-1 site as its core . These results indicate that two distinct classes of transcription factors can recognize common regulatory sequences, a phenomenon we refer to as cross-coupling.

Nature, 1990 May 3, 345(6270), 84 - 6
Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients; Ensoli B et al.; Kaposi's sarcoma (KS) is frequently associated with human immunodeficiency virus-1 (HIV-1) infection . Supernatants from HIV-1-infected T cells carrying the CD4 antigen promote the growth of cells derived from KS lesions of AIDS patients (AIDS-KS cells), and the HIV-1 tat gene, introduced into the germ line of mice, induces skin lesions closely resembling KS . Here we report that the tat gene product (Tat) is released from both HIV-1-acutely infected H9 cells and tat-transfected COS-1 cells . These Tat-containing supernatants specifically promote growth of AIDS-KS cells which are inhibited by anti-Tat antibodies; recombinant Tat has the same growth-promoting properties . Therefore a viral regulatory gene product can be released as a biologically active protein and directly act as a growth stimulator . These and previous data indicate that extracellular Tat could be involved in the development or progression, or both, of KS in HIV-1-infected individuals.

Biochim Biophys Acta, 1990 May 2, 1052(2), 264 - 72
Leukotriene B4 generation by human monocytes and neutrophils stimulated by uropathogenic strains of Escherichia coli; Steadman R et al.; The generation of the 5-lipoxygenase product, leukotriene B4 (LTB4) by human mononuclear phagocytes (monocytes) following incubation with 25 different uropathogenic strains of Escherichia coli correlated with the haemolytic activity of the strains (r = 0.572, P less than 0.01) . LTB4 generation by human neutrophils (PMN), however, was unrelated to this haemolytic potential (r = 0.164) . In contrast, both prelabelled monocytes and PMN were stimulated by haemolytic strains of E . coli and by haemolytic culture supernatants to release significant amounts of {3H}arachidonic acid . There was a significant correlation between haemolytic activity and {3H}arachidonic acid release generated by individual strains from monocytes (r = 0.804, P less than 0.001) and PMN (r = 0.888, P less than 0.001) . In addition, nonhaemolytic strains but not their culture supernatants were capable of causing slow release of both {3H}arachidonic acid and LTB4 from PMN and mononuclear cells . These results suggest that both the possession of haemolytic activity, and the direct interaction of bacteria with the leukocyte surface are mechanisms by which uropathogenic strains of E . coli may cause the release and metabolism of arachidonic acid . In addition, there was synergistic augmentation by nonhaemolytic bacteria of the PMN LTB4 response to haemolytic culture supernatants or to low doses of the calcium ionophore A23187 . These results support an ionophore-like mechanism for the activation of the cell by haemolysin . LTB4 generation by PMN incubated with haemolytic supernatants was also augmented by particulate zymosan in a manner dependent on the dose of zymosan, suggesting that the direct interaction of E . coli with PMN may involve an activation mechanism similar to that for zymosan . These results demonstrate differing responses of peripheral mononuclear cells and PMN from the same donors to identical strains of E . coli and suggest that the generation of the potent chemotactic agent LTB4 in response to E . coli infection in vivo need not depend solely on the elaboration of cytotoxic haemolysins by individual strains.

Biol Chem Hoppe Seyler, 1990 May, 371 Suppl, 157 - 60
Mutations in the QVVAG region of the cysteine proteinase inhibitor stefin B; Jerala R et al.; Variants of human stefin B were constructed by cassette mutagenesis . Val47 as the constituent of highly conserved QVVAG sequence was substituted by hydrophobic amino acids of increasing size - Ala, Ile and Phe . Recombinant proteins were expressed in E . coli and Ki values for papain were determined . Substitutions did not cause a major change in Ki value and we conclude that the interaction with the proteinases is not the reason for the conservation of the pentapeptide.

Mol Gen Genet, 1990 May, 221(3), 395 - 402
Rice chloroplast RNA polymerase genes: the absence of an intron in rpoC1 and the presence of an extra sequence in rpoC2; Shimada H et al.; The chloroplast genome contains sequences homologous to the Escherichia coli rpoA, rpoB and rpoC genes . The chloroplast rpoC gene is divided into rpoC1 and rpoC2, of which rpoC1 contains an intron . Comparison of the rice rpo genes with those from tobacco, spinach and liverwort revealed unique features of the rice genes; the lack of an intron in rpoC1 and the presence of an extra sequence of 381 bp in rpoC2 . The intron in rpoC1 is thus optional, and possible intron boundary sites in split rpoC1 genes can be estimated by comparison with rice rpoC1 . The extra sequence is located in the middle of rpoC2 and has repeated structures . The amino acid sequence deduced from this sequence is extremely hydrophilic and anionic . The origin and function of this sequence are discussed.

Mol Gen Genet, 1990 May, 221(3), 371 - 8
Cloning and sequencing of the hemA gene of Rhodobacter capsulatus and isolation of a delta-aminolevulinic acid-dependent mutant strain; Hornberger U et al.; The Rhodobacter capsulatus hemA gene, coding for the enzyme delta-aminolevulinic acid synthase (ALAS), was isolated from a genome bank by hybridization with a hemT probe from Rhodobacter sphaeroides . Subcloning of the initial 3.9 kb HindIII fragment allowed the isolation of a 2.5 kb HindIII-BglII fragment which was able to complement the delta-aminolevulinic acid-requiring (ALA-requiring) Escherichia coli mutant SHSP19 . DNA sequencing revealed an open reading frame coding for a protein with 401 amino acids which displayed similarity to the amino acid sequences of other known ALASs . However, no resemblance was seen to the HemA protein of E . coli K12 . Based on the sequence data, an ALA-requiring mutant strain of R . capsulatus was constructed by site-directed insertion mutagenesis . Introduction of a plasmid, containing the hemA gene of R . capsulatus on the 3.9 kb HindIII fragment, restored ALA-independent growth of the mutant indicating that there is only one gene for ALA biosynthesis in R . capsulatus . Transfer of the R' factor pRPS404 and hybridization analysis revealed that the ALAS gene is not located within the major photosynthetic gene cluster.

FEMS Microbiol Lett, 1990 May, 57(1-2), 73 - 7
A procedure for enrichment and isolation of mutants of the salt-tolerant yeast Debaryomyces hansenii having altered glycerol metabolism; Morales C et al.; The salt-tolerant yeast Debaryomyces hansenii produces and accumulates glycerol when subjected to salt stress, whereby the buoyant density of the cells is changed . This property allows for enrichment of mutants with altered glycerol metabolism by density gradient centrifugation . Colonies derived from cells with rapidly changing density following an osmotic shock were screened for increased glycerol production by observing their ability to support growth of a glycerol-requiring strain of Escherichia coli . The glycerol overproducting phenotype of two isolates was confirmed by chemical analysis.

Br Vet J, 1990 May-Jun, 146(3), 205 - 10
Changes in plasma composition in calves surviving or dying from diarrhoea; Groutides CP et al.; With the growing variety of solutions available for oral and parenteral fluid therapy it is increasingly important to define the adverse changes in plasma associated with diarrhoea, particularly those associated with a fatal outcome . The effects of E . coli-induced diarrhoea in week-old Jersey calves were measured, comparing survivors with those that died . The main effects of diarrhoea were dehydration, metabolic acidosis, pre-renal uraemia and hyponatraemia . Hypernatraemia was unusual and mild . Calves which survived tended to be hypokalaemic whereas those which died showed intensifying metabolic acidosis and hyperkalaemia . Hypoglycaemia developed, but it was not generally worse in calves which failed to survive, though there were exceptions.

Antimicrob Agents Chemother, 1990 May, 34(5), 889 - 95
Prolonged endotoxemia enhances the renal injuries induced by gentamicin in rats; Auclair P et al.; The aim of this study was to evaluate the role of chronic endotoxemia in the nephrotoxicity of gentamicin (GM) . Saline or Escherichia coli lipopolysaccharide (LPS) was administered to conscious rats by continuous intravenous perfusion (1 mg/kg per day for 7 days) from a subcutaneously implanted osmotic pump . Twenty-four hours after surgery (day zero), treatment with saline or GM (15 mg/kg; intraperitoneally, twice a day) was started for 5 days . Levels of LPS in plasma measured by Limulus amoebocyte lysate activity decreased significantly from days 1 through 8 . At days 5 and 8, the cortical concentrations of GM were higher in the LPS-perfused and GM-treated group (LPS plus GM) than they were in the saline-perfused and GM-treated group (saline plus GM) (P less than 0.05) . Blood urea nitrogen and serum creatinine remained at normal levels throughout the experiment . A significant increase of cortical tubular cell regeneration was observed in the LPS plus GM animals as compared with regeneration observed in the other groups (saline plus saline, LPS plus saline, and saline plus GM), as measured by {3H}thymidine incorporation into DNA . Moreover, histopathological nephrotoxicity scores showed a synergistic toxic effect between LPS and GM . These results demonstrate that chronic perfusion of low doses of LPS potentiates the nephrotoxicity of GM.

Curr Genet, 1990 May, 17(5), 409 - 11
Transformation of the rice blast fungus Magnaporthe grisea to hygromycin B resistance; Leung H et al.; Low frequency, integrative transformation of three fertile hermaphroditic strains of Magnaporthe grisea has been achieved using plasmid pAN7-1 and cosmid pAN7-2, which contain an Escherichia coli hygromycin B phosphotransferase gene linked to Aspergillus nidulans regulatory sequences.

DNA Cell Biol, 1990 May, 9(4), 243 - 50
Cloning and expression of a cDNA encoding human placental protein 11, a putative serine protease with diagnostic significance as a tumor marker; Grundmann U et al.; The placental protein 11 (PP11) can act as a tumor marker because of its specific association with various forms of cancer . A lambda gt11 cDNA library prepared from human placenta was screened with a polyclonal anti-PP11 antiserum . Out of 10(6) independent clones, only one clone reacted with the anti-PP11 antiserum . The isolated cDNA coded only for the carboxy-terminal part of PP11 and was subsequently used to rescreen a lambda gt10 placental cDNA library . Two cDNA clones out of 10(6) screened were identified encoding the entire protein of 369 amino acids, including a typical hydrophobic signal sequence of 18 amino acids . Expression of the PP11 cDNA coding sequence in Escherichia coli resulted in the synthesis of a protein with the expected size which can be specifically immunoprecipitated with anti-PP11 antiserum . Fractionation experiments revealed that two forms of the protein are present in the bacterial cell: a higher-molecular-weight form of approximately 42 kD in the cytoplasm and a smaller-molecular-weight form of approximately 42 kD in the periplasm . This result indicates that PP11 can be synthesized in E . coli and is process by removal of the hydrophobic signal sequence . Both the placental and the processed recombinant PP11 protein exhibit a protease activity.

Yeast, 1990 May-Jun, 6(3), 245 - 54
Cloning and sequencing of the malate synthase gene from Hansenula polymorpha; Bruinenberg PG et al.; We have cloned the MAS gene, encoding the microbody matrix enzyme malate synthase (EC 4.1.3.2.) from the methylotrophic yeast Hansenula polymorpha . The gene was isolated by screening of a genomic library with a mixed-sequence probe, based on the partial amino acid sequence of the purified enzyme . The nucleotide sequence of a 2.4-kilobase stretch of DNA covering the MAS gene was determined . The gene contains an open reading frame of 555 amino acids, amounting to a calculated molecular mass of 63,254 for the encoded protein . Comparison of the amino acid sequence with the malate synthase sequences of Escherichia coli, Brassica napus L . and Cucumis sativus L . clearly establishes the homology of all four proteins . Compared to the soluble enzyme from E . coli, the malate synthases from H . polymorpha and both plant species, which are located in the microbodies, have a short carboxy-terminal extension . In the plant malate synthases, the extension is probably involved in routing to the microbodies, since it contains the potential peroxisomal targeting signal, Ser-Arg/Lys-Leu, at the carboxy terminus . The H . polymorpha enzyme terminates with similar amino acids, but their sequence, Ser-Leu-Lys, does not conform to any of the known peroxisomal targeting signals.

J Leukoc Biol, 1990 May, 47(5), 475 - 9
Interleukin-4 downregulates interleukin-6 production in human peripheral blood mononuclear cells; Lee JD et al.; We report that recombinant human interleukin-4 (IL-4) downregulates interleukin-6 (IL-6) production by human peripheral blood mononuclear cells (PBMC) . PBMC were preincubated for up to 24 hr in the presence of IL-4 (100 U/ml) and then activated with lipopolysaccharide B Escherichia coli 026:B6 (LPS, 10 micrograms/ml), recombinant human tumor necrosis factor-alpha (TNF-alpha, 200 U/ml), or Concanavalin A (Con A, 10 micrograms/ml) . Although all these signals induced IL-6 production, IL-4-treated cells produced significantly reduced levels of IL-6 protein . This effect was dose and time dependent . We conclude that IL-4 is a potent downregulatory modulator of IL-6 expression in human PBMC.

Plasmid, 1990 May, 23(3), 248 - 51
Physical and genetic analyses of IncI2 plasmid R721: evidence for the presence of shufflon; Komano T et al.; A physical map of the 75.1-kb IncI2 plasmid R721 was constructed by using 15 restriction enzymes, and the regions of several genetic determinants including the origins of replication and of conjugal DNA transfer were located on the physical map . It was found that R721 bears a DNA region which undergoes DNA rearrangement similar to the shufflon of R64.

J Biochem (Tokyo), 1990 May, 107(5), 708 - 13
Purification and properties of recombinant rat catalase produced in Escherichia coli; Furuta S et al.; Catalase is a characteristic enzyme of peroxisomes . To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes . The concentration of recombinant catalase produced in E . coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein . The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations . The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents . The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected . The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase . Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.

Mol Microbiol, 1990 May, 4(5), 715 - 27
The arcB gene of Escherichia coli encodes a sensor-regulator protein for anaerobic repression of the arc modulon; Iuchi S et al.; The arcA (dye) and arcB genes of Escherichia coli are responsible for anaerobic repression of target operons and regulons of aerobic function (the arc modulon) . The amino acid sequence of ArcA (Dye) indicated that it is the regulator protein of a two-component control system . Here we show that ArcB is a membrane sensor protein on the basis of its deduced amino acid sequence (778 residues), hydropathicity profile, and cellular distribution . On the carboxyl end of the ArcB sequence there is an additional domain showing homology with conserved regions of regulator proteins . Deletion into this domain destroyed ArcB function . ArcB conserved a histidine residue for autophosphorylation of the sensor proteins, and aspartic residues important for the regulator proteins.

Mol Microbiol, 1990 May, 4(5), 697 - 705
The 'Bayer bridges' confronted with results from improved electron microscopy methods; Kellenberger E; In electron micrographs of conventionally prepared thin sections of Escherichia coli one observes (i) a wavy appearance of the two membranes showing frequent appositions (named adhesion sites) and (ii) intermembrane bridges after plasmolysis which, it is claimed, occur at the adhesion sites and are related to intermembrane protein transport (transmigration) . When chemical fixation is replaced by cryofixation, the observations are very different . (a) The two membranes are equally spaced and no contacts, adhesions or other sorts of connections are visible . (b) After plasmolysis the protoplast is shrunken, but the typical bridges are no longer produced . (c) In addition, when peptidoglycan is stained on conventionally prepared sections, it is revealed as a 7-nm-thick sacculus which is not interrupted at the sites of apposition . In view of the new observations, the structural concepts derived from conventionally prepared material must be revised . It is proposed that the intermembrane space is entirely filled by a gel, the outer part of which is the 7 nm thick, very stable, chemically resistant peptidoglycan (or murein) . The inner part is much less stable and is proposed to undergo rapid autolytic changes upon cell death . The large 'Bayer bridges' might then tentatively be explained as an artificial post-mortem enhancement of either a stream of proteins transmigrating across the periplasm or of a pre-existing, but not yet resolved, structure . This enhancement probably occurs during the 7-10 min between plasmolysis and fixation that are prescribed for the procedure necessary for revealing 'Bayer bridges'.

Farmakol Toksikol, 1990 May-Jun, 53(3), 20 - 2
{New possibilities in searching for immunomodulators among compounds with a steroid structure}; Kuz'mitskii BB et al.; It was shown that a phytosteroid ecdisterone which is the principle of a new tonic drug ecdisten in doses of 5-20 mg/kg is able to stimulate the primary immune reaction slightly effecting the indices of T-cell immunity activity and phagocyte functions . On increasing ecdisterone dose to 50 mg/kg inhibition of the number of antibody cells in the mouse spleen was marked.

Mol Gen Genet, 1990 May, 221(3), 491 - 4
Analysis of a mutated phage T6 receptor protein of Escherichia coli K 12; Maier C et al.; The tsx-206 allele encodes an altered Tsx protein, Tsx-206, that can no longer function as the T6 receptor . We show here that this allele also confers resistance to the Tsx-specific phages H1, H3, H8, K9, K18 and Ox1 but not to colicin K . The Tsx-206 protein still mediates the efficient permeation of deoxyadenosine across the outer membrane at low substrate concentration . A host-range mutant of phage T6, T6h3.1, was isolated which can use both the Tsx-206 and the Tsx wild-type protein as its receptor . Cloning and DNA sequence analysis of the tsx-206 allele showed that the phage resistant phenotype was associated with an Asn to Tyr substitution at position 254 of the 272-residue Tsx protein.

Mol Gen Genet, 1990 May, 221(3), 466 - 74
Export incompatibility of N-terminal basic residues in a mature polypeptide of Escherichia coli can be alleviated by optimising the signal peptide; MacIntyre S et al.; Export of the outer membrane protein, OmpA, across the cytoplasmic membrane of Escherichia coli was severely inhibited by the presence of two, three, four or six additional basic residues at the N-terminus of the mature polypeptide, but not by three similarly positioned acidic residues . Because a few bacterial proteins do possess basic residues close to the leader peptidase cleavage site and because the type of inhibition described here could pose problems in the construction of hybrid secretory proteins, we also studied means of alleviating this form of export incompatibility . Inhibition was abolished when basic residues were preceded by acidic ones . Also, the processing rates of the mutants with two or six basic residues could be partially restored by increasing the length of the hydrophobic core of the signal peptide . Taking this as a precedent, it is suggested that the structure of the signal peptide is an important feature for maintenance of a reasonable rate of translocation of those exported proteins which possess basic residue(s) at the N-terminus of the mature polypeptide.

Mol Gen Genet, 1990 May, 221(3), 331 - 8
Induction of -2 frameshift mutations within alternating GC sequences by carcinogens that bind to the C8 position of guanine residues: development of a specific mutation assay; Bintz R et al.; Using a forward mutation assay we have previously found that N-2-acetylaminofluorene (AAF), a strong chemical carcinogen, induces a majority of frameshift mutations located at specific sequences called mutation hot spots . Among these hot spot sequences, the NarI sequence (GGCGCC), is specific for -2 frameshifts (GGCGCC)----GGCC) . Interestingly, these frameshift mutations occur independently of a functional umuDC locus . Being interested in elucidating this mutation pathway we have developed a reversion assay that is specific for this class of mutations . The assay is based on the reversion of a +2 frameshift mutant of plasmid pBR322 from tetracycline sensitivity to tetracycline resistance . It is shown that only "true" reversion events lead to tetracycline resistance . The carcinogen AAF induces this reversion event at a frequency that is increased four- to fivefold over the background frequency . A series of chemical carcinogens which, like AAF, bind covalently to the C8 position of guanine, are compared for their efficiency to induce this specific mutation event . Large variations in the mutagenic efficiency of these chemicals are observed and discussed in terms of the anti/syn conformation of the carcinogen-modified guanine residue . Based on this test, we describe a convenient spot assay that this presently used in our laboratory to isolate Escherichia coli mutants affected in this mutation pathway.

Mol Gen Genet, 1990 May, 221(3), 299 - 305
A soybean gene encoding delta 1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated; Delauney AJ et al.; We have isolated several cDNA clones encoding delta 1-pyrroline-5-carboxylate reductase (P5CR, L-proline: NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) which catalyzes the terminal step in proline biosynthesis, by direct complementation of a proC mutation in Escherichia coli with an expression library of soybean root nodule cDNA . The library was constructed in the lambda ZapII vector, converted to a plasmid library by in vivo excision of recombinant pBluescript phagemids, and used for transformation of the E . coli mutant . Complementing plasmids contained inserts of about 1.2 kb which hybridized to a 1.3 kb RNA transcript in nodules, uninfected roots and leaves . DNA sequence analysis of one full length cDNA clone showed that it encodes a 28 586 Mr polypeptide with 39% amino acid identity to the E . coli P5CR sequence . Genomic analysis showed that there are two to three copies of the P5CR gene in the soybean genome . The steady-state level of P5CR mRNA in root nodules was twice as high as in uninfected roots and about five times higher than in leaves . Subjecting young seedlings to osmotic stress by watering with 400 mM NaCl resulted in an almost six-fold increase in the level of root P5CR mRNA, suggesting that this gene may be osmoregulated.

J Gen Microbiol, 1990 May, 136 ( Pt 5), 811 - 8
Cloning and expression of an alpha-amylase gene from Streptomyces thermoviolaceus CUB74 in Escherichia coli JM107 and S . lividans TK24; Bahri SM et al.; A gene coding for a thermostable extracellular alpha-amylase, carried by a 5.7 kb BamHI chromosomal DNA fragment isolated from Streptomyces thermoviolaceus strain CUB74, was cloned into Escherichia coli JM107 using, as a cloning vector, the high-copy-number plasmid pUC8 . E . coli containing a recombinant plasmid pQR300 expressed the amylase gene and exported the enzyme into the periplasmic space and the culture medium . The amylase protein expressed by E . coli had the same molecular mass (50 kDa) as that expressed by the Streptomyces parent strain, which suggests that the enzyme is processed similarly by both strains . The amylase gene was also cloned into Streptomyces lividans TK24 using pIJ702 as vector . The enzyme was stable at 70 degrees C when CaCl2 was present.

FEMS Microbiol Lett, 1990 May, 57(1-2), 83 - 6
A comparison of HEp-2 cell invasion by enteropathogenic and enteroinvasive Escherichia coli; Donnenberg MS et al.; In order to further characterize cellular invasion by enteropathogenic Escherichia coli (EPEC), we compared invasion of HEp-2 cells by EPEC and enteroinvasive E . coli (EIEC) . We used a gentamicin HEp-2 cell assay and measured bacterial recovery under conditions of varying incubation time and temperature, and in the presence or absence of inhibitors of cellular microfilaments and microtubules . We found that, unlike EIEC, EPEC did not rapidly multiply within HEp-2 cell but invaded well at 32 degrees C . While microfilament inhibitors reduced invasion by both EIEC and EPEC, microtubule inhibitors reduced invasion by EPEC only . These results suggest that EPEC and EIEC differ in their mechanisms of epithelial cell invasion.

FEMS Microbiol Lett, 1990 May, 57(1-2), 19 - 26
Acid shock proteins of Escherichia coli; Heyde M et al.; Synthesis of total cellular proteins of Escherichia coli was studied after transfer of cultures from pH 6.9 to pH 4.3 . Proteins induced by such an external pH shift down were identified by mono- and bi-dimensional electrophoresis . 30 to 45 min after an acid shift, a group of at least sixteen polypeptides was markedly induced . Four of these polypeptides corresponded to the well known heat shock proteins GroEL, DnaK, HtpG and HtpM . Their pH induction was RpoH-dependent . Three other pH-induced proteins were previously identified as stress proteins induced either by osmolarity or aerobiosis or low temperature (proteins 32 (defined in this paper), C70.0 and C62.7) . Seven other proteins were specifically induced after an acid shift and were called acid shock proteins (ASP) . The induction of one of these proteins was RpoH-dependent, whereas that of others was RpoH-independent.

FEMS Microbiol Lett, 1990 May, 57(1-2), 181 - 5
HEp-2 cell adherence and Vero cell cytotoxin production by EPEC strains isolated from children with diarrhoea in New Zealand; Gunzburg ST et al.; A total of 112 EPEC strains isolated from children with diarrhoea in New Zealand were examined for mannose-resistant HEp-2 cell adherence and production of exotoxins . Enterotoxin production was not detected in any of the strains examined . Verotoxin production was detected in 13 (11.6%) strains and of these 4 were also found to adhere to HEp-2 cells . HEp-2 cell adherence was displayed by a total of 29 (25.8%) strains of which 22 were diffusely adherent . Only 3 (2.7%) strains were shown to belong to the new virulence phenotype, entero-aggregative adherence, when examined in the adherence assay . We identified one strain with the novel characteristic of causing detachment of HEp-2 cells from glass coverslips and are further investigating this possible virulence mechanism . These results suggest that if EPEC strains are to be considered as a cause of diarrhoea, the search for new virulence factors must be extended.

FEMS Microbiol Lett, 1990 May, 57(1-2), 135 - 8
Conjugative transfer of a shuttle plasmid from Escherichia coli to Mycobacterium smegmatis {corrected}; Lazraq R et al.; The chimeric plasmid pMY10 containing the origin of replication of pAL5000, the origin of replication of pBR322, the origin of transfer of pRK2 and a kanamycin resistance gene was constructed and successfully transferred by conjugation from Escherichia coli harbouring the helper plasmid pRK4.24 into Mycobacterium smegmatis . This is the first report of conjugtive transfer of plasmid between E . coli and an acid fast organism.

Yakugaku Zasshi, 1990 May, 110(5), 293 - 303
{Molecular mechanism of N4-aminocytidine mutagenesis}; Negishi K; N4-Aminocytidine is strongly mutagenic towards E . coli, S . typhimurium, B . subtilis and coliphages phi X174 and M13mp2 . It also causes mutations in mammalian cell lines and somatic cell mutations in D . melanogaster . The sequence analysis of deoxyribonucleic acid (DNA) from mutated phages revealed that N4-aminocytidine induces both adenine-thymine (AT) to guanine-cytosine (GC) and GC to AT transitions . No transversions are detectable . When E . coli and the mammalian cells were cultured in the presence of {3H}-N4-aminocytidine, {3H}-N4-aminodeoxycytidine was found in their DNA . It is likely that N4-aminocytidine is metabolized within the cells into N4-aminodeoxy-cytidine 5'-triphosphate (dCamTP), which is then incorporated into DNA, thereby causing base-pair transitions . To prove this hypothesis, we studied the incorporation of dCamTP into polynucleotides in the in vitro DNA synthesis catalyzed by E . coli DNA polymerase I large fragment (Klenow enzyme) and DNA polymerase alpha from a mouse cell line . Both polymerases catalyze incorporation of dCamTP into DNA efficiently in place of dCTP opposite guanine, and less efficiently, but to a significant extent, in place of dTTP opposite adenine . These observations prove the erroneous nature of dCamTP as a substrate for DNA synthesis . DNA containing N4-aminocytosine was prepared by the incorporation of dCamTP into single-stranded phage DNA annealed to complementary oligonucleotides . The DNA was transfected to E . coli cells . The analysis of progeny phages indicates that N4-aminocytosine residue in DNA causes A to G or G to A mutation in the position opposite to the site where N4-aminocytosine should be incorporated.

Int J Hyperthermia, 1990 May-Jun, 6(3), 597 - 605
Effects of heat and other agents on amino acid uptake in Escherichia coli; Wainberg RH et al.; In Escherichia coli K1060 grown at 37 degrees C we observed that the uptake of both L-{3H}leucine and L-{35S}methionine was inhibited by exposure of the cells to 48 degrees C . The calcium channel blockers diltiazem and verapamil, and the anti-arrhythmic agent quinidine, inhibited the uptake of L-{3H}leucine at both 37 degrees C and 48 degrees C . Verapamil also inhibited the uptake of L-{35S}methionine at 37 degrees C, but at 48 degrees C protected against some of the heat-induced decrease in the uptake of this amino acid . The local anaesthetic procaine markedly inhibited the uptake of both labelled amino acids at temperatures between 37 degrees C and 48 degrees C . Amino acid uptake and cell killing were not correlated.

Protein Eng, 1990 May, 3(6), 523 - 6
Protein engineering of alcohol dehydrogenases: effects of amino acid changes at positions 93 and 48 of yeast ADH1; Creaser EH et al.; By protein engineering we have investigated changes to two amino acid residues (Trp93 and Ser48) in the substrate pocket of yeast alcohol dehydrogenase 1 . Upon changing Thr48 to serine we produced an enzyme which has markedly greater activity towards aliphatic alcohols with chain length up to 8, together with a general increase in catalytic activity (V/K) . Changes at position 93 were less pronounced, with the Phe enzyme being more active than the parent towards the range of alcohols but with the alanine enzyme showing very little difference from the wild-type . Enzymes with the double changes at 48 and 93 showed increased activity towards alcohols with 3-8 carbons but the increases were not additive over the single changes . The enzymes with changes at the two positions would metabolize both stereoisomers of 2-octanol whereas the parent ADH would attack only one of them . None of the engineered enzymes would attack cyclohexanol or aromatic alcohols . The results are in general agreement with the prediction that reducing the size of amino acids in the substrate pocket would enhance the ability to oxidize alcohols larger than ethanol.

Protein Eng, 1990 May, 3(6), 515 - 21
Site-directed mutagenesis of aspartic acid 372 at the ATP binding site of yeast phosphoglycerate kinase: over-expression and characterization of the mutant enzyme; Minard P et al.; A new phosphoglycerate kinase over-expression vector, pYE-PGK, has been constructed which greatly facilitates the insertion and removal of mutant enzyme genes by cleavage at newly introduced BamHI sites . This vector has been used to prepare mutant protein in appreciable (100 mg) quantities for use in kinetic, crystallographic and NMR experiments . Aspartate 372 is an invariant amino acid residue in genes known to code for a functionally active PGK . The function of this acidic residue appears to be to help desolvate the magnesium ion complexed with either ADP or ATP when this substrate binds to the enzyme . Both crystallographic and nuclear magnetic resonance experiments show that the replacement of the residue with asparagine has only minimal effects on the overall structure . The substitution of the charged carboxyl group with that of the neutral amide affects the binding of the nucleotide substrate as predicted but not, as might have been expected, the binding of 3-phosphoglycerate . The overall velocity of the enzymic reaction (Vmax) is reduced 10-fold by the substitution of aspartic acid 372 by an asparagine residue (D372N) . This reduction in Vmax is considerably less than one would expect from its known position within the structure of the enzyme . This result therefore poses questions about our understanding of charged groups at the active centres of enzymes and of the reason for their apparent conservation.

J Virol Methods, 1990 May, 28(2), 207 - 16
Automated electrokinetic analysis; description and application in virology and cell biology; Sloyer JL Jr et al.; The electrophoretic mobility (EPM) of selected macromolecules in solution was shown to be accurately determined using an automated electrokinetic analyzer, the PenKem S3000 . In addition, the S3000 was used to monitor the effects of T4 phage infection on the EPM of Escherichia coli B . EPM, expressed as the ratio of velocity in microns/sec to field strength in V/cm, was measured for calf thymus DNA, for pneumococcal capsular polysaccharide serotype 3 (PCP-3), and for bovine serum albumin (BSA) unbound in solution; values of -3.05, -2.736 and -1.176, respectively, were obtained . The EPM of these macromolecules remained the same when they were bound to latex beads . The S3000 may therefore be suitable for measurement of the EPM of unbound macromolecules . The EPM of T4 phage in solution was measured to be -1.203 . However, both the zwitterionic latex-bound T4 phage as well as T4 phage disrupted by ultrasonication exhibited an EPM of approximately -2.50, suggesting to us that binding to zwitterionic latex may cause release of phage DNA . The notion that phage DNA is responsible for the increased negative charge was supported by the observation that the EPM of E . coli B increased to the level of free DNA within 5 min when E . coli B (the host cell for phage T4) had been exposed to 10 phage particles per cell . Electronmicrographs of phage infected E . coli B cells showed numerous strands of free DNA at the bacterial surface . It is concluded that the S3000 not only measures the EPM of macromolecules in solution but that the instrument can be used also to monitor the behavior of the host cell surface in response to attachment of viral particles.

J Appl Bacteriol, 1990 May, 68(5), 453 - 9
Inactivation of Legionella pneumophila by monochloramine; Cunliffe DA; Chloramination which is used in South Australia to control the growth of Naegleria fowleri, was investigated to see if it would also control that of Legionella pneumophila . It was found that L . pneumophila was more sensitive than Escherichia coli to monochloramine . At 1.0 mg/l, a 99% kill of L . pneumophila was achieved in 15 min compared with 37 min for a 99% kill of E . coli . Combined with the stability of monochloramine, even at elevated temperatures, the results suggest that this disinfectant would control the growth of L . pneumophila in water distribution systems.

Anal Biochem, 1990 May 1, 186(2), 316 - 9
Lucigenin chemiluminescence as a probe for measuring reactive oxygen species production in Escherichia coli; Peters TR et al.; Addition of oxygen to whole cells of Escherichia coli suspended in the presence of the chemiluminescent probe bis-N-methylacridinium nitrate (lucigenin) resulted in a light emission increase of 200% of control . Addition of air to cells showed a chemiluminescent response far less than the response to oxygen . The redox cycling agents paraquat and menadione, which are known to increase intracellular production of O2- and H2O2, were also found to cause a measurable increase in lucigenin chemiluminescence in E . coli cells when added at concentrations of 1 and 0.1 mM, respectively . The oxygen-induced chemiluminescent response was not suppressed by extracellularly added superoxide dismutase or catalase . Further, the lucigenin-dependent chemiluminescent response of aerobically grown E . coli to oxygen was significantly greater than that of cells grown anaerobically . Heat-killed cells showed no increase in chemiluminescence on the addition of either oxygen, paraquat, or menadione . These results show that lucigenin may be used as a chemiluminescent probe to demonstrate continuous intracellular production of reactive oxygen metabolites in E . coli.

Anal Biochem, 1990 May 1, 186(2), 219 - 21
A microtiter plate assay for aspartate transcarbamylase; Else AJ et al.; A discontinuous, colorimetric method for the assay of aspartate transcarbamylase has been adapted for use with 96-well microtiter plates . The method is based on that of L.M . Prescott and M.E . Jones (1969 Anal . Biochem . 32, 408-419) for the detection of ureido compounds, using monoxime and antipyrine . The enzymatic reaction is carried out in a volume of 150 microliters and is stopped by the addition of 100 microliters of a color mix . After development, the absorbance at 460 nm is directly proportional to the quantity of N-carbamyl-L-aspartate up to at least 0.125 mumol and to the quantity of Escherichia coli aspartate transcarbamylase up to about 7 ng . Kinetic parameters obtained from saturation curves for L-aspartate in 50 mM Tris-acetate, pH 8.0, are indistinguishable from those previously obtained: Vmax = 26,225 mumol h-1 mg-1; S0.5 = 14.7 mmol liter-1; hill constant = 2.5.

Biochemistry, 1990 May 1, 29(17), 4188 - 93
Protein surface charges and Ca2+ binding to individual sites in calbindin D9k: stopped-flow studies; Martin SR et al.; The kinetics of calcium dissociation from two groups of site-specific mutants of calbindin D9k--a protein in the calmodulin superfamily with two Ca2+ sites and a tertiary structure closely similar to that of the globular domains of troponin C and calmodulin--have been studied by stopped-flow kinetic methods, using the fluorescent calcium chelator Quin 2, and by 43Ca NMR methods . The first group of mutants comprises all possible single, double, and triple neutralizations of three particular carboxylate groups (Glu-17, Asp-19, and Glu-26) that are located on the surface of the protein . These carboxylates are close to the two EF-hand calcium binding sites, but are not directly liganded to the Ca2+ ions . Conservative modification of these negative carboxylate side chains by conversion to the corresponding amides results in a marked reduction in the Ca2+ binding constants for both sites, as recently reported {Linse et al . (1988) Nature 335, 651-652} . The stopped-flow kinetic results show that this reduction in Ca2+ affinity derives primarily from a reduction in the Ca2+ association rate constant, kon . The estimated maximum value of the association rate constant (kon(max) for Ca2+ binding to the wild-type protein is ca . 10(9) M-1 s-1 . In contrast, for the mutant protein with three charges neutralized the maximum association rate constant is estimated to be only 2 X 10(7) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 May 1, 29(17), 4129 - 36
Three-dimensional solution structure of the reduced form of Escherichia coli thioredoxin determined by nuclear magnetic resonance spectroscopy; Dyson HJ et al.; The three-dimensional solution structure of reduced (dithiol) thioredoxin from Escherichia coli has been determined with distance and dihedral angle constraints obtained from 1H NMR spectroscopy . Reduced thioredoxin has a well-defined global fold consisting of a central five-strand beta-sheet and three long helices . The beta-strands are packed in the sheet in the order beta 1 beta 3 beta 2 beta 4 beta 5, with beta 1, beta 3, and beta 2 parallel and beta 2, beta 4, and beta 5 arranged in an antiparallel fashion . Two of the helices connect strands of the beta-sheet: alpha 1 between beta 1 and beta 2 and alpha 2 between beta 2 and beta 3 . Strands beta 4 and beta 5 are connected by a short loop that contains a beta-bulge . Strands beta 3 and beta 4 are connected by a long loop that contains a series of turn-like or 3(10) helical structures . The active site Cys-Gly-Pro-Cys sequence forms a protruding loop between strand beta 2 and helix alpha 2 . The structure is very similar overall to that of oxidized (disulfide) thioredoxin obtained from X-ray crystal structure analysis but differs in the local conformation of the active site loop . The distance between the sulfurs of Cys 32 and Cys 35 increases from 2.05 A in the disulfide bridge to 6.8 +/- 0.6 A in the dithiol of reduced thioredoxin, as a result of a rotation of the side chain of Cys 35 and a significant change in the position of Pro 34 . This conformational change has important implications for the mechanism of thioredoxin as a protein disulfide oxidoreductase.

Antimicrob Agents Chemother, 1990 May, 34(5), 786 - 91
Potentiation of susceptibility to aminoglycosides by salicylate in Escherichia coli; Aumercier M et al.; Susceptibility of Escherichia coli to kanamycin and seven other aminoglycosides has been found to be strongly potentiated by salicylate . At pH 7.5, in the presence of 15 mM salicylate and 0.5 micrograms of kanamycin per ml, the efficiency of plating of the bacteria was 2 x 10(-5), whereas there was no significant killing in the presence of kanamycin or salicylate alone . With 0.75 micrograms of kanamycin per ml, the addition of 2.5 mM salicylate was sufficient to reduce the efficiency of plating by more than 10(4)-fold . Synergistic effects were found also at pHs 6.5 and 8.5 . To determine whether the action of salicylate resulted from its behavior as a weak acid or its salicyl structure, similar experiments were carried out with acetate and salicyl alcohol . Acetate, a membrane-permeating weak acid, showed a synergistic effect on kanamycin susceptibility at pH 6.5 that was comparable to the effect seen with salicylate at pH 6.5 . However, acetate had no synergistic effect with kanamycin at pH 7.5 or 8.5 . This is consistent with the ability of acetate to increase the membrane potential of cells and the dependence of susceptibility to kanamycin and other aminoglycosides on the membrane potential . Salicyl alcohol, which has a hydroxyl group in the place of the carboxyl group that is present in salicylate, was an effective synergist with kanamycin . It was equally effective at pHs 6.5 and 7.5 and somewhat more effective at pH 8.5 . These results support the hypothesis that two effects are involved in the synergy between aminoglycosides and salicylate: a weak acid effect, possibly to increase the membrane potential, and an uncharacterized effect related to the salicyl structure.

Trends Biochem Sci, 1990 May, 15(5), 191 - 4
Logic of the Escherichia coli cell cycle; D'Ari R et al.; The logic of Escherichia coli's responses to environmental changes gives hope that its cell cycle will be equally well designed . During growth in a constant environment, internal signals trigger cell-cycle events such as replication initiation and cell division . Internal signals must also provide the cell with information about its present state, enabling it to coordinate the synthesis of cytoplasm, DNA and cell wall and maintain proper cell shape and composition . How the cell regulates these aspects of its growth is a fascinating--and as yet unfinished--story.

J Surg Res, 1990 May, 48(5), 476 - 80
Hepatic protein synthesis in a modified septic rat model; von Allmen D et al.; The aim of this study was to develop a reproducible and sustained sepsis model in rats, lasting 3-4 days and characterized by appropriate metabolic changes, including increased hepatic protein synthesis, consistent with an acute-phase response . The rat cecal ligation and puncture (CLP) model was modified by decreasing the size and number of cecal punctures and increasing fluid resuscitation, which resulted in a 60% survival rate at 96 hr compared to 20% for standard CLP . Cultures of blood and peritoneal fluid 96 hr following induction of sepsis were positive in all septic animals with a mixed aerobic and anaerobic flora but with predominant growth of Escherichia coli . Septic rats demonstrated increased serum lactate levels and leukocytosis, while serum glucose and resting energy expenditure were not different from controls . Hepatic protein synthesis, measured in vivo by flooding dose technique, was increased by 74% in septic animals . Synthesis of the acute-phase proteins alpha 1-acid glycoprotein, complement component C3, and transferrin, measured by incorporation of {14C}leucine into proteins during a 120-min isolated liver perfusion, was increased twofold in septic animals . The present modified CLP model in rats may be useful in studies on the regulation of acute-phase protein synthesis during prolonged sepsis and in experiments aimed at modulating the septic response in liver by different treatments.

JPEN J Parenter Enteral Nutr, 1990 May-Jun, 14(3), 250 - 4
The effect of high lipid diet on in vitro prostaglandin E2 and thromboxane B2 production by splenic macrophages; Ogle CK et al.; Feeding animals a diet high in linoleic acid for 7 days had no effect on the in vitro production of PGE2 by unstimulated macrophages . Feeding animals the high linoleic acid diet for 30 days greatly increased the production of PGE2 by macrophages when they were unstimulated, but decreased the production of PGE2 when they were stimulated with LPS . Feeding animals a diet high in linoleic acid for 30 days increased the production of TxB2 by macrophages when they were unstimulated, but decreased the production of TxB2 when they were stimulated . Normal, unstimulated splenic macrophages produced almost 80 times more TxB2 than PGE2 . However, when the macrophages were stimulated the ratio decreased to six or less because of a greater increase in PGE2 production . The high linoleic acid diet did not inhibit the antibody dependent cellular cytotoxicity of macrophages.

DNA Cell Biol, 1990 May, 9(4), 273 - 8
High-level expression of Escherichia coli tRNA (m5U54)-methyltransferase; Gu XR et al.; A cloning and high-expression system for tRNA (m5U54)-methyltransferase (RUMT) is described . Polymerase chain reaction (PCR) was used to replicate the coding sequence and create flanking restriction sites for cloning . The PCR product was then inserted into expression vectors containing the tac and PL promoters . With the PL promoter, induced cells produced about 1.5% of their soluble protein as catalytically active RUMT . With the tac promoter, up to 8% of the total cell protein was active enzyme, and RUMT was purified to near homogeneity in three steps.

J Gen Virol, 1990 May, 71 ( Pt 5), 1029 - 37
Characterization of the DA26 gene in a hypervariable region of the Autographa californica nuclear polyhedrosis virus genome; O'Reilly DR et al.; A region of the baculovirus Autographa californica nuclear polyhedrosis virus genome that is frequently found to be altered after serial passage of the virus in cell culture was characterized . Sequence analysis of this region of the genome in wild-type and mutant viruses revealed that some of the mutations affected a 675 bp open reading frame, designated DA26 . The DA26 gene was disrupted both by deletion and by insertion of sequences that resembled transposable elements . Northern blot analysis of DA26 showed that it was expressed very early after infection . DA26-specific transcripts could be detected after the 1 h viral adsorption period upon infection of cultured Trichoplusia ni cells . These transcripts were mapped by nuclease protection assays . A recombinant virus was constructed in which DA26 was disrupted by insertion of the Escherichia coli lacZ gene . This virus was viable in both T . ni and Spodoptera frugiperda cells and analysis of the kinetics of protein synthesis revealed no differences between wild-type and recombinant viruses . The disruption of DA26 also did not interfere with the ability of the virus to infect T . ni or S . frugiperda larvae.

Am J Obstet Gynecol, 1990 May, 162(5), 1282 - 3
Pneumatometra; Muram D et al.; We report a patient who had a significant amount of gas distending the uterine cavity . Severe cervical stenosis was noted at the time that curettage was performed . Because of the cervical stenosis, gas was trapped within the endometrial cavity.

Proc Natl Acad Sci U S A, 1990 May, 87(10), 3987 - 91
Reactivity of primary biliary cirrhosis sera with Escherichia coli dihydrolipoamide acetyltransferase (E2p): characterization of the main immunogenic region; Fussey SP et al.; Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial autoantibodies in the serum . The major antigens recognized by the antibodies are the E2 components of the 2-oxo acid dehydrogenase complexes, all of which possess covalently attached lipoic acid cofactors . A bacterial etiology has been proposed for the disease, and patients' antibodies are known to recognize the E2 subunits (E2p) of both mammalian and bacterial pyruvate dehydrogenase complexes . Immunoblotting and ELISA inhibition techniques using extracts of Escherichia coli deletion strains, genetically restructured E2 polypeptides, and isolated lipoyl domains demonstrate that (i) the E2o subunit of the E . coli 2-oxoglutarate dehydrogenase complex is recognized by patients' antibodies; (ii) the main immunogenic region of E2p lies within the lipoly domains; (iii) the presence of a lipoly residue within the domain is crucial for effective recognition by the antibodies; and (iv) octanoylated E2p, octanoylated E2o, and octanoylated lipoyl domain, produced by a mutant deficient in lipoate biosynthesis, are recognized by patients' antibodies but not as effectively as their lipoylated counterparts . These findings indicate that antibodies in PBC patients' sera bind to a unique peptide-cofactor conformation within the lipoyl domains of the E2 polypeptides and that this epitope is partially mimicked by substituting the lipoyl cofactor with an octanoyl group.

Proc Natl Acad Sci U S A, 1990 May, 87(10), 3957 - 61
Escherichia coli thymidylate synthase: amino acid substitutions by suppression of amber nonsense mutations; Michaels ML et al.; By using site-directed oligonucleotide mutagenesis, amber nonsense stop codons (5'-TAG-3') have been introduced at 20 sites in the Escherichia coli thymidylate synthase gene . By transforming the thyA mutant plasmids into 13 strains, each of which harbor different amber suppressor tRNAs, we were able to generate over 245 amino acid substitutions in E . coli thymidylate synthase (EC 2.1.1.45) . Growth characteristics of these mutants have been studied, yielding a body of information that includes some surprising results in light of the recently published crystal structure of the enzyme.

Proc Natl Acad Sci U S A, 1990 May, 87(10), 3713 - 7
The gamma subunit of DNA polymerase III holoenzyme of Escherichia coli is produced by ribosomal frameshifting; Flower AM et al.; The tau and gamma subunits of DNA polymerase III holoenzyme are both products of the dnaX gene . Since tau and gamma are required as stoichiometric components of the replicative complex, a mechanism must exist for the cell to coordinate their synthesis and ensure that both subunits are present in an adequate quantity and ratio for assembly . We have proposed that gamma is produced by a translational frameshift event . In this report, we describe the use of dnaX-lacZ fusions in all three reading frames to demonstrate that gamma, the shorter product of dnaX, is generated by ribosomal frameshifting to the -1 reading frame of the mRNA within an oligo(A) sequence that is followed by a sequence predicted to form a stable secondary structure . Immediately after frameshifting a stop codon is encountered, leading to translational termination . Mutagenesis of the oligo(A) sequence abolishes frameshifting, and partial disruption of the predicted distal secondary structure severely impairs the efficiency . Comparison of the expression of lacZ fused to dnaX distal to the site of frameshifting in the -1 and 0 reading frames indicates that the efficiency of frameshifting is approximately 40%.

Proc Natl Acad Sci U S A, 1990 May, 87(10), 3665 - 9
Existence of two forms of rat liver arginyl-tRNA synthetase suggests channeling of aminoacyl-tRNA for protein synthesis; Sivaram P et al.; Arginyl-tRNA synthetase (arginine-tRNA ligase, EC 6.1.1.19) is found in extracts of mammalian cells both as a free protein (Mr = 60,000) and as a component (Mr approximately 72,000) of the high molecular weight aminoacyl-tRNA synthetase complex (Mr greater than 10(6) . Several pieces of evidence indicate that the low molecular weight free form is not a proteolytic degradation product of the complex-bound enzyme but that it preexists in vivo: (i) the endogenous free form differs in size from the active proteolytic fragment generated in vitro, (ii) conditions expected to increase or decrease the amount of proteolysis do not alter the ratio of the two forms of the enzyme, and (iii) the free form contains an NH2-terminal methionine residue . A model is presented that provides a rationale for the existence of two forms of arginyl-tRNA synthetase in cells . In this model the complexed enzyme supplies arginyl-tRNA for protein synthesis, whereas the free enzyme provides arginyl-tRNA for the NH2-terminal arginine modification of proteins by arginyl-tRNA:protein arginyltransferase . This latter process targets certain proteins for removal by the ubiquitin-dependent protein degradation pathway . The necessity for an additional pool of arginyl-tRNA for the modification reaction leads to the conclusion that the arginyl-tRNA destined for protein synthesis (and/or protein modification) is channeled and unavailable for other processes . Other evidence supporting channeling in protein synthesis is discussed.

Am J Physiol, 1990 May, 258(5 Pt 2), H1498 - 506
Systemic and local effects of endotoxin on canine gracilis muscle vascular conductance; Bond RF et al.; To define the site and mechanism of action that endotoxin has on the peripheral vasculature, an in situ constant-flow double-canine gracilis muscle (GM) preparation was utilized . During systemic endotoxemia, one GM was innervated and the other was denervated during a 30-min intravenous infusion of 2 mg/kg endotoxin . Significantly increased vascular conductance (URP) in the denervated GM (106 +/- 26%) occurred compared with the innervated GM (50 +/- 7%), which suggests that decompensation is not totally dependent on neural depression . During local endotoxemia, with both GMs either intact or denervated, one GM was infused intra-arterially for 30 min with a dose of endotoxin calculated to provide a blood concentration similar to that achieved during systemic endotoxemia, whereas the other GM was infused with the vehicle . The URPs did not change significantly in either the saline or endotoxin GMs . Therefore, endotoxin does not act directly on peripheral vasculature or totally through depression of the autonomic nervous system . It apparently interacts with a systemically dependent mechanism to release a vasodepressor substance that is transported to the peripheral vasculature causing relaxation of vascular tone.

Urology, 1990 May, 35(5), 423 - 7
Electron microscopic study of acute retrograde pyelonephritis in mice; Deguchi T et al.; A strain of Escherichia coli O6:H-, which was isolated from a patient with acute pyelonephritis, was used to produce ascending pyelonephritis in mice . The histologic features at the early stage of acute pyelonephritis and the pathways of bacterial invasion and distribution in the infected kidneys were studied by using transmission electron microscope . The histologic changes were characterized by degeneration and destruction of renal pelvic and tubular epithelial cells, and a massive infiltration of polymorphonuclear leukocytes . Bacteria invaded the pelvic surface and spread over the renal medulla and cortex by contiguity twelve hours after infection . It was also shown that bacteria ascended the tubules, multiplied in the tubular lumina, destroyed the tubular epithelial cells, and spread over the renal cortex by twelve hours . In addition, bacteria and leukocytes phagocytizing bacteria were present in the capillaries in the renal interstitium twelve hours after infection.

Proc Natl Acad Sci U S A, 1990 May, 87(9), 3522 - 5
Molecular mechanisms of DNA repair inhibition by caffeine; Selby CP et al.; Caffeine potentiates the mutagenic and lethal effects of genotoxic agents . It is thought that this is due, at least in some organisms, to inhibition of DNA repair . However, direct evidence for inhibition of repair enzymes has been lacking . Using purified Escherichia coli DNA photolyase and (A)BC excinuclease, we show that the drug inhibits photoreactivation and nucleotide excision repair by two different mechanisms . Caffeine inhibits photoreactivation by interfering with the specific binding of photolyase to damaged DNA, and it inhibits nucleotide excision repair by promoting nonspecific binding of the damage-recognition subunit, UvrA, of (A)BC excinuclease . A number of other intercalators, including acriflavin and ethidium bromide, appear to inhibit the excinuclease by a similar mechanism--that is, by trapping the UvrA subunit in nonproductive complexes on undamaged DNA.

Proc Natl Acad Sci U S A, 1990 May, 87(9), 3508 - 12
Cloning and expression of a mammalian peptide chain release factor with sequence similarity to tryptophanyl-tRNA synthetases; Lee CC et al.; The termination of protein synthesis is encoded by in-frame nonsense (stop) codons . Most organisms use three nonsense codons: UGA, UAG, and UAA . In contrast to sense codons, which are decoded by specific tRNAs, nonsense codons are decoded by proteins called release factors (RFs) . Here we report the cloning of a mammalian RF cDNA by the use of monoclonal antibodies specific for rabbit RF . Functional studies showed that, when expressed in Escherichia coli, the protein encoded by this cDNA has in vitro biochemical characteristics similar to those of previously characterized mammalian RFs . DNA sequencing of this eukaryotic RF cDNA revealed a remarkable sequence similarity to bacterial and mitochondrial tryptophanyl-tRNA synthetases, with the greatest similarity confined to the synthetase active site, and no obvious similarity to bacterial RFs.

Proc Natl Acad Sci U S A, 1990 May, 87(9), 3464 - 8
Transcriptional effects of polyamines on ribosomal proteins and on polyamine-synthesizing enzymes in Escherichia coli; Huang SC et al.; We find that the transcription of various ribosomal proteins can be differentially affected by polyamines and by changes in growth rates . Using strain MG1655 of Escherichia coli K-12 (F-, lambda-), we have determined the effects of polyamines and changes in growth rate on the transcription of several ribosomal genes and the polyamine-synthesizing enzymes ornithine decarboxylase (L-ornithine carboxy-lyase; EC 4.1.1.17) and arginine decarboxylase (L-arginine carboxylyase; EC 4.1.1.19) . Ribosomal proteins S20 and L34 can be differentiated from the other ribosomal proteins studied; the transcription of S20 and L34 is especially sensitive to polyamines and less sensitive to changes in growth rates . In contrast, the transcription of S10, S15, S19, L2, L4, L20, L22, and L23 is insensitive to polyamines although it is particularly sensitive to changes in growth rates . Like S20 and L34, the transcription of ornithine decarboxylase and arginine decarboxylase is especially sensitive to polyamines . Polyamines specifically enhance the transcription of ribosomal proteins S20 and L34, and decrease that of ornithine decarboxylase and arginine decarboxylase . It is evident that polyamines can exert both positive and negative regulation of gene expression in E . coli that can be differentiated from the effects caused by changes in growth rates.

Proc Natl Acad Sci U S A, 1990 May, 87(9), 3396 - 400
Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites; Lenz J et al.; Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins . Repair depends on the ability of cellular factors to distinguish the damaged sites . Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites . A binding assay based on the use of beta-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins . Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA . These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins.

Proc Natl Acad Sci U S A, 1990 May, 87(9), 3314 - 8
The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor; Eliasson R et al.; Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2+ ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E . coli . After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions . One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several defined protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein B1) of the aerobic reductase but not by monoclonal anti-B1 antibodies . Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component . Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established . The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction . One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal . We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP.

Mutat Res, 1990 May, 241(1), 83 - 93
Genotoxicity of benzo{a}pyrene, 2-nitrofluorene and airborne particulates in the DNA-repair host-mediated assay; Heussen GA et al.; The genotoxic activity of benzo{a}pyrene (BAP), 2-nitrofluorene (NF) and airborne particulate matter was evaluated in the DNA-repair host-mediated assay after intraperitoneal or intratracheal administration . Dimethylnitrosamine (DMNA), used as a positive control, showed a genotoxic effect after both intraperitoneal and intratracheal administration, the strongest effect being found in liver, followed by lungs and kidneys, whereas a weak effect was observed in the spleen . In general no difference in genotoxicity was found between the 2 administration routes used . For BAP, although clearly positive in vitro, a moderate dose-dependent effect was found only in the liver after intraperitoneal administration . NF, which was positive in vitro both with and without a metabolizing system, produced no genotoxic effect in any of the organs tested after intraperitoneal administration . Extracts of airborne particulate matter which were genotoxic in vitro failed to cause a genotoxic effect in vivo by either route of administration . Possible explanations for the differences between the data obtained in vitro and in vivo are discussed.

J Bacteriol, 1990 May, 172(5), 2802 - 3
Characterization of the defect in the Escherichia coli mutT1 mutator gene; Bhatnagar SK et al.; With a probe constructed from the wild-type gene, a DNA fragment containing the entire mutT1 mutator gene was isolated and cloned into pUC18 . Nucleotide sequence analysis revealed that the mutator defect was most likely due to an IS1 insertion into the wild-type gene.

J Bacteriol, 1990 May, 172(5), 2779 - 81
Starvation-induced cross protection against osmotic challenge in Escherichia coli; Jenkins DE et al.; Stationary-phase Escherichia coli cultures showed enhanced osmotic resistance as compared with cultures in mid-logarithmic growth or preadapted to osmotic stress . The osmotolerance that developed during starvation or osmotic adaptation required de novo protein synthesis . Of the 22 polypeptides induced during osmotic shock, five were also starvation proteins.

J Bacteriol, 1990 May, 172(5), 2762 - 4
Second-site revertants of the P1 copN22 copy mutant; Froehlich BJ et al.; Miniplasmids with the P1 copN22 mutation have a copy number about seven times that of the wild type . Selection for reduced copy number from this plasmid led to the isolation of second-site pseudorevertants, called poc mutants . DNA sequence analysis showed that all six independent poc mutants have a single base change in the same codon of the repA gene . This implicates the amino acid at this location, either directly or indirectly, in interactions important for copy number control.

J Bacteriol, 1990 May, 172(5), 2688 - 92
Constitutive synthesis of a transport function encoded by the Thiobacillus ferrooxidans merC gene cloned in Escherichia coli; Kusano T et al.; Mercuric reductase activity determined by the Thiobacillus ferrooxidans merA gene (cloned and expressed constitutively in Escherichia coli) was measured by volatilization of 203Hg2+ . (The absence of a merR regulatory gene in the cloned Thiobacillus mer determinant provides a basis for the constitutive synthesis of this system.) In the absence of the Thiobacillus merC transport gene, the mercury volatilization activity was cryptic and was not seen with whole cells but only with sonication-disrupted cells . The Thiobacillus merC transport function was compared with transport via the merT-merP system of plasmid pDU1358 . Both systems, cloned and expressed in E . coli, governed enhanced uptake of 203Hg2+ in a temperature- and concentration-dependent fashion . Uptake via MerT-MerP was greater and conferred greater hypersensitivity to Hg2+ than did uptake with MerC . Mercury uptake was inhibited by N-ethylmaleimide but not by EDTA . Ag+ salts inhibited mercury uptake by the MerT-MerP system but did not inhibit uptake via MerC . Radioactive mercury accumulated by the MerT-MerP and by the MerC systems was exchangeable with nonradioactive Hg2+.

J Bacteriol, 1990 May, 172(5), 2642 - 9
Regulation of the glyoxylate bypass operon: cloning and characterization of iclR; Sunnarborg A et al.; In Escherichia coli, expression of the glyoxylate bypass operon appears to be controlled, in part, by the product of iclR+ . Mutations in iclR have been found to yield constitutive expression of this operon, suggesting that iclR+ encodes a repressor protein . We have cloned iclR+ by taking advantage of its tight genetic linkage with the glyoxylate bypass operon . The clone complemented a mutant allele of iclR in trans, restoring an inducible phenotype for this operon . Deletion analysis identified a region of ca . 900 base pairs that was necessary and sufficient for complementation . The nucleotide sequence of the insert was then determined . Translation of this sequence revealed an open reading frame capable of encoding a protein with Mr 29,741 preceded by a potential Shine-Dalgarno ribosome-binding site . The deduced amino acid sequence includes a region at the amino terminus that may form a helix-turn-helix motif, a structure found in many DNA-binding domains.

J Bacteriol, 1990 May, 172(5), 2535 - 40
Site-directed mutations in the repA C-terminal region of plasmid Rts1: pleiotropic effects on the replication and autorepressor functions; Zeng H et al.; We induced site-directed mutations near the 3' terminus of the gene repA, which encodes the protein of 288 amino acid residues essential for plasmid Rts1 replication, and obtained seven repA mutants . Three of them contained small deletions at the 3' terminus . Mutant repAz delta C4, which encodes a RepA protein that lacks the C-terminal four amino acids, expressed a high-copy-number phenotype and had lost both autorepressor and incompatibility functions . Deletion of one additional amino acid residue to form the RepAz delta C5 protein caused restoration of the wild-type copy number and strong incompatibility . Studies of the remaining four repA mutants, each of which contained a single amino acid substitution near the RepA C terminus, suggested that Lys-268 is involved in both ori(Rts1) activation and autorepressor-incompatibility activities and that Arg-279 contributes to ori(Rts1) activation but not to incompatibility . Lys-268 is part of a dual-lysine sequence with Lys-267 and is located 21 amino acids upstream of the RepA C terminus . A dual-lysine sequence is also found at a similar position in both mini-F RepE and mini-P1 RepA proteins.

J Bacteriol, 1990 May, 172(5), 2421 - 6
A new model for the penetration of prey cells by bdellovibrios; Tudor JJ et al.; Bdellovibrio bacteriovorus 109J and most other bdellovibrios cause prey cells to round following penetration . Bdellovibrio sp . strain W does not cause rounding of the prey . Analysis of enzyme activities during the early stages of bdellovibrio attack indicated that strain W differs from most other bdellovibrios in that there is no glycanase activity produced during penetration . Likewise, heat-killed prey were penetrated normally by strain 109J, but the resulting bdelloplast did not become round and no glycanase was detected, indicating that glycanase is not essential for penetration . Peptidoglycan from prey cells penetrated by strain W was sensitive to lysozyme, but these cells were not susceptible to attack and penetration by strain 109J, indicating that peptidoglycan deacetylation is not the primary exclusion mechanism . We propose a model in which it is the peptidase activity of the bdellovibrios which allows them to breach the peptidoglycan of their prey and in which the glycanase activity exhibited by strain 109J and other bdellovibrios is responsible for the rounding of the bdelloplast.

J Bacteriol, 1990 May, 172(5), 2367 - 71
Differential mRNA stability controls relative gene expression within the plasmid-encoded arsenical resistance operon; Owolabi JB et al.; The arsenical resistance (ars) operon of the conjugative plasmid R773 encodes an ATP-driven anion extrusion pump, conferring bacterial resistance to arsenicals . The operon contains a regulatory gene, arsR, and three structural genes, arsA, arsB, and arsC . The hydrophilic ArsA and ArsC proteins are produced in large amounts, but the hydrophobic ArsB protein, an integral membrane polypeptide, is synthesized in limited quantities . Northern (RNA-DNA) hybridizations provide evidence that the inducible operon is regulated at the level of transcription . The genes were transcribed in the presence of an inducer (arsenite) as a single polycistronic mRNA with an approximate size of 4.4 kilobases (kb) . This transcript was processed to generate relatively stable mRNA species: one of 2.7 kb, encoding the ArsR and ArsA proteins, and a second of 0.5 kb, encoding the ArsC protein . Segmental differences in stability within the polycistronic transcript are proposed to account for the differential expression of the ars genes . In addition, analysis of the mRNA structure at the 5' end of arsB suggests a potential translational block to the synthesis of this membrane protein.

J Bacteriol, 1990 May, 172(5), 2320 - 7
Analysis of regulation of the ilvGMEDA operon by using leader-attenuator-galK gene fusions; Lawther RP et al.; Five of the genes for the biosynthesis of isoleucine and valine form the ilvGMEDA operon of Escherichia coli K-12 . Expression of the operon responds to changes in the availability of isoleucine, leucine, and valine (ILV) . Addition of an excess of all three amino acids results in reduced expression of the operon, whereas limitation for one of the three amino acids causes an increase in expression . The operon is preceded by a leader-attenuator which clearly regulates the increased expression that occurs due to reduced aminoacylation of tRNA . To assess the factors that result in the reduced expression of this operon upon the addition of ILV, a series of plasmids were constructed in which the ilv regulatory region was fused to galK . In response to addition of the amino acids, expression of the galK gene fused to the leader-attenuator decreased five- to sevenfold, instead of the twofold observed for the chromosomal operon . A deletion analysis with these plasmids indicated that the ILV-specific decrease in expression required an intact leader-attenuator but not ilvGp2 or the DNA that precedes this promoter . This conclusion was supported by both S1 nuclease analysis of transcription initiation and determination of galK mRNA levels by RNA-RNA hybridization.

Virology, 1990 May, 176(1), 178 - 83
Functional substitution of the basic domain of the HIV-1 trans-activator, Tat, with the basic domain of the functionally heterologous Rev; Subramanian T et al.; The tat gene of HIV is a strong activator of the viral LTR . The Tat protein contains a highly basic domain that is important for its transport to the nuclear/nucleolar locations . The Tat basic domain when fused to Escherichia coli beta-galactosidase directed the chimeric protein to the nucleus and nucleolus . Tat mutants lacking the entire basic domain were severely defective in trans-activation . Substitution of the basic domain of Tat with that of the functionally unrelated HIV-1 Rev protein targeted the chimeric protein to the nucleolus and restored the function of Tat . In contrast, substitution with the nuclear targeting signal (NLS) of SV40 T antigen targeted the chimeric protein to the nucleus and accumulation in the nucleolar region was excluded . The Tat-NLS chimeric protein did not restore the trans-activation function of Tat efficiently . These results indicate that the arginine-rich basic domain of the trans-activator, Tat, and post-transcriptional trans-regulator, Rev, are functionally similar with regard to trans-activation of HIV-1 LTR.

EMBO J, 1990 May, 9(5), 1665 - 72
Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure; Hendrickson WA et al.; An expression system has been established for the incorporation of selenomethionine into recombinant proteins produced from plasmids in Escherichia coli . Replacement of methionine by selenomethionine is demonstrated at the level of 100% for both T4 and E . coli thioredoxins . The natural recombinant proteins and the selenomethionyl variants of both thioredoxins crystallize isomorphously . Anomalous scattering factors were deduced from synchrotron X-ray absorption measurements of crystals of the selenomethionyl proteins . Taken with reference to experience in the structural analysis of selenobiotinyl streptavidin by the method of multiwavelength anomalous diffraction (MAD), these data indicate that recombinant selenomethionyl proteins analyzed by MAD phasing offer a rather general means for the elucidation of atomic structures.

Endocrinology, 1990 May, 126(5), 2359 - 68
Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate; Husmann DA et al.; We have developed polyclonal antibodies to two synthetic peptides corresponding to the amino-(N-)terminal or carboxyl-(C-)terminal segments of the human androgen receptor (hAR) protein, as deduced from the nucleic acid sequence of the androgen receptor cDNA . Immunoreactive antisera were identified by solid phase enzyme-linked immunosorbent assay and purified by peptide affinity chromatography . Specific immunoreactivity with the hAR was confirmed by immunoblotting, using both a fusion protein produced in E . coli that contains the C-terminal 880-amino acid sequence of hAR and the full-length receptor protein produced in COS cells after transfection with a plasmid containing the entire hAR-coding region . Immunohistological evaluation of rat and human prostatic tissue using anti-C-terminal or anti-N-terminal antibodies demonstrated similar patterns of specific staining of the nuclei of epithelial and stromal cells . Castration resulted in a decrease in the amount of nuclear AR detected in the rat prostate after a short time of exposure to anti-C-terminal antibodies (less than 4 h), but did not alter the level of specific staining obtained with anti-N-terminal antibodies . This decrease in nuclear staining using anti-C-terminal antibodies could be reversed by treating castrated animals with dihydrotestosterone . When longer times of exposure to the primary antibodies were used, high levels of nuclear staining were obtained with both types of antibodies in prostate specimens from castrate as well as as intact rats . This immunohistochemical staining pattern contrasts with receptor measurements in rat prostate homogenates that indicate the partition of AR binding into the low salt (cytosolic) fraction in the castrate animal and into the high salt (nuclear) fraction in the intact animal . Our results suggest that the AR is predominantly a nuclear protein even in the absence of ligand and that dihydrotestosterone serves to tighten its association with the nucleus . These data also suggest that the immunoreactivity of anti-C-terminal antibodies is influenced by the presence of dihydrotestosterone, presumably via an alteration in the physical state of the receptor protein.

Arch Biochem Biophys, 1990 May 1, 278(2), 373 - 80
Alkylation of isocitrate lyase from Escherichia coli by 3-bromopyruvate; Ko YH et al.; The inactivation of tetrameric isocitrate lyase from Escherichia coli by 3-bromopyruvate, exhibiting saturation kinetics, is accompanied by the loss of one sulfhydryl per subunit . The substrates glyoxylate and isocitrate protect against inactivation whereas the substrate succinate does not . The modification by 3-bromopyruvate (equimolar to subunits) imparts striking resistance to digestion of isocitrate lyase by trypsin, chymotrypsin, and V8 protease as well as a major decrease in the intensity of tryptophan fluorescence . After alkylation, the sequence Gly-His-Met-Gly-Gly-Lys is found following the modified Cys residue in the tryptic peptide representing positions 196-201 . Thus Cys195 is alkylated by 3-bromopyruvate.

Mol Cell Biol, 1990 May, 10(5), 1908 - 14
Genetic exploration of interactive domains in RNA polymerase II subunits; Martin C et al.; The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase . These homologous regions may represent separate protein domains with unique functions . We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae . A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1 . All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2 . The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2 . A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2 . These suppressor mutations were not clustered in a particular region . Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.

J Infect Dis, 1990 May, 161(5), 1020 - 2
Oxygen-derived free radicals mediate the cutaneous necrotizing vasculitis induced by epinephrine in endotoxin-primed rabbits; Ward PH et al.; To assess the possible role of oxygen-derived free radical in the pathogenesis of cutaneous necrotizing vasculitis (Thomas reaction) induced by epinephrine in endotoxin-primed rabbits, animals were treated with superoxide dismutase (intraperitoneal or local), catalase, allopurinol, or ibuprofen 2 h before inducing the reaction . This is done by an earlobe intravenous injection (10 micrograms) of endotoxin followed by an intradermal injection (100 micrograms) of epinephrine in the shaved abdomen; 18-24 h later, a hemorrhagic-necrotic lesion can be observed at the site of the epinephrine injection . The reaction was completely inhibited by superoxide dismutase, catalase, and ibuprofen, while allopurinol afforded only partial protection . Thus, oxyradicals seem to participate in the pathogenesis of this reaction.

Infect Immun, 1990 May, 58(5), 1337 - 43
Net fluid secretion and impaired villous function induced by colonization of the small intestine by nontoxigenic colonizing Escherichia coli; Schlager TA et al.; The role of colonizing bacteria in the small bowel in causing diarrhea remains unclear . We examined whether colonizing, nontoxigenic Escherichia coli could alter small bowel function by determining net water and electrolyte fluxes and sucrase activity in colonized and noncolonized ileal segments by using the reversible-ileal-tie adult rabbit model . Colonization of the ileum with nontoxigenic E . coli for greater than or equal to 72 h at greater than or equal to 10(4)/cm2 was associated with significant functional derangements, as follows: (i) overt liquid diarrhea in 50% of animals colonized at greater than 10(4)/cm2; (ii) reversal of normal net ileal absorption to net secretion of water, sodium, and chloride; and (iii) significant decrease in mucosal sucrase activity . We conclude that small bowel colonization by colonizing, nontoxigenic E . coli impairs water and electrolyte absorption and sucrase activity in the absence of recognized enterotoxin, cytotoxin, invasion, or effacement traits.

Infect Immun, 1990 May, 58(5), 1232 - 9
Purification and characterization of an Escherichia coli Shiga-like toxin II variant; MacLeod DL et al.; A Shiga-like toxin II variant was purified to homogeneity from Escherichia coli TB1(pCG6), which contained the toxin genes cloned in multicopy plasmid pUC18 . The purification scheme involved polymyxin B extraction of the toxin from bacterial cells, followed by differential (NH4)2SO4 precipitation, anion- and cation-exchange fast-protein liquid chromatography, and immunoaffinity exclusion chromatography . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified toxin revealed three protein bands that migrated with calculated molecular weights of 33,000, 27,500, and 7,500 . These bands correspond to values for the A, A1, and B subunits, respectively, that would be expected on the basis of the nucleotide sequence and comparison with data for Shiga toxin and other Shiga-like toxins . Electrophoresis under nonreducing conditions resulted in disappearance of the 27,000-molecular-weight band . Western blot (immunoblot) analysis revealed three protein bands with molecular weights of 33,000, 27,500, and 7,500 . The purified toxin induced typical signs of edema disease in pigs injected intravenously with doses as small as 3 ng/kg of body weight . The 50% cytotoxic doses for Vero, PK15, and Madin-Darby bovine and canine kidney cells were 0.5, 2.0, 8.0, and 8.0 pg, respectively . The 50% lethal dose of purified toxin for mice was 0.9 pg by the intraperitoneal route . Approximately 75 micrograms of purified toxin was required to induce a 1-ml/cm fluid response in rabbit ileal loops . Antiserum to the Shiga-like toxin II variant neutralized homologous toxin, Shiga-like toxin II, and Verotoxin 2 but not Shiga-like toxin I.

Infect Immun, 1990 May, 58(5), 1159 - 66
Construction of a nontoxic fusion peptide for immunization against Escherichia coli strains that produce heat-labile and heat-stable enterotoxins; Clements JD; The 5' terminus of the gene that codes for the heat-stable enterotoxin of Escherichia coli (ST) was genetically fused to the 3' terminus of the gene that codes for the binding subunit of the heat-labile enterotoxin of E . coli (LT-B) . The ST-encoding gene used for these studies was constructed synthetically with appropriate restriction sites to permit in-frame, downstream insertion of the oligomer . For this construction, maximum expression of ST antigenicity was obtained when a seven-amino-acid, proline-containing linker was included between the LT-B and ST moieties . The LT-B-ST fusion peptide was purified by affinity chromatography and consisted of a single polypeptide chain with an apparent molecular weight of 18,000 when examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis . There was no evidence of multimer formation and no change in the mobility of the fusion peptide when it was boiled in SDS or in SDS with dithiothreitol . The LT-B-ST fusion peptide was nontoxic, and immunologic determinants of both LT and ST were recognized by antibodies to the native toxins . More importantly, the LT-B-ST fusion peptide was immunogenic . Animals immunized with crude or purified preparations containing the hybrid molecule produced antibodies that were able to recognize native toxin in vitro . Significantly, these antibodies were able to neutralize the biological activity of native ST.

Biochem Cell Biol, 1990 May, 68(5), 832 - 8
Delta Lys120, a mutation which destabilizes the ribosome-binding domain of ribosomal protein L7/L12; Laughrea M et al.; Five-residue-long deletions centered on Ala63, Ala75, and Glu118 of ribosomal protein L7/L12 gave low mutant yields (5% or less) when the mutant genes were cloned in phage M13mp18 and controlled by the L10 promotor . Deletions of Glu118-Lys120 or Lys120 (the COOH-terminus of L7/L12) gave higher mutant yields, up to 50% with L7/L12 delta Lys120 . L7/L12 delta Lys120 was not preferentially found in the S100 and not preferentially removed by LiCl washing, but was preferentially extracted from 70S ribosomes in the presence of 28-35% ethanol in 0.25-0.5 M NH4Cl . It follows that delta Lys120 destabilizes the ribosome-binding domain of ribosomal protein L7/L12 in an ethanol-containing solvent, which raises the question whether Lys120 is part of the ribosome-binding domain of L7/L12 during some step of protein synthesis or whether it is essential to preserve the conformation of the physiological ribosome-binding domain under structurally stressful conditions.

Genetika, 1990 May, 26(5), 842 - 6
{Characteristics of lysogenization of Escherichia coli K-12 strains by phage MupAp1 during zygotic induction}; Oskolkova OB et al.; Genetic proofs of the fact that lysogenization of recipient cells by Mu phage in the course of zygotic induction is normally a slowed process were obtained . Stabilization of lysogenic state seems to occur after the stage of induced excision of Mu DNA during the process of conjugational crossing followed by integration as such of Mu DNA-containing recombinant structure into the recipient chromosome.

Genetika, 1990 May, 26(5), 833 - 41
{The role of transposons in replication: Tn5 suppresses ts-mutation for plasmid RP1 replication in Escherichia coli cells}; Baumanis GE et al.; Effect of restoration by transposon Tn5 of genetic damage in RP1 plasmid replication (named transposon suppression) was described . Hybrid plasmid, a derivative of RP1 and RP4, having ts mutation for replication--tsr12 and deletion in the aphA gene controlling kanamycin resistance, was constructed . Five of derivatives of this plasmid containing transposon Tn5 were made, and the strains containing both the Tn5 integrated into the chromosome and intact hybrid plasmid or the parental plasmid with the replication ts mutation, were constructed . It was shown that transposon Tn5 comprised within the hybrid plasmid or in the chromosome promotes maintenance of these replication defective plasmids in the bacterial culture at a non-permissive temperature and thus suppresses plasmid mutation tsr12 . It was determined that the extent of suppression of plasmid replication ts mutation depends on the localization of transposon Tn5.

FEMS Microbiol Lett, 1990 May, 57(1-2), 97 - 9
Plasmid RP4 host-lethal function kilA and its repressor korA sequences are part of the cryptic tellurite resistance transposon Tn521; Grewal KK; The normally silent 4.5 kb tellurite resistance transposon Tn521 of RP4 has been shown to carry sequences from both the flanking kilA and korA loci of this broad host range plasmid . The major portion of both of these sequences were used as probes to examine DNA homology in Southern transfer hybridization experiments with plasmid recipients of Tn521 chosen from varying incompatibility groups . In the case of every recipients molecule analyzed using either probe, DNA homology was observed.

Biotechniques, 1990 May, 8(5), 488 - 92
An Escherichia coli expression vector for high-level production of heterologous proteins in fusion with CMP-KDO synthetase; Bolling TJ et al.; The construction of a vector which overproduces the enzyme, CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl-transferase (CMP-KDO synthetase or CKS) and its use as an expression vector for producing heterologous proteins in E . coli is described . The vector, which includes a modified lac promoter and synthetic ribosome binding site upstream of the native kdsB gene (encoding CKS), produces CKS at levels as high as 70% of the total cellular proteins . Several heterologous gene sequences have been fused to the 3'-end of the kdsB gene with resulting protein fusions produced at a level of up to 40% of the total cellular proteins.

Immunology, 1990 May, 70(1), 133 - 5
The selective augmentation by recombinant human tumour necrosis factor-alpha of neutrophil responses to pathogenic Escherichia coli; Steadman R et al.; Endotoxin release may amplify the neutrophil (PMN) responses to bacterial infection through the release of monocyte-derived tumour necrosis factor (TNF) . The present study was designed to assess the effect of recombinant human TNF-alpha (rhTNF-alpha) on the in vitro response of human PMN to two defined strains of pathogenic Escherichia coli . In the absence of rhTNF-alpha, a P-fimbriate strain caused significant release of the PMN secondary granule marker vitamin B12-binding protein (B12 BP), and a low level of release of leukotriene B4 (LTB4) . Type 1-fimbriate strain 504, however, stimulated the release of the primary granule marker myeloperoxidase (MPO) and PMN chemiluminescence (CL), in addition to B12 BP and LTB4 release . Following rhTNF-alpha (10(-9) M) pretreatment, the release of LTB4 by PMN stimulated with the P-fimbriate strain was synergistically augmented, while B12 BP and MPO release were additively increased . In contrast, rhTNF-alpha did not significantly affect any of the responses by the type 1-fimbriate strain . These results suggest selectivity in the priming of PMN by rhTNF-alpha and confirm the independence of PMN responses to phagocytic stimuli.

Xenobiotica, 1990 May, 20(5), 549 - 54
Role of free radicals produced during the metabolism of mitomycin C in Escherichia coli inactivation; Schiavano GF et al.; 1 . The primary objective of this study was to assess whether reactive oxygen species produced during the metabolism of mitomycin C are responsible for, or contribute to, the induction of the cytotoxic response . 2 . Escherichia coli wild-type and superoxide dismutase mutants were grown either anoxically or euoxically, in K medium or minimal glucose medium, and treated with mitomycin C in K medium or M9 salts . The cytotoxic response did not vary significantly between the wild-type and the SOD mutants . 3 . The toxicity of mitomycin C was not affected by the hydroxyl radical scavengers, dimethyl sulphoxide or ethanol, whereas protection was afforded by thiourea . 4 . It is concluded that oxygen radical species do not play a significant role in mediating mitomycin C inactivation of normally growing bacteria.

J Leukoc Biol, 1990 May, 47(5), 440 - 8
Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescin; Rothe G et al.; Hydroethidine (HE) and 2',7'-dichlorofluorescin (DCFH) were used for the flow cytometric measurement of reactive oxygen metabolites in leukocytes . Hydroethidine and DCFH were both rapidly oxidized in a cell-free cuvette assay to ethidium bromide (EB) and 2',7'-dichlorofluorescein (DCF) by H2O2 and peroxidase, but not by H2O2 alone, while only HE was oxidized by KO2, a source of O2- . Quiescent lymphocytes, monocytes, and neutrophils spontaneously oxidized HE to EB, while DCFH was only oxidized to a low degree . Neutrophils increased 6.9-fold in EB red fluorescence and 12.5-fold in DCF green fluorescence during the respiratory burst induced by phorbol 12-myristate 13-acetate or 6.1-fold and 4.7-fold, respectively, during the respiratory burst induced by Escherichia coli bacteria . The HE or DCFH oxidation during the respiratory burst, unlike the spontaneous HE oxidation, was not inhibitable by 10 mM NaNe indicating a non-mitochondrial source of cellular oxidants during the respiratory burst such as NADPH oxidase, which produces O2- . The oxidation of DCFH, but not of HE, was decreased in stimulated neutrophils, which were simultaneously loaded with HE and DCFH . Intracellular DCFH oxidation induced by incubation of resting neutrophils with extracellular H2O2 was not influenced by the presence of HE . This indicates that HE is oxidized at an earlier step in the reactive oxygen metabolism of neutrophils than DCFH, i.e., by early oxygen metabolites like O2-, while DCFH is oxidized in part by H2O2 and phagosomal peroxidases . The differential oxidation of HE and DCFH during simultaneous cellular staining permits the analysis of up to three functionally different neutrophil populations in septic patients . This is of interest for the determination of disease-related alterations of oxygen metabolism in quiescent and stimulated leukocytes.

Am J Physiol, 1990 May, 258(5 Pt 1), C902 - 12
Immune-related intestinal chloride secretion . II . Effect of adenosine on T84 cell line; Barrett KE et al.; The inflammatory mediator adenosine caused sustained Cl- secretion across monolayers of T84 cells . The effect was promptly reversed by the adenosine receptor antagonist 8-phenyltheophylline and appeared to be mediated through an adenosine A2-receptor {rank order of potency: 5'-(N-ethyl)-carboxamido-adenosine (NECA) greater than adenosine greater than (-)-N6-(phenylisopropyl)adenosine (PIA) greater than or equal to (+)-PIA} . High doses of adenosine and its analogues increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) but not guanosine 3',5'-cyclic monophosphate (cGMP) or free cytosolic Ca2+ . However, lower concentrations of adenosine had maximal effects on Cl- secretion with little or no effect on cAMP . In other respects, Cl- secretion resembled that induced by cAMP-mediated secretagogues such as vasoactive intestinal peptide (VIP) . Addition of both low and high doses of NECA activated basolateral K+ and apical Cl- channels, exhibited synergism with Ca2(+)-mediated secretagogues, did not produce additive effects with VIP or Escherichia coli heat-stable enterotoxin, and was associated with cAMP-dependent protein kinase-mediated protein phosphorylation . The results suggest that either adenosine mobilizes an intracellular pool of cAMP that is extremely efficiently coupled to the cAMP-dependent protein kinase and is thereafter rapidly destroyed or that second messenger(s) other than cAMP, cGMP, or Ca2+ are able to activate Cl- secretion in the T84 cell line . In the latter case, such messenger(s), as yet unidentified, might represent a final common pathway for cyclic nucleotide-activated Cl- secretion.

J Bacteriol, 1990 May, 172(5), 2814 - 6
Mutagenesis of dimeric plasmids by the transposon gamma delta (Tn1000); Liu L et al.; The Escherichia coli F factor mediates conjugal transfer of a plasmid such as pBR322 primarily by replicative transposition of transposon gamma delta (Tn1000) from F to that plasmid to form a cointegrate intermediate . Although resolution of this cointegrate always yields a plasmid containing a single gamma delta insertion, the occasional recovery of transposon-free plasmids after conjugal transfer has led to alternative hypotheses for F mobilization . We show here that gamma delta-free plasmids are found after F-mediated conjugal transfer only when the donor plasmid is a dimer and the recipient is Rec+.

J Bacteriol, 1990 May, 172(5), 2793 - 7
Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a "mini" mycobacterium-Escherichia coli shuttle vector; Ranes MG et al.; Functional domains of pAL5000 were determined by gene disruption and deletion analysis . Of the five plasmid open reading frames (ORFs), ORF1 to ORF5, and a putative origin of replication previously identified (J . Rauzier, J . Moniz-Pereira, and B . Gicquel-Sanzey, Gene 71:315-321), two of the ORFs (ORF3 and ORF4) were deemed dispensable for plasmid replication . A "mini" mycobacterium-Escherichia coli shuttle plasmid applicable for general recombinant DNA studies in mycobacteria was constructed by using the gene for Kanr (Tn903) as a selective marker . Heterologous expression of the gene for Kanr was confirmed by Western blotting (immunoblotting) analysis.

J Bacteriol, 1990 May, 172(5), 2728 - 35
Induction of the nag regulon of Escherichia coli by N-acetylglucosamine and glucosamine: role of the cyclic AMP-catabolite activator protein complex in expression of the regulon; Plumbridge JA; The divergent nag regulon located at 15.5 min on the Escherichia coli map encodes genes necessary for growth on N-acetylglucosamine and glucosamine . Full induction of the regulon requires both the presence of N-acetylglucosamine and a functional cyclic AMP (cAMP)-catabolite activator protein (CAP) complex . Glucosamine produces a lower level of induction of the regulon . A nearly symmetric consensus CAP-binding site is located in the intergenic region between nagE (encoding EIINag) and nagB (encoding glucosamine-6-phosphate deaminase) . Expression of both nagE and nagB genes is stimulated by cAMP-CAP, but the effect is more pronounced for nagE . In fact, very little expression of nagE is observed in the absence of cAMP-CAP, whereas 50% maximum expression of nagB is observed with N-acetylglucosamine in the absence of cAMP-CAP . Two mRNA 5' ends separated by about 100 nucleotides were located before nagB, and both seem to be similarly subject to N-acetylglucosamine induction and cAMP-CAP stimulation . To induce the regulon, N-acetylglucosamine or glucosamine must enter the cell, but the particular transport mechanism used is not important.

J Bacteriol, 1990 May, 172(5), 2427 - 32
Secretion of methanol-insoluble heat-stable enterotoxin (STB): energy- and secA-dependent conversion of pre-STB to an intermediate indistinguishable from the extracellular toxin; Kupersztoch YM et al.; The methanol-insoluble, heat-stable enterotoxin of Escherichia coli synthesized by clinical strains or strains that harbor the cloned gene was shown to be an extracellular polypeptide . The toxin (STB) was first detected as an 8,100-Mr precursor (pre-STB) that was converted to a transiently cell-associated 5,200-Mr form . Proteolytic conversion of pre-STB to STB was shown to be inhibited by the proton motive force uncoupler carbonyl cyanide m-chlorophenylhydrazone and did not occur in a secA background . After STB was detected as a cell-associated molecule, an extracellular form with identical electrophoretic mobility became apparent . The results suggest that there is no proteolytic processing during the mobilization of STB from the periplasm to the culture supernatant . The determined amino acid sequence of STB coincides fully with the 48 carboxy-terminal amino acids inferred from the DNA sequence . The 23 amino-terminal residues inferred from the DNA sequence were absent in the mature toxin.

J Bacteriol, 1990 May, 172(5), 2245 - 9
Overproduction of acetate kinase activates the phosphate regulon in the absence of the phoR and phoM functions in Escherichia coli; Lee TY et al.; A DNA fragment of Escherichia coli cloned on pBR322 elevated the production of alkaline phosphatase and phosphate-binding protein in a phoR phoM strain . Nucleotide sequence analysis and enzyme assays revealed that the DNA fragment contained the ackA gene, which codes for acetate kinase . A high gene dosage of ackA was needed to induce the production of alkaline phosphatase and phosphate-binding protein in this strain . Overexpression of ackA elevated the intracellular ATP concentration, an effect that might be related to activation of the phosphate regulon in the phoR phoM strain.

Virology, 1990 May, 176(1), 16 - 24
Cloning and sequencing the cytosine methyltransferase gene M . CviJI from Chlorella virus IL-3A; Shields SL et al.; The Chlorella virus IL-3A gene encoding the DNA methyltransferase M.CviJI, which methylates the internal cytosine in (G/A)GC(T/C/G) sequences, was cloned and expressed in Escherichia coli . The region containing the M.CviJI gene was sequenced and a single open reading frame of 1101 bp was identified that could code for a polypeptide of 367 amino acids with a predicted molecular weight of 41,864 . M.CviJI contained regions of amino acids which were similar to bacterial cytosine methyltransferases . Eighteen other Chlorella viruses, of 36 tested, contained DNA sequences which hybridized to the M.CviJI gene; DNA from some, but not all, of these 18 viruses also contained 5-methylcytosine in (G/A)GC(T/C/G) sequences.

J Virol, 1990 May, 64(5), 2369 - 79
Definition of the sequence requirements for binding of the EBNA-1 protein to its palindromic target sites in Epstein-Barr virus DNA; Ambinder RF et al.; Interaction between the trans-acting DNA-binding protein EBNA-1 and cis-acting sequences in the ori-P region of Epstein-Barr virus DNA is required for maintenance of the viral plasmid state in latently infected B cells and is involved in the regulation of transcription during latency . In the Epstein-Barr virus genome, a total of 26 EBNA-1-binding sites occur within three clustered loci referred to as the family of repeats and dyad symmetry locus of ori-P and the separate BamHI-Q locus . Incubation of a bacterially expressed carboxy-terminal domain of EBNA-1 (28,000-molecular-weight EBNA-1 {28K EBNA-1}) with synthetic monomer and dimer consensus binding sites gave characteristic DNA-protein complexes in a mobility retardation assay . A similar approach with the naturally occurring Q locus confirmed that it contains two distinct but low-affinity binding sites . We then examined the precise sequence requirements for EBNA-1 binding, using a set of 30-base-pair oligonucleotides designed to contain symmetric point mutations within both halves of the palindromic target site . Analysis of all possible single substitutions between positions 1 and 10 in the consensus half-palindrome sequence revealed that positions 9 and 10 did not contribute to EBNA-1 binding and that considerable flexibility could be tolerated at positions 1 and 2 . Positions 3 through 8 of the recognition site had the most stringent requirements, with transversions at these positions either reducing or eliminating binding . The relative spacing of the halves of the palindrome was also critical, since the addition or removal of 2 base pairs at the center of the sequence abolished binding . Similar results were obtained when a partially purified preparation of intact Raji EBNA-1 was substituted for the 28K EBNA-1, and the results were further supported by methylation interference studies which indicated contact points between EBNA-1 and the guanine residues at positions -8, -7, and +3 of the binding site . The three naturally occurring EBNA-1-binding loci have previously been shown to differ in their relative affinities for EBNA-1 . The present study indicates that the sequence variations occurring within the family of repeats would not affect binding affinity, whereas certain base substitutions within the Q and dyad symmetry sites would be predicted to contribute to the observed lower affinities of these sites . An apparent Kd of 1.5 x 10(-11) M for binding of 28K EBNA-1 to a consensus recognition site was calculated from Scatchard analysis.

J Virol, 1990 May, 64(5), 2327 - 36
Preferred crossover sites on polyomavirus DNA; Bourgaux P et al.; RmI is a hybrid replicon consisting of polyomavirus (Py) and mouse sequences that yields unit-length polyomavirus DNA via recombination between two directly repeated viral sequences of 182 base pairs (S repeats) . To define the contribution of the S repeats in this intramolecular recombination, we derived from RmI a series of replicons containing the original S repeats as well as additional direct viral repeats which were 1 to 2 kilobases in length (L repeats) . After mouse 3T6 cells were transfected with these constructs, recombination products that displayed the physical properties of homologous recombinants were detected . The structures of these recombinants indicated that whereas repeat length influences the likelihood of recombination, crossover occurs preferentially near the S repeats, provided that one of them is proximal to the viral origin of replication . This finding suggests that recombination near the S repeats depends on a process initiated near the viral origin of replication.

J Virol, 1990 May, 64(5), 2208 - 16
Isolation and characterization of herpes simplex virus mutants containing engineered mutations at the DNA polymerase locus; Marcy AI et al.; We have derived Vero cell lines containing the herpes simplex virus DNA polymerase (pol) gene that complement temperature-sensitive pol mutants . These cell lines were used to recover viruses containing new mutations at the pol locus . Two spontaneously arising host-range mutants, 6C4 and 7E4, were isolated . These mutants did not grow efficiently on Vero cells or synthesize late polypeptides but formed plaques on a cell line containing the pol gene (DP6 cells) . Whereas mutant 6C4 specified a wild-type-size Pol protein, we detected no full-length Pol protein in 7E4-infected cell extracts . Complementation studies demonstrated that 6C4 and 7E4 contain different mutations and indicated that 6C4 is in a complementation group different from that of pol temperature-sensitive mutant tsC7 or tsD9 . A mutant in which 2.2 kilobases of pol sequences were replaced with the Escherichia coli lacZ gene under the control of the herpes simplex virus thymidine kinase promoter was constructed . This mutant formed blue plaques on DP6 cells in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactoside . Using this virus in marker rescue experiments, we engineered three mutants containing deletions in the pol coding region which grew efficiently on DP6 cells but not on Vero cells and which differed in their synthesis of Pol polypeptides . The lacZ insertion virus was also used to introduce a deletion in the region upstream of the pol long open reading frame, which removes a short open reading frame that could encode a 10-amino-acid peptide . This mutant grew to similar titers on Vero and DP6 cells, indicating that these sequences are not essential for growth of the virus in tissue culture.

J Virol, 1990 May, 64(5), 1946 - 55
The export pathway of the pseudorabies virus gB homolog gII involves oligomer formation in the endoplasmic reticulum and protease processing in the Golgi apparatus; Whealy ME et al.; The pseudorabies virus gII gene shares significant homology with the gB gene of herpes simplex virus type 1 . Unlike gB, however, gII is processed by specific protease cleavage events after the synthesis of its precursor . The processed forms are maintained in an oligomeric complex that includes disulfide linkages . In this report, we demonstrate the kinetics of modification, complex formation, and subsequent protease processing . In particular, we suggest that gII oligomer formation in the endoplasmic reticulum is an integral part of the export pathway and that protease cleavage occurs only after oligomers have formed . Furthermore, through the use of glycoprotein gene fusions between the gIII glycoprotein and the gII glycoprotein genes of pseudorabies virus, we have mapped a functional cleavage domain of gII to an 11-amino-acid segment.

J Photochem Photobiol B, 1990 May, 5(3-4), 505 - 17
Photodynamic action of methylene blue: repair and mutation in Escherichia coli; Menezes S et al.; The effects of the photodynamic action of methylene blue (MB-PDA) on strains of Escherichia coli were investigated to determine whether the dye could be used in photodynamic therapy (PDT) . Using the method of alkaline sucrose gradient sedimentation, it was shown that in darkness MB induces a type of prelesion in DNA that transforms into single-strand breaks in alkaline conditions, provided that the dye is present during the processing of the gradient . This prelesion is completely reversible if the cells are washed immediately to remove the dye . However, after illumination with white light, the prelesions become "fixed" stable lesions, irreversible even after successive washings . The lethal damage induced by MB-PDA in E . coli can be repaired by the excision-repair system (about 30%) and by the recA-dependent repair system (about 70%) . Polymerase I enzyme participates actively in the repair of the damage . MB-PDA is a weak mutagen and the induction of mutations by this treatment is restricted to high survival rates . Moreover, MB-PDA does not induce the SOS system (an inducible repair system dependent of the recA and loxA genes products), as measured by Weigle reactivation . However, it seems that this treatment can impair the repair systems in E . coli.

Proc Natl Acad Sci U S A, 1990 May, 87(10), 3919 - 23
Enhanced resolution of DNA restriction fragments: a procedure by two-dimensional electrophoresis and double-labeling; Yi M et al.; A probe-free method was developed to detect DNA rearrangement in bacteria based on the electrophoretic separation of twice-digested restriction fragments of genomic DNA into a two-dimensional (2-D) pattern . The first restriction enzyme digestion was done in solution, followed by electrophoresis of the restriction fragments in one dimension . A second restriction enzyme digestion was carried out in situ in the gel, followed by electrophoresis in a second dimension perpendicular to the first electrophoresis . The 2-D pattern provides for the resolution of 300-400 spots, which are defined and indexed by an "x,y" coordinate system with size markers . This approach has greatly increased the resolution power over conventional one-dimensional (1-D) electrophoresis . To study DNA rearrangement, a 2-D pattern from a test strain was compared with the 2-D pattern from a reference strain . After the first digestion, genomic DNA fragments from the test strain were labeled with 35S, while those from the reference strain were labeled with 35P . This was done to utilize the difference in the energy emission of 35S and 32P isotopes for autoradiography when two x-ray films were exposed simultaneously on top of the gel after the 2-D electrophoresis . The irradiation from the decay of 35S exposed only the lower film, whereas the irradiation from the decay of 32P exposed both the lower and upper films . Different DNA fragments existed in the test DNA compared with the reference DNA can be identified unambiguously by the differential two 2-D patterns produced on two films upon exposure to the 35S and 32P fragments in the same gel . An appropriate photographic procedure further simplified the process, allowing only the difference in DNA fragments between these two patterns to be shown in the map . We have utilized the difference map obtained from Escherichia coli strains HB101 and HB101 (lambda) genomic DNA to show the incorporation of one copy of phage lambda DNA without the use of a lambda DNA probe . This is the same test system that was used previously.

Proc Natl Acad Sci U S A, 1990 May, 87(10), 3708 - 12
Activation of ara operons by a truncated AraC protein does not require inducer; Menon KP et al.; The araC gene of Escherichia coli encodes a protein that binds the inducer L-arabinose to activate the transcription of three ara operons . In a study to determine the functional domains within the AraC protein, we have generated a set of overlapping deletions from the proximal end of the araC gene . We found that the removal of up to nearly 60% of the coding sequence of this protein still allows transcriptional activation of the ara operons in vivo, up to 27% that of the wild type . These truncated proteins, however, no longer require arabinose for induction . The ligand-induced conformational change apparently either releases or unmasks an existing functional domain within AraC, rather than generating a new conformation that is required for activation of the promoter of araBAD . Since the truncated protein of the mutant C154 (which lacks 153 amino acid residues from the N terminus) retains DNA binding specificity, the DNA-recognition domain is localized in the C-terminal half of the AraC protein . Truncated proteins were unable to repress araBAD or araC in vivo, even though they were able to bind all ara operators . We propose that the N-terminal half of AraC is essential for the formation of the DNA loops that are responsible for repression of araBAD and for autoregulation of araC.

J Clin Invest, 1990 May, 85(5), 1694 - 7
Biological properties of recombinant human monocyte-derived interleukin 1 receptor antagonist; Arend WP et al.; Human monocytes cultured on adherent IgG produce a specific IL-1 inhibitor that functions as a receptor antagonist (IL-1ra) . This molecular has been purified, sequenced, cloned as a cDNA, and expressed in Escherichia coli . Recombinant IL-1ra has 17,000 mol wt and binds to IL-1 receptors on T lymphocytes, synovial cells, and chondrocytes with an affinity nearly equal to that of IL-1 . These studies have examined some biological properties of purified recombinant human IL-1ra . This protein exhibits a dose-responsive inhibition of Il-1 alpha and Il-1 beta augmentation of PHA-induced murine thymocyte proliferation . The recombinant IL-1ra also blocks IL-1 alpha and IL-1 beta stimulation of PGE2 production in human synovial cells and rabbit articular chondrocytes, and of collagenase production by the synovial cells . A 50% inhibition of these IL-1-induced biological responses requires amounts of IL-1ra up to 100-fold in excess of the amounts of IL-1 alpha or IL-1 beta present . IL-1ra may play an important role in normal physiology or in pathophysiological states by functioning as a natural IL-1 receptor antagonist in the cell microenvironment.

J Bacteriol, 1990 May, 172(5), 2782 - 4
An upstream uncD sequence modulates translation of Escherichia coli uncC; Dunn SD et al.; The effect of upstream uncD sequences on expression of the Escherichia coli uncC gene, encoding the epsilon subunit of F1-ATPase, was studied . uncC expression was reduced severalfold in plasmid constructs bearing, in addition to uncC, a region of uncD located between 85 and 119 bases upstream from the uncC initiation codon . This reduction was independent of in-frame translation of the uncD sequences . An mRNA stem-loop structure in which sequences located within the inhibitory region of uncD base pair with the uncDC intercistronic region is suggested to function in modulating uncC expression.

J Bacteriol, 1990 May, 172(5), 2736 - 46
Surface topology of the Escherichia coli K-12 ferric enterobactin receptor; Murphy CK et al.; Monoclonal antibodies (MAb) were raised to the Escherichia coli K-12 ferric enterobactin receptor, FepA, and used to identify regions of the polypeptide that are involved in interaction with its ligands ferric enterobactin and colicins B and D . A total of 11 distinct FepA epitopes were identified . The locations of these epitopes within the primary sequence of FepA were mapped by screening MAb against a library of FepA::PhoA fusion proteins, a FepA deletion mutant, and proteolytically modified FepA . These experiments localized the 11 epitopes to seven different regions within the FepA polypeptide, including residues 2 to 24, 27 to 37, 100 to 178, 204 to 227, 258 to 290, 290 to 339, and 382 to 400 of the mature protein . Cell surface-exposed epitopes of FepA were identified and discriminated by cytofluorimetry and by the ability of MAb that recognize them to block the interaction of FepA with its ligands . Seven surface epitopes were defined, including one each in regions 27 to 37, 204 to 227, and 258 to 290 and two each in regions 290 to 339 and 382 to 400 . One of these, within region 290 to 339, was recognized by MAb in bacteria containing intact (rfa+) lipopolysaccharide (LPS); all other surface epitopes were susceptible to MAb binding only in a strain containing a truncated (rfaD) LPS core, suggesting that they are physically shielded by E . coli K-12 LPS core sugars . Antibody binding to FepA surface epitopes within region 290 to 339 or 382 to 400 inhibited killing by colicin B or D and the uptake of ferric enterobactin . In addition to the FepA-specific MAb, antibodies that recognized other outer membrane components, including Cir, OmpA, TonA, and LPS, were identified . Immunochemical and biochemical characterization of the surface structures of FepA and analysis of its hydrophobicity and amphilicity were used to generate a model of the ferric enterobactin receptor's transmembrane strands, surface peptides, and ligand-binding domains.

J Bacteriol, 1990 May, 172(5), 2693 - 703
Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein; Chevalier C et al.; The gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri, was cloned in Escherichia coli as a 5-kilobase-pair (kbp) DNA fragment . The complete nucleotide sequence of the 5.0-kbp spiroplasmal DNA fragment was determined (GenBank accession no . M31161) . The spiralin gene was identified by the size and amino acid composition of its translational product . Besides the spiralin gene, the spiroplasmal DNA fragment was found to contain five additional open reading frames (ORFs) . The translational products of four of these ORFs were identified by their amino acid sequence homologies with known proteins: ribosomal protein S2, elongation factor Ts, phosphofructokinase, and pyruvate kinase, respectively encoded by the genes rpsB, tsf, pfk, and pyk . The product of the fifth ORF remains to be identified and was named protein X (X gene) . The order of the above genes was tsf--X--spiralin gene--pfk--pyk . These genes were transcribed in one direction, while the gene for ribosomal protein S2 (rpsB) was transcribed in the opposite direction.

J Bacteriol, 1990 May, 172(5), 2287 - 93
Aerobic regulation of the Escherichia coli tonB gene by changes in iron availability and the fur locus; Postle K; The tonB gene is required for the transport of several different iron-siderophore complexes across the Escherichia coli outer membrane . In this study, transcriptional regulation of the tonB gene was investigated by using three different tonB-lacZ fusions to monitor tonB expression under aerobic conditions and in the presence of a wild-type tonB gene . Prior work by other laboratories suggests that tonB is expressed at low constitutive levels regardless of changes in iron availability or the fur locus . In contrast, these data show that tonB transcription is repressed threefold by growth in the presence of FeCl3 compared with growth in the presence of the iron chelator dipyridyl and that this repression requires the fur locus . A 168-base-pair DNA fragment carrying the tonB promoter was sufficient for the observed transcriptional regulation . In addition, the tonB gene appeared to have a substantially stronger promoter than previously recognized . The inability of other laboratories to detect tonB transcription regulation appears to be due to the extremely slow growth of iron-starved tonB strains and the use of Mu d1(lac Apr)- or lambda plac Mu53-generated fusions that encode a thermolabile TrpA-LacZ hybrid protein . The data also suggest that the previously reported growth phase regulation of tonB occurs only in media with intermediate levels of available iron and is due to iron starvation-induced derepression as the culture approaches stationary phase.

Plasmid, 1990 May, 23(3), 237 - 41
Use of the tyrosinase gene from Streptomyces to probe promoter sequences for Escherichia coli; Sugiyama M et al.; We have constructed a promoter-probe vector utilizing the expression of a promoter-less tyrosinase derived from Streptomyces plasmid pIJ702 . The vector, pMX100, has single sites for EcoRI, KpnI, BamHI, XbaI, SalI, and SphI for cloning promoter sequences . When the tac promoter was inserted into pMX100, E . coli harboring the chimeric plasmid produced the melanin pigment.

Plasmid, 1990 May, 23(3), 183 - 93
Mechanism of integration of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus; Jaoua S et al.; The site-specific recombination mechanism through which the plasmid RP4 has been previously shown to integrate into the chromosome of Myxococcus xanthus has been investigated further . Once integrated in one of the numerous chromosomal sites from two different strains, through a precise site on the plasmid, the latter can be excised either precisely or after a definite 14.5-kb deletion . In some cases, the integration is followed by different DNA rearrangements that yield a higher rate of excision and integration . A model for the site-specific integration and excision of the plasmid is proposed.

Res Microbiol, 1990 May, 141(4), 407 - 23
Nucleotide sequence of the gene encoding the 35-kDa protein of Mycobacterium tuberculosis; O'Connor SP et al.; A 35-kilodalton (kDa) protein of Mycobacterium tuberculosis is expressed by recombinant Escherichia coli which possess a plasmid that contains a 2.4-kilobase fragment of M . tuberculosis chromosomal DNA . The nucleotide sequence of this fragment was determined by the dideoxynucleotide chain-termination method . Analysis of the sequence revealed four open reading frames that could encode proteins greater than 250 amino acids in length . The reading frame for the 35-kDa protein was identified by subcloning DNA fragments into expression vectors pTTQ18 and pTTQ19, and assaying for production of the 35-kDa protein by Western blotting . A protein with a primary structure of 270 amino acids and a predicted molecular weight of 29,260 daltons was deduced from the nucleotide sequence . A computer-aided search of nucleic and amino acid sequence databases did not identify any proteins with significant sequence similarity to this protein . The organization of the gene encoding this protein was compared with other mycobacterial genes that have been sequenced . Information obtained from the investigation of this protein may aid in the development of reagents to diagnose and control mycobacterial disease.

Genes Dev, 1990 May, 4(5), 822 - 34
The Drosophila Fos-related AP-1 protein is a developmentally regulated transcription factor; Perkins KK et al.; Drosophila AP-1 consists of two proteins (dFRA and dJRA) that have functional and structural properties in common with mammalian Fos and Jun proto-oncogene products . Here, we report the isolation and characterization of cDNAs encoding the full-length dFRA and dJRA proteins . The predicted amino acid sequences reveal that both proteins contain a bipartite DNA-binding domain consisting of a leucine repeat and an adjacent basic region, which are characteristic of members of the AP-1 family . By using protein translated in vitro or expressed in Escherichia coli, we demonstrate that dFRA, in contrast to the mammalian cFos proteins, recognizes the AP-1 site on its own and activates transcription in vitro in the absence of dJRA or Jun . Heteromeric complexes formed between dFRA and dJRA bind the AP-1 site better than either protein alone, and the two proteins activate transcription synergistically in vitro . In the developing embryo, dFRA mRNA is first expressed in a limited set of cells in the head and is later restricted to a subset of peripheral neurons, several epidermal cells near the muscle attachment sites, and a portion of the gut . In contrast, dJRA appears to be uniformly expressed at a low level in all cell types . These results indicate that dFRA is a developmentally regulated transcription factor and suggest that its potential interplay with dJRA plays an important role in cell-type-specific transcription during Drosophila embryonic development.

Z Naturforsch {C}, 1990 May, 45(5), 538 - 43
Molecular characterization of genes coding for wild-type and sulfonylurea-resistant acetolactate synthase in the cyanobacterium Synechococcus PCC7942; Friedberg D et al.; We present here the isolation and molecular characterization of acetolactate synthase (ALS) genes from the cyanobacterium Synechococcus PCC7942 which specify a sulfonylurea-sensitive enzyme and from the sulfonylurea-resistant mutant SM 3/20, which specify resistance to sulfonylurea herbicides . The ALS gene was cloned and mapped by complementation of an Escherichia coli ilv auxotroph that requires branched-chain amino acids for growth and lacks ALS activity . The cyanobacterial gene is efficiently expressed in this heterologous host . The ALS gene codes for 612 amino acids and shows high sequence homology (46%) at the amino acid level with ALS III of E . coli and with the tobacco ALS . The resistant phenotype is a consequence of proline to serine substitution in residue 115 of the deduced amino acid sequence . Functional expression of the mutant gene in wild-type Synechococcus and in E . coli confirmed that this amino-acid substitution is responsible for the resistance . Yet the deduced amino-acid sequence as compared with other ALS proteins supports the notion that the amino-acid context of the substitution is important for the resistance.

Vet Immunol Immunopathol, 1990 May, 25(1), 83 - 97
Immunogenicity of subcellular fractions of Brucella abortus: measurement by in vitro lymphocyte proliferative responses; Smith R 3rd et al.; Five groups of heifers were immunized with various subcellular fractions of Brucella abortus and tested for their responsiveness in lymphocyte proliferative responses in vitro . The five subcellular fractions used as immunogens were: (1) a mixture of recombinant outer membrane proteins fused to Escherichia coli beta-galactosidase, (2) a mixture of outer membrane proteins BaomI, BaomIIB1, and BaomIII1, (3) a mixture of outer membrane proteins 7.5 kDa and 8.8 kDa, (4) a complex of smooth lipopolysaccharide and proteins, and (5) a complex of outer membranes and peptidoglycan (OM-PG complex) from a rough strain . All immunogens were emulsified in adjuvant and administered twice at a 61-day interval . Two other groups of cows were included; one immunized with strain 19 and the other with adjuvant only . Strain 19 and the rough OM-PG complex induced responsiveness in lymphocyte proliferation assays in a high percentage of immunized cows . The smooth lipopolysaccharide-protein complex induced responsiveness in fewer cows . The lowest frequencies of responding cows were found in groups that received either recombinant proteins or purified protein mixtures . Based on these results, we concluded: (1) cellular immunity, as measured by in vitro lymphocyte proliferative responses, can be induced with subcellular fractions of B . abortus and (2) the more complex the immunogen, the greater the frequency of responding cows.

Neuron, 1990 May, 4(5), 797 - 805
The product of rab2, a small GTP binding protein, increases neuronal adhesion, and neurite growth in vitro; Ayala J et al.; The rab genes code for small GTP binding proteins that share with p21ras the ability to bind and hydrolyze GTP . They present significant sequence homologies with the products of YPT1 and SEC4, two small GTP binding proteins involved in the regulation of secretion in the yeast . Several rab genes are expressed in the developing and adult mouse brain . To test directly the possible involvement of these genes in neuronal differentiation, purified rab proteins produced in E . coli were introduced into neurons dissociated from E15 rat midbrain . The most striking effects were obtained with rab2 protein (rab2p) . Compared with untreated cells, neurons loaded with rab2p presented an enhanced adhesion to the culture substratum . This phenomenon was visible 3 hr after seeding and was followed within 24 hr by a dramatic increase in neurite growth . Loading the same population of neurons with the products of four other rab genes either decreased neuronal adhesion and neurite growth or had no effect . These experiments suggest that the expression of rab2p plays an important role in neuronal differentiation.

Mutat Res, 1990 May, 230(1), 1 - 7
Investigation of the SOS response of Escherichia coli after gamma-irradiation by means of the SOS chromotest; Kozubek S et al.; The kinetics of SOS system induction in Escherichia coli PQ37 cells by gamma-irradiation has been studied by the SOS chromotest technique . It was shown that the synthesis of constitutive alkaline phosphatase is not immediately stopped in cells that suffered lethal damages from gamma-irradiation . The production of DNA damages inducing the SOS system was 0.021/Gy per genome . The SOS system was switched off approximately 200 min after gamma-irradiation . A correction is proposed to the calculation of the SOS system induction factor.

Proc Natl Acad Sci U S A, 1990 May, 87(10), 3763 - 7
Autogenous regulation of the Bordetella pertussis bvgABC operon; Roy CR et al.; The bvgABC operon of the bacterial pathogen Bordetella pertussis encodes a sensory transduction system that regulates the expression of several virulence genes in response to environmental stimuli . In this study we have examined the transcriptional regulation of the bvgABC operon . Transcriptional bvg::lacZYA fusions in Escherichia coli show that the bvgABC operon is autogenously activated . Autoactivation is inhibited by the same environmental stimuli that result in the lack of expression of bvg-activated genes in B . pertussis . These observations were confirmed in B . pertussis using a chromosomal chloramphenicol acetyltransferase transcriptional fusion in bvgC . Transcriptional initiation sites upstream of bvgA were mapped by primer extension analysis in E . coli and B . pertussis . Two differentially regulated bvg promoters were identified . The bvgP1 promoter is a positively autoregulated promoter located 90 base pairs upstream of bvgA . The bvgP2 promoter is located 141 base pairs upstream of bvgA and does not appear to require any positive regulatory factors for activity . These results suggest a model describing the regulatory events that take place upstream of the bvgABC operon.

Proc Natl Acad Sci U S A, 1990 May, 87(10), 3635 - 9
Purification and properties of Myxococcus xanthus C-factor, an intercellular signaling protein; Kim SK et al.; C-factor, a Myxococcus xanthus protein that restores the developmental defects of a class of nonautonomous mutants resulting from mutation of the csgA gene, has been purified approximately 1000-fold from starved wild-type cells . The monomeric form of C-factor is a single polypeptide with a molecular mass of 17 kDa that can be solubilized by detergent from membrane components . Characterization by gel filtration and denaturing gel electrophoresis suggests that biologically active C-factor is a dimer composed of two 17-kDa monomers . Antibodies against a form of the M . xanthus csgA gene product overexpressed in Escherichia coli react with purified C-factor.

Genomics, 1990 May, 7(1), 88 - 96
Characterization and chromosomal mapping of a cDNA encoding tryptophan hydroxylase from a mouse mastocytoma cell line; Stoll J et al.; A cDNA library was constructed from RNA prepared from P815 mouse mastocytoma cells and screened for tryptophan hydroxylase . An essentially full-length clone that recognizes a major mRNA species of 1.9 kb in mastocytoma cell lines and in pineal gland, duodenum, and brainstem of the mouse was obtained . The predicted amino acid sequence of this mouse mastocytoma clone showed 97 and 87% identity, respectively, with tryptophan hydroxylase clones isolated from rat and rabbit pineal glands, but the mouse clone contains an unusual 3-amino-acid duplication near the N-terminus and lacks a phosphorylation site . A fragment of the cDNA produced an enzymatically active protein when expressed in Escherichia coli, thus demonstrating that the catalytic domain is included in the C-terminal 380 amino acids . The mouse tryptophan hydroxylase locus, termed Tph, was mapped by Southern blot analysis of somatic cell hybrids and by an interspecific backcross to a position in the proximal half of chromosome 7 . Because TPH has been mapped to human chromosome 11, this assignment further defines regions of homology between these mouse and human chromosomes.

Carcinogenesis, 1990 May, 11(5), 781 - 5
Induction of specific frameshift and base substitution events by benzo{a}pyrene diol epoxide in excision-repair-deficient Escherichia coli; Bernelot-Moens C et al.; We have determined the DNA alterations recovered after treatment with (+-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene {(+-)-anti-BPDE} in the lacI gene of excision-repair-deficient (Uvr-) Escherichia coli . The high induction of -(G:C) frameshifts, G:C----T:A and A:T----T:A transversions, and the presence of complex mutations of a particular motif, distinguish the mutational distribution recovered in the Uvr- strain, although other mutational classes, including -(A:T) frameshifts and duplications, were also moderately induced . The great majority of -(G:C) frameshifts, the predominant mutation recovered, occurred in runs of G residues . The G:C----T:A transversion was found to occur more frequently (10/12 occurrences) at 5'-Y-G-3' sites, sequences at which the labile BPDE:N7G adduct has been predicted to occur . Moreover, the relative proportions of G:C----T:A, A:T----T:A and G:C----A:T mutations correlate well with the expected proportions of alkali-labile lesions at G, A and C residues . These results support the model that (+-)-anti-BPDE-induced base substitution mutagenesis in E.coli proceeds through abasic intermediates across from which adenine is preferentially incorporated during replication.

J Bacteriol, 1990 May, 172(5), 2313 - 9
Relationship between DNA cycle and growth rate in Synechococcus sp . strain PCC 6301; Binder BJ et al.; Flow cytometry was used to examine cell cycle regulation in Synechococcus sp . strain PCC 6301 under a variety of growth conditions . The DNA frequency distributions of exponentially growing and dark-blocked populations confirmed that this cyanobacterium contains multiple chromosome copies even at very slow growth rates . Furthermore, the presence of major peaks corresponding to other than 2" chromosome copies strongly suggests that DNA replication is initiated asynchronously . Although this suggestion is at odds with the standard formulation of the procaryotic cell cycle model, it is similar to recent observations of asynchrony in Escherichia coli replication mutants.

Virology, 1990 May, 176(1), 39 - 47
Functional domains of the HIV-1 rev gene required for trans-regulation and subcellular localization; Venkatesh LK et al.; The rev gene of human immunodeficiency virus type 1 (HIV-1) encodes a 116 amino acid nuclear regulatory protein (Rev) that increases the cytoplasmic expression of viral mRNAs containing the Rev response element (RRE) and coding for the structural proteins, Gag and Env . To identify the functional domains of Rev, amino acid deletion and chain termination mutations were introduced in the Rev coding region . The ability of these mutants to increase the cytoplasmic expression of a Rev-test plasmid (pSV-AR), containing the RRE cloned into the 3' noncoding region of the CAT gene in plasmid pSV2CAT, was examined in transient expression assays in HeLa cells . Our results indicate that three distinct regions mapping within the N-terminal 98 amino acids of Rev are essential for its activity . The subcellular localization of the various Rev proteins was examined in COS cells by indirect immunofluorescence . Rev was found to localize predominantly in the nucleolus of transfected cells . All mutant Rev proteins, with the exception of a deletion mutant (rev delta 41-44) lacking four Arg residues of a highly basic domain, were found to localize in the nucleolus . Mutant rev delta 41-44 exhibited weak diffuse fluorescence in the nucleus with a tendency to accumulate in the cytoplasm . A 15 amino acid region encompassing this basic domain (38-52) when fused to the Escherichia coli beta-galactosidase gene efficiently directed the fusion gene product to the nucleus and nucleolus, suggesting a role for this domain in the nucleolar localization of Rev.

Plant Mol Biol, 1990 May, 14(5), 775 - 83
Alcohol dehydrogenase gene expression in potato following elicitor and stress treatment; Matton DP et al.; A cDNA clone corresponding to a mRNA that rapidly accumulates during the hypersensitive-like response induced by elicitor treatment of potato (Solanum tuberosum L.) tuber was characterized . The clone encodes a polypeptide (Mr = 41,097) having 83%-85% amino acid identity with known plant alcohol dehydrogenase sequences (ADH; EC 1.1.1.1) . The identity of the clone was confirmed by measuring the ADH enzyme activity in extracts of Escherichia coli transformed with the cDNA clone . In potato tuber disks, a wide range of stresses, including treatment with fatty acid elicitors, salicylic acid, UV light and anaerobiosis, was shown to induce accumulation of Adh transcripts . In stems, a high constitutive level of Adh transcripts could be detected in 4-week old plants, but not in 8-week old plants . However, the mRNA could be induced to accumulate in stems of 8-week old plants by treatment with arachidonic acid elicitor or by anaerobiosis . Induction in leaves was also obtained during anaerobiosis and after treatment with a Phytophthora infestans mycelial homogenate.

Cell Growth Differ, 1990 May, 1(5), 233 - 9
DNA-binding activity of retinoblastoma protein is intrinsic to its carboxyl-terminal region; Wang NP et al.; The retinoblastoma (RB) gene encodes a nuclear phosphoprotein with a molecular weight of 110,000 (pp110RB) associated with DNA-binding activity . This sequence-nonspecific DNA binding activity was further studied by Southwestern and DNA-cellulose chromatography using purified fusion proteins expressed in Escherichia coli . Three fusion proteins, containing amino acids 612-775, 776-928, and 612-928 of pp110RB, bound to DNA; the binding affinity of the latter was approximately 20-fold higher than those of either smaller region . Other regions of pp110RB had no detectable binding activity, indicating that the carboxyl-terminal region of the RB protein is the major domain responsible for interacting with DNA . Since several potential phosphorylation sites reside within this region, isoforms of RB protein from cellular lysates with various degrees of phosphorylation were compared with respect to their DNA-binding affinity . The hyperphosphorylated form was eluted from DNA-cellulose columns at 0.1-0.3 M NaCl, whereas the hypophosphorylated form appeared in the eluates only at salt concentrations of 0.4-0.7 M, implying that phosphorylation of RB protein may affect its DNA-binding activity . That pp110RB can bind DNA intrinsically, and that this activity can be modulated by phosphorylation, is consistent with the proposed regulatory role of the RB protein in cell growth and differentiation.

Mol Microbiol, 1990 May, 4(5), 747 - 58
Integrity of Escherichia coli P pili during biogenesis: properties and role of PapJ; Tennent JM et al.; The papJ gene of uropathogenic Escherichia coli is required to maintain the integrity of Gal alpha (1-4)Gal-binding P pili . Electron microscopy and ELISA have established that strains carrying the papJ1 mutant allele have a large amount of pilus antigen free of the cells . In contrast to the whole pili released by strains unable to produce the PapH pilus anchor, the free papJ1 pili consist of variably sized segments that appear to result from internal breakages to the pilus . The DNA sequence of papJ is presented and its gene product identified as an 18kD periplasmic protein that possesses homology with nucleotide-binding proteins . PapJ may function as a 'molecular chaperone' directly or indirectly establishing the correct assembly of PapA subunits in the P pilus.

Antibiot Khimioter, 1990 May, 35(5), 7 - 10
{Structural changes in the composition of amplifying sequence AUD-Sr1 of Streptomyces rimosus after cloning into Escherichia coli}; Grigor'ev AE et al.; The presence of four EcoRV sites in the composition of the amplifying sequence AUD-Sr1 of S . rimosus resulting from its introduction on vector pUC19 into E . coli was studied . It was shown that the sequence had no EcoRV sites in a S . lividans strain while a number of plasmid and phage DNAs and chromosomal DNA of the strain were to be affected by EcoRV . The following regularities were observed: practically all the investigated hybrid plasmids from E . coli contained EcoRV sites in the composition of the AUD, EcoRV sites were present in strictly specified loci of the AUD sequence, appearance of an EcoRV site was accompanied by an increase in the size of the AUD fragment which contained it, the inserts detected in the SacI-PvuII fragment of the AUD were small in size (0.1 to 0.8 kb) and varied in different hybrid plasmids . The following mechanisms are discussed: insertion of the E . coli IS element into the AUD-Sr1 and high mutability of the amplifying sequence AUD-Sr1 in E . coli.

J Gen Microbiol, 1990 May, 136 ( Pt 5), 897 - 903
Growth-rate-dependent synthesis of K99 fimbrial subunits is regulated at the level of transcription; van der Woude MW et al.; Increase in the production of the fimbrial adhesion K99 by enterotoxigenic Escherichia coli in continuous cultures at specific growth rates above 0.25 h-1 was shown to be independent of the nature of the growth-limiting nutrient . The correlation between specific growth rate and K99 production was also found to be independent of the copy number of the K99 operon . Introduction of additional copies of the K99 regulatory region did not affect growth-rate-dependent K99 production in wild-type strains, indicating that no hypothetical regulatory host factor is titrated by the K99 regulatory region . Regulation at the transcriptional level was measured with galactokinase gene fusions . The transcription of the fimbrial subunit gene increased with an increase in specific growth rate . This growth-rate-dependent transcription was found to originate from the strong promoter PA . Transcription originating from the weaker promoter PB was independent of growth rate . The results indicated that transcriptional regulation at PA is involved in the growth-rate-dependent regulation of K99 production.

Mol Immunol, 1990 May, 27(5), 429 - 33
Nucleotide sequence of the VH, VL regions of an anti-idiotopic antibody reacting with a private idiotope of the anti-lysozyme D1.3 antibody; Souchon H et al.; Antibody E225 reacts with a private idiotope of the anti-lysozyme antibody D1.3 . A complex between the Fab fragments from these BALB/c monoclonal antibodies has been crystallized and the determination of the three-dimensional structure of this idiotope-anti-idiotope complex is under way . The nucleotide VH and VL sequences of E225 presented here have been determined to provide the amino acid sequence information necessary for the interpretation of the high resolution electron density maps of the complex, obtained by X-ray crystallography . The cDNAs synthesized from the Vkappa and VH mRNAs were cloned in E . coli . Both cDNA strands were sequenced by the dideoxy termination method . The translated amino acid sequence shows that Vkappa, VH correspond to groups five (V) and II(b) of mouse immunoglobulin light and heavy chains, respectively . Sequence alignments between the complementarity determining regions of E225 and the antigenic determinant of lysozyme recognized by D1.3 do not indicate whether or not the anti-idiotopic antibody structurally mimics the external antigen.

Proc Natl Acad Sci U S A, 1990 May, 87(10), 3874 - 8
Rat mitochondrial and cytosolic 3-hydroxy-3-methylglutaryl-CoA synthases are encoded by two different genes; Ayte J et al.; We report the isolation and characterization of a 1994-base-pair cDNA that encompasses the entire transcription unit of the mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (EC 4.1.3.5.) gene from rat . Analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 508 residues and 56,918-Da molecular mass . Identify of the cDNA clone isolated as mitochondrial HMG-CoA synthase was confirmed by the following criteria: (i) Amino acid residues are 65% homologous with hamster cytosolic HMG-CoA synthase . (ii) A 19-amino acid sequence probably corresponding to the catalytic site is highly homologous (90%) to that reported for chicken liver mitochondrial HMG-CoA synthase . (iii) The expression product of the cDNA in Escherichia coli has HMG-CoA synthase activity . (iv) The protein includes a sequence of 37 amino acid residues at the N terminus that is not present in the cytosolic enzyme . The predominantly basic, hydrophobic, and hydroxylated nature of the residues of this sequence suggests that it is a leader peptide to target HMG-CoA synthase inside mitochondria . These data plus the hybridization pattern in genomic Southern blot analysis, the different transcript size (2.0 kilobases versus 3.4 kilobases for the cytosolic enzyme), and the different expression pattern shown in RNA blot experiments suggest the presence of two HMG-CoA synthase genes, one for the cytosolic and another for the mitochondrial enzyme.

J Bacteriol, 1990 May, 172(5), 2547 - 57
Identification of the promoter, operator, and 5' and 3' ends of the mRNA of the Escherichia coli K-12 gene aroG; Baseggio N et al.; The promoter, operator, and 5' and 3' ends of the mRNA of the Escherichia coli gene aroG (encoding the phenylalanine-sensitive 3-deoxy-arabinoheptulosonate-7-phosphate synthase) were located . Primer extension analysis and nuclease S1 mapping of in vivo transcripts were used to determine the 5' and 3' ends, respectively, of the mRNA . Both ends exhibited some heterogeneity with respect to length . The 3' end of the major molecular species was located within a region that has structural homology with known rho-independent terminators . The location of the aroG promoter was identified in both strands of the DNA by in vitro DNase I footprinting and methylation protection experiments with RNA polymerase . In these experiments, a region of up to 80 base pairs (bp) was protected by the binding of RNA polymerase . The location of the aroG operator was also identified in both strands of the DNA by in vitro DNase I footprinting with pure TyrR . TyrR protected 26 to 28 bp of DNA containing a 22-bp palindrome (TYR R box) and overlapping the -35 region of the promoter . Mutations in the aroG regulatory DNA were isolated by site-directed mutagenesis and cloned in a low-copy-number plasmid to generate aroG-lac fusions . The effects of the mutations on the regulation of aroG expression were determined by measuring the beta-galactosidase activities of the fusions in strains with tyrR, tyrR+, and multicopy tyrR+ genotypes . The results of this mutant analysis confirmed that the aroG operator contains a single TYR R box.

J Bacteriol, 1990 May, 172(5), 2241 - 4
Protein-bound choline is released from the pneumococcal autolytic enzyme during adsorption of the enzyme to cell wall particles; Markiewicz Z et al.; The inactive precursor form of the pneumococcal autolytic enzyme cloned in Escherichia coli was isolated by affinity chromatography on Sepharose-linked choline . The enzyme was recovered in an electrophoretically pure and activated form by elution from the affinity column with radioactive choline solution . When radioactive choline was used for elutions, the enzyme protein isolated contained protein-bound choline, at approximately 1 mol of choline per mol of enzyme protein, indicating the presence of a single choline recognition site . Radioactive choline remained bound to the enzyme protein during dialysis, precipitation by trichloroacetic acid or ammonium sulfate, and during gel filtration, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Incubation of the choline-labeled autolysin with pneumococcal cell walls at 0 degrees C resulted in the adsorption of the enzyme to the wall particles and a simultaneous release of free choline from the enzyme protein . It is suggested that the choline molecules that became bound to the enzyme protein during the activation of autolysin are expelled from the choline-binding site and replaced by choline residues from the wall teichoic acid as the autolysin molecules adsorb to their insoluble substrate before the onset of enzymatic wall hydrolysis.

J Bacteriol, 1990 May, 172(5), 2230 - 5
Transfer region of IncI1 plasmid R64 and role of shufflon in R64 transfer; Komano T et al.; To locate the transfer region of the 122-kiloase plasmid R64drd-11 belonging to incompatibility group I1, a series of deletion derivatives was constructed by in vitro recombinant DNA techniques followed by double homologous recombination in vivo . A plasmid designated pKK609 and bearing a 56.7-kilobase R64 sequence was the smallest transferable plasmid . A plasmid designated pKK610 and no longer possessing the 44-base-pair sequence of the R64 transfer system is located at one end . The other end of the R64 transfer region comprises a DNA segment of about 19 kilobases responsible for pilus formation . Shufflon, DNA with a novel rearrangement in R64, was found to be involved in pilus formation.

EMBO J, 1990 May, 9(5), 1471 - 6
Selective binding of ligands to beta 1, beta 2 or chimeric beta 1/beta 2-adrenergic receptors involves multiple subsites; Marullo S et al.; The molecular basis of ligand binding selectivity to beta-adrenergic receptor subtypes was investigated by designing chimeric beta 1/beta 2-adrenergic receptors . These molecules consisted of a set of reciprocal constructions, obtained by the exchange between the wild-type receptor genes of one to three unmodified transmembrane regions, together with their extracellular flanking regions . Eight different chimeras were expressed in Escherichia coli and studied with selective beta-adrenergic ligands . The evaluation of the relative effect of each chimeric exchange on ligand binding affinity was based on the analysis of delta G values, calculated from the equilibrium binding constants, as a function of the number of substituted beta 2-adrenergic receptor transmembrane domains . The data showed that the contribution of each exchanged region to subtype selectivity varies with each ligand; moreover, while several regions are critical for the pharmacological selectivity of all ligands, others are involved in the selectivity of only some compounds . The selectivity displayed by beta-adrenergic compounds towards beta 1 or beta 2 receptor subtypes thus results from a particular combination of interactions between each ligand and each of the subsites, variably distributed over the seven transmembrane regions of the receptor; these subsites are presumably defined by the individual structural properties of the ligands.

Infect Immun, 1990 May, 58(5), 1402 - 7
Phorbol esters enhance the cyclic GMP response of T84 cells to the heat-stable enterotoxin of Escherichia coli (STa); Weikel CS et al.; We examined the effect of protein kinase C (PKC) activation on the cyclic GMP response to heat-stable enterotoxin (STa) in a colonic carcinoma intestinal epithelial cell line, T84 cells . Our results demonstrate that the active phorbol ester analog, phorbol dibutyrate, but not the inactive alpha-phorbol dibutyrate, acts synergistically with STa to elevate cyclic GMP in intact T84 cells . The effect is observed in the absence or presence of the phosphodiesterase inhibitor, isobutylmethylxanthine, which suggests that phorbol dibutyrate modifies cyclic GMP synthesis rather than cyclic GMP degradation . In contrast to several systems in which prolonged treatment with phorbol ester desensitizes PKC-mediated responses, the cyclic GMP response in T84 cells is not diminished by prolonged treatment of T84 cells with phorbol dibutyrate . Also, transient treatment of T84 cells with phorbol dibutyrate enhances subsequent STa-stimulated cyclic GMP accumulation . These observations suggest that PKC activation produces a long-lived signal in T84 cells which enhances cyclic GMP accumulation in response to STa . Second messenger "cross talk" {T . Yoshimasa, D . R . Sibley, M . Bouvier, R . J . Lefkowitz, and M . G . Caron, Nature (London) 327:67-70, 1987} may be important in the pathogenesis of diarrheal disease.

Acta Virol, 1990 May, 34(3), 256 - 62
Immunological characterization of BLV proteins synthesized in Escherichia coli; Siakkou H et al.; Hybrid proteins composed of beta-galactosidase and polypeptides of the bovine leukaemia virus (BLV) including those of the main core protein p24, the envelope protein gp51 and the transmembrane protein gp30 were produced in Escherichia coli and immunologically characterized . The hybrid proteins were immunologically reactive with sera from cattle naturally infected with BLV, demonstrating a possible use for diagnosis of BLV infection . Detection of antibodies was most sensitive with the p24 derivative.

Microb Pathog, 1990 May, 8(5), 343 - 52
K88 fimbriae as carriers of heterologous antigenic determinants; Bakker D et al.; The K88 fimbriae of enterotoxigenic Escherichia coli are strongly immunogenic antigens that can be used to evoke protective immunity . To find out whether these fimbriae can be used as carriers for foreign epitopes, a highly variable region present in the primary structure of the different K88 variants was replaced with five different heterologous epitopes to investigate to what extent these insertions affected the expression, assembly (biogenesis), stability and immunogenic properties of the resulting hybrid fimbriae . Amino acid residues 163-173, were replaced using site-directed in vitro mutagenesis and the hybrid fimbriae were tested for these aspects using ELISA, immunoelectronmicroscopy and immunoblotting . Replacement of this highly variable region did not affect the biosynthesis of fimbriae, although all mutations tested resulted in a reduced expression depending on the epitope inserted . Testing of the different hybrid fimbriae with a panel of monoclonal antibodies raised against the various K88 serotypes K88ab, K88ac and K88ad indicated that replacement of amino acid sequence 163-173 did not affect conserved or K88ab specific epitopes but the K88ac and K88ad specific conformation was lost . Immunization with hybrid fimbriae raises antibodies specific for the inserted heterologous epitopes.

Tissue Antigens, 1990 May, 35(5), 229 - 33
Monoclonal antibodies detecting discrete epitopes of human perforin; Geisberg M et al.; Perforin is a cytolytic protein of natural killer (NK) cells and cytotoxic T cells (CTL) . Purified perforin has been shown to cause cell lysis and to form stable pores in the target cell membrane, but its relevance to cytolysis in vivo is not clear . The gene for human perforin has been cloned, but monoclonal antibodies (mabs) have not been available . In order to study further its role in cytotoxicity, we have generated mabs to different regions of human perforin . Four mabs were produced from mice immunized with hybrid proteins comprising E . coli TrpE protein at the N-terminus and different regions of human perforin at the C-terminus . These proteins were made using the pATH expression plasmids into which fragments of perforin cDNA were subcloned . Monoclonal antibody PA1 was made from a mouse immunized with a hybrid protein containing the C-terminal 240 amino acids (AA) of perforin, PE1 - the N-terminal 118 AA, and PB1 and PB2 - the central 199 AA . The three plasmid constructs contained non-overlapping cDNA segments which covered the entire sequence of perforin . All mabs reacted with the immunizing hybrid protein, but not with the other hybrid proteins, indicating that at least three epitopes are recognized by this set of mabs . All mabs immunoprecipitated a molecule of about 68 kd from lysates of metabolically-labelled cytolytic large granular lymphocytic leukemia cells, but not from control lysates of non-cytolytic promyelocytic U937 cells . These mabs should be of use in determining structure-function relationships for perforin.

Izv Akad Nauk SSSR Biol, 1990 May-Jun, (3), 359 - 65
{Alu-like sequences in the regions of replication initiation in plasmids p15A and R6K}; Korotkov EV; A method of computer analysis of DNA sequences has been proposed . It is based on information similarity of compared sequences and it significantly increases the usefulness of the computer analysis . This approach has been applied to the search of interconnected areas of Alu-repeats and replication origins of p15A and R6K plasmids . An Alu-like region located in the first stem of the secondary structure of RNA-1 and E . coli RNA-polymerase binding site has been found in the p15A . On R6K replication origin, Alu-like repeats have been found in the area of tandem 22 bp repeats . This comparison also allowed to reveal hidden periodicity of the sequence of human Alu-repeat . A hypothesis that explained the data obtained has been proposed . The proposed approach may be used as a method for revealing DNA sequences that have similar genetic functions.

Mol Gen Genet, 1990 May, 221(3), 421 - 6
Apurinic endonuclease activity from wild-type and repair-deficient mei-9 Drosophila ovaries; Venugopal S et al.; An endonuclease which acts on apurinic (AP) sites in DNA was partially purified from Drosophila ovaries . The enzyme present in the female germ line has a molecular weight of 63,000 daltons, is Mg++ dependent, and produces a site upon cleaving depurinated DNA that supports DNA repair synthesis . Although the same characteristics are shared by the enzyme present in the excision-deficient mutant mei-9, specific activity for the AP endonuclease is reduced 98% when compared with that found for its wild-type counterpart . Moreover, cross-reactivity toward an antibody that recognizes the wild-type AP endonuclease protein is reduced roughly 90% for partially purified preparations from mei-9 . Mixing experiments between extracts of mei-9 and wild type suggest that the mei-9 structural gene somehow alters or influences the levels of the AP endonuclease protein, but in view of the complex phenotype of this mutant the endonuclease is probably not the product of the gene itself.

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