Escherichia coli

Tsitologiia, 1986 Dec, 28(12), 1345 - 50
{Transformation of bone marrow cells in rodents by recombinant plasmid pBRSV}; Kazakova TB et al.; Bone marrow cells (mouse strain CBA/Ca and Syrian hamster cells) were transformed with pBRSV DNA containing T-antigen of the SV40 virus . The SV40 T-antigen in transformed cell was detected in 0.5% cases by immunofluorescence with specific antibodies . Extrachromosomal localization of recombinant DNA was shown by means of retransformation of E . coli cells with cytoplasmic spleen DNA from mice previously injected intravenously the transformed bone marrow cells.

EMBO J, 1986 Dec 1, 5(12), 3401 - 6
Initiation of Escherichia coli minichromosome replication at oriC and at protein n' recognition sites . Two modes for initiating DNA synthesis in vitro; Seufert W et al.; The start sites for leading and lagging DNA strands were determined in vitro with minichromosomes as templates . Fragments from replication intermediates were analyzed by hybridization to single-stranded probes . Leading strand synthesis in the counterclockwise direction was found to originate in or close to (position 248 to -44) the minimal origin . Complementary lagging strand synthesis started several positions to the left outside of oriC . The results suggest in addition a concerted synthesis of leading and lagging strands following the dnaA directed assembly of initiation proteins at double-stranded oricC DNA (pre-replisome) . In addition, DNA synthesis could initiate at protein n' recognition sequences located within and clockwise to the asnA gene . Initiation at n' sites was dependent on protein i activity, whereas leading and lagging strand initiation in the oriC region was not affected by protein i . Our results argue against an involvement of the phi X174-type primosome in the initiation of discontinuous DNA synthesis at oriC . An alternative function is suggested.

Bioorg Khim, 1986 Dec, 12(12), 1612 - 24
{Variants of phage M13 DNA containing a fragment of the beta-galactosidase gene--a convenient mutation system for the study of oligonucleotide-directed mutagenesis}; Petrenko VA et al.; A model system is developed to test oligonucleotide-directed mutations: T----C transition, T and C deletions (delta T and delta C), C insertion, double mutations (A----G, delta T), (T----C, A----G), and large oligonucleotide deletions (36 or 44 nucleotides) . The system includes 9 variants of the phage M13 DNA carrying fragment of beta-galactosidase gene, and oligodeoxyribonucleotides partially noncomplementary to DNA sequence of this gene . Six variants are obtained by the site-localized mutagenesis, the other were described earlier . Induced mutations are easily tested by phenotype change of transformed bacteria (Lac+----Lac-); by formation or loss of the sites for BamHI and EcoRI restrictases; by DNA hybridization with 32P-labeled oligonucleotides; and by DNA sequencing by the Sanger method . The system is used to study the role of some factors, such as completeness of RF DNA synthesis, thermal stability of the oligonucleotide: DNA complex, quality of enzymes and substrates used in polymerase reaction, mutation type or the efficiency of mutagenesis . A number of unexpected mutations were observed in the course of oligonucleotide-directed mutagenesis . Lower yields of some mutants induced by oligonucleotides are shown to be due to the action of repair systems of bacteria.

Arch Biochem Biophys, 1986 Dec, 251(2), 606 - 15
Comparison of the energetics of lactose active transport: artificial versus enzyme-associated energy source; Chen LI et al.; To further consider the thermochemical method as a useful approach for active transport research and to investigate the characteristic of a proton electrochemical potential (delta mu H+) across the membrane, the energetics of lactose active transport across Escherichia coli membrane vesicles coupled with an artificial electron donor (phenazine methosulfate-ascorbate) has been investigated . The results were compared with those obtained with an enzyme-associated electron donor (lactate dehydrogenase-D-lactate) . The oxidation of an electron donor provided the energy necessary for the transport process . The observed higher heat of ascorbate oxidation reaction in the presence of a proton ionophore (carbonyl cyanide m-chlorophenylhydrazone) further confirmed the formation of delta mu H+ across the membrane . Part of the oxidation energy was utilized to form delta mu H+ . Comparison of the energetics revealed that the magnitudes of delta Hox (the enthalpy of the oxidation reaction) and delta Hm (the enthalpy of the formation of delta mu H+) in the two energy sources were comparable (-46 kcal/mol of ascorbate to -40 kcal/mol of D-lactate for delta Hox and 9.6 kcal/mol of ascorbate to 14 kcal/mol of D-lactate for delta Hm) . Comparable and low value (about 1%) was also found in the free energy transfer (defined by delta Gm/delta Gox) from the oxidation reaction to the formation of delta mu H+ . These results, in combination with the close values of delta mu H+ observed in the two systems, suggested that the characteristic of the created delta mu H+ was independent of the energy source . Examination of delta Hm might provide the information on the ratio of the number of protons produced, as 1 mol of two different electron donors was oxidized . The oxidation reaction in the presence of membrane vesicles was discussed.

Mol Cell Biol, 1986 Dec, 6(12), 4396 - 408
A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps; Sadowski I et al.; Proteins encoded by oncogenes such as v-fps/fes, v-src, v-yes, v-abl, and v-fgr are cytoplasmic protein tyrosine kinases which, unlike transmembrane receptors, are localized to the inside of the cell . These proteins possess two contiguous regions of sequence identity: a C-terminal catalytic domain of 260 residues with homology to other tyrosine-specific and serine-threonine-specific protein kinases, and a unique domain of approximately 100 residues which is located N terminal to the kinase region and is absent from kinases that span the plasma membrane . In-frame linker insertion mutations in Fujinami avian sarcoma virus which introduced dipeptide insertions into the most stringently conserved segment of this N-terminal domain in P130gag-fps impaired the ability of Fujinami avian sarcoma virus to transform rat-2 cells . The P130gag-fps proteins encoded by these transformation-defective mutants were deficient in protein-tyrosine kinase activity in rat cells . However v-fps polypeptides derived from the mutant Fujinami avian sarcoma virus genomes and expressed in Escherichia coli as trpE-v-fps fusion proteins displayed essentially wild-type enzymatic activity, even though they contained the mutated sites . Deletion of the N-terminal domain from wild-type and mutant v-fps bacterial proteins had little effect on autophosphorylating activity . The conserved N-terminal domain of P130gag-fps is therefore not required for catalytic activity, but can profoundly influence the adjacent kinase region . The presence of this noncatalytic domain in all known cytoplasmic tyrosine kinases of higher and lower eucaryotes argues for an important biological function . The relative inactivity of the mutant proteins in rat-2 cells compared with bacteria suggests that the noncatalytic domain may direct specific interactions of the enzymatic region with cellular components that regulate or mediate tyrosine kinase function.

Appl Environ Microbiol, 1986 Dec, 52(6), 1394 - 7
Location of plasmid-mediated citrate utilization determinant in R27 and incidence in other H incompatibility group plasmids; Taylor DE et al.; Citrate utilization (Cit+) is encoded by a specific subgroup of incompatibility HI plasmids, viz., IncHI1 plasmids . Only one IncHI1 plasmid, pRG1271, which originated in a Mexican typhoid outbreak in 1972, did not specify Cit+ . All other Cit+ plasmids hybridized to a Cit+ probe, a 2-kilobase BglII fragment derived from the Cit+ transposon Tn3411 . The position of the Cit+ determinant was mapped to a 13.5-kilobase ApaI fragment within the prototype IncHI1 plasmid R27 . No other functions have been mapped within this region . Citrate utilization mediated by IncHI1 was observed only after a prolonged lag period of approximately 150 h, and certain Escherichia coli strains, e.g., E . coli K-12 J53-1, were not able to utilize citrate specified by the H plasmids . Most E . coli strains harboring the multicopy Cit+ plasmid pOH2, a derivative of pBR322, required only 18 to 24 h to express the Cit+ phenotype, but E . coli J53-1 (pOH2) required at least 72 h for expression.

Proc Natl Acad Sci U S A, 1986 Dec, 83(23), 9070 - 4
Escherichia coli K-12 restricts DNA containing 5-methylcytosine; Raleigh EA et al.; We have observed that plasmids containing certain cloned modification methylase genes of type II restriction-modification systems cannot be transformed into many laboratory strains of Escherichia coli K-12 . The investigation of this phenomenon, reported here, has revealed (i) DNA containing 5-methylcytosine is biologically restricted by these strains, while DNA containing 6-methyladenine is not; (ii) restriction is due to two genetically distinct systems that differ in their sequence specificities, which we have named mcrA and mcrB (for modified cytosine restriction) . Since 5-methylcytosine containing DNA is widespread in nature, the Mcr systems probably have a broad biological role . Mcr restriction may seriously interfere with molecular cloning of 5-methylcytosine-containing foreign DNAs . The Mcr phenotypes of some commonly used strains of E . coli K-12 are reported.

J Surg Res, 1986 Dec, 41(6), 645 - 52
The effect of enkephalins and prostaglandins on O-2 release by neutrophils; Simpkins CO et al.; Derivatives of superoxide (O-2), produced by phagocytic cells, are thought to play a role in the adult respiratory distress syndrome (ARDS) and other disease states . Control of the release of O-2 may prove beneficial . Using human neutrophils as a source of O-2, and an assay for O-2 based upon the reduction of cytochrome C, we found that prostaglandin D2 (PGD2), leucine enkephalin (LE), and methionine enkephalin (ME) inhibited O-2 release . The Escherichia coli product, N-formyl methionyl leucyl phenylalanine (FMLP), was employed to stimulate O-2 release . PGD2 was most potent while there was no significant difference between LE and ME . Another peptide, thyrotropin releasing hormone (TRH), had no effect on O-2 release . There was no correlation between the potency of the inhibitory effect on O-2 release and the effect of these agents on the binding of {3H} FMLP to human neutrophils . Comparison of different but structurally related prostaglandins (PGD2, PGE2, and PGF2 alpha) revealed that PGD2 was more potent than PGE2 in inhibiting O-2 and that PGF2 alpha had no effect . This result suggested that the presence and position of the carbonyl group was an important determinant of the magnitude of inhibition.

J Virol, 1986 Dec, 60(3), 902 - 9
Strain-specific transcription and translation of the BamHI Z area of Epstein-Barr Virus; Seibl R et al.; The expression of the 1,800-base-pair BamHI Z region of Epstein-Barr virus DNA was analyzed by hybrid-selected translation with several DNA subclones and RNA from different cell lines . Furthermore, large segments of the three reading frames extending in this area were expressed as fusion proteins into Escherichia coli . The fusion proteins were partially purified and used to immunize rabbits . These sera were used to confirm our mapping assignments and to identify the respective posttranslationally modified proteins in in vivo labeling experiments . The reading frame BRLF1 (the first reading frame starting in the BamHI R fragment in leftward orientation) encoded a 93- to 96-kilodalton (kDa) protein depending on the cell line . The molecular weight of in vivo-labeled proteins was increased relative to that of in vitro-translated proteins, indicating that a posttranslational modification had occurred . The BZLF1 reading frame encoded a 35-kDa protein . It was posttranslationally cleaved from a 38-kDa precursor in induced B95-8 and induced Raji cells and from a 40-kDa precursor in induced P3HR1 cells . In Raji cells superinfected with virus derived from P3HR1 cells, the protein seemed to be expressed both from endogenous Raji genomes and from infecting genomes . The transcripts for the 93- to 96-kDa and the 35-kDa protein overlapped partially . The serum against the expressed third reading frame BZLF2 specifically precipitated a 140-kDa protein . This reading frame contains only 650 nucleotides, and therefore further coding sequences were presumably spliced to BZLF2 . The latter is deleted in the Raji cell line; therefore, the observed 140kDa protein in superinfected Raji cells was expressed from infecting P3HR1 genomes.

J Virol, 1986 Dec, 60(3), 1018 - 26
Partition of E1A proteins between soluble and structural fractions of adenovirus-infected and -transformed cells; Chatterjee PK et al.; The partition of E1A proteins between soluble and structural framework fractions of human cells infected or transformed by subgroup C adenoviruses was investigated by using gentle cell fractionation conditions . A polyclonal antibody raised against a trpE-E1A fusion protein (K.R . Spindler, D.S.E . Rosser, and A . J . Berk, J . Virol . 132-141, 1984) synthesized in Escherichia coli was used to measure the steady-state levels of E1A proteins recovered in the various fractions by immunoblotting . The relative concentration of E1A proteins recovered in the soluble fraction of adenovirus type 2-infected cells was at least fivefold greater than the relative concentration in the corresponding fraction of transformed 293 cells . The observed distribution of E1A proteins was not altered by the sulfhydryl-blocking reagent N-ethylmaleimide . E1A proteins were recovered in nuclear matrix, chromatin, and cytoskeleton fractions after further fractionation of the structural framework fraction . However, the E1A protein species that could be identified by one-dimensional gel electrophoresis were not uniformly distributed among the subcellular fractions examined . The results obtained when fractionation was performed in the presence of the oxidation catalysts Cu2+ or (ortho-phenanthroline)2 Cu2+ indicate that E1A proteins can be efficiently cross-linked, via disulfide bonds, to the structural framework of both adenovirus-infected and adenovirus-transformed cells.

J Immunol, 1986 Dec 1, 137(11), 3632 - 8
Immunosuppression in viral oncogenesis . III . Effects of virus infection on interleukin 1 and interleukin 2 generation and responsiveness; Strayer DS et al.; Malignant rabbit fibroma virus (MV) is an oncogenic immunosuppressive leporipoxvirus . We studied the effects of MV infection and MV-associated tumor-induced suppressor factor (TISF) on the production of and responsiveness to interleukins 1 and 2 . Adherent cells from MV tumor-bearing rabbits elaborate adequate amounts of IL 1 in response to E . coli endotoxin . Neither live virus nor TISF alters the production or the responsiveness to IL 1 . However, when we examined spleen cells from rabbits 7 days after MV inoculation, we noted that their ability to produce and respond to IL 2 is deficient . Despite their relatively poor capacity to produce IL 2, these spleen cells express receptor for IL 2 in normal amounts, as measured by the monoclonal antibody 7D4 . TISF derived from T lymphocytes from MV tumor-bearing rabbits is by itself capable of inhibiting partially normal secretion of IL 2 and also the response of the cloned murine T cell line HT-2 to added IL 2 . Full expression of the immunosuppressive capacity of spleen cells from MV tumor-bearing rabbits requires cell-cell contact, however, and cannot be replaced by either live virus or spleen cell supernatants . Such spleen cells inhibit normal mitogen responsiveness, a defect not remedied by adding exogenous IL 2 . Immunologic dysfunction induced by MV infection is transient, and by 11 days after virus inoculation, actively mediated recovery from immunosuppression is observed . We found that spleen cells from rabbits studied 11 days postinoculation secreted IL 2 normally . Thus, immunologic dysfunction secondary to infection with malignant rabbit fibroma virus reflects deficiencies in both elaboration of and response to IL 2, and return of immune function later in the course of the infection is associated with return of the ability of lymphocytes to secrete IL 2.

J Bacteriol, 1986 Dec, 168(3), 1457 - 8
Genetic mapping in Escherichia coli of tmk, the locus for dTMP kinase; Binkley JP et al.; The genetic location of tmk, the gene for dTMP kinase, has been mapped at min 24.0 on the Escherichia coli map.

J Bacteriol, 1986 Dec, 168(3), 1228 - 33
Molecular cloning and analysis of genes for production of K5, K7, K12, and K92 capsular polysaccharides in Escherichia coli; Roberts I et al.; With a DNA fragment from within the region encoding the transport functions for K1 production as a hybridization probe in Southern blot experiments, homologous DNA sequences were detected in the DNA from Escherichia coli strains producing K5, K7, K92, and K100 capsular polysaccharides . No homology with the laboratory strain LE392 was detected . The same DNA probe was used to prescreen cosmid libraries in LE392 by colony hybridization, as a rapid method to isolate clones encoding the genes for K5, K7, K12, and K92 antigen production . Clones carrying sequences homologous to the probe that also produced capsular material were identified by using polyclonal and monoclonal antibodies raised against the K antigen in question and K antigen-specific phages . By restriction enzyme mapping of the appropriate cosmid clones it was possible to align the genes for the production of different K antigens in terms of common restriction endonuclease cleavage sites . A DNA fragment encoding the postulated transport functions for K7 antigen production could complement deletion mutations in the transport functions for K1 antigen production . Thus the transport to the cell surface of chemically distinct polysaccharides may be by a common process . Analysis in E . coli of the proteins produced by plasmids carrying the likely transport functions for K1, K5, and K7 antigen production revealed that each region coded for a similar polypeptide.

Endocrinol Jpn, 1986 Dec, 33(6), 843 - 9
Endotoxin-induced ATP depletion in thyrotoxic rats; Ando K et al.; Effect of endotoxin from E . coli on the ATP content in heart muscle, the liver and the kidney of thyrotoxic rats was studied . When endotoxin (200-400 micrograms) was intravenously injected to rats taking drinking water containing 2-7.5 micrograms T3 per ml, body temperature rose and the heart rate increased . At the same time, a marked decrease in the ATP content in heart muscle and the kidney was observed together with an increase in Na+-K+-ATPase activity . Such changes were not observed or seen only to a small extent in euthyroid rats after endotoxin administration . Endotoxin-induced ATP depletion in T3-treated rats was prevented by administration of 5 mg hydrocortisone just prior to endotoxin injection . These findings indicate that endotoxin easily causes ATP depletion in some tissue or organs in thyrotoxicosis, even if the dose of endotoxin is not enough to produce such an effect in the euthyroid . These observations are of interest in relation to thyroid storm associated with bacterial infection.

Mol Gen Genet, 1986 Dec, 205(3), 546 - 9
In vivo cloning of DNA into multicopy cosmids by mini-Mu-cosduction; de Mendoza D et al.; A general in vivo procedure for cloning Escherichia coli genes into cosmids has been developed . The method we describe here uses a deleted Mu phage (a mini-Mu) to transpose E . coli genes into cosmids during mini-Mu replication . The resulting cosmids clones are packaged in vivo into lambda phage particles . Plasmids carrying a particular DNA sequence can be selectively recovered after infection of a new host with the in vivo constructed genomic cosmid library . This system was used successfully to clone several E . coli genes.

Genetika, 1986 Dec, 22(12), 2775 - 83
{Precise mapping of the gpp gene involved in guanosine tetraphosphate synthesis and ilvC-gpp deletion in the region of the Escherichia coli chromosome}; Belitskii BR et al.; Using the set of transducing lambda phages the gpp gene, responsible for pppGpp to ppGpp conversion, was localized between rep and trxA genes on 85 min of the Escherichia coli genetic map . Taking advantage of the Tn10 transposon inserted into the adjacent ilvY locus, we deleted the region of E . coli chromosome covering ilvC, rep and gpp genes . The metabolism of (p)ppGpp in the deletion-containing cells confirms that the product of the gpp gene, guanosine pentaphosphatase, is not the only enzyme, responsible for pppGpp degradation and ppGpp synthesis.

Arch Biochem Biophys, 1986 Dec, 251(2), 465 - 70
ATPase activity of biotin carboxylase provides evidence for initial activation of HCO3- by ATP in the carboxylation of biotin; Climent I et al.; When we incubated biotin carboxylase from Escherichia coli with ATP in absence of biotin we observed HCO3- -dependent ATP hydrolysis, which was activated by 10% ethanol in the same proportion as the activity of D-biotin carboxylation assayed in the presence of biotin . The two activities exhibited identical heat stability and were protected equally by glycerol; both required Mg2+ and K+ and showed similar dependency on the concentration of ATP . Biotin assay excluded potential contamination by traces of biotin as a cause of the observed ATP hydrolysis, and this was confirmed by the findings that carboxybiotin did not accumulate and that avidin was uninhibitory . Therefore we concluded that this HCO3- -dependent ATPase was genuinely a partial activity of biotin carboxylase . This partial activity supports a sequential mechanism for enzymatic carboxylation of biotin in which HCO3- is activated by ATP in a first step . It is consistent with the initial formation of the carbonic-phosphoric anhydride (HOCO2PO3(2-)), and it does not agree with models where biotin is phosphorylated by ATP prior to reaction with HCO3- . It appears that enzymes that use HCO3- for carboxylation, including biotin-dependent carboxylases, phosphoenolpyruvate carboxylase, and carbamoyl phosphate synthetase, activate HCO3- by a common mechanism involving the initial formation of the carbonic-phosphoric anhydride.

J Gen Virol, 1986 Dec, 67 ( Pt 12), 2781 - 4
Lambdoid coliphages conferring a novel pattern of phage sensitivity on Escherichia coli K12; Poon AP et al.; Seven temperate coliphages recovered from naturally occurring lysogenic strains of Escherichia coli were found to lyse E . coli C but not K12 . Four of these C-specific phages produced mutants (hrk) able to grow on K cells . The K cells harbouring HK253hrk and HK183hrk were converted so that they could adsorb and be lysed by three other non-mutant C-specific phages . HK253, HK183 and two other phages were shown to recombine with phage lambda.

J Surg Res, 1986 Dec, 41(6), 609 - 19
The role of endorphins and vasopressin in canine endotoxin shock; Cronenwett JL et al.; Chemical antagonists were used to assess the role of beta-endorphin and arginine-vasopressin (AVP) in canine endotoxin shock . Fifteen awake dogs were given Escherichia coli endotoxin IV . Within 5 min, CO decreased to 28%, LV dP/dt to 46%, and MAP to 52% baseline . Fifteen minutes after endotoxin, five dogs each received naloxone, AVP antagonist, or no treatment . Control (untreated) animals exhibited persistent cardiovascular depression, with CO 49%, LV dP/dt 69%, and MAP 91% of baseline after 45 min . Naloxone improved CO to 69%, LV dP/dt to 94%, and MAP to 91% by 30 min after treatment . AVP blockade improved CO to 105%, LV dP/dt to 107%, and MAP to 95% of baseline by 30 min after treatment, and caused significant tachycardia . Plasma cortisol and AVP increased markedly in all groups after endotoxin administration . AVP antagonist treatment increased mean survival from 1.4 to 4 days . These data suggest that abnormally elevated AVP contributes to cardiovascular depression in canine endotoxin shock and that AVP blockade is therapeutic in the animal model studied.

J Bacteriol, 1986 Dec, 168(3), 1343 - 51
Mutations in an integration host factor-binding site: effect on lambda site-specific recombination and regulatory implications; Thompson JF et al.; The manner in which integration host factor (IHF) regulates lambda site-specific recombination has been analyzed by examining the behavior of both wild-type and mutant DNAs in integrative and excisive recombination as well as in protein binding . While integrative recombination of an attP with two base changes in the H1 site required 8-fold more IHF than did wild type, binding to this site was lowered at least 500-fold, suggestive of cooperative interactions . A mutant attP with nine base changes did not integrate at all in vitro, with the defect being less severe in vivo . IHF inhibition of excisive recombination was relieved by both mutations in vitro and in vivo . These results imply that occupancy of the H1 site is critical for determining the direction of recombination . It is proposed that IHF inhibition of excision provides a monitor of the strength of the induction stimulus and the nutritional state of the cell; this would allow the prophage to excise selectively in conditions which favor successful completion of the lytic cycle.

J Biomol Struct Dyn, 1986 Dec, 4(3), 463 - 76
CC/GG contacts facilitate the B to A transition of DNA in solution; Minchenkova LE et al.; Self-complementary decadeoxynucleotides, CCGATATCGG, CCAGATCTGG, CCCTGCAGGG, GGGGGCCCCC, were designed and synthesized to estimate the A-philic free energy of CC/GG contacts . First, regions of temperature-stability of the double-stranded conformation were determined for each 10-mer . Then, circular dichroism spectra were recorded for the B-family forms at different temperatures, counter-ion concentrations and trifluoroethanol contents . A cooperative change typical of the B-A transition is observed in the CD spectra at a trifluoroethanol content specific for each duplex . The positions of half-transition points were functions not only of the nucleotide sequence but of the duplex length as well: the B to A transitions were hindered in these 10-mers in comparison with a lengthy DNA . The B-phility value was estimated to be 3 kcal/mol of 10-mer . The B-A transition point was shown to drop with an increase in the number of CC/GG contacts in a duplex . The designed 10-mers made it possible to estimate quantitatively the A-phility of CC/GG contact as compared with an average DNA: (FA-FB)CC = 0.2 Kcal/mol, (FA-FB)DNA = 0.7 Kcal/mol.

Microb Pathog, 1986 Dec, 1(6), 533 - 47
Influence of cloned Escherichia coli hemolysin genes, S-fimbriae and serum resistance on pathogenicity in different animal models; Hacker J et al.; The virulence of the uropathogenic E . coli strain 536 (O6:K15:H31) which produces the S-fimbrial adhesin (Sfa+), is serum-resistant (Sre+) and hemolytic (Hly+) and its derivatives were assessed in five different animal models . Cloned hemolysin (hly) determinants from the chromosomes of O6, O18 and O75 E . coli strains and from the plasmid pHly152 were introduced into the spontaneous Sfa-, Sre-, Hly- mutant 536-21 and its Sfa+, Sre+, Hly- variant 536-31 . As already demonstrated for the 536-21 strains (Infect . Immun . 42: 57-63) the O18-hly determinant but not the plasmid-encoded hly determinant of pHly152 transformed into 536-31 contribute to lethality in a mouse peritonitis model . Similar results were obtained with both Hly- host strains and their Hly+ transformants in a chicken embryo test and in a mouse nephropathogenicity assay in which the renal bacterial counts were measured 15 min to 8 hours after i.v . infection . S-fimbriae and serum resistance had only a marginal influence in these three in vivo systems . In contrast all three factors, S-fimbriae, serum resistance and hemolysin, were necessary for full virulence in a respiratory mouse infection assay . In a subcutaneously-induced sepsis model in the mouse restoration of S-fimbriae and serum resistance and separately chromosomally-encoded hemolysis increased virulence to a level comparable to that of the parental 536 strain.

Mol Biol Med, 1986 Dec, 3(6), 495 - 508
Fimbrial phase variation and DNA rearrangements in uropathogenic isolates of Escherichia coli; Abraham JM et al.; Having previously shown that the oscillating on-off expression (phase variation) of type 1 fimbriae in Escherichia coli is regulated genetically by an invertible element of DNA, we wished to determine whether E . coli isolates recovered from infected humans behaved in similar fashion . We examined four different clinical isolates that expressed type 1 fimbriae, P fimbriae, or both . Using, in Southern blot analysis, a DNA probe from the type 1 fimbrial switch, that hybridized to one DNA band from phase-on bacteria and to two DNA bands from phase-off bacteria, we found that the three clinical isolates expressing type 1 fimbriae contained the same invertible switch previously seen in the K-12 isolate . Employing a similar approach to characterize the on-off expression of P fimbriae, we used a DNA probe containing the known transcriptional signals for P fimbriae . Although we detected DNA rearrangement in the two strains expressing P fimbriae, unlike the case for type 1 fimbriae, the rearrangement did not correlate with the on-off state of the P fimbriae . Rather, the DNA rearrangement correlated with the environmental conditions of growth of the bacteria from which the DNA was isolated . These results confirm the notion that P fimbriae expression and type 1 fimbriae expression are controlled differently.

Arch Biochem Biophys, 1986 Dec, 251(2), 458 - 64
Escherichia coli H+-ATPase: loss of the carboxyl terminal region of the gamma subunit causes defective assembly of the F1 portion; Miki J et al.; Mutant genes for the gamma subunit of H+-translocating ATPase (H+-ATPase) were cloned from eight different strains of Escherichia coli isolated in this laboratory . Determination of their nucleotide sequences revealed that they are amber nonsense mutations: a Gln codon at position 15, 158, 227, 262, and 270, respectively, was replaced by a termination codon in these strains . As terminal Met is missing in the gamma subunit, these results indicate that these strains are capable of synthesizing fragments of gamma subunits of 13, 156, 225, 260, and 268 amino acid residues, respectively . Studies on the properties of membranes of these strains suggested the importance of the region between Gln 269 and the carboxyl terminus (residue 286) for forming a stable F1 complex with ATPase activity and the region between Gln 226 and Gln 261 for normal interaction of F1 with F0 . The sequence from Gln 261 to Gln 269 also seemed to be important for stability of F1 assembly on the membranes . The high frequency of the nonsense mutations suggested that the number of essential residues is limited in this subunit . Comparison of the homologies of the amino acid sequences of the gamma subunits from four different sources confirmed this notion: 19% of amino acid residues are identically conserved in these four strains, and the conserved regions are the amino terminal and carboxyl terminal regions.

J Bacteriol, 1986 Dec, 168(3), 1332 - 5
gamma-Glutamyltranspeptidase from Escherichia coli K-12: formation and localization; Suzuki H et al.; Escherichia coli cells showed maximum activity of gamma-glutamyltranspeptidase (EC 2.3.2.2) when they were grown at 20 degrees C, 14% of maximum activity at 37 degrees C, and none at 43 degrees C . The enzyme activity of intact cells grown at 20 degrees C was stably maintained after the temperature was changed to 45 degrees C . The activity increased during the exponential phase, and maximum activity was found at stationary phase . Its intracellular localization in the periplasmic space was confirmed.

J Bacteriol, 1986 Dec, 168(3), 1325 - 31
gamma-Glutamyltranspeptidase from Escherichia coli K-12: purification and properties; Suzuki H et al.; gamma-Glutamyltranspeptidase (GGT) (EC 2.3.2.2) was purified from the periplasmic fraction of Escherichia coli K-12 to electrophoretic homogeneity . The final purification step, chromatofocusing, gave two protein peaks showing GGT activity (fractions A and B) . The major heavy fraction (fraction A) consisted of two different subunits, with molecular weights of 39,200 and 22,000 . The minor light fraction (fraction B) consisted of those with molecular weights of 38,600 and 22,000 . Fraction A catalyzes the hydrolysis and transpeptidation of all gamma-glutamyl compounds tested, but it prefers basic amino acids and aromatic amino acids as acceptors . The apparent Km values for glutathione and gamma-glutamyl-p-nitroanilide as gamma-glutamyl donors in the transpeptidation reaction were both 35 microM, and those for glycylglycine and L-arginine as acceptors were 0.59 and 0.21 M, respectively . The enzyme was inhibited by some amino acids and by protease inhibitors and affinity-labeling reagents for GGT . The temperature stability of the purified GGT supports our hypothesis that E . coli GGT is synthesized only at lower temperature rather than that the synthesized GGT is degraded or inactivated at higher temperature.

J Bacteriol, 1986 Dec, 168(3), 1234 - 42
Identification of two new hemagglutinins of Escherichia coli, N-acetyl-D-glucosamine-specific fimbriae and a blood group M-specific agglutinin, by cloning the corresponding genes in Escherichia coli K-12; Rhen M et al.; Genes encoding the Escherichia coli IH11165 hemagglutinins with specificity for terminal N-acetyl-D-glucosamine and blood group M antigen, respectively, were cloned by a cosmid cloning procedure . A 22-kilobase-pair subclone expressed both hemagglutination specificities in the nonhemagglutinating E . coli HB101 recipient strain . Derivatives obtained by insertion and deletion mutagenesis expressed either one of the two hemagglutination specificities . Both agglutinins were purified; the agglutinin recognizing terminal N-acetyl-D-glucosamine was associated with a new type of fimbria (G fimbria) with an apparent subunit molecular mass of 19.5 kilodaltons, whereas the blood group M agglutinin (M agglutinin) was nonfimbrial and had an apparent subunit mass of 21 kilodaltons.

Eur J Biochem, 1986 Dec 1, 161(2), 513 - 8
Column centrifugation generates an intersubunit disulfide bridge in Escherichia coli F1-ATPase; Tozer RG et al.; Passage of F1-ATPase through a centrifuge column {Penefsky, H . S . (1979) Methods Enzymol . 56, 527-530} caused formation of a product with a relative molecular mass of 72,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis . The product was identified as cross-linked alpha and delta subunits by using Western blots and subunit-specific monoclonal antibodies . The cross-link was reversed by 50 mM dithiothreitol implying that it was a disulfide bridge . Formation of the cross-link was inhibited by 2 mM EDTA and was stimulated in some buffers by the addition of 10 microM CuCl2 . Time course experiments indicated that the majority of the cross-link formed while the enzyme was passing through the column . Thus the cross-link induced by column centrifugation arose from the rapid, heavy-metal-ion-catalysed oxidation of two sulfhydryl groups, one on the alpha subunit and one on the delta subunit, to a disulfide . These results demonstrate that care must be exercised when running proteins through centrifuge columns as potentially deleterious disulfide formation can result . An anti-beta monoclonal antibody was capable of immunoprecipitating the entire enzyme including the cross-linked subunits, implying that the cross-linked alpha and delta subunits were still a part of F1 . The formation of the cross-link affected neither the hydrolytic activity of the enzyme nor its susceptibility to inhibition by epsilon subunit . The cross-linked enzyme was unable to bind to F1-depleted membranes in experiments in which soluble F1 and membranes were separated by centrifugation . Column centrifugation did not generate the cross-link on membrane-bound enzyme . These results indicate that the alpha-delta cross-link results in a loss of binding affinity between F1 and F0.

J Gen Microbiol, 1986 Dec, 132 ( Pt 12), 3261 - 8
The relationship of the delta transfer factor and KColIb to the ColIb plasmid; Walia SK et al.; The structures of the colicin Ib plasmid (ColIb), the delta transfer factor and a plasmid determining kanamycin resistance and colicin Ib production called KColIb, were compared . Radiolabelled mini-ColIb plasmids and isolated DNA fragments of ColIb were used as probes for nitrocellulose blots of digests of the other two large plasmids . The structure of delta was consistent with it having one large deletion of about 10 MDa in the SB fragment and two insertions of approximately 6 MDa and 12 MDa in the SB and SA fragments of the ColIb plasmid . It was hypothesized that KColIb had six small insertions in SA, SB, SE and near the junction of the SB and SD fragments . However, ColIb, KColIb and delta were homologous for at least 70% of their lengths . The highly conserved regions in the three plasmids were the regions that corresponded to fragments SA, SC and SD of ColIb . In addition, delta and KColIb differed from ColIb at similar sites . The possible evolution of these plasmids is discussed.

Arch Int Physiol Biochim, 1986 Dec, 94(5), S35 - 8
Singlet oxygen mutagenicity induced in the lac operon; Decuyper-Debergh D et al.; We have studied the specificity of singlet oxygen (1O2) mutagenesis in single-stranded DNA phage by analysing 1O2-induced mutations in the lac insert of the M13 mp 19 hybrid phage . 107 lac mutants were analysed showing mainly single-base substitutions with a total of 93% and 7% of 40-50 base deletion mutations . Most of the substitutions are G----T and C----A transversions with respectively 27 and 54% of the mutations . The replicative form of the M13 mp 19 DNA (RFDNA) was used as substrate for the 1O2 reactions, there are then two types of progeny phages DNA's . As guanine residues are the targets of the oxidation, it appears that both types of transversions are provided by one type of lesion: the guanine oxidised by 1O2 is read like a thymine by E . coli DNA polymerase-I.

Br J Pharmacol, 1986 Dec, 89(4), 635 - 40
Feline endotoxin shock: effects on tissue histamine and histidine decarboxylase activity; Parratt JR et al.; Tissue histamine levels as well as specific and non-specific histidine decarboxylase were examined before and at various times (5 and 10 min) after the intravenous injection of a lethal dose (2 mg kg-1) of E . coli endotoxin in anaesthetized cats . Histamine levels were increased 5 min after endotoxin, especially in the skin, liver, lung and stomach . There was evidence, in most of the cats, for a rapid and substantial activation of specific histidine decarboxylase especially in the lungs, liver, heart and gastrointestinal tract 5-10 min after endotoxin administration . It is suggested that endotoxin induces the local formation of histamine and that this formation and local release may contribute to the pathophysiology of endotoxin shock in this species.

Int J Radiat Biol Relat Stud Phys Chem Med, 1986 Dec, 50(6), 973 - 81
Differential effects of procaine and phenethyl alcohol on excision repair of DNA in u.v.-irradiated Escherichia coli; Tomiyama H et al.; Experiments were performed to investigate the involvement of the cell membrane in the excision DNA repair process in Escherichia coli . Two membrane-binding drugs, procaine and phenethyl alcohol (PEA), inhibited liquid-holding recovery (LHR) in u.v.-irradiated E . coli wild-type and recA strains . In uvrB and polA strains where, after u.v.-irradiation, LHR was absent the two drugs had no effect . Both drugs markedly reduced the removal of u.v.-induced thymine dimers in the DNA of wild-type cells (H/r30) . Analysis by alkaline sucrose gradients revealed that PEA inhibited the incision step in excision repair . In contrast, procaine had no effect on incision but apparently inhibited the late steps in excision repair . PEA dissociated DNA from the cell membrane, whereas procaine did not . The results suggest that the two drugs PEA and procaine inhibit LHR and the excision repair process operating on u.v.-induced damage in E . coli by at least two different mechanisms each of which may involve the cell membrane.

J Surg Res, 1986 Dec, 41(6), 620 - 6
Lung permeability and hemodynamics during endotoxemia: effect of aprotinin; Winn R et al.; To test the hypothesis that the broad spectrum protease inhibitor, aprotinin, can prevent early pathophysiology of sepsis, we administered endotoxin (0.1-0.75 microgram/kg) by a 30-min infusion to awake goats . Animals were used as their own controls receiving endotoxin with no treatment on one day and treatment with a bolus injection (10 trypsin inhibitory units, TIU, per kg) followed by a 6-hr infusion (5 TIU/kg/hr) of aprotinin on another . The effect on systemic and pulmonary hemodynamics, lung lung lymph flow (QL), lymph plasma protein ratio (L/P), and systemic eicosanoid levels were assessed . QL quickly reached 28 ml/hr (four times baseline) in both groups then slowly returned toward baseline . L/P ratio of both groups decreased by about 10% then returned to baseline . QL and L/P were not different between groups . Likewise, vascular parameters were not different between groups . Mean pulmonary artery pressure increased approximately 150% to a peak of 58 cm H2O in both groups while pulmonary artery wedge pressure doubled from a baseline of 8 cm H2O then both groups returned to baseline . Systemic arterial pressure decreased over the 6 hr experimental period by 15 Torr to 70 Torr in both groups . Cardiac output declined from 4.3 to 3 liter/min after the endotoxin, remaining at the level for 2 hr then progressively increased to about 5 liter/min in both groups . We conclude that aprotinin, in doses similar to those reported to give protection from acute lung injury of various origins, fails to modify the early cardiopulmonary pathophysiology of endotoxin.

J Bacteriol, 1986 Dec, 168(3), 1155 - 8
Heat shock regulatory gene rpoH mRNA level increases after heat shock in Escherichia coli; Tilly K et al.; The Escherichia coli rpoH gene product sigma 32 is essential for the increase in heat shock gene transcription found after exposure of the bacteria to a sudden temperature increase . It is not known how the concentration of active sigma 32 is modulated . We showed that rpoH transcript levels increased after heat shock and that the magnitude of the increase in the level of mRNA was correlated with the magnitude of the temperature shift . The increase in the level of rpoH mRNA was still found in rpoH mutants so the mechanism of induction differed from that of the set of previously identified heat shock genes . The increased concentration of rpoH mRNA should result in a higher level of sigma 32, which is likely to be important for increasing heat shock gene transcription.

J Bacteriol, 1986 Dec, 168(3), 1120 - 7
Endonuclease IV (nfo) mutant of Escherichia coli; Cunningham RP et al.; A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA . The sedimentation coefficient of the enzyme was similar to that of endonuclease IV . An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome . nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin . The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate . It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA . These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active . However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not . The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested.

Nucleic Acids Res, 1986 Nov 25, 14(22), 8979 - 95
Differential repair of DNA damage in specific nucleotide sequences in monkey cells; Leadon SA; An immunological method was developed that isolates DNA fragments containing bromouracil in repair patches from unrepaired DNA using a monoclonal antibody that recognizes bromouracil . Cultured monkey cells were exposed to either UV light or the activated carcinogen aflatoxin B1 and excision repair of damage in DNA fragments containing the integrated and transcribed E . coli gpt gene was compared to that in the genome overall . A more rapid repair, of both UV and AFB1 damage was observed in the DNA fragments containing the E . coli gpt genes . The more efficient repair of UV damage was not due to a difference in the initial level of pyrimidine dimers as determined with a specific UV endonuclease . Consistent with previous observations using different methodology, repair of UV damage in the alpha sequences was found to occur at the same rate as that in the genome overall, while repair of AFB1 damage was deficient in alpha DNA . The preferential repair of damage in the gpt gene may be related to the functional state of the sequence and/or to alterations produced in the chromatin conformation by the integration of plasmid sequences carrying the gene.

Nucleic Acids Res, 1986 Nov 25, 14(22), 8919 - 32
Mechanism of intramolecular recyclization and deletion formation following transformation of Escherichia coli with linearized plasmid DNA; Conley EC et al.; The deletion end-points of a number of type I (less than monomeric) plasmid deletants obtained by transforming recA+ or recA- E . coli with linear pBR322 DNA were determined by DNA sequencing . In both monodirectional and bidirectional deletions the recyclization point was normally characterized by recombination between directly repeated sequences of between 4 and 10 bp present on each arm of the linearized pBR322 molecule . Frequently, short tracts of uninterrupted homology involved in recombinational recircularization were embedded in regions of relative non-homology . A model predicting the probability of matching sequences in either end of a linear plasmid molecule is presented . It is proposed that exonucleolytic processing of the exposed termini of linear plasmid molecules generates substrates for subsequent recombinational recyclization and deletion . The activity of host recombination and repair functions in recircularizing linear DNA molecules explains the generation of many of the aberrant recombinant DNA constructs obtained during gene cloning procedures.

J Biol Chem, 1986 Nov 25, 261(33), 15772 - 7
Structure and expression of the alkB gene of Escherichia coli related to the repair of alkylated DNA; Kondo H et al.; When the alkB gene of Escherichia coli that controls sensitivity of bacteria to methyl methanesulfonate was placed under the control of the lac regulatory region on a multicopy plasmid, the gene product, AlkB protein, was overproduced . By monitoring the band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was purified to near physical homogeneity . An amino-terminal sequence and total amino acid composition of the purified AlkB protein were in accord with the amino acid sequence deduced from the nucleotide sequence of the alkB gene, determined by the phage M13 dideoxy method . It was concluded that the AlkB protein is comprised of 216 amino acids and has a molecular weight of 23,900 . The nucleotide sequence analysis also revealed that the ada and alkB genes are adjacent on the E . coli chromosome and that the first initiation codon for AlkB protein overlaps with the termination codon for Ada protein . We constructed hybrid plasmids carrying an alkB'-lacZ' fusion, with or without the ada control region, and investigated expression of the alkB gene in response to the alkylating agent . We obtained evidence that the ada and alkB genes constitute an operon.

J Biol Chem, 1986 Nov 25, 261(33), 15761 - 6
Purification and structure of 3-methyladenine-DNA glycosylase I of Escherichia coli; Sakumi K et al.; We constructed a recombinant plasmid carrying a gene that suppresses tag mutation . To overproduce its gene product, a 0.8-kilobase DNA fragment which carries the gene was placed under the control of the lac promoter in pUC8 . 3-Methyladenine-DNA glycosylase activity in cells carrying such plasmids (pCY5) was 450-fold higher than that of wild type strain, on exposure to isopropyl-beta-D-thiogalactopyranoside . From an extract of such cells, the enzyme was purified to apparent physical homogeneity, and the amino acid composition and the amino-terminal amino acid sequence of the enzyme were determined . The data were in accord with nucleotide sequence of the gene, determined by the dideoxy method . It was deduced that 3-methyladenine-DNA glycosylase I comprises 187 amino acids and its molecular weight is 21,100, consistent with the value estimated from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein . Only 3-methyladenine was excised from methylated DNA by the purified glycosylase . These results show that the tag is the structural gene for 3-methyladenine-DNA glycosylase I.

J Biol Chem, 1986 Nov 25, 261(33), 15474 - 9
A sulfhydryl presumed essential is not required for catalysis by an aminoacyl-tRNA synthetase; Profy AT et al.; Several aminoacyl-tRNA synthetases are sensitive to reagents that modify sulfhydryl groups . We report here the significance of N-ethylmaleimide (NEM)-mediated inactivation of Escherichia coli glycyl-tRNA synthetase, and alpha 2 beta 2 enzyme . We confirmed earlier observations that NEM abolishes synthetase-catalyzed aminoacylation with pseudo-first order kinetics and provided a second method of proof that the site of inactivation is located in the beta-subunit . Using oligonucleotide-directed mutagenesis of the glyS gene, each beta-subunit cysteine codon (positions 98, 395, and 450) was replaced, individually, by an alanine codon . The three resulting mutant proteins are each active in vivo, and their in vitro aminoacylation activities are comparable to that of the native enzyme . A mutant incorporating all three amino acid substitutions is also active in vivo and in vitro . These results establish conclusively that a beta-subunit cysteine thiol is not required for the catalysis of aminoacylation . The Cys98----Ala and Cys450----Ala mutants are inactivated by NEM with the same kinetics as the wild-type protein . However, the Cys395----Ala mutant is refractory to NEM . This suggests that NEM inactivation of the native enzyme is due to alkylation of Cys395 . Aware that inactivation may result from steric effects, we constructed a mutant with a bulkier amino acid residue at position 395 (Cys395----Gln) . The aminoacylation activity of this protein is less than 10% of that of the wild-type enzyme . The glutamine substitution affects only the tRNA-dependent step of the reaction--the rate of glycyl adenylate synthesis is not lowered . In these features, the mutant resembles the NEM-inactivated protein . We propose that the NEM sensitivity of glycyl-tRNA synthetase, and possibly of other synthetases, arises from steric or conformational effects of the alkylated cysteine side chain.

J Biol Chem, 1986 Nov 25, 261(33), 15390 - 5
Channeling of 3-hydroxy-4-trans-decenoyl coenzyme A on the bifunctional beta-oxidation enzyme from rat liver peroxisomes and on the large subunit of the fatty acid oxidation complex from Escherichia coli; Yang SY et al.; Rates of the NAD+-dependent oxidation of 2-trans,4-trans-decadienoyl-CoA, a metabolite of trans-omega-6-unsaturated fatty acids, catalyzed by the mitochondrial enoyl-CoA hydratase plus 3-hydroxyacyl-CoA dehydrogenase and by the corresponding enzymes from peroxisomes, as well as Escherichia coli, were compared . The study of the mitochondrial system revealed that the conventional kinetic theory of coupled enzyme reactions cannot be applied to systems in which the primary reaction has a small equilibrium constant, and/or the concentration of coupling enzyme is higher than 0.01 Km for the intermediate and higher than the steady-state concentration of the intermediate . In contrast to the results obtained with the mitochondrial beta-oxidation system of unlinked enzymes, the steady-state velocities of 2-trans,4-trans-decadienoyl-CoA degradation catalyzed by either the peroxisomal bifunctional enzyme or by the E . coli fatty acid oxidation complex were found to be equal to the activities of enoyl-CoA hydratase even though the concentration of coupling enzyme was equal to that of the primary enzyme, and the quotient of Vmax/Km for the dehydration of 3-hydroxy-4-trans-decenoyl-CoA is much larger than the Vmax/Km for its dehydrogenation . The extraordinarily high efficiencies of these two multifunctional proteins in catalyzing the degradation of 2-trans,4-trans-decadienoyl-CoA is best explained by the direct transfer of the 3-hydroxy-4-trans-decenoyl-CoA intermediate from the active site of enoyl-CoA hydratase to that of 3-hydroxyacyl-CoA dehydrogenase . The discovery of an intermediate channeling mechanism on the peroxisomal bifunctional enzyme explains on the molecular level why the peroxisomal beta-oxidation system is well suited for the degradation of trans-fatty acids.

J Biol Chem, 1986 Nov 25, 261(33), 15349 - 52
Domain structure of rat liver carbamoyl phosphate synthetase I; Powers-Lee SG et al.; Independently folded structural domains of rat liver carbamoyl phosphate synthetase I have been identified by partial proteolytic cleavage under nondenaturing conditions . The pattern of fragments produced was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The NH2-terminal sequences of the fragments were determined by automated Edman degradation . Comparison of these fragment sequences with the sequence of the intact protein allowed alignment of the fragments . The hydrolysis of carbamoyl phosphate synthetase I (Mr 160,000) by either trypsin or elastase proceeded in two stages, with two alternative routes of degradation for elastase . The alignment of the final tryptic fragments from the NH2 terminus to the COOH terminus was: Mr 87,000 fragment-Mr 62,000 fragment-group of small peptides . The alignment of the final elastase fragments was: Mr 37,000 fragment-Mr 108,000 fragment-group of small peptides . The rates of cleavage were affected by the presence of the substrate ATP or the positive allosteric effector N-acetylglutamate; the preferred route of elastase cleavage was also affected . In addition to providing a map of the carbamoyl phosphate synthetase I domains and preliminary information on the interaction of substrates with these domains, the present studies provide further support for the proposal that domains serve as units of protein evolution since the 37-kDa fragment encompasses the region of the rat liver synthetase that is homologous to the 40-kDa subunit of the Escherichia coli synthetase.

Nucleic Acids Res, 1986 Nov 25, 14(22), 8905 - 17
Deletion and rearrangement of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules; Conley EC et al.; When E . coli was transformed with linearized pBR322 DNA, many transformants contained recircularized plasmids bearing deletions and other rearrangements . Most aberrant molecules were less than monomeric length and had lost the restriction site used for linearization, with the deleted region extending mono- (type Ia) or bi-directionally (type Ib) . Type II deletants were greater than monomeric but less than dimeric and contained the pBR322 sequence in direct repeat with deletion at one or both junctions (type IIa) or in inverted repeat with loss of sequence at both junctions (type IIb) . Type III deletants were greater than dimeric but less than trimeric, consisting of pBR322 sequences in both direct and inverse repeat with deletions at two or more junctions . Transformation frequencies for linear DNA were drastically reduced in xth-1- bacteria with type IIb deletants predominating in transformants . This indicates that exonuclease III is important for perfect recyclization of plasmids and the generation of type I deletants . In vivo recyclization of in vitro ligation products explains many of the aberrant DNA molecules that are encountered during gene cloning.

J Biol Chem, 1986 Nov 25, 261(33), 15402 - 9
Covalent modification of the inhibitor-binding site(s) of Escherichia coli ADP-glucose synthetase . Isolation and structural characterization of 8-azido-AMP-incorporated peptides; Larsen CE et al.; The photoaffinity inhibitor analog {2-3H}8-azido-AMP is specifically and covalently incorporated into Escherichia coli ADP-glucose synthetase . The reaction site(s) of {2-3H}8-azido-AMP with the enzyme was identified by reverse phase high performance liquid chromatography isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease-generated peptides containing the labeled analog . Three regions of modification, represented by six labeled peptides, accounted for over 85% of the covalently bound label . The major binding region of the azido analog, composed of residues 108-128, contained approximately 55% of the recovered covalently bound radioactivity . A single residue, Tyr-113, contained between 50 and 75% of the label found in the major binding region . This site is the same as the major binding region of the substrate site-specific probe, 8-azido-ADP-{14C}glucose (Lee, Y . M., and Preiss, J . (1986) J . Biol . Chem . 261, 1058-1064) . Conformational analysis of this region predicts that it is a part of a Rossmann fold, the supersecondary structure found in many adenine nucleotide-binding proteins . Two minor reaction regions of the enzyme with {2-3H}8-azido-AMP were also identified by chemical characterization . One region, containing 20% of the covalently bound label, was composed of residues 11-68 . This region contains Lys-38, the previously determined pyridoxal phosphate-modified allosteric activator site (Parsons, T . F., and Preiss, J . (1978) J . Biol . Chem . 253, 7638-7645) . The third minor region of modification, residues 222-254, contained approximately 15% of the covalently bound label . The three modified peptide regions may be juxtaposed in the enzyme's tertiary structure.

J Biol Chem, 1986 Nov 25, 261(33), 15345 - 8
A mutated bovine prochymosin zymogen can be activated without proteolytic processing at low pH; McCaman MT et al.; As a first step towards understanding how the zymogen structure of prochymosin contributes to the process by which active enzyme is produced, we altered the nucleotide sequence which encodes the amino-terminal (or propeptide) region of the protein . Of the two sites for autoproteolysis of prochymosin, one where pseudochymosin is formed at a pH of 2 and the other where chymosin is formed at pH 4-5, we changed the former by removing one codon and changing two other codons . This genetically modified prochymosin was proteolytically processed and activated normally at pH 4.5 . However, at pH 2.0 we observed only partial activation of the zymogen and found no evidence of proteolytic processing . The properties of this engineered prochymosin suggest that zymogen activation does not require proteolysis and that the two different zymogen processing sites can function independently from one another.

Nucleic Acids Res, 1986 Nov 25, 14(22), 9035 - 49
The E4 promoter of adenovirus type 2 contains an E1A dependent cis-acting element; Gilardi P et al.; To study how the E1A polypeptides of adenovirus type 2 regulate transcription, we have constructed chimeric plasmids containing the bacterial gene encoding chloramphenicol acetyl transferase (CAT) under the control of either the wild type or the deleted E4 promoter of adenovirus type 2 . Our previous results showed that promoter sequences located upstream from position -158, as measured from the cap site, are essential to the transactivation process . From a new set of deletion mutants, we now show that two regions, located between positions -239 and -218 and between positions -179 and -158, are involved in the E1A transactivation process . The deletion of only one of them does not significantly alter the E1A induction process compared with the wild type . Moreover, we show that these two regions lie within a DNA fragment which possesses the properties of an E1A-inducible "enhancer-like" element . In addition, the DNA fragment which contains this enhancer element is also able to confer the E1A inducibility to a heterologous promoter.

FEBS Lett, 1986 Nov 24, 208(2), 189 - 93
Fluorescence labeling of an aminoacyl-tRNA at the 3'-end and its interaction with elongation factor Tu.GTP; Joshi RL et al.; A new approach for the fluorescence labeling of an aminoacyl-tRNA at the 3'-end is applied to study its interaction with bacterial elongation factor Tu (EF-Tu) and GTP at equilibrium . The penultimate cytidine residue in yeast tRNATyr-C-C-A was replaced by 2-thiocytidine (s2C) . The resulting tRNATyr-C-s2C-A was aminoacylated and then alkylated at the s2C residue with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS) . A greater than 100% increase in the intensity of fluorescence emission of the modified Tyr-tRNATyr-C-s2C(AEDANS)-A was observed upon interaction with EF-Tu.GTP . A ternary complex dissociation constant of 1.27 X 10(-8) M was calculated from this direct interaction . Using such fluorescent aminoacyl-tRNA, the affinity of any unmodified aminoacyl-tRNA can be determined by competition experiments . By this approach, we show here that the affinity of unmodified Tyr-tRNATyr-C-C-A is identical to that of the modified Tyr-tRNATyr . This indicates that the fluorescence labeling procedure applied does not alter the affinity of the aminoacyl-tRNA for EF-Tu.GTP . The introduction of 2-thiocytidine into nucleic acids and their labeling with spectroscopic reporter groups may provide a unique means of investigating various types of nucleic acid-protein interactions.

FEBS Lett, 1986 Nov 24, 208(2), 183 - 8
RNA structural elements for expression in Escherichia coli . Alpha 1-antitrypsin synthesis using translation control elements based on the cII ribosome-binding site of phage lambda; Tessier LH et al.; Analysis of a series of lambda cII::alpha 1-antitrypsin (alpha 1AT) gene fusions of different sizes showed that increased alpha 1AT expression correlated with the stabilisation of a particular computer-predicted RNA secondary structure . Moreover, significant synthesis of unfused alpha 1AT was achieved by reconstruction of this conformation to permit interaction between the upstream region of the ribosome-binding site and the first part of the alpha 1AT coding sequence . This high-level expression was dependent upon certain silent point mutations in the coding sequence, indicating that RNA primary and secondary structure determinants can operate in concert to dictate the efficiency of protein synthesis.

FEBS Lett, 1986 Nov 24, 208(2), 194 - 8
Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by in vitro gene fusion; Sanchez J et al.; Two different lengths of the gene encoding Escherichia coli heat-stable toxin (STa) were fused to the carboxy end of the gene coding for the E . coli heat-labile toxin A-subunit (LTA) . The hybrid genes directed expression of chimeric LTA-STa proteins . Association of these chimeras with native heat-labile toxin B-subunit (LTB) resulted in protein complexes that bound to GM1 ganglioside and thereby could be assayed in a GM1 ELISA . The complexes reacted with monoclonal antibodies against either LTA, LTB or STa indicating that the STa and LT epitopes remained immunologically intact after fusion . Genetically constructed chimeric proteins exhibiting LT and STa antigens on the same molecule may represent a promising approach to development of broadly protective immunoprophylactic agents and/or useful immunodiagnostic reagents for diarrhoeal diseases caused by enterotoxinogenic E . coli.

Biochim Biophys Acta, 1986 Nov 21, 874(2), 227 - 34
The secondary structure of salt-extracted ribosomal proteins from Escherichia coli as studied by circular dichroic spectroscopy; Dijk J et al.; Ribosomal proteins from Escherichia coli MRE600 have been obtained by a new, mild purification procedure . This involves extraction of the subunits with salt followed by chromatographic fractionation in the presence of salt . The use of urea or other denaturing agents and conditions is avoided . A survey of the secondary structure of the 30 S and 50 S proteins, as observed by circular dichroic spectroscopy, is presented . The spectra have been analysed by a new procedure which uses a library of 16 circular dichroic spectra of proteins with a known three-dimensional structure . This method provides a more reliable analysis, especially of the contribution from beta-sheet . The results show that most of the 30 S proteins have a high alpha-helix content, whereas the 50 S proteins are more diverse . The latter group shows a larger contribution from beta-sheet . The data presented here are compared with those already published for a number of proteins which were, with one exception, prepared in the presence of urea . In most cases we find higher alpha-helix and beta-sheet values for the salt-extracted proteins than for the corresponding urea-treated proteins . In those cases, however, where special care was taken to renature the urea-treated proteins agreement is found to within the expected experimental error . The results show that salt-extracted ribosomal proteins have a well-defined secondary structure with a relatively small contribution from unordered structure.

J Mol Biol, 1986 Nov 20, 192(2), 389 - 417
Residue-specific assignments of resonances in the 1H nuclear magnetic resonance spectrum of ribosomal protein E-L30 by systematic application of two-dimensional Fourier transform nuclear magnetic resonance methods; van de Ven FJ et al.; A two-dimensional Fourier transform nuclear magnetic resonance study of the ribosomal protein E-L30 is reported . Five two-dimensional techniques, namely: nuclear magnetic resonance J-resolved spectroscopy, correlated spectroscopy, double quantum spectroscopy, relayed coherence transfer and nuclear Overhauser enhancement spectroscopy were used . Qualitative inspection of the spectra obtained by these techniques provided evidence that the E-L30 molecule has a well-defined structure in solution . This analysis indicated that, despite the fact that the protein is stable only at moderate temperatures and neutral pH, a structural analysis of the molecule would be feasible . A detailed analysis of the spectra permitted unambiguous discrimination between the spin systems of different amino acids, resulting in residue-specific resonance assignments . We were able to assign all resonances of all six threonine, four valine, five alanine, two histidine, two serine, one phenylalanine, one asparagine and one aspartic acid residue of E-L30 . Complete resonance assignment was obtained for two glycine residues . Partial assignments became available for all six isoleucine, three glycine and one glutamine residue . These results form a sound basis for the structure determination of the protein described in the accompanying paper.

J Mol Biol, 1986 Nov 20, 192(2), 351 - 60
Pre-steady-state kinetics of ribosomal translocation; Robertson JM et al.; The two partial reactions of elongation factor G dependent translocation, the release of deacylated tRNA from the P site and the displacement of peptidyl tRNA from the A to the P site, have been studied with the stopped-flow technique . The experiments were performed with poly(U)-programmed ribosomes from Escherichia coli carrying deacylated tRNAPhe in the P site and N-AcPhe-tRNAPhe in the A site in the presence of GTP . The kinetics of the reaction were followed by monitoring either the intensity or the polarization of the fluorescence of both wybutine and proflavine located in the anticodon loop or of proflavine located in the D loop of yeast tRNAPhe or N-AcPhe-tRNAPhe . Both displacement and release fluorescence changes could be described by three exponentials, exhibiting apparent first-order rate-constants (20 degrees C) of 2 to 5 s-1 (15 s-1, 35 degrees C), 0.1 to 0.3 s-1, and 0.01 to 0.02 s-1, measured with a saturating concentration of elongation factor G (1 microM) . The activation energy for the fast process of both reactions was found to be 70 kJ/mol (17 kcal/mol), while the intermediate process exhibits an activation energy of 30 kJ/mol (7 kcal/mol) . The fast step is assigned to the displacement of the N-AcPhe-tRNAPhe from the A to the P site, and to the release of the tRNAPhe from the P site . The reactions take place simultaneously to form an intermediate post-translocation complex . The latter, in the intermediate step, rearranges to form a post-translocation complex carrying the deacylated tRNAPhe in an exit site and N-AcPhe-tRNAPhe in the P site, both in their equilibrium states . In parallel, or subsequently, the deacylated tRNAPhe spontaneously dissociates from the ribosome, thus completing the translocation process . The slow process has not been assigned.

J Mol Biol, 1986 Nov 20, 192(2), 291 - 322
Negatively stained 50 S ribosomal subunits of Escherichia coli; Hoppe W et al.; Ribosomes are large nucleoproteins of approximately 3 X 10(6) Mr . In contrast to helical or spherical nucleoproteins (viruses) of similar size (which consist of several hundred small asymmetric units reproduced by symmetry), ribosomes are completely asymmetric; therefore, the amount of structural information (defined by the number of independent image elements) necessarily increases from about 10 to 20 to about 1000 to 2000 (at resolutions of the order of 2 nm) . With present techniques, only stained single particles can be studied in the electron microscope . Our published work on the 30 S subunit and on the 50 S subunit has demonstrated that three-dimensional reconstructions of stained single particles show a great number of structural details that are reproducible if the particles have the same orientation . One of the main results of this paper is the final proof of this reproducibility from detailed comparisons of individual 50 S subunits and of independent averages over a few (3 to 5) particles in the kidney or crown orientation; in the latter case, even after a chemical modification . The 50 S subunit is non-uniformly stained along the optical axis . It displays a complicated, stain-filled channel-like structure, within which is approximately the partial volume expected for the RNA . The particle shows an irregular but reproducible boundary surface against the stain . At several sites, the channel structure protrudes to the surface . Since the secondary structure of the RNA is well known, one might try to locate it in the subunit after chemical identification of its surface contacts (the 3' end of 23 S RNA and the 3' end of the 5 S RNA have been localized) . Most interesting is a groove on the surface, which might accommodate the mRNA.

J Immunol Methods, 1986 Nov 20, 94(1-2), 51 - 5
Two-site column enzyme immunoassay for neuron-specific enolase (NSE) in human serum using monoclonal antibodies; Kimura S et al.; A new method for the rapid determination of neuron-specific gamma-enolase (NSE), gamma-subunit of alpha gamma- and gamma gamma-enolase in human serum was developed by employing monoclonal antibodies for the separation method . The assay system consists of 0.1 ml Sepharose 4B column with immobilized rabbit anti-mouse IgG antibodies for the separation of bound label, Fab' fragments of rabbit anti-bovine gamma gamma-enolase IgG labeled with beta-D-galactosidase from Escherichia coli, and F(ab')2 fragments of two mouse monoclonal antibodies to gamma gamma-enolase . Serum samples or standard NSE solutions were incubated at 30 degrees C with the monoclonal antibody fragments . 10 min later, the galactosidase-labeled antibody fragments were added to the mixture, and incubated at 30 degrees C for 30 min . Then the reaction mixture was applied to a micro-column of Sepharose 4B with immobilized anti-mouse IgG antibodies . From the galactosidase activity bound in the column, NSE concentration in the samples could be estimated within 2 h . The minimum detection limit of the assay system was 30 pg/tube, being sufficiently sensitive for the assay of serum NSE with a satisfactory precision . Serum concentrations of NSE determined by the present method correlated well with that by the colorimetric solid-phase immunoassay method.

J Mol Biol, 1986 Nov 20, 192(2), 419 - 41
Sequential resonance assignments as a basis for the determination of a three-dimensional structure of protein E-L30 of Escherichia coli; van de Ven FJ et al.; Nuclear Overhauser enhancement spectra of ribosomal protein E-L30 were searched for interresidual connectivities involving peptide bond amide protons in order to establish sequential neighbourships between amino acid residues . By comparing these data with the actual amino acid sequence of the protein, sequential resonance assignments became available for almost 90% for the amino acids in E-L30 . With the aid of these assignments, some 30 nuclear Overhauser connectivities could be interpreted in terms of short interproton distances involving remote sites in the polypeptide chain . It turned out that these contacts between residues generated enough constraints to permit construction of a three-dimensional structure for the protein.

J Mol Biol, 1986 Nov 20, 192(2), 257 - 74
Specific endonucleolytic cleavage sites for decay of Escherichia coli mRNA; Cannistraro VJ et al.; The polycistronic lac mRNA of Escherichia coli contains three messages . The rate of degradation of the second (lacY) message was observed to be equal to that of the third (lacA), and each decayed twice as fast as did the first (lacZ) . Specific 5'- and 3'-ended lacY mRNA molecules could be recovered from cells; most likely, they are generated from endonucleolytic cleavages that are a part of the degradative process . They were observed by S1 nuclease mapping, and the exact 5'- and 3'-end oligonucleotides of many of them were identified by direct sequencing . Almost all of the molecules started with a 5' adenosine that would be preceded by a pyrimidine . The specificity was further restricted by neighboring nucleotides, and analysis of the data suggested that 5'-U-U decreases-A-U- is especially vulnerable . Also, computer analyses predicted the most stable secondary structures of selected segments of the mRNA and suggested that cleavages may only occur in regions of single strandedness . A model of mRNA degradation is proposed based on these observations and earlier ones . There is no unique target on a message for the initial inactivating attack: any region free of ribosomes is vulnerable, but for statistical reasons the initial attack of most molecules is near the ribosome-loading site . With no further ribosome loading, the newly unprotected 5' ends are "chopped off" at one of the next preferred target sites almost as fast as the last ribosomes moves down the mRNA.

J Mol Biol, 1986 Nov 20, 192(2), 235 - 55
Actions of the anticodon arm in translation on the phenotypes of RNA mutants; Yarus M et al.; In previous publications, we have shown that it is practical to study the translational activity of tRNAs by replacement and alteration of the anticodon arm sequence of the genus on a plasmid clone . Experiments in which the anticodon arm sequence is transplanted between tRNA genes suggest that the translational activity is determined by these sequences . We have therefore made every variant of the anticodon loop and the three base-pairs of the stem proximal to the loop, in order to resolve the relation between the structure of Su7Am tRNATrp, and its function . All derivatives conserved the normal secondary structure of the molecule, which was known to be essential for translational activity . The probability of translation of the amber codon by these suppressors is measured in this work . This translational activity in vivo is rationalized in terms of data on the copy numbers of the plasmid clones, the nucleotide modifications of the tRNAs, the steady-state level of the mature tRNA, and the aminoacylation of these molecules . Nucleotide modification levels vary among these tRNAs, giving information about the specificities of modification systems that make O-methylribose, pseudouridine, and modified A in the anticodon arm . However, for this series of tRNAs, none of these modifications has a strong effect on translational efficiency of the tRNAs . A few of the substitutions reduce aminoacylation of the tRNAs with glutamine, as determined by comparison of suppression in normal strains and related strains, which have 25-fold elevated levels of the glutaminyl-tRNA synthetase (GlnRS) . The substitutions that have the largest effect on GlnRS action are, unexpectedly, purines for conserved pyrimidines on the 5' side of the anticodon loop . Data on the concentrations of tRNA in vivo suggest that the anticodon loop and helix contribute similarly to the determination of the steady-state level of the tRNAs . This level varies sevenfold, though all tRNAs are processed from a homologous precursor made from the same transcription unit . Effects on levels appear to be mediated by changes in anticodon arm structure . A robust equation that relates aminoacyl-tRNA levels to suppressor efficiency is developed in order to resolve effects on tRNA levels and on ribosomal steps: E = A/(K + A), where E is efficiency, A is aminoacyl-tRNA concentration, and K is the effective concentration, or cellular tRNA content required for an individual tRNA to have an efficiency of 0.50 . The tRNAs vary in their intrinsic ability to function on the ribosome (represented by K), after other influences have been normalized.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1986 Nov 18, 25(23), 7774 - 81
Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli . 3 . Secondary and tertiary structure as determined by NMR; Klevit RE et al.; Sequence-specific resonance assignments of the 1H NMR spectrum of the 85-residue histidine-containing phosphocarrier protein (HPr) are complete {Klevit, R . E., Drobny, G . P., & Waygood, E . B . (1986) Biochemistry (first paper of three in this issue)} . Additional side-chain assignments have been made with long-range coherence transfer experiments {Klevit, R . E., & Drobny, G . P . (1986) Biochemistry (second paper of three in this issue)} . In this paper, the NMR assignments were used to determine the secondary structure and the tertiary folding of HPr in solution . The secondary structural elements of the protein were determined by visual inspection of the pattern of nearest-neighbor nuclear Overhauser effects (NOEs) and the presence of persistent amide resonances . Escherichia coli HPr consists of four beta-strands, three alpha-helices, four reverse turns, and several regions of extended backbone structure . Long-range NOEs, especially among side-chain protons, were used to determine the tertiary structure of the protein by use of the secondary structural components . The four beta-strands form a single antiparallel beta-pleated sheet . The hydrophobic faces of the alpha-helices interact to form a hydrophobic core and sit above the hydrophobic face of the beta-sheet, forming an open-face beta-sheet sandwich structure . The active site histidine, His-15, is on a short kinked segment of backbone that is accessible to the solvent . The positively charged phosphorylation site (His-15 and Arg-17) interacts with the negatively charged carboxyl terminus of the protein (Glu-85).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Nov 18, 25(23), 7770 - 3
Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli . 2 . Leucine resonance assignments by long-range coherence transfer; Klevit RE et al.; Sequence-specific assignments of the NH, C alpha H, and C beta H resonances in the NMR spectrum of the histidine-containing protein (HPr) from Escherichia coli are complete {Klevit, R . E., Drobny, G . P., & Waygood, E . B . (1986) Biochemistry (first paper of three in this issue)} . In addition, the C gamma H3 resonances of valyl, threonyl, and isoleucyl residues have been assigned by two-dimensional relayed coherence transfer (RELAY) experiments . In order to rigorously assign the resonances from longer side chains such as leucines, long-range transfer experiments have been applied to HPr . Coherence transfers via isotropic mixing within large spin systems were accomplished by multiple pulse trains applied during the mixing time of a two-dimensional experiment.

Biochemistry, 1986 Nov 18, 25(23), 7760 - 9
Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli . 1 . Sequential resonance assignments; Klevit RE et al.; Two-dimensional NMR studies at 500 MHz have been performed on the histidine-containing protein (HPr) from Escherichia coli . HPr is one of the phosphocarrier proteins involved in the bacterial phosphoenolpyruvate:sugar phosphotransferase system that is responsible for the concomitant phosphorylation and translocation of a number of sugars . Sequential resonance assignments of HPr are complete . The conventional method of sequential assignments involving J-correlated spectroscopy (COSY) and nuclear Overhauser spectroscopy (NOESY) has been supplemented by optimized relayed coherence transfer spectroscopy (RELAY) to help overcome the spectral overlap that is inevitable in the spectra of proteins the size of HPr . RELAY experiments were performed in H2O to obtain NH-C beta H connectivities and in D2O to obtain C alpha H-C gamma H connectivities . The abundance of relayed coherence transfer peaks in the two experiments greatly aided in the assignment process of the complicated protein spectrum . The assignments lay the groundwork for the determination of the solution structure of HPr, as described in the accompanying paper {Klevit, R . E., & Waygood, E . B . (1986) Biochemistry (third paper of three in this issue)}.

Biochemistry, 1986 Nov 18, 25(23), 7560 - 6
Equilibrium and kinetic measurements of the conformational transition of thioredoxin in urea; Wilson J et al.; Addition of urea to solutions of Escherichia coli thioredoxin results in a cooperative unfolding of the protein centered at 6.7 M urea at 25 degrees C and 5.1 M urea at 2 degrees C and neutral pH as judged by changes in tryptophan fluorescence emission, far-ultraviolet circular dichroism, and exclusion chromatography . Kinetic profiles of changes in tryptophan fluorescence emission intensity were analyzed following either manual or stopped-flow mixing to initiate unfolding or refolding . Unfolding of the native protein occurs in a single kinetic phase whose time constant is markedly dependent on urea concentration . Refolding of the urea-denatured protein occurs in a multiplicity of kinetic phases whose time constants and fractional amplitudes are also dependent upon urea concentration . Urea gradient gel electrophoretic and exclusion chromatographic measurements suggest the transient accumulation of at least one and likely two compact nativelike intermediate conformations during refolding . Simulations of both electrophoretic and chromatographic results suggest that the intermediate conformations are generated by the concerted action of the middle and fast refolding phases.

Biochemistry, 1986 Nov 18, 25(23), 7477 - 83
Uniform preparations of large unilamellar vesicles containing anionic lipids; Li W et al.; A general procedure for the preparation of large unilamellar vesicles of selected sizes has been developed . The procedure consists of dissolving the lipid in organic solvent, washing with mild acid, removing the solvent, adding salt (0.15 M KCl) solution, and adjusting the pH (raising it to about pH 10 and lowering it immediately to pH 7.55) . The procedure takes less than 30 min . The resulting unilamellar vesicles are of a single size with a rather low standard deviation . The sizes of these preparations range between 150 and 1000 nm in diameter . Sizes and polydispersities were measured to within 1-2% by photon correlation spectroscopy . Vesicle size varies with the phospholipid structure, the composition of the phospholipid mixture, the ionic strength of the medium, the alkyl chain composition, the cholesterol content of the phospholipid mixture, and the timing of the pH adjustment procedure . Uniform preparations of vesicles have been obtained from the dioleoyl esters of phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine, from diphytanyl ethers of glycolipid sulfate, phosphatidylglycerol, phosphatidylglycerol phosphate, and phosphatidylglycerol sulfate, from bovine liver phosphatidylinositol, from Escherichia coli phosphatidylethanolamine, from membrane lipid extracts from E . coli and Holabacterium cutirubrum, and from dodecanesulfonate-alkanol mixtures and free oleic acid . The preparation of unilamellar vesicles from oleic acid is novel, and the size range is 600-3000 nm; the preparations are relatively uniform . Vesicles of phospholipids in which sucrose and trehalose replace salt as the impermeant do not differ significantly from those prepared in pentaerythritol.

Biochemistry, 1986 Nov 18, 25(23), 7386 - 92
Effect of magnesium ion on the structure of the 5S RNA from Escherichia coli . An imino proton magnetic resonance study of the helix I, IV, and V regions of the molecule; Leontis NB et al.; The imino proton nuclear magnetic resonance spectrum of Escherichia coli 5S ribonucleic acid (RNA) changes when the Mg2+ ion concentration drops below physiological levels . The transition between the physiological and low magnesium spectral forms of 5S RNA has a midpoint at approximately 0.3 mM Mg2+ . Many of the most conspicuous changes observed in the downfield spectrum of 5S RNA as the magnesium concentration is reduced are due to adjustments in the structures of helices I and IV and the disappearance of resonances originating in helix V . The binding of ribosomal protein L25 to 5S RNA in the absence of magnesium stabilizes helix V structures.

Biochemistry, 1986 Nov 18, 25(23), 7368 - 74
The dnaB protein of Escherichia coli: mechanism of nucleotide binding, hydrolysis, and modulation by dnaC protein; Biswas EE et al.; The mechanism of nucleotide binding and hydrolysis by dnaB protein and dnaB X dnaC protein complex has been studied by using fluorescent nucleotide analogues . Binding of trinitrophenyladenosine triphosphate (TNP-ATP) or the corresponding diphosphate (TNP-ADP) results in a blue shift of the emission maximum and a severalfold amplification of the fluorescence emission of the nucleotide analogues . Scatchard analysis of TNP-ATP binding indicates that TNP-ATP binds with a high affinity (Kd = 0.87 microM) and a 8.5-fold enhancement of fluorescence emission of the nucleotide . Only three molecules of TNP-ATP or TNP-ADP bind per hexamer of dnaB protein in contrast to six molecules of ATP or ADP binding to a dnaB hexamer . TNP-ATP and TNP-ADP are both competitive inhibitors of single-stranded (SS) DNA-dependent ATPase activity of dnaB protein . TNP-AMP neither binds to dnaB protein nor inhibits the ATPase activity . Formation of dnaB X dnaC complex by dnaC protein results in diminution of the TNP-ATP fluorescence enhancement and a concomitant decrease in the SS DNA-dependent ATPase activity . Kinetic analysis of the ATPase activity of dnaB X dnaC complex indicates that the decrease in the ATPase activity on complex formation is due to a reduction of the maximal velocity (Vmax) . The dnaB protein hydrolyzes both TNP-ATP and dATP, however, with an extremely slow rate in the presence of single-stranded M13 DNA . The 2'-OH group of the nucleotide most likely plays an important role in the hydrolysis reaction but not in the nucleotide binding.

Biochemistry, 1986 Nov 18, 25(23), 7344 - 54
Energetics of cooperative protein-DNA interactions: comparison between quantitative deoxyribonuclease footprint titration and filter binding; Senear DF et al.; Using the binding of cI repressor protein to the lambda right and left operators as a model system, we have analyzed the two common experimental techniques for studying the interactions of genome regulatory proteins with multiple, specific sites on DNA . These are the quantitative DNase footprint titration technique {Brenowitz, M., Senear, D . F., Shea, M . A., & Ackers, G . K . (1986) Methods Enzymol . 130, 132-181} and the nitrocellulose filter binding assay {Riggs, A., Suzuki, H., & Bourgeois, S . (1970) J . Mol . Biol . 48, 67-83} . The footprint titration technique provides binding curves that separately represent the fractional saturation for each site . In principle, such data contain the information necessary to determine the thermodynamic constants for local site binding and cooperativity . We show that in practice, this is not possible for all values of the constants in multisite systems, such as the lambda operators . We show how these constants can nevertheless be uniquely determined by using additional binding data from a small number of mutant operators in which the number of binding sites has been reduced . The filter binding technique does not distinguish binding to the individual sites and yields only macroscopic binding parameters which are composite averages of the various local site and cooperativity constants . Moreover, the resolution of even macroscopic constants from filter binding data for multisite systems requires ad hoc assumptions as to a relationship between the number of ligands bound and the filter retention of the complex . Our results indicate that no such relationship exists . Hence, the technique does not permit determination of thermodynamically valid interaction constants (even macroscopic) in multisite systems.

Biochemistry, 1986 Nov 18, 25(23), 7375 - 85
Relationship of the physical and enzymatic properties of Escherichia coli recA protein to its strand exchange activity; Roman LJ et al.; We have shown that performing the recA protein catalyzed strand exchange reaction in the presence of acetate anions, rather than chloride which is commonly used, greatly increases the rate of the reaction . The initial rate of the reaction in an acetate-based buffer is approximately 3-4 times higher in the presence of Escherichia coli single-stranded DNA binding protein (SSB protein) and 2 times higher in its absence than the initial rate in chloride . To determine the enzymatic basis for this stimulatory effect of acetate buffer, we investigated the relationship between a number of physical and enzymatic properties of recA protein and the strand exchange reaction . We have found that although the acetate anion has some effect on the aggregation properties and the single-stranded DNA-dependent ATPase activity of recA protein, these effects cannot explain the enhanced strand exchange activity in an acetate-based buffer . We do find, however, that two aspects of recA protein activity closely parallel the ability of this protein to catalyze strand exchange . The first is the ability of recA protein to displace SSB protein from single-stranded DNA, an event critical to presynaptic complex formation . RecA protein is able to resist displacement by SSB protein at a lower magnesium concentration in acetate than in chloride buffer . The magnesium ion concentration dependence of strand exchange coincides exactly with this behavior . The second activity correlated to strand exchange is the duplex DNA-dependent ATPase activity of recA protein . We find that over a wide variety of sodium chloride and sodium acetate concentrations, this duplex DNA-dependent ATPase activity is linearly related to the amount of product formed in the strand exchange reaction . We postulate that this duplex DNA-dependent ATPase activity is important in the denaturation of the duplex DNA during the branch migration step of strand exchange and have also determined that this reaction is quite efficient, with the number of ATP molecules hydrolyzed per base pair exchanged being 0.75 +/- 0.25 . In addition, recA protein catalyzed strand exchange between circular single-strand and linear duplex DNA molecules is shown to be irreversible, and a possible explanation for this irreversibility is presented.

Eur J Biochem, 1986 Nov 17, 161(1), 191 - 6
Solution structure of a short DNA fragment studied by neutron scattering; Lederer H et al.; The solution structure of a DNA fragment of 130 base pairs and known sequence has been investigated by neutron small-angle scattering . In 0.1 M NaCl, the overall structure of the DNA fragment which contains the strong promoter A1 of the Escherichia coli phage T7 agrees with that expected for B-DNA . The neutron scattering curve is well fitted by that of a rigid rod with a length of 44 nm and a diameter of 2 nm . The results were confirmed by quasi-elastic light scattering and analytical centrifugation . The neutron measurements in H2O and D2O buffer reveal a cross-sectional inhomogeneity not detected by X-ray small-angle scattering . This inhomogeneity is caused by the hydration layer around the DNA core and not by the helical structure . The primary solvent shell has a density increased by at least 4-9% compared to bulk water.

Biochim Biophys Acta, 1986 Nov 17, 862(2), 343 - 51
Phosphatidylserine decarboxylase: generation of asymmetric vesicles and determination of the transbilayer distribution of fluorescent phosphatidylserine in model membrane systems; Denkins YM et al.; Large unilamellar vesicles (LUV) that contained a fluorescent analog of phosphatidylserine (NBD-PS) were used in model systems to determine the feasibility of employing phosphatidylserine decarboxylase (PS-decarboxylase) to generate asymmetric vesicles and to determine the transbilayer distribution of PS . PS-decarboxylase prepared by sonication of Escherichia coli JA 200 pLC 8-47 was found to be stable in detergent-free buffers and catalyzed the conversion of NBD-PS to NBD-phosphatidylethanolamine (NBD-PE) . PS-decarboxylase was capable of decarboxylating virtually all of the NBD-PS present in the outer leaflet of LUV containing a symmetric or asymmetric (outside only) distribution of NBD-PS, but not NBD-PS present in the inner leaflet of the vesicles . The ability of PS-decarboxylase to decarboxylate only NBD-PS located in the outer leaflet of the vesicles was independently verified by resonance energy transfer (between NBD-PS and (lissamine) rhodamine B-labeled phosphatidylethanolamine) and by derivatization with trinitrobenzenesulfonic acid (TNBS) . These techniques revealed that the exchangeable pool (the fraction of NBD-PS on the outer leaflet) and the respective fraction of Tnp-(NBD-PS) formed were equivalent to the extent of PS-decarboxylase-mediated decarboxylation of NBD-PS to NBD-PE . These results show that PS-decarboxylase can be used to generate asymmetric vesicles (i.e., PS inside, PE outside) and determine the intrabilayer distribution of PS in model membranes.

Eur J Biochem, 1986 Nov 17, 161(1), 225 - 31
Accessibility of F0 subunits from Escherichia coli ATP synthase . A study with subunit specific antisera; Deckers-Hebestreit G et al.; Antisera have been raised against denatured and non-denatured subunits a, b and c of the F0 complex of the ATP synthase from Escherichia coli . The subunit specificity of the antibodies has been established with immunoblot analysis or enzyme-linked immunosorbent assay (ELISA) . In inside-out oriented membrane vesicles the binding avidities of both sets of antisera, against denatured and non-denatured subunits of F0, were similar in the presence as well as in the absence of the F1 part . F1-depleted everted membrane vesicles always produced an efficient binding of the different antisera . In the presence of F1 no antibody recognition could be observed with the anti-a antisera, while anti-b and anti-c antisera showed strong binding . However, a higher membrane protein concentration was necessary for the same antibody binding as in F1-stripped vesicles . In membrane vesicles with right-side-out orientation the recognition of the three F0 subunits was dependent on the antisera set used . Antisera raised against denatured subunits showed no binding to the membrane vesicles, only in case of anti-(dodecylsulfate-denatured b) antiserum could a slight affinity be detected . An antigen-antibody recognition with all three F0 subunits occurred when the antisera against non-denatured subunits were incubated with membrane vesicles of right-side-out orientation . The membrane protein concentration which was necessary to produce a significant binding was 10-100-fold higher compared to that of F1-depleted everted membrane vesicles.

Eur J Biochem, 1986 Nov 17, 161(1), 163 - 9
Overproduction and high-yield purification of phospholipase A from the outer membrane of Escherichia coli K-12; de Geus P et al.; The cloned gene for the outer-membrane-bound phospholipase A from Escherichia coli was placed under control of the strong PL promoter of phage lambda . Induction of PL resulted in a 250-fold overexpression up to about 2% total cellular protein . This overproduced enzyme was indistinguishable from the wild-type enzyme . A homogeneous phospholiphase A preparation was obtained in high yield from overproducing bacteria, using the zwitterionic detergent C12-Sulfobetaine and anion-exchange chromatography . Detergent gradients were found to exert great influence on the elution characteristics . Considerations for the choice of optimal detergent gradients are discussed . The purified enzyme migrated as a single 29-kDa band in SDS/polyacrylamide gels, and required Ca(II) for activity . Maximum activity was displayed by enzyme samples taken from solutions with detergent concentrations near the critical micelle concentration . However, upon switching from high to optimal detergent concentration, maximum activity was restored in several hours, probably reflecting a slow conformational transition of the protein . Because the final pure protein was found to hydrolyze phospholipids in the intact erythrocyte membrane, a densely packed bilayer, we assume that this protein is in its biological native state.

Eur J Biochem, 1986 Nov 17, 161(1), 119 - 26
Characterization of the thioredoxin system in the facultative phototroph Rhodobacter sphaeroides Y; Clement-Metral JD et al.; This paper reports the purification and characterization of a thioredoxin system (thioredoxin, thioredoxin reductase, NADPH) from the facultative phototroph Rhodobacter sphaeroides Y . Rhodobacter sph . Y thioredoxin was purified to homogeneity with an assay based on the reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by NADPH and Escherichia coli thioredoxin reductase . Rhodobacter sph . Y thioredoxin reductase was purified with the same assay using NADPH and E . coli thioredoxin . Rhodobacter sph . Y thioredoxin contained 102 amino acid residues and had a single intrachain disulfide bond . The two half-cystine residues are part of the active site made up of the sequence -Ala-Glu-Trp-Cys-Gly-Pro-Cys-Arg- which is identical to that of E . coli thioredoxin except for the presence of an Arg instead of a Lys . Rhodobacter sph . Y thioredoxin contains two tryptophan residues . The fluorescence intensity of the tryptophan residues is quenched in oxidized thioredoxin; on reduction, a much smaller increase is observed with Rhodobacter sph . Y thioredoxin than with the E . coli protein . However, the presence of 5 M guanidine X HCl results in the complete exposure of the two tryptophan residues . Rhodobacter sph . Y thioredoxin reductase has structural and functional similarities to E . coli thioredoxin reductase: it has a molecular mass of 68 kDa, and consists of two, probably identical, subunits . Each subunit has one bound FAD molecule . The enzyme is highly specific for NADPH; it is also highly specific for Rhodobacter sph . Y thioredoxin with a Km value of 3.3 +/- 0.6 microM . A kinetic study of the two thioredoxin systems shows that they have a high degree of cross-reactivity.

Biochem J, 1986 Nov 15, 240(1), 273 - 6
Purification, N-terminal amino acid sequence and properties of hydroxymethylbilane synthase (porphobilinogen deaminase) from Escherichia coli; Hart GJ et al.; Hydroxymethylbilane synthase (porphobilinogen deaminase) was purified to apparent homogeneity from Escherichia coli . The enzyme is a monomer of Mr approx . 40,000 . The Km for porphobilinogen and relative Vmax . values have been obtained at various pH values over the range 6.2-8.8, enabling pK values for ionizable groups important for activity to be determined . The N-terminal amino acid sequence is presented.

Anal Biochem, 1986 Nov 15, 159(1), 143 - 9
Determination of thiol proteins using monobromobimane labeling and high-performance liquid chromatographic analysis: application to Escherichia coli thioredoxin; Chinn PC et al.; A highly sensitive and specific assay for Escherichia coli thioredoxin was developed using the thiol-specific reagent monobromobimane . Treatment of dithiothreitol-reduced thioredoxin with an excess of monobromobimane in Tris buffer (pH 8.0, 23 degrees C) for 30 min resulted in the formation of a stable derivative which was quantitated by reverse-phase high-performance liquid chromatography with fluorescence detection providing sensitivity in the low picomole range . This method was applied to the determination of intracellular levels of thioredoxin in E . coli . Cell extracts were heated, treated with dithiothreitol, reacted with monobromobimane, and desalted to give a solution which was analyzable for thioredoxin using the chromatographic procedure.

J Biol Chem, 1986 Nov 15, 261(32), 15307 - 9
Crystallization and preliminary X-ray study of AMP nucleosidase; Giranda VL et al.; Adenosine-5'-monophosphate nucleosidase from Escherichia coli has been crystallized in the presence of its strong competitive inhibitor formycin 5'-monophosphate and its allosteric activator adenosine 5'-triphosphate . Crystals are tetragonal bipyramids which grow to 1.2 mm in the longest dimension, are resistant to radiation damage, and diffract to a resolution of 3.5 A . The space group is P4(1)2(1)2 or P4(3)2(1)2, and the unit cell dimensions are a = 120.1 A and c = 243.7 A . The asymmetric unit is estimated to contain four subunits of 52,000 daltons . The crystals appear suitable for single crystal x-ray structure investigation.

J Biol Chem, 1986 Nov 15, 261(32), 15252 - 6
Purification and characterization of the OmpR protein, a positive regulator involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli; Jo YL et al.; The OmpR protein is a positive regulator involved in osmoregulatory expression of the ompF and ompC genes, which respectively code for major outer membrane proteins OmpF and OmpC of Escherichia coli . The OmpR protein has been purified to homogeneity from an overproducing strain harboring an ompR gene-carrying plasmid . Throughout the purification the OmpR protein behaved as a single entity . The molecular weight determined on sodium dodecyl sulfate-polyacrylamide gel, the total amino acid composition, and the NH2-terminal amino acid sequence of the purified protein were essentially the same as those deduced from the nucleotide sequence of the ompR gene . Molecular weight determination and cross-linking study on the native protein revealed that the purified protein exists as a monomer . The purified OmpR protein was specifically bound to the promoter regions of the ompC and ompF genes . Experiments with a series of upstream deletions of the ompC and ompF promoters revealed that the region upstream from the -35 region was indispensable for OmpR binding to both the ompC and the ompF promoters . Although it has been proposed that depending on the medium osmolarity the OmpR protein may exist in two alternative structures, which respectively regulate functioning of the ompC and the ompF promoters, the purified OmpR protein appeared to be homogeneous and interacted with both promoters to the same extent.

J Biol Chem, 1986 Nov 15, 261(32), 15075 - 80
Kinetic analysis of lamB mutants suggests the signal sequence plays multiple roles in protein export; Stader J et al.; We have developed a quantitative assay to measure the rate of processing of precursor LamB into mature protein and have used this assay to characterize 10 previously isolated and 3 new lamB signal sequence mutants . The data suggest that the LamB signal sequence serves a complex function . Our assay has revealed five types of signal sequence defect: 1) a strong kinetic defect resulting from alteration of the secondary structure in the putative alpha-helical region in the hydrophobic core, 2) a strong, or 3) a weak kinetic defect due to placement of a charged residue in the hydrophobic core, 4) decreased synthesis of LamB, and 5) both a decrease in synthesis and a strong kinetic defect . The effect of an extragenic suppressor, prlA4 on the rate of processing pLamB containing signal sequence mutations was also examined and compared to the rates in wild-type strains . It was found that prlA4 increases the rate of processing in some, but not all, mutants having a kinetic defect while having no effect on the decreased synthesis seen in mutants of types 4 and 5.

J Biol Chem, 1986 Nov 15, 261(32), 15049 - 52
A role for proteins S3 and S14 in the 30 S ribosomal subunit; Ramakrishnan V et al.; Small ribosomal subunits prepared by the method of Kirillov et al . (Kirillov, S . V., Makhno, V . I., Peshin, N . N., and Semenkov, Yu . P . (1986) Nucleic Acids Res . 5, 4305-4315) are active but fail to reconstitute . The inability to reconstitute is due to a deficiency in proteins S3 and S14 . Supplementation of the protein component with pure S3 and S14 leads to an enhancement of the activity of the reconstituted product . Our results provide evidence that these two proteins are involved in assembly but may not be required once the 30 S subunit has been properly assembled.

J Biol Chem, 1986 Nov 15, 261(32), 14997 - 5005
The role of thioredoxin in filamentous phage assembly . Construction, isolation, and characterization of mutant thioredoxins; Russel M et al.; Filamentous phage assembly in vivo shows an absolute requirement for thioredoxin and a partial requirement for thioredoxin reductase . Mutants in which one or both of the active site cysteine residues of thioredoxin were changed to alanine or serine were constructed and shown to support filamentous phage assembly . Some of the mutants were almost as effective as wild-type thioredoxin, while others supported phage assembly only when high levels of the mutant protein were present in the infected cell . The mutant proteins were all inactive in an assay which couples oxidation of NADPH to reduction of 5,5'-dithiobis-2-nitrobenzoic acid) via thioredoxin reductase and thioredoxin . These active site mutants make phage assembly completely independent of thioredoxin reductase, which suggests that the phage needs, and the active site mutants provide, the proteins in the reduced conformation . Other mutants were isolated on the basis of their failure to support filamentous phage growth . These specified mutant thioredoxin proteins with varying levels of redox activity in vivo and in vitro . The locations of these mutations suggest that the surface of thioredoxin thought to interact with thioredoxin reductase also interacts with the filamentous phage assembly machinery . An in vivo assay for thioredoxin redox function, based on the ability of cells to utilize methionine sulfoxide, was developed . Met- cells containing mutant thioredoxins that are inactive in vitro do not form colonies on plates containing methionine sulfoxide as the sole methionine source.

J Biol Chem, 1986 Nov 15, 261(32), 14987 - 90
Use of an azido-ubiquinone derivative to identify subunit I as the ubiquinol binding site of the cytochrome d terminal oxidase complex of Escherichia coli; Yang FD et al.; The radiolabeled, photoreactive azido-ubiquinone derivative (azido-Q), 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl-{3H}octyl)- 1,4-benzoquinone, was used to investigate the active site of ubiquinol oxidase activity of the cytochrome d complex, a two-subunit terminal oxidase of Escherichia coli . The azido-Q, when reduced by dithioerythritol, was shown to support enzymatic oxygen consumption by the cytochrome d complex that was 8% of the rate observed with ubiquinol-1 . This observation provided the rationale behind further studies of the possible photoinactivation and labeling of the active site by this azido-Q . Ten min of photolysis of the purified cytochrome d complex in the presence of the azido-Q resulted in a 60% loss of the ubiquinol-1 oxidase activity . Uptake of the radiolabeled azido-Q by the cytochrome d complex was correlated to the photoinactivation of the ubiquinol-1 oxidase activity . Both increased linearly during the first 4 min of photolysis and reached 90% of the maximum within 10 min . Photolysis times longer than 10 min resulted in no increase in the maximum of 2 mol of azido-Q incorporated per mol of enzyme . The rate of azido-Q uptake by subunit I, but not subunit II, correlated well with the rate of loss of ubiquinol oxidase activity . Use of ubiquinol-0, which is not oxidized by the enzyme, to competitively inhibit radiolabeling of nonspecific binding sites, resulted in a significant decrease (42%) of azido-Q labeling of subunit II while it did not affect the labeling of subunit I . After photolysis for 4 min, the ratio of radiolabeled azido-Q in subunits I to II of the complex was 4.3 to 1.0 . These observations support the conclusion that the ubiquinol substrate binding site is located on subunit I of the cytochrome d complex.

J Biol Chem, 1986 Nov 15, 261(32), 14875 - 7
A mutation in Escherichia coli tRNA nucleotidyltransferase that affects only AMP incorporation is in a sequence often associated with nucleotide-binding proteins; Zhu LQ et al.; Escherichia coli strain 5C15 contains a mutation in the cca gene that decreases AMP incorporation by tRNA nucleotidyltransferase while leaving CMP incorporation unaffected . Earlier studies of the purified mutant enzyme suggested that the mutation was localized to the AMP-incorporating site . In order to analyze this mutation in more detail, the cca gene from strain 5C15 was cloned into plasmid pUC8 . Analysis of tRNA nucleotidyltransferase activity in extracts of a strain transformed with this plasmid demonstrated an elevated level of CMP incorporation, but low AMP incorporation, as expected from the properties of the original mutant . Sequence analysis of the mutant cca gene revealed only a single G to A point mutation leading to a glycine to aspartic acid substitution at position 70 of the peptide chain . The amino acid change was localized to one of two Gly-X-Gly-X-X-Gly sequences present in the protein . This sequence has been identified previously near the nucleotide-binding domain of various proteins, but it has not been noted in enzymes that incorporate nucleotide residues . However, other sequences often associated with ATP-binding domains are not found in tRNA nucleotidyltransferase . The implications of these findings for our understanding of nucleotide-binding domains are discussed.

J Biol Chem, 1986 Nov 15, 261(32), 15062 - 9
sn-1,2-Diacylglycerol kinase of Escherichia coli . Structural and kinetic analysis of the lipid cofactor dependence; Walsh JP et al.; The lipid cofactor requirement of Escherichia coli sn-1,2-diacylglycerol kinase was studied using a beta-octylglucoside mixed micellar assay (Walsh, J . P., and Bell, R . M . (1986) J . Biol . Chem . 261, 6239-6247) . The enzyme was shown to have an absolute requirement for a lipid activator . sn-1,2-Dioleoylglycerol was both an activator and a substrate for the enzyme, 1,3-dioleoylglycerol was an activator but not a substrate, and sn-1,2-dioctanoylglycerol was a substrate but not an activator . Activation was observed with a large number of phospholipids, sulfolipids, neutral lipids, and detergents . Lipids with longer alkyl/acyl chains stimulated activity to a greater extent and at lower concentrations than their shorter chain homologs . Anionic lipids were the best activators, and neutral lipids were somewhat less effective . Cationic lipids were poor activators . Lipid activation was cooperative in all cases, with Hill coefficients ranging from 2.9 to 4.7 . Lipid activators stabilized the enzyme against inactivation induced by diacylglycerols . The effectiveness of several lipids in stabilizing the enzyme correlated with their effectiveness as kinetic activators, suggesting a common mechanism . Kinetic analyses also suggested that a lipid cofactor-induced conformational change occurs as a part of the activation process . beta-Octylglucoside was shown not to function as a lipid cofactor for diacylglycerol kinase . The requirement for detergent in the assay was related, instead, to the need to disperse and deliver water-insoluble substrates and cofactors to the enzyme . beta-Octylglucoside also provided an inert matrix to which lipid substrates and cofactors could be added, enabling study of their concentration dependencies.

J Biol Chem, 1986 Nov 15, 261(32), 15022 - 9
The primary structure and the functional domains of an elongation factor-1 alpha from Mucor racemosus; Linz JE et al.; We have determined the complete nucleotide sequence for TEF-1, one of three genes coding for elongation factor (EF)-1 alpha in Mucor racemosus . The deduced EF-1 alpha protein contains 458 amino acids encoded by two exons . The presence of an intervening sequence located near the 3' end of the gene was predicted by the nucleotide sequence data and confirmed by alkaline S1 nuclease mapping . The amino acid sequence of EF-1 alpha was compared to the published amino acid sequences of EF-1 alpha proteins from Saccharomyces cerevisiae and Artemia salina . These proteins shared nearly 85% homology . A similar comparison to the functionally analogous EF-Tu from Escherichia coli revealed several regions of amino acid homology suggesting that the functional domains are conserved in elongation factors from these diverse organisms . Secondary structure predictions indicated that alpha helix and beta sheet conformations associated with the functional domains in EF-Tu are present in the same relative location in EF-1 alpha from M . racemosus . Through this comparative structural analysis we have predicted the general location of functional domains in EF-1 alpha which interact with GTP and tRNA.

J Biol Chem, 1986 Nov 15, 261(32), 14929 - 35
Escherichia coli exonuclease VII . Cloning and sequencing of the gene encoding the large subunit (xseA); Chase JW et al.; We have determined the sequence of the gene encoding the large subunit of Escherichia coli exonuclease VII (xseA) and the amino acid sequence of the protein it encodes . The coding region of the xseA gene is 1368 base pairs . The protein encoded by the gene contains 456 amino acids and has a calculated molecular weight of 51,823 . The promoter for xseA is close to that for guaB, and these two genes are transcribed in opposite directions: xseA clockwise and guaB counterclockwise on the standard E . coli genetic map . The cloned xseA gene can complement an xseA deletion mutant strain . In an xseA+ genetic background production of large quantities of the xseA gene product appeared to decrease the amount of exonuclease VII activity in cell extracts . In fact, no exonuclease VII activity at all could be detected following induction of strains in which the xseA gene was under lambda pL regulation . These observations suggest that the proper ratio of the large and small exonuclease VII subunits must be maintained in order to produce active enzyme.

Anal Biochem, 1986 Nov 15, 159(1), 17 - 23
Rapid isolation and characterization of hybridization selected recombinants from lambda genomic libraries; Porteous DJ; This paper describes an efficient protocol for the screening of lambda genomic libraries, plaque and DNA purification, and probe characterization by a combination of new and recently described techniques . The protocol has allowed large numbers of human subchromosome-specific probes to be rapidly generated from an EMBL3 library of human-mouse somatic cell hybrid DNA . The protocol affords considerable savings in time and effort over previous procedures.

J Biol Chem, 1986 Nov 15, 261(32), 15126 - 33
Nuclear genes encoding the yeast mitochondrial ATPase complex . Analysis of ATP1 coding the F1-ATPase alpha-subunit and its assembly; Takeda M et al.; Mitochondria prepared from the yeast nuclear pet mutant N9-84 lack a detectable F1-ATPase activity . Genetic complementation of this mutant with a pool of yeast genomic DNA in the yeast Escherichia coli shuttle vector YEp13 restored its growth on a nonfermentable carbon source . Mitochondria prepared from the transformed host contained an 8-fold higher than normal level of the F1 alpha-subunit and restored ATPase activity to 50% that of the wild-type strain . Deletion and nucleotide sequence analysis of the complementing DNA on the plasmid revealed a coding sequence designated ATP1 for a protein of 544 amino acids which exhibits 60 and 54% direct protein sequence homology with the proton-translocating ATPase alpha-subunits from tobacco chloroplast and E . coli, respectively . In vitro expression and mitochondrial import experiments using this ATP1 sequence showed that additional amino-terminal sequences not present in the comparable plant and bacterial subunits function as transient sequences for import.

J Biol Chem, 1986 Nov 15, 261(32), 14896 - 901
Primary structure of the Neurospora plasma membrane H+-ATPase deduced from the gene sequence . Homology to Na+/K+-, Ca2+-, and K+-ATPase; Addison R; The gene for the Neurospora crassa plasma membrane H+-ATPase has been cloned and sequenced . The gene encodes for a protein of 920 amino acids with a molecular weight of 100,002 . The coding region is interrupted by four introns: three near the amino terminus and one near the carboxyl terminus . The deduced amino acid sequence of the N . crassa plasma membrane H+-ATPase exhibits 75% homology to the amino acid sequence of the Saccharomyces cerevisiae plasma membrane H+-ATPase . Also, an amino acid comparison with the Na+/K+-ATPase from sheep kidney, Ca2+-ATPase from rabbit muscle, and K+-ATPase from Escherichia coli reveals that certain regions are highly conserved and suggest that these regions may serve essential functions which are common to the various cation-motive ATPases . This observation suggests that the phosphorylatable, cation-motive ATPases may function via a similar energy transduction mechanism.

J Biol Chem, 1986 Nov 15, 261(32), 14878 - 81
Conformation-specific antiserum raised against subunit c of ATP synthase (F1F0) from Escherichia coli; Deckers-Hebestreit G et al.; Subunit c of the membrane-integrated, proton-translocating F0 portion of the ATP synthase (F1F0) from Escherichia coli has been isolated under nondenaturing conditions (Schneider, E., and Altendorf, K . (1985) EMBO J . 4, 515-518) and antibodies have been raised in rabbits . The primary antisera did not recognize the antigen when present in the same buffer as used for the immunization . Surprisingly, in one of the three antisera a strong antibody binding was observed when intact F0, a.c complex or reconstituted subunit c was provided as the antigen . Incorporation of subunit c into liposomes together with subunits a and b forming an active, H+-translocating complex was not required for the recognition by the antiserum . Subunit c prepared by chloroform/methanol extraction or by chromatography in the presence of sodium dodecyl sulfate was not recognized by the anti-c antiserum when incorporated into liposomes.

Anal Biochem, 1986 Nov 15, 159(1), 35 - 42
Removal of the epsilon subunit from Escherichia coli F1-ATPase using monoclonal anti-epsilon antibody affinity chromatography; Dunn SD; The usefulness of two monoclonal antibodies, epsilon-1 and epsilon-4, which recognize the epsilon subunit of Escherichia coli F1-ATPase, for removing that subunit from ATPase was assessed . The epsilon subunit is a tightly bound, but dissociable, inhibitor of the ATPase . epsilon-1 binds epsilon with 10-fold higher affinity than epsilon-4 . epsilon-1 recognizes a site on epsilon which is hidden by the quaternary structure of ATPase, while epsilon-4 can recognize epsilon when it is part of ATPase . Each antibody was purified and coupled to Sepharose to generate affinity columns . Solutions of ATPase in a buffer which was designed to reduce the affinity of epsilon for the enzyme were pumped through the columns and the degree of epsilon depletion was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blotting . Neither column retained ATPase significantly . At low ATPase concentrations and low flow rates, the epsilon-1 column was more efficient than the epsilon-4 column, removing in excess of 95% of the epsilon in a single passage compared with 93% removal by the epsilon-4 column . At higher protein concentrations or flow rates, however, the performance of the epsilon-1 column was substantially poorer, while that of the epsilon-4 column was much less affected . Very little epsilon emerged from the epsilon-4 column before most of the measured epsilon-binding capacity was filled . A second passage through the epsilon-4 column reduced residual epsilon to less than 2% of that which was originally present . Pure, active epsilon was eluted from either column by 1 M NH4OH, pH 11.(ABSTRACT TRUNCATED AT 250 WORDS)

J Immunol, 1986 Nov 15, 137(10), 3354 - 60
Alterations in the amino-terminal third of mouse interleukin 2: effects on biological activity and immunoreactivity; Zurawski SM et al.; In this report, we describe plasmids that direct the expression of active mouse interleukin 2 (mIL 2) in Escherichia coli, and the use of this expression system to perform a mutational analysis of the N-terminal region of the mIL 2 protein . We found that the N-terminus was tolerant to the addition of a few amino acids, and even the addition of 20 amino acids resulted in only a modest decrease in activity of the protein . The bioactivity of mIL 2 as defined by its ability to sustain the proliferation of cloned T cells was also only minimally affected by deletion of up to 13 N-terminal amino acids, or of the entire poly-GLN stretch (amino acids 15-26) . Deletion of the 30 N-terminal amino acids drastically reduced but did not abolish activity . Deletion of the 41 N-terminal amino acids completely abolished activity, whereas certain changes in the initial 37 amino acids drastically reduced the biological activity of the protein . We also analyzed the immunoreactivity of the mutant proteins with the anti-IL 2 monoclonal antibodies S4B6 and DMS-1 . This analysis showed that the determinant recognized by S4B6 required that the N-terminal mIL 2 amino acids 26-45 be intact, whereas the DMS-1 determinant was located to the C-terminal side of amino acid 46.

Biochim Biophys Acta, 1986 Nov 14, 879(2), 149 - 56
Endogenous suppression of neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes; Marki F et al.; Phospholipase A2 activity was measured in homogenized and acid-extracted human polymorphonuclear leukocytes using {1-14C}oleate-labelled autoclaved Escherichia coli as substrate . In whole homogenate and in the supernatant and particular fractions separated by centrifugation at 150,000 X g, phospholipase activity was barely detectable (1-4 pmol/h per 10(6) cell equivalents) . By contrast, acid extracts of these fractions contained over 10-times as much phospholipase activity in the dialyzed supernatants (20-300 pmol/h per 10(6) cell equivalents), whereas phospholipase inhibitor(s) were found in the sediment . The acid-solubilized phospholipase A2 activity was absolutely Ca2+-dependent and optimal at pH 7.0-7.5 with 1.0 mM added Ca2+ . Addition of the resuspended sediment of the acid extract dose-dependently suppressed phospholipase activity in the supernatant; less than equivalent amounts were sufficient to inhibit 95% . Suppressor activity was lipid-extractable . After thin layer chromatography of lipid extracts, the bulk of inhibitory activity was recovered from the free fatty acid region . Analysis of the fatty acids by gas liquid chromatography showed that 63% were unsaturated . All unsaturated fatty acids tested were potent inhibitors of phospholipase A2 activity (IC50 3-10 microM) . Oleoyl-CoA, hydroxyeicosatetraenoic acids and leukotriene D4 were also inhibitory, while methyl oleate, saturated fatty acids and the prostaglandins E2 and F2 alpha had no effect . These in vitro data indicate that neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes is largely suppressed by endogenous inhibitors and suggest that unsaturated fatty acids and some of their metabolites may partly account for this suppressor activity.

Nucleic Acids Res, 1986 Nov 11, 14(21), 8583 - 94
Complete nucleotide sequence of recD, the structural gene for the alpha subunit of Exonuclease V of Escherichia coli; Finch PW et al.; Intracellular amplification of the Escherichia coli RecB and RecC proteins does not result in an increase in Exonuclease V activity unless the level of a third protein, encoded between the recB and argA genes, is also amplified . Nucleotide sequence analysis of this region reveals a 1,824 nucleotide open reading frame which would encode a protein of 608 amino acids with a calculated molecular weight of 66,973 . This is assumed to be the structural gene for the alpha subunit of Exonuclease V, recently designated recD . The proposed initiation codon of the recD gene overlaps the termination codon of the upstream recB gene by one nucleotide, suggesting that these genes may form an operon . The deduced amino acid sequence of the RecD protein contains a region which is homologous to highly conserved sequences in adenine nucleotide binding proteins.

Nucleic Acids Res, 1986 Nov 11, 14(21), 8573 - 82
Complete nucleotide sequence of the Escherichia coli recB gene; Finch PW et al.; The complete nucleotide sequence of the Escherichia coli recB gene which encodes a subunit of the ATP-dependent DNase, Exonuclease V, has been determined . The proposed coding region for the RecB protein is 3543 nucleotides long and would encode a polypeptide of 1180 amino acids with a calculated molecular weight of 133,973 . The start of the recB coding sequence overlaps the 3' end of the upstream ptr gene, and the recB termination codon overlaps the initiation codon of the downstream recD gene, suggesting that these genes may form an operon . No sequences which reasonably fit the consensus for an E . coli promoter could be identified upstream of the proposed recB translational start . The predicted RecB amino acid sequence contains regions of homology with ATPases, DNA binding proteins and DNA repair enzymes.

Nucleic Acids Res, 1986 Nov 11, 14(21), 8489 - 99
Chromogenic identification of oligonucleotide-directed mutants; Wright CF et al.; We describe a simple plaque color assay for identifying oligonucleotide-directed mutations in cloned DNA fragments . The basis of the method is to: fuse the sequence of interest in-frame to the E.coli lacZ gene to produce a blue plaque phage, mutate the site of interest to a stop codon to generate a white plaque phage, and revert the stop codon and surrounding nucleotides to give a blue plaque phage containing one or more desired amino acid changes . The advantages of this cyclic method are that it produces truncated as well as amino acid substituted protein molecules, it can be repeated to introduce additional mutations, and it eliminates the need for labor intensive screening . Essentially any piece of DNA can be mutated using this method if the fragment has one open reading frame . If there is an open reading frame between the site and the lacZ gene, ATG codons can be inserted at the target site . We have used this method to produce termination and amino acid substitution mutants in the yeast CUP1 gene.

Nucleic Acids Res, 1986 Nov 11, 14(21), 8387 - 97
Effect of site-specific DNA methylation and mutagenesis on recognition by methylated DNA-binding protein from human placenta; Zhang XY et al.; Methylated DNA-binding protein (MDBP) from human placenta is the first protein shown to bind specifically to certain DNA sequences only when they are methylated at cytosine residues . Among the sites recognized by MDBP is pB site 1, a pBR322-derived sequence which has a high affinity for MDBP when methylated at all CpG positions . We have substituted pB site 1 with 5-methyl-cytosine (m5C) residues at one to three of its CpG dinucleotides on one strand by the use of m5C-containing oligonucleotides . MDBP binds best when all three CpG dinucleotides in the region 5'-ATCGTCACGGCGAT-3' are methylated . Even more binding is obtained when both strands are methylated . Alteration of various residues in this binding site by oligonucleotide-directed mutagenesis decreased the binding . However, two mutations which increased the dyad symmetry of part of the binding site yielded ligands with a higher affinity for MDBP.

Nucleic Acids Res, 1986 Nov 11, 14(21), 8625 - 35
Nucleotide sequence of the AAD(2'') aminoglycoside adenylyltransferase determinant aadB . Evolutionary relationship of this region with those surrounding aadA in R538-1 and dhfrII in R388; Cameron FH et al.; The nucleotide sequence of the aadB gene which confers resistance to kanamycin, gentamicin, and tobramycin has been determined . The size of the longest reading frame is 747 bases encoding a protein of predicted size 27,992 daltons . A segment of the aadB gene sequence (including the promoter region) was found upstream of the aadA gene in R538-1 and of the dhfrII gene in R388 and the proposed promoters for these genes coincide with the aadB promoter region . The sequence homology extends upstream to the end of the sequenced regions of R388 and R538-1 . Almost perfect homology was also found between the sequences 3'- to the aadB gene and 3'- to the aadA genes of R538-1 and pSa . This segment includes a 59 base element previously found flanking the Tn7 aadA gene . A model is presented for the evolution of this region of the plasmid genomes in which the 59- base element functions as an insertional "hot spot" and the possibility that this region is analogous to the aadA/aadB region of the Tn21- like transposon family is considered.

Nucleic Acids Res, 1986 Nov 11, 14(21), 8615 - 24
Efficient construction of cDNA libraries in plasmid expression vectors using an adaptor strategy; Haymerle H et al.; We describe a method for the construction of large DNA fragment libraries in plasmid vectors, in which complementary, single-stranded extensions are ligated onto both vector and insert DNA using un-phosphorylated adaptor oligonucleotides . Special consideration has been taken of the requirements of expression screening as follows: cDNA synthesis using random oligonucleotide primers is described which maximises the probability of obtaining open reading frame fragments from large mRNA molecules, the adaptors use codons found in high abundance E . coli proteins to minimise problems of premature termination when using strong promoters, and the sequence encoded by the adaptors, when cloned into the bacterial expression vector pEX1, promotes a surface location for the foreign antigenic determinant where it is accessible to antibodies used for screening.

Nucleic Acids Res, 1986 Nov 11, 14(21), 8557 - 71
The effect of Escherichia coli Uvr protein binding on the topology of supercoiled DNA; Oh EY et al.; The effects of the binding of the E . coli UvrA and UvrB proteins on the linking number (delta L) of superhelical DNA has been measured . The effects of cofactor ATP structure on UvrAB-nucleoprotein complex formation revealed that nucleotide binding, not hydrolysis, is sufficient to locally unwind the DNA helix of both ultraviolet light-damaged as well as undamaged DNAs . The extent of this unwinding is of the same order of magnitude as the nucleotide distances of the double incision sites generated by the UvrABC endonucleolytic reaction.

Nucleic Acids Res, 1986 Nov 11, 14(21), 8231 - 45
Insertion elements and transitions in cloned mouse mammary tumour virus DNA: further delineation of the poison sequences; Brookes S et al.; The provirus of mouse mammary tumour virus (MMTV) is reputed to contain sequences within the viral gag gene that prevent or inhibit its propagation as a recombinant DNA clone in Escherichia coli . Here we report the successful isolation of several lambda and plasmid clones comprising the 5' virus-host DNA junction fragments from integrated MMTV proviruses in BR6 mice . Although the lambda clones appeared intact, almost all of the plasmids were found to contain the bacterial insertion sequences IS1 or IS2 within a small region of the gag gene . One nondisrupted clone was recovered which had undergone multiple G to A transitions, some of which created stop codons in gag . These results have provided more precise information as to the location of the poison sequences and are discussed in relation to possible explanations for the phenomenon.

FEBS Lett, 1986 Nov 10, 208(1), 155 - 7
Differential reactivity of 9-NH2-ellipticine on apurinic and apyrimidinic sites in circular DNA; Malvy C et al.; Endonucleases for apurinic sites as well as chemical compounds reacting with aldehydes do not generally differentiate between apurinic and apyrimidinic sites . We have studied the effect of the apurinic site reagent, 9-NH2-ellipticine, on apyrimidinic sites enzymatically generated on PBR322 DNA and compared it to its' action on apurinic PM2 and PBR322 DNAs . In conditions where this compound induces breakage of apurinic sites, it does not display any action on apyrimidinic sites.

FEBS Lett, 1986 Nov 10, 208(1), 1 - 6
Structure of the nucleotide-binding domain in the beta-subunit of Escherichia coli F1-ATPase; Duncan TM et al.; We propose a working model for the tertiary structure of the nucleotide-binding domain of the beta-subunit of E . coli F1-ATPase, derived from secondary structure prediction and from comparison of the amino acid sequence with the sequences of other nucleotide-binding proteins of known three-dimensional structure . The model is consistent with previously published results of specific chemical modification studies and of analyses of mutations in the beta-subunit and its implications for subunit interactions and catalytic mechanism in F1-ATPases are discussed.

Biochim Biophys Acta, 1986 Nov 6, 862(1), 57 - 64
Channel-closing activity of porins from Escherichia coli in bilayer lipid membranes; Xu GZ et al.; The opening and closing of the ompF porin from Escherichia coli JF 701 was investigated by reconstituting the purified protein into planar bilayer membranes . The electrical conductance changes across the membranes at constant potential were used to analyze the size and aggregate nature of the porin channel complexes and the relative number of opening and closing events . We found that, when measured at pH 5.5, the channel conductance diminished and the number of closing events increased when the voltage was greater than 100 mV . The results suggest that the number of smaller sized conductance channels increases above this potential . There was also an increase in the smaller subunits and in the closing events when the pH was lowered to 3.5, and these changes were further enhanced by increasing the voltage . We propose that both lowering the pH and elevating the potential across the membrane stabilize the porin in a conformation in which the subunits are less tightly associated and the subunits open in a non-cooperative manner . These same conditions also appear to stabilize the closed state of the pore.

J Biol Chem, 1986 Nov 5, 261(31), 14697 - 703
Cloning and characterization of a mouse cysteine proteinase; Portnoy DA et al.; cDNA clones encoding a mouse cysteine proteinase were isolated from a cDNA library constructed from mRNA derived from the macrophage-like cell line J774 . The DNA sequence predicts a protein that is closely related to, but distinct from, the lysosomal enzyme cathepsin H . Alignment of the predicted amino acid sequence with the known protein sequences for seven other cysteine proteinases suggests that the cloned DNA encodes a 334-residue protein containing both a 17-amino acid pre-region and a 96-amino acid pro-region . Consistent with this prediction, antiserum raised to a recombinant fusion protein expressed in Escherichia coli immunoprecipitated multiple forms of the cysteine proteinase in mouse peritoneal macrophages and fibroblasts . In pulse-chase experiments, a 36-kDa precursor, presumedly the pro-form, was converted intracellularly into a 28-kDa protein and subsequently into a 21-kDa protein . Indirect immunofluorescence microscopy results suggested that the cysteine proteinase was localized to lysosomes . Western blot analysis detected significantly more of the proteinase in thioglycolate-elicited peritoneal macrophages than in resident peritoneal macrophages . Northern blot analysis revealed that several cell lines failed to express mouse cysteine proteinase mRNA.

J Biol Chem, 1986 Nov 5, 261(31), 14582 - 6
Reactivity of a sulfhydryl group of the ras oncogene product p21 modulated by GTP binding; Hattori S et al.; We have studied the sensitivity of sulfhydryl groups of a highly purified p21 protein of the v-rasH oncogene to a thiol-specific reagent, N-ethylmaleimide (NEM) . Approximately 70% of GTP binding and autokinase activities of p21 were inactivated by NEM, and excessive amounts of GTP or GDP protected p21 activities . Thiol titration revealed the presence of one fast reactive cysteine residue, the susceptibility of which is modulated by GTP binding . A total of 4 and 6 residues, respectively, became titratable upon denaturation and reduction, suggesting the presence of a disulfide bond . This GTP-modulated sulfhydryl group was identified as Cys-80 in the following tryptic peptide sequence: NH2-Thr-Gly-Glu-Gly-Phe-Leu-Cys-Val-Phe-Ala-Ile-Asn-Asn-Thr-Lys-COOH . This is based on the comparative tryptic peptide mapping of {14C}NEM-modified p21 in the presence and absence of GTP . The GTP-modulated peptide co-chromatographed with a synthetic peptide of the predicted sequence . Amino acid analysis of the purified {14C}NEM-modified peptide from tryptic digests of p21 also confirmed its identity . This region of p21 shares an extensive sequence homology with various G-proteins and appears to be in the vicinity of the GTP-binding domain of these proteins.

J Biol Chem, 1986 Nov 5, 261(31), 14506 - 14
tRNA binding sites on the subunits of Escherichia coli ribosomes; Gnirke A et al.; Programmed 30 S subunits expose only one binding site, to which the different classes of tRNA (deacylated tRNAPhe, Phe-tRNAPhe, and N-acetylphenylalanyl (AcPhe)-tRNAPhe) bind with about the same affinity . Elongation factor Tu within the ternary complex does not contribute to the binding of Phe-tRNA . Binding of acylated or deacylated tRNA to 30 S depends on the cognate codon; nonprogrammed 30 S subunits do not bind tRNA to any significant extent . The existence of only one binding site/30 S subunit (and not, for example, two sites in 50% of the subunits) could be shown with Phe-tRNAPhe as well as deacylated tRNAPhe pursuing different strategies . Upon 50 S association the 30 S-bound tRNA appears in the P site (except the ternary complex which is found at the A site) . Inhibition experiments with tetracycline demonstrated that the 30 S inhibition pattern is identical to that of the P site but differs from that of the A site of 70 S ribosomes . In contrast to 30 S subunits the 50 S subunit exclusively binds up to 0.2 and 0.4 molecules of deacylated tRNAPhe/50 S subunit in the absence and presence of poly(U), respectively, but neither Phe-tRNA nor AcPhe-tRNA . Noncognate poly(A) did not stimulate the binding indicating codon-anticodon interaction at the 50 S site . The exclusive binding of deacylated tRNA and its dependence on the presence of cognate mRNA is reminiscent of the characteristics of the E site on 70 S ribosomes . 30 and 50 S subunits in one test tube expose one binding site more than the sum of binding capacities of the individual subunits . The results suggest that the small subunit contains the prospective P site and the large subunit the prospective E site, thus implying that the A site is generated upon 30 S-50 S association.

J Biol Chem, 1986 Nov 5, 261(31), 14496 - 505
Activities and incision patterns of ABC excinuclease on modified DNA containing single-base mismatches and extrahelical bases; Thomas DC et al.; ABC excision nuclease of Escherichia coli is a DNA repair enzyme that recognizes major helical distortions caused by bulky base adducts and incises on both sides of the adduct, thus removing the modified nucleotides in the form of a 12-13-base long oligomer . We tested the enzyme with substrates that contained unusual helical structures caused by single-base mismatches or one, three, or four extrahelical bases (loops) . We find that the enzyme does not cut DNAs containing helical perturbations caused by these structures . However, when the mismatched or extrahelical bases are modified with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide, a reagent specific for unpaired G and T residues, the enzyme incises at the modified nucleotides in the regular manner . In addition, we find that when mismatches and loops are located near pyrimidine dimers and (6-4) photoproducts they do not inhibit incision at the photoproducts by the excinuclease but sometimes affect the incision pattern . Our results indicate that ABC excinuclease may be a useful enzymatic reagent to probe the structural changes caused by mismatches and deletions in DNA and provide additional information on the requirements for incision by this repair enzyme.

J Mol Biol, 1986 Nov 5, 192(1), 1 - 15
Structure and function of the F plasmid genes essential for partitioning; Mori H et al.; The F plasmid in Escherichia coli has its own partition mechanism controlled by the sopA and sopB genes, and by the cis-acting sopC region . The DNA sequence of the entire partition region and its flanking regions is described here . Two large open reading frames coding for 43,700 Mr and 35,400 Mr proteins correspond to sopA and sopB, respectively . The sopB reading frame is located immediately downstream from the sopA reading frame . Twelve 43 base-pair direct repeats exist in the sopC region without any spacer regions, and one pair of seven base-pair inverted repeats exists in each of the direct repeats . Analysis of deletions in the sopC region showed that the direct repeats play an important role in plasmid partition and IncD incompatibility . IncG incompatibility is exhibited by pBR322 derivatives carrying the sopB gene alone . When compared with the partition genes parA and parB of plasmid P1, homology in amino acid sequence was found between the SopA protein of F and the ParA protein of P1, and also between SopB protein of F and ParB protein of P1 . In addition, homology was found between Rep proteins of F and P1.

J Biol Chem, 1986 Nov 5, 261(31), 14771 - 80
Sequence-dependent S1 nuclease hypersensitivity of a heteronomous DNA duplex; Evans T et al.; Using cloned (dG-dA)n X (dC-dT)n DNA duplexes {GA)n) as models of homopurine-homopyrimidine S1-hypersensitive sites, we show that cleavage of the alternate (non-B, non-Z) DNA structure by S1 nuclease is length-dependent, in both supercoiled and linear forms, which are similar because of the identity of their nicking profiles . However, the length of flanking sequences, the presence of borders, and the DNA topology affect the equilibrium between the alternate structure and B-DNA . The B form of (GA)38 has a 10.4-base pair helical repeat, but the two phosphodiester backbones have different conformations (heteronomous DNA with a dinucleotide repeat unit) . Extension experiments reveal that the alternate structure is also heteronomous, in agreement with the nicking patterns generated by S1 and mung bean nucleases and by venom phosphodiesterase . Sensitivity to the latter enzyme at pH 9.0 indicates that the alternate DNA does not appear only in the low pH of the S1 nuclease reaction . Moreover, Hoogsteen G-CH+ base-pairing does not seem to be a prerequisite for the appearance of sensitivity because S1 still recognizes the structure even when all Gs are methylated at N-7 . This is consistent with the results of chemical probing of the structure using dimethyl sulfate and diethyl pyrocarbonate at various pH values, which show absence of protection at guanine N-7 . However, diethyl pyrocarbonate treatment at low pH results in hyper-reactivity of A residues.

J Mol Biol, 1986 Nov 5, 192(1), 27 - 38
Interaction of mutant lambda repressors with operator and non-operator DNA; Nelson HC et al.; We have described a set of mutations that alter side-chains on the operator binding surface of lambda repressor . In this paper, we study the interactions of 12 purified mutant repressors with operator and non-operator DNA . The mutant proteins have operator affinities that are reduced from tenfold to greater than 10,000-fold compared to wild-type . Nine of the mutants have affinities for non-operator DNA that are similar to wild-type, two mutants show decreased non-specific binding, and one mutant has increased affinity for non-operator DNA . We discuss these findings in terms of the structural and energetic contributions of side-chain--DNA interactions, and show that certain contacts between the repressor and the operator backbone contribute both energy and specificity to the interaction.

J Biol Chem, 1986 Nov 5, 261(31), 14760 - 3
Transfer of functional adenovirus E1A transcription activator proteins into mammalian cells by protoplast fusion; Ferguson B et al.; Human adenovirus 2/5 E1A proteins were used to evaluate protoplast fusion as a method of transferring functional proteins into mammalian cells . Both the E1A 13 and 12 S mRNA products expressed in Escherichia coli are shown to activate in trans adenovirus gene expression following transfer into monkey kidney cells by protoplast fusion . Approximately 20% of the recipient mammalian cells exhibited positive nuclear E1A-specific immunofluorescence following fusion with protoplasts containing E1A protein . E . coli-expressed E1A protein was modified post-translationally in Vero cells following protoplast fusion, as evidenced by its shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility . These results establish protoplast fusion as a simple rapid method for examining the functional activity, intracellular distribution, and post-translational modification of E . coli-expressed proteins in intact mammalian cells.

Biochemistry, 1986 Nov 4, 25(22), 7229 - 35
Nuclear magnetic resonance study of the state of protonation of inhibitors bound to mutant dihydrofolate reductase lacking the active-site carboxyl; London RE et al.; 13C nuclear magnetic resonance spectra have been obtained for complexes of {2-13C}methotrexate and {2-13C}trimethoprim with wild-type dihydrofolate reductase (DHFR) from Escherichia coli and with two mutant enzymes in which aspartic acid-27 is replaced by asparagine and by serine, respectively . In both the wild-type and mutated enzymes, exchange between the free inhibitor and the enzyme-complexed inhibitor is slow on the NMR time scale; hence, despite the considerably increased dissociation constants for binary complexes with the enzymes, the dissociation rate remains small relative to the frequency separation of the resonances . In all cases but one, the pKa of an inhibitor that is complexed to enzyme differs greatly from that of the free inhibitor . However, while the pKa of both inhibitors in complexes with the wild-type enzyme is elevated to above 10, the pKa of the inhibitors complexed with the Asn-27 and Ser-27 enzymes is lowered to a value below 4 . Exact determinations of bound pKa values are limited by the solubility of the enzyme and the dissociation constants of the complexes . The single exception to these general conclusions is the ternary complex of the Ser-27 DHFR with trimethoprim and NADPH . In this complex, both free and enzyme-complexed trimethoprim exhibit similar pKa values (approximately equal to 7.6) . However, both the exchange between free and enzyme-complexed inhibitor and the protonation of the enzyme-complexed inhibitor are slow in the NMR time scale, so that the spectra reveal three resonances corresponding to free inhibitor, to protonated enzyme-complexed inhibitor, and to unprotonated enzyme-complexed inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Nov 4, 25(22), 7174 - 8
Chemical modification of tryptophan residues in Escherichia coli succinyl-CoA synthetase . Effect on structure and enzyme activity; Ybarra J et al.; Succinyl-CoA synthetase of Escherichia coli is an alpha 2 beta 2 protein containing active sites at the interfaces between alpha- and beta-subunits . The alpha-subunit contains a histidine residue that is phosphorylated during the reaction . The beta-subunit binds coenzyme A and probably succinate {see Nishimura, J . S . (1986) Adv . Enzymol . Relat . Areas Mol . Biol . 58, 141-172} . Chemical modification studies have been conducted in order to more clearly define functions of each subunit . Tryptophan residues of the enzyme were modified by treatment with N-bromosuccinimide at pH 7 . There was a linear relationship between loss of enzyme activity and tryptophan modified . At one tryptophan residue modified per beta-subunit, 100% of the enzyme activity was lost . In this enzyme sample, one methionine residue in each alpha- and beta-subunit was oxidized to methionine sulfoxide, although loss of enzyme activity could not be related in a linear manner to the formation of this residue . Subunits were prepared from enzyme that was inactivated 50% by N-bromosuccinimide with 0.5 tryptophan modified per beta-subunit but with insignificant modification of methionine residues in either subunit . Small decreases in the tyrosine and histidine content were observed in the alpha-subunit but not in the beta-subunit . In this case, modified beta-subunit when mixed with unmodified alpha-subunit gave a population of molecules that was 50% as active as the refolded, unmodified control but was only slightly changed with respect to phosphorylation capacity and unchanged with respect to rate of phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Nov 4, 25(22), 7168 - 74
Comparison of aspartate transcarbamoylases from wheat germ and Escherichia coli: functionally identical histidines in nonhomologous local sequences; Cole SC et al.; Aspartate transcarbamoylase (ATCase) from wheat germ and the catalytic subunit of the enzyme from Escherichia coli are trimers of similar size . The former is a regulatory enzyme in its trimeric state, while the latter is a component of a complex regulatory dodecamer . In a comparison of the two enzymes, reaction with diethyl pyrocarbonate revealed a highly active, essential histidine residue in each case . The two histidines (i.e., one in each enzyme) behaved nearly identically with respect to the following functional properties: kinetics of acylation (ethoxyformylation) and concomitant inactivation; kinetics of deacylation by hydroxylamine and concomitant reactivation; hyperbolic dependence of the apparent first-order rate constant (kapp) on diethyl pyrocarbonate concentration; pH dependence of kapp; failure of active-center ligands to protect the residue against diethyl pyrocarbonate, producing instead near-identical increases in the inactivation rate . These similarities point to an essential, highly conserved histidine in each enzyme, in a functional microenvironment that has changed relatively little since the divergence of plants and bacteria . Ethoxyformylated peptides were isolated from tryptic digests of the two inactivated enzymes . Sequencing of the major labeled peptide in each case showed the wheat and E . coli histidines embedded in nonhomologous primary segments, suggesting that, contrary to expectation, these segments are not part of the conserved microenvironment . In the case of the E . coli enzyme, the essential residue was identified as His-134 in the known sequence, which has a potential catalytic role on crystallographic evidence {Krause, K . L., Volz, K . W., & Lipscomb, W . N . (1985) Proc . Natl . Acad . Sci . U.S.A . 82, 1643-1647} . A second, much less reactive histidine was identified as His-64.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Nov 4, 25(22), 6866 - 75
Intramolecular cleavage of LexA and phage lambda repressors: dependence of kinetics on repressor concentration, pH, temperature, and solvent; Slilaty SN et al.; LexA repressor of Escherichia coli and phage lambda repressor are inactivated in vivo and in vitro by specific cleavage of an Ala-Gly peptide bond in reactions requiring RecA protein . At mildly alkaline pH, the in vitro cleavage reaction also proceeds spontaneously, suggesting that peptide bond hydrolysis is an activity of the repressors rather than of RecA . The spontaneous cleavage reaction, termed "autodigestion", has been characterized for the LexA and lambda repressors . The results show that the reaction is intramolecular . The rate of LexA autodigestion was studied over the pH range 7.15-11.77 and over the temperature range 4-46 degrees C . The logarithm of the rate constant increased linearly with pH and reached a plateau value (2.5 X 10(-3) s-1 at 37 degrees C) at pH above 10 . The data closely followed a model in which a single residue side chain (apparent pK = 9.8 at 37 degrees C) must be deprotonated for the protein to show activity . Analysis of the temperature dependence gave the heat of proton dissociation as 19.9 kcal/mol and the heat of activation for hydrolysis as 15.3 kcal/mol at 25 degrees C . Autodigestion of lambda repressor, studied over the pH range 8.65-10.70 at 37 degrees C, was similar to the LexA reaction in its pH dependence, yielding a pK of 9.8 . The maximum rate at 37 degrees C for lambda repressor, 6.1 X 10(-5) s-1, was 40 times slower than for LexA, a difference similar to that previously observed in vivo and in vitro for RecA-dependent cleavage reactions . There was no significant solvent deuterium isotope effect on the autodigestion of LexA . Changes in buffer composition, including high concentrations of glycine for lambda repressor and of imidazole or hydroxylamine for LexA, indicated that solvent components other than water do not participate in the rate-determining step . Removal or addition of metal ions did not significantly affect LexA autodigestion . These and other observations suggest that the deprotonated form of an amino acid side chain plays a central role in the chemistry of the cleavage reaction . The above observations establish repressor autodigestion as a member of an emerging set of biologically important self-processing reactions.

Biochemistry, 1986 Nov 4, 25(22), 7142 - 54
Reaction energetics of a mutant triosephosphate isomerase in which the active-site glutamate has been changed to aspartate; Raines RT et al.; The essential catalytic base at the active site of the glycolytic enzyme triosephosphate isomerase is the carboxylate group of Glu-165, which directly abstracts either the 1-pro-R proton of dihydroxyacetone phosphate or the 2-proton of (R)-glyceraldehyde 3-phosphate to yield the cis-enediol intermediate . Using the methods of site-directed mutagenesis, we have replaced Glu-165 by Asp . The three enzymes chicken isomerase from chicken muscle, wild-type chicken isomerase expressed in Escherichia coli, and mutant (Glu-165 to Asp) chicken isomerase expressed in E . coli have each been purified to homogeneity . The specific catalytic activities of the two wild-type isomerases are identical, while the specific activity of the mutant enzyme is reduced by a factor of about 1000 . The observed kinetic differences do not derive from a change in mechanism in which the aspartate of the mutant enzyme acts as a general base through an intervening water molecule, because the D2O solvent isotope effects and the stoichiometries of inactivation with bromohydroxyacetone phosphate are identical for the wild-type and mutant enzymes . Using the range of isotopic experiments that were used to delineate the free-energy profile of the wild-type chicken enzyme, we here derive the complete energetics of the reaction catalyzed by the mutant protein . Comparison of the reaction energetics for the wild-type and mutant isomerases shows that only the free energies of the transition states for the two enolization steps have been seriously affected . Each of the proton abstraction steps is about 1000-fold slower in the mutant enzyme . Evidently, the excision of a methylene group from the side chain of the essential glutamate has little effect on the free energies of the intermediate states but dramatically reduces the stabilities of the transition states for the chemical steps in the catalyzed reaction.

Eur J Biochem, 1986 Nov 3, 160(3), 515 - 20
Glutamate decarboxylase side reactions catalyzed by the enzyme; Choi SY et al.; A homogeneous glutamate decarboxylase isolated from pig brain contains 0.8 mol of tightly bound pyridoxal 5-phosphate/enzyme dimer . Upon addition of exogenous pyridoxal 5-phosphate (pyridoxal-5-P), the enzyme acquires maximum catalytic activity . Preincubation of the enzyme with L-glutamate (10 mM) brings about changes in the absorption spectrum of bound pyridoxal-5-P with the concomitant formation of succinic semialdehyde . However, the rate of this slow secondary reaction, i.e . decarboxylative transamination, is 10(-4) times the rate of normal decarboxylation . It is postulated that under physiological conditions enzymatically inactive species of glutamate decarboxylase, generated by the process of decarboxylative transamination, are reconstituted by pyridoxal-5-P produced by the cytosolic enzymes pyridoxal kinase and pyridoxine-5-P oxidase . The catalytic activity of resolved glutamate decarboxylase is recovered by preincubation with phospho-pyridoxyl-ethanolamine phosphate . The experimental evidence is consistent with the interpretation that the resolved enzyme binds the P-pyridoxyl analog, reduces the stability of the covalent bond of the phospho-pyridoxyl moiety, and catalyzes the formation of pyridoxal-5-P.

Eur J Biochem, 1986 Nov 3, 160(3), 459 - 67
Lactate dehydrogenase and glyceraldehyde-phosphate dehydrogenase are single-stranded DNA-binding proteins that affect the DNA-polymerase-alpha-primase complex; Grosse F et al.; Affinity chromatography on double-stranded (ds) and single-stranded (ss) DNA-cellulose columns was employed to find analogs of the Escherichia coli and T4 single-stranded DNA binding proteins (SSB proteins) in calf thymus . The interaction of several purified SSB proteins with the pure DNA-polymerase-alpha--primase complex on DNA synthesis on activated DNA and on primase-initiated M13 DNA served as a criterion for a possible involvement of one of these proteins in the process of DNA replication . Two SSB proteins were purified to essential homogeneity . These most abundant proteins exhibited apparent relative molecular masses of 35,000 (SSB-35) and 37,000 (SSB-37) for the protomers and 140,000 and 80,000 for the native enzymes . Both proteins resisted elution with 0.5 mg/ml dextran sulfate and were eluted from the ssDNA-cellulose with 0.2 M and 1 M NaCl, respectively . SSB-35 stimulated the DNA-polymerase-alpha--primase complex from the same organism up to fivefold over a broad range of DNA covering . By contrast, SSB-37 inhibited the primase-initiated replication of M13 DNA . Like most eukaryotic SSB proteins, these proteins showed a 300-fold preference for binding to ssDNA over dsDNA in a nitrocellulose filter binding assay, as well as strong binding to several DNA and RNA homopolymers . Furthermore, we provide evidence for a cooperative mode of binding for SSB-37 . Although SSB-35 and SSB-37 behave as typically eukaryotic SSB proteins in all assays employed, we tested these SSB proteins for dehydrogenase activities as well . SSB-35 was found to be identical with lactate dehydrogenase and SSB-37 was identical with a dimeric form of glyceraldehyde-3-phosphate dehydrogenase . These results imply that further studies are mandatory in order to prove the authenticity of eukaryotic SSB proteins.

Eur J Biochem, 1986 Nov 3, 160(3), 491 - 7
Purification and characterization of human interleukin-1 beta expressed in recombinant Escherichia coli; Wingfield P et al.; The high-level expression of human interleukin-1 beta in Escherichia coli is described . The protein contributes about 12% of the total cell protein and is associated with the soluble cytoplasmic fraction of the cell . A method for the purification of the protein is given which is based on anion- and cation-exchange chromatographies . The isolated protein, shown to be homogeneous by several analytical methods, has been characterized by amino acid analysis, N- and C-terminal sequence analysis and analytical centrifugation . The protein is biologically active as demonstrated by two different in vitro assays, namely, the mononuclear cell factor (IL-1/MCF) assay and lymphocyte activating factor (IL-1/LAF) assay . The specific activities determined with the IL-1/MCF and IL-1/LAF assays, are 2 X 10(7) and 4 X 10(7) units mg-1, respectively.

Eur J Biochem, 1986 Nov 3, 160(3), 593 - 7
Conformational changes induced by domain assembly within the beta 2 subunit of Escherichia coli tryptophan synthase analysed with monoclonal antibodies; Friguet B et al.; The effects of domain assembly on the conformation of the F1 (N-terminal) and F2 (C-terminal) domains of the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) were analysed using six monoclonal antibodies which recognize six different epitopes of the native beta 2 subunit (five carried by the F1 domain and one carried by the F2 domain) . For this purpose, the affinity constant of each monoclonal antibody for the isolated domains F1 or F2, the associated domains in the trypsin-nicked apo-beta 2 and in the native apo-beta 2 subunits were determined, both with the intact immunoglobulin and the Fab fragment . It was found that the association of the F1 and F2 domains within beta 2 is accompanied by structural changes of the two domains, as detected by variations of their affinity constants for the monoclonal antibodies.

Jpn J Surg, 1986 Nov, 16(6), 412 - 7
Plasma endotoxin levels and functions of peripheral granulocytes in surgical patients with respiratory distress syndrome; Miyata T et al.; Plasma endotoxin levels and granulocyte functions (chemiluminescence and chemotaxis) were determined in fifty-two patients with postoperative sepsis . Seventeen had concurrent respiratory distress syndrome (RDS group) and the remaining thirty-five were free of the syndrome (non-RDS group) . The plasma endotoxin concentrations were higher in the RDS group than in the non-RDS group (p less than 0.001) . All nine patients with particularly high levels (greater than 80 pg) belonged to the RDS group . We noted a positive correlation in chemiluminescence (p less than 0.001, r = 0.67) and a negative correlation in chemotactic activity (p less than 0.001, r = 0.69) . To determine whether endotoxin alters normal granulocyte functions in vitro, healthy granulocytes were treated by the endotoxin (E . coli 0111:B4) . There was an increase in chemiluminescence and a decrease in chemotactic activity, as observed in vivo . Furthermore, normal granulocytes chemiluminescence was increased by pretreatment of RDS plasma showing high endotoxin levels in vitro (n = 4, p less than 0.05) . Thus, endotoxin in the plasma probably plays an important role in marked changes in peripheral granulocyte functions in patients with respiratory distress syndrome.

Anal Biochem, 1986 Nov 1, 158(2), 371 - 6
In situ autoradiographic detection of folylpolyglutamate synthetase activity; Sussman DJ et al.; The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells . A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase . Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate . As a result, the conversion of {6-3H}deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA . In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of {6-3H}deoxyuridine into DNA . Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs . Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E . coli FPGS enzyme can provide pteroylpolyglutamates which function in mammalian cells.

Mol Biol (Mosk), 1986 Nov-Dec, 20(6), 1488 - 98
{Study of the function of low molecular weight nuclear RNA in the cell-free system of isolated nuclei}; Evtushenko VI et al.; Possible involvement of small nuclear RNAs in transcription was explored . The fraction of small (4-8S) nuclear or cytoplasmic RNAs was isolated by Sepharose 4B gel chromatography and its influence on the RNA synthesis in isolated nuclei was studied . Small nuclear or cytoplasmic RNAs stimulated RNA synthesis in isolated nuclei both in the presence and in the absence of cytosol . On the contrary, the influence of E . coli tRNA or high molecular weight cytoplasmic RNA varied depending on the presence of cytosol . The data indicate that small nuclear RNAs may participate in transcription although the mechanisms of their involvement demand further investigation.

Equine Vet J, 1986 Nov, 18(6), 472 - 4
Influence of vitamin E and selenium supplement on antibody production in horses; Baalsrud KJ et al.; Fifteen horses used for serum production were maintained on low vitamin E and selenium diets . They were divided into four groups receiving: Group 1 no supplements, Group 2 vitamin E, Group 3 selenium and Group 4 both vitamin E and selenium . The humoral immune response to novel antigens, such as tetanus toxoid and equine influenza virus, was increased in groups receiving either vitamin E or selenium/vitamin E . No effects were recorded on the titres against Escherichia coli or the levels of immunoglobulin G.

J Pediatr Gastroenterol Nutr, 1986 Nov-Dec, 5(6), 902 - 6
Prolonged and recurring diarrhea in the northeast of Brazil: examination of cases from a community-based study; McAuliffe JF et al.; From prospective daily surveillance of diarrhea in a poor rural area of northeastern Brazil, this study of prolonged diarrheal episodes identified the 3% of diarrheal episodes that lasted 15 days or longer . These episodes also defined a subpopulation of children who spent over 16% of their days with diarrhea . Such children warrant further attention in an attempt to define potentially treatable causes as well as to assure appropriate nutritional support . There was no single season for these prolonged illnesses, but they appeared to involve both the wet, slightly warmer season of peak enterotoxigenic Escherichia coli diarrhea as well as the dry, slightly cooler season of peak rotaviral infections . Limited etiologic data support this idea that multiple pathogens are found, often in combination with each other, that may work together to contribute to the important problem of chronic diarrhea . Future studies should focus attention on further defining risk factors, mechanisms, and appropriate therapy for the subset of children who experience prolonged diarrhea in this type of setting.

J Endocrinol, 1986 Nov, 111(2), 329 - 34
Biochemical characterization of circulating Met-enkephalins in canine endotoxin shock; Watson JD et al.; The alterations in arterial, venous and adrenal vein levels of immunoreactive Met-enkephalins following endotoxin administration have been investigated in dogs by direct measurement and gel filtration chromatography . Animals were anaesthetized with alpha-chloralose and allowed to breath spontaneously . The left lumbar adrenal vein, limb vein and femoral artery were cannulated for blood sampling . Severe shock was produced by the administration of a large bolus of E . coli endotoxin followed by a continuous infusion . The production of endotoxin shock was associated with significant increases in adrenal vein and systemic venous plasma immunoreactive Met-enkephalin levels . Forty-five minutes after induction of endotoxin shock arterial immunoreactive Met-enkephalin levels were generally higher than baseline values . In resting anaesthetized animals a large 31,000 molecular weight form of Met-enkephalin, presumably proenkephalin, was found in plasma obtained from the adrenal vein, systemic and pulmonary circulations . Following endotoxin this enkephalin-containing peptide still predominated in arterial and venous plasma, whereas in the adrenal vein the proportion of Met-enkephalin immunoreactivity attributable to this large peptide fell . This was associated with the appearance of increasing amounts of smaller molecular forms (18,000, 8000, 3-5000 molecular weight and the pentapeptide itself) . In this model enkephalin-containing peptides were not biochemically modified by their passage through the lungs.

Scand J Immunol, 1986 Nov, 24(5), 615 - 9
Hepatic production of biliary IgM in the rat; Dahlgren UI et al.; Drainage of the thoracic duct resulted in a decrease in the IgA level in rat bile, but at the same time there was an increase in both the total IgM level and the specific IgM antibody activity to Escherichia coli 06 in the bile of animals immunized in the Peyer's patches with these bacteria . The increase in total IgM was significantly higher in animals immunized with the E . coli 06 than in unimmunized rats . The level of total IgG was not altered during the drainage . IgM antibodies to E . coli 04 given intravenously during lymph drainage did not appear in the bile, whereas specific IgM antibodies to E . coli 06 occurring after active immunization increased in the bile of the same animal . The data elucidate two aspects of the hepatic IgM turnover . First IgM could take the place of IgA in cases of IgA deficiency, and second the IgM might originate from intrahepatic production.

J Bacteriol, 1986 Nov, 168(2), 923 - 30
Heat shock protein synthesis during development in Caulobacter crescentus; Gomes SL et al.; Caulobacter crescentus cells respond to a sudden increase in temperature by transiently inducing the synthesis of several polypeptides . Two of the proteins induced, Hsp62 and Hsp70, were shown to be analogous to the heat shock proteins of Escherichia coli, GroEL and DnaK, respectively, by immunological cross-reactivity with antibodies raised against the E . coli proteins . Two-dimensional gel electrophoretic resolution of extracts of cells labeled with {35S}methionine during heat shock led to the identification of 20 distinct Hsps in C . crescentus which are coordinately expressed, in response to heat, at the various stages of the cell division cycle . Thus, a developmental control does not seem to be superimposed on the transient activation of the heat shock genes . Nonetheless, under normal temperature conditions, four Hsps (Hsp70, Hsp62, Hsp24b, and Hsp23a) were shown to be synthesized, and their synthesis was cell cycle regulated.

Arch Biochem Biophys, 1986 Nov 1, 250(2), 461 - 8
Spectroscopic studies on heme d in the visible and infrared; Vavra MR et al.; Heme d has been isolated from the terminal oxidase complex of Escherichia coli strain MR43L/F152 and purified by high-pressure liquid chromatography . The infrared spectrum indicated that carbonyls in the chlorin skeleton of this isolated heme existed as carboxylic acids . Earlier work on the iron-free chlorin had demonstrated the presence of a spirolactone substituent . This may have arisen from a cyclization reaction from a dicarboxylic acid, diol precursor . Although the free heme in extracts can exist as a diol, this does not prove that the diol as such is the precise form in the enzyme complex . Visible and fluorescence spectra are reported for a variety of derivatives and complexes of heme d to establish a spectral library that may be used to prove the presence of this structure in other enzymes or cells . Association constants have been measured for complexes of heme d with cyanide, imidazole, and pyridine and are contrasted to available data for protoheme.

Acta Chir Scand, 1986 Nov, 152, 641 - 5
Metabolic burst and motility in granulocytes following trauma and endotoxinaemia in the anaesthetized pig; Andersen OK et al.; The early effects of trauma and endotoxinaemia in pigs have been studied with regard to granulocyte motility and metabolic burst (chemiluminescence) . Increased spontaneous chemiluminescence was observed following a moderate aseptic trauma (osteotomies) or endotoxin infusion in healthy pigs . Zymosan-opsonized chemiluminescence was increased post trauma . Endotoxin infusion, however, led to unchanged values during infusion but a significant decreased Zymosan-opsonized chemiluminescence 3 h after ended infusion . Migration capacity was unchanged by trauma, but markedly reduced following endotoxin infusion . When traumatized pigs had an endotoxin infusion 24 h post trauma, the increase in spontaneous chemiluminescence was significantly more pronounced than following endotoxin infusion in healthy pigs, and Zymosan-opsonized chemiluminescence less decreased . Motility, however, was equally reduced as following endotoxin infusion in healthy pigs . These findings correspond well with the known decreased phagocytic capacity of granulocytes in septicaemia, but also indicate a rapid post-traumatic reaction which might be an attempt to reduce the lethal danger of a subsequent infection.

Tohoku J Exp Med, 1986 Nov, 150(3), 317 - 25
Stability of endotoxin detected in human plasma against endotoxin-inactivating factor (EIF): quantitative analysis of EIF using chromogenic endotoxin assay; Yajima Y et al.; Using a quantitative blood endotoxin assay utilizing chromogenic substrate coupled with perchloric acid pretreatment (PCA-LCT), we showed the presence of endotoxin-inactivating factor (EIF) in human plasma in vitro . EIF activity inactivated added endotoxin to about 10(-4) of the initial level within 20 min, followed by a stable phase where the residual endotoxin became resistant to EIF and was not further inactivated . The residual endotoxin may represent the endotoxin in patient plasma which is also EIF resistant . We postulate that endotoxin, upon entering the blood, is rapidly inactivated by chemical modification of its active site, lipid A, through EIF . Subsequently, inactivated endotoxin, mainly consisting of polysaccharide, is gradually removed from circulation by endocytosis in the reticuloendothelial system.

Injury, 1986 Nov, 17(6), 407 - 9
Post-traumatic meningitis in children; Lau YL et al.; A retrospective survey over a 66-month period of children admitted with head injury who subsequently developed meningitis within the same period yielded six cases (five boys, one girl), giving an incidence of 0.38 per cent . Two of the six died, and four survived with no sequelae . Four cases occurred within the first week . One patient, who had received prophylactic antibiotics, developed Escherichia coli meningitis after 14 days and one had meningitis 2 years after the head injury . The most common organism was pneumococcus (four cases) . Three patients had periorbital haematomas and none had cerebrospinal fluid leakage . Increasing drowsiness and fever were the most consistent features . Radiography of the skull was of little use in demonstrating fracture of the base of the skull . Two of the four surviving patients had craniotomy with successful dural repair.

Z Gastroenterol, 1986 Nov, 24(11), 691 - 9
{Increase of plasma cholecystokinin by Escherichia coli endotoxin-induced shock in swine}; Riepl R et al.; The gastrointestinal tract is the source of numerous peptide hormones . Since the gut will be altered severely during prolonged general circulatory low flow states, the reactions of the gut hormones are of great interest . In this study 18 anesthetized pigs were put into shock states to get first informations about the changes of the plasma levels of cholecystokinin (CCK) . 12 pigs (group I and II) were exposed to a general circulatory shock state by a 2-hr intravenous infusion of a sublethal dose of Escherichia coli endotoxin . 6 of them (gr . II) first received a gastroenterectomy apart from a small duodenal remnant proximal and distal to the papilla of Vater . The remaining 6 pigs (gr . III) suffered a severe hemorrhagic-hypovolemic shock over a 150-min period by arterial bleeding . Plasma CCK increased significantly (p less than 0.05) in the aorta (gr . I, II), the portal vein (gr . I, II), the superior caval vein (gr . I), and the internal jugular vein (gr . I) at the end of the 2-hr endotoxin infusion . In group II, the rise of CCK levels in the superior caval vein was also marked, but insignificant . The CCK-concentrations in the internal jugular vein were measured only in group I . By contrast, no changes in plasma CCK were seen in hemorrhagic shock (gr . III) . Within each group plasma samples taken from the various blood vessels at identical time points showed no significant differences . Since the plasma concentrations of CCK remained unchanged during hemorrhagic shock, the release of CCK by E . coli endotoxin is not due to the general circulatory low flow state.(ABSTRACT TRUNCATED AT 250 WORDS)

Vet Pathol, 1986 Nov, 23(6), 712 - 7
Lymphocytic depletion of bursa of Fabricius and thymus in chickens inoculated with Escherichia coli; Nakamura K et al.; Specific-pathogen-free 10-week-old chickens were inoculated via the air sac with Escherichia coli and showed lymphocytic depletion of bursa of Fabricius and thymus . In experiment I, chickens were necropsied at 12 and 24 hours, 2, 3, and 5 days after inoculation . At 12 hours after inoculation there was lymphocytic depletion in the medulla of lymphoid follicles of the bursa . At 24 hours after inoculation there was lymphocytic depletion also in the cortex of follicles and edema in interfollicular interstitium and follicular medulla . At 2 and 3 days after inoculation there were more marked lymphocytic depletion in medulla and cortex, and fibrosis in interfollicular interstitium . Partial repopulation of follicles with lymphocytes was seen at 5 days after inoculation . In the thymus, lymphocytic depletion occurred in the cortex . At 12 hours after inoculation, lymphocytic necrosis increased in number more than that of control chickens . The width of the cortex and medulla decreased . At 24 hours after inoculation, lymphocytic necrosis increased further . At 2 to 5 days after inoculation, the boundary between the cortex and medulla of lobules was obscure and cellular elements of the cortex and medulla were mingled . In experiment II, chickens were necropsied as in experiment I and also at 8 and 14 days after inoculation . The relative weights of the bursa and thymus reduced rapidly to minimal relative weights at 8 days after inoculation . At 14 days after inoculation, both bursa and thymus had normal relative weights and histological structures . These findings indicate that E . coli infection may induce transient lymphocytic depletion of lymphoid tissues in the chicken.

Plasmid, 1986 Nov, 16(3), 168 - 74
Functioning of the F-plasmid origin of replication in an Escherichia coli K12 Hfr strain during exponential growth; Lycett GW et al.; The pattern of chromosome replication in the Escherichia coli K12 Hfr strain KL99 was investigated during exponential growth by DNA-DNA hybridization . The levels of chromosomal markers close to the point of insertion of F (near pyrC) were raised in relation to other markers by comparison with the situation in an isogenic F- strain . The data are shown to be consistent with the proposal that the integrated F plasmid was regulating its copy number by a mass-titration mechanism.

Mol Biol (Mosk), 1986 Nov-Dec, 20(6), 1631 - 7
{Plasmid R1drd-19-mediated superoligomerization of the plasmid pACYC184in the recB mutant of Escherichia coli K12}; Terent'ev MA et al.; It was found that monomers of the pACYC184 plasmid undergo superoligomerization in a recB mutant of Escherichia coli K12 which is deficient in ATP-dependent RecBC nuclease and carries the drug resistance plasmid R1drd-19 . The observed effect is specifically related to the ability of R1drd-19 to determine an ATP-dependent exonucleolytic activity which is functionally similar but not identical to the RecBC nuclease . The oligomerization of pACYC184 is accompanied by the formation of high-order circular structures, and this leads to elimination of the plasmid from cells growing under non-selective conditions.

Mol Gen Genet, 1986 Nov, 205(2), 349 - 52
Alteration by mutation of the control by oxygen of the nar operon in Escherichia coli; Bonnefoy V et al.; A nar-lac operon fusion was used to isolate a mutant in which the expression of the nar operon was no longer repressed by oxygen . The nard mutation, located upstream of the nar structural genes, was found to be cis dominant; it led to independence from the Fnr protein which, in the wild-type strain, exerts a strict positive control on the nar operon . Both other known controls, nitrate induction and autoregulation, were unaffected . It is proposed that molecular oxygen controls the expression of nar via Fnr and that the nard mutation affects the Fnr binding site of the narGHI control region.

Mol Gen Genet, 1986 Nov, 205(2), 240 - 7
Functional interactions in vivo between suppressor tRNA and mutationally altered ribosomal protein S4; Kirsebom LA et al.; Ribosomal mutants (rpsD) which are associated with a generally increased translational ambiguity were investigated for their effects in vivo on individual tRNA species using suppressor tRNAs as models . It was found that nonsense suppression is either increased, unaffected or decreased depending on the codon context and the rpsD allele involved as well as the nature of the suppressor tRNA . Missense suppression of AGA and AGG by glyT(SuAGA/G) tRNA as well as UGG by glyT(SuUGG-8) tRNA is unaffected whereas suppression of UGG by glyT(SuUGA/G) or glyV(SuUGA/G) tRNA is decreased in the presence of an rpsD mutation . The effects on suppressor tRNA are thus not correlated with the ribosomal ambiguity (Ram) phenotype of the rpsD mutants used in this study . It is suggested that the mutationally altered ribosomes are changed in functional interactions with the suppressor tRNA itself rather than with the competing translational release factor(s) or cognate aminoacyl tRNA . The structure of suppressor tRNA, particularly the anticodon loop, and the suppressed codon as well as the codon context determine the allele specific functional interactions with these ribosomal mutations.

Kosm Biol Aviakosm Med, 1986 Nov-Dec, 20(6), 73 - 6
{Direct and mediated action of a permanent magnetic field on biological objects}; Pantev TP et al.; The effect of a constant magnetic field (CMF) of H = 2.3 X 10(5) A/m (2900 Oe) on the viability and radiosensitivity of E . coli B and the effect of magnetically activated water (MAW) on the radioresistance of rats were examined . The exposure did not influence the growth kinetics of E . coli B . Cell cultivation in the magnetically pretreated nutrient medium enhanced the bacterial growth . Preliminary exposure of bacterial cells to a CMF for 24 and 48 hrs increased and that for 72 hrs decreased their radioresistance . Twice a day the experimental weanlings were given MAW and the controls--tap water . The postradiation longevity of the MAW rats proved extended as compared to that of the controls . The MAW rats showed a greater osmotic stability of erythrocytes, a higher concentration of nucleic acids, and a larger count of leucocytes.

Genetika, 1986 Nov, 22(11), 2658 - 63
{Mutants of Escherichia coli K-12 with increased resistance to ionizing radiation . VI . Increased radioresistance and heat shock proteins}; Verbenko VN et al.; By means of one-dimensional electrophoresis, it is shown that in radiation-resistant Gamr444 and Gamr445 mutants of Escherichia coli K-12 high-molecular weight heat shock proteins are hyperproduced at 32-37 degrees C and are induced more intensively during heat shock (in comparison to the parental wild-type strain AB1157) . When the missense htpR15 mutation of the positive regulatory htpR gene for heat shock proteins was introduced by transduction into the genome of the Gamr444 mutant, its enhanced radiation-resistance disappeared but could be restored upon introduction of pKV3 plasmid bearing the htpR+ gene . These data show that heat shock proteins are participating in the enhanced radioresistance of Gamr mutants.

Genetika, 1986 Nov, 22(11), 2560 - 71
{The genetic Rec system of homologous recombination in Escherichia coli}; Lantsov VA; A review of modern data on genetic and biochemical bases of the Rec system is presented . Taking into consideration the final result of recombination, that is the preferential way of integration of donor DNA into recipient chromosome, it is proposed to distinguish three main pathways of homologous recombination RecBC (recF- recipient genotype), RecF (recBC- sbcB- sbcC-) and SOS . A recBC nuclease unwinds a donor linear DNA duplex to create the 3'-single-stranded end which was shown to be responsible for the initiation of recombination with the help of RecA protein . As a result, integration of single- and double-stranded donor fragments into recipient chromosome takes place in full accordance with the Meselson-Radding model . The peculiarity of the RecF pathway is its ability to promote recombination between two DNA closed ring structures . The SOS way is characterized by frequent single-stranded DNA exchanges . Both pathways are interconnected . The SOS way is regarded here as an extreme situation of the RecF one.

Biokhimiia, 1986 Nov, 51(11), 1858 - 67
{Study of the surface of Escherichia coli ribosomes and ribosomal particles by the tritium bombardment method}; Iusupov MM et al.; A new technique of atomic tritium bombardment has been used to study the surface topography of Escherichia coli ribosomes and ribosomal subunits . The technique provides for the labeling of proteins exposed on the surface of ribosomal particles, the extent of protein labeling being proportional to the degree of exposure . The following proteins were considerably tritiated in the 70S ribosomes: S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L1, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27 . A conclusion is drawn that these proteins are exposed on the ribosome surface to an essentially greater extent than the others . Dissociation of 70S ribosomes into the ribosomal subunits by decreasing Mg2+ concentration does not lead to the exposure of additional ribosomal proteins . This implies that there are no proteins on the contacting surfaces of the subunits . However, if a mixture of subunits has been subjected to centrifugation in a low Mg2+ concentration at high concentrations of a monovalent cation, proteins S3, S5, S7, S14, S18 and L16 are more exposed on the surface of the isolated 30S and 50S subunits than in the subunit mixture or in the 70S ribosomes . The exposure of additional proteins is explained by distortion of the native quaternary structure of ribosomal subunits as a result of the separation procedure . Reassociation of isolated subunits at high Mg2+ concentration results in shielding of proteins S3, S5, S7 and S18 and can be explained by reconstitution of the intact 30S subunit structure.

Arch Microbiol, 1986 Nov, 146(2), 151 - 8
Ethylene formation by cell-free extracts of Escherichia coli; Ince JE et al.; The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated . Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme . 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway . KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe3+ chelated to EDTA, and oxygen . In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose . The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth . As in the plant system, ethylene produced by E . coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different . It seems possible that ethylene production by bacteria might generally occur via the route seen in E . coli.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1986 Nov, 262(4), 474 - 82
Uterine secretions induce phagocytosis of Escherichia coli by neutrophils; Matsuda H et al.; Phagocytosis of Escherichia coli by neutrophils was induced in the presence of uterine secretions . The phagocytosis-inducing activity of uterine secretions was not changed by heat-treatment (56 degrees C for 30 min) . The phagocytosis-inducing activity of uterine secretions from rabbits treated with estradiol-17 beta was slightly higher than that from control rabbits treated with vehicle . On the other hand, administration of progesterone significantly depressed phagocytosis-inducing activity of uterine secretions, and the inhibitory effect of progesterone was not affected by estradiol-17 beta . Neutrophils pretreated with uterine secretions did not phagocytize intact bacteria; in contrast, bacteria pretreated with uterine secretions were phagocytized by intact neutrophils . The present results suggest that there are heat-stable opsonins in uterine secretions which induce phagocytosis of bacteria by neutrophils, and that estrogen slightly enhances the opsonic activity of uterine secretions but progesterone depresses it and inhibits the promoting effect of estrogen.

Virus Res, 1986 Nov, 6(2), 123 - 32
In vitro encapsidation of plasmid DNA into human adenovirus empty capsids; Kosturko LD et al.; Plasmid DNA, added to extracts of human adenovirus type 3-infected HeLa cells, binds to empty viral capsids and can be purified using cesium density gradient centrifugation . The fraction of DNA bound depends on the amount of DNA added to the extract, and the capsid partially protects the bound DNA from digestion by DNase I . This capsid binding of plasmid DNA does not require the presence of the adenovirus DNA packaging sequence . However, the presence of the adenovirus packaging sequence in the plasmid results in better protection of the bound plasmid molecule from cellular nucleases.

EMBO J, 1986 Nov, 5(11), 3001 - 6
Altered translation initiation factor 2 in the cold-sensitive ssyG mutant affects protein export in Escherichia coli; Shiba K et al.; The Escherichia coli gene secY (pr1A) codes for an integral membrane protein that plays an essential role in protein export . We previously isolated cold-sensitive mutations (ssy) as extragenic suppressors of temperature-sensitive secY24 mutation . Now we show that the ssyG class of mutations are within infB coding for the translation initiation factor IF2 . The mutants produce altered forms of IF2 with a cold-sensitive in vitro activity to form a translation initiation complex . The mutation suppresses not only secY24 but also other secretion-defective mutations such as secA51 and rp10215 . The beta-galactosidase enzyme activity of the MalE-LacZ 72-47 hybrid protein is strikingly reduced in the ssyG mutant at the permissive high temperature, while the hybrid protein itself is normally synthesized . This effect, which was observed only for the hybrid protein with a functional signal sequence, may result from some alteration in the cellular localization of the protein . These results suggest that IF2 or the translation initiation step can modulate protein export reactions . The isolation of cold-sensitive ssyG mutations in infB provides genetic evidence that IF2 is indeed essential for normal growth of E . coli cells.

EMBO J, 1986 Nov, 5(11), 2995 - 3000
Functional dissection of Escherichia coli promoters: information in the transcribed region is involved in late steps of the overall process; Kammerer W et al.; After binding to a promoter Escherichia coli RNA polymerase is in contact with a region of about 70 bp . Around 20 bp of this sequence are transcribed . Information encoded within this transcribed region is involved in late steps of the functional program of a promoter . By changing such 'down-stream' sequences promoter strength in vivo can be varied more than 10-fold . By contrast, information for early steps of the promoter program such as recognition by the enzyme and formation of a stable complex resides in a central core region of about 35 bp . Our data show that the strength of a promoter can be limited at different levels of the overall process . Consequently promoters of identical strength can exhibit different structures due to an alternate optimization of their program.

EMBO J, 1986 Nov, 5(11), 2987 - 94
Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures; Deuschle U et al.; The strength in vivo of 14 promoters was determined in a system which permits the quantitation of RNA synthesis with high accuracy . Up to 75-fold differences in promoter strength were measured and the most efficient signals are promoters from coliphages T7 and T5 . Their activity approaches the strength of fully induced promoters of the rRNA operons which may be close to the functional optimum of a single sequence . By contrast, a synthetic 'consensus promoter' belongs to the less efficient signals . Our data show that optimal promoter function can be achieved by alternate structures and strongly suggest that information outside of the 'classical' promoter region contributes to promoter activity.

Chem Biol Interact, 1986 Nov, 60(2), 207 - 15
Toxicity, mutagenicity and induction of recA protein in Escherichia coli treated with cis-diamminedichloroplatinum(II) and cis-diamminetetrachloroplatinum(IV); Razaka H et al.; After exposure of bacteria to equal concentrations of cis-diamminedichloroplatinum(II) (DDP) and cis-diamminetetrachloroplatinum(IV) (DTP), the intracellular concentration of DTP was an order of magnitude greater than DDP . However, at identical intracellular drug concentrations, the Pt(IV) compound formed only half as many platinum-DNA lesions . For equal numbers of DNA lesions, the toxicity of both agents was identical whereas the mutagenicity of DTP was 7 times less than for DDP and its capacity to induce recA protein was less than DDP by a factor of 3.5 . Bioreduction of Pt(IV) compounds to their corresponding Pt(II) analogues has been proposed as a mechanism for the reaction of Pt(IV) compounds with cellular DNA . According to this hypothesis, DTP would be reduced to DDP in the cell prior to its reaction with DNA and the platinum-DNA lesions of the two compounds should be identical . Our results suggest that reductive elimination can not entirely account for DNA damage caused by PT(IV) compounds in bacteria.

Mol Biochem Parasitol, 1986 Nov, 21(2), 189 - 97
Identification of proteins encoded by variant surface glycoprotein expression site-associated genes in Trypanosoma brucei; Cully DF et al.; The variant surface glycoprotein (VSG) genes of Trypanosoma brucei may be transcribed from several distinct telomeric expression sites (ESs) . The mechanism responsible for regulating potential expression sites is unknown . Two members of a pleomorphic family of expression site associated genes (ESAGs) have been cloned and sequenced . By examination of the DNA sequences we inferred that ESAGs encode amphiphilic glycoproteins . Fragments of two ESAGs were inserted into the Escherichia coli expression vectors pATH and pEX . Antisera to the resulting anthranilate synthetase ESAG protein (ESAGP) fusion protein immune precipitated a 46 kDa glycoprotein from detergent extracts of T . brucei . In the presence of tunicamycin, the size of the immune-precipitated protein was reduced to 36 kDa, corresponding to the molecular weight predicted by the ESAG sequence . The 36 kDa and 46 kDa proteins were absent from procyclic culture forms of T . brucei.

J Bacteriol, 1986 Nov, 168(2), 901 - 10
Location of functional regions of the Escherichia coli RecA protein by DNA sequence analysis of RecA protease-constitutive mutants; Wang WB et al.; In previous work (E . S . Tessman and P . K . Peterson, J . Bacteriol . 163:677-687 and 688-695, 1985), we isolated many novel protease-constitutive (Prtc) recA mutants, i.e., mutants in which the RecA protein was always in the protease state without the usual need for DNA damage to activate it . Most Prtc mutants were recombinase positive and were designated Prtc Rec+; only a few Prtc mutants were recombinase negative, and those were designated Prtc Rec- . We report changes in DNA sequence of the recA gene for several of these mutants . The mutational changes clustered at three regions on the linear RecA polypeptide . Region 1 includes amino acid residues 25 through 39, region 2 includes amino acid residues 157 through 184, and region 3 includes amino acid residues 298 through 301 . The in vivo response of these Prtc mutants to different effectors suggests that the RecA effector-binding sites have been altered . In particular we propose that the mutations may define single-stranded DNA- and nucleoside triphosphate-binding domains of RecA, that polypeptide regions 1 and 3 comprise part of the single-stranded DNA-binding domain, and that polypeptide regions 2 and 3 comprise part of the nucleoside triphosphate-binding domain . The overlapping of single-stranded DNA- and nucleoside triphosphate-binding domains in region 3 can explain previously known complex allosteric effects . Each of four Prtc Rec- mutants sequenced was found to contain a single amino acid change, showing that the change of just one amino acid can affect both the protease and recombinase activities and indicating that the functional domains for these two activities of RecA overlap . A recA promoter-down mutation was isolated by its ability to suppress the RecA protease activity of one of our strong Prtc mutants.

J Bacteriol, 1986 Nov, 168(2), 828 - 32
Effects of nucleotides on ATP-dependent protein translocation into Escherichia coli membrane vesicles; Chen L et al.; We have shown previously that Escherichia coli can translocate the same protein either co- or posttranslationally and that ATP hydrolysis is essential for the posttranslational translocation of the precursors of alkaline phosphatase and OmpA protein into inverted E . coli membrane vesicles . ATP-dependent protein translocation has now been further characterized . In the absence of exogenous Mg2+, dATP, formycin A-5'-triphosphate, ATP-alpha-S, and N1-oxide-ATP could replace ATP, but many other nucleotides were not only ineffective but inhibited ATP-dependent translocation . The inhibitors included nonhydrolyzable ATP analogs, ATP-gamma-S, 8-azido-ATP, AMP, ADP, cyclic AMP, PPi, and tripolyphosphate . On the other hand, adenosine, adenosine 5'-tetraphosphate, and N1,N6-etheno-ATP neither supported nor inhibited translocation . Moreover, photoaffinity labeling of azido-adenine nucleotides rendered membranes inactive for subsequent ATP-dependent protein translocation . These results suggest that protein translocation involves at least an ATP-binding site in the membrane and hydrolysis of ATP and that both the adenosine and phosphate moieties of ATP play a role.

J Bacteriol, 1986 Nov, 168(2), 775 - 9
Binding specificity of the periplasmic oligopeptide-binding protein from Escherichia coli; Guyer CA et al.; The structural properties required for the binding of peptide substrates to the Escherichia coli periplasmic protein involved in oligopeptide transport were surveyed by measuring the ability of different peptides to compete for binding in an equilibrium dialysis assay with the tripeptide Ala-Phe-{3H}Gly . The protein specifically bound oligopeptides and failed to bind amino acids or dipeptides . Acetylation of the peptide amino terminus of (Ala)3 severely impaired binding, whereas esterification of the carboxyl terminus significantly reduced but did not completely eliminate binding . Peptides composed of L-amino acids competed more effectively than did peptides containing D-residues or glycine . Experiments with a series of alanyl peptide homologs demonstrated a decrease in competitive ability with increasing chain length beyond tripeptide . Competition studies with tripeptide homologs indicated that a wide variety of amino acyl side chains were tolerated by the periplasmic protein, but side-chain composition did affect binding . Fluorescence emission data suggested that this periplasmic protein possesses more than one substrate-binding site capable of distinguishing peptides on the basis of amino acyl side chains.

J Bacteriol, 1986 Nov, 168(2), 746 - 51
Expression of Escherichia coli infC: identification of a promoter in an upstream thrS coding sequence; Pramanik A et al.; infC, the gene which codes for translation initiation factor 3, is situated in a cluster in the genome of Escherichia coli with genes for several other components of the translation apparatus . Only three nucleotides separate the termination codon of thrS from the initiation codon of infC . This implies that infC is either cotranscribed with thrS from a thrS promoter or that the transcriptional signals for infC are embedded within the upstream thrS coding region . In the present work, several plasmids have been constructed which encompass infC and various amounts of the upstream thrS sequence . The ability of the plasmid DNA, or derived restriction fragments, to direct the synthesis of initiation factor 3 was tested in an in vitro DNA-dependent coupled transcription-translation system and in plasmid-transformed maxicells . The results indicate that initiation factor 3 is synthesized in the absence of the thrS promoter . A promoter whose presence is sufficient for the expression of infC has been localized to an 89-base-pair region which lies 178 to 267 base pairs upstream of the infC initiation codon . S1 nuclease mapping of in vivo transcripts confirms that a transcription initiation site is located in this region . These studies demonstrate that infC can be transcribed from a promoter within the upstream thrS coding sequence.

J Bacteriol, 1986 Nov, 168(2), 642 - 7
Amplified expression of the tag+ and alkA+ genes in Escherichia coli: identification of gene products and effects on alkylation resistance; Kaasen I et al.; We have constructed plasmids which overproduce the tag and alkA gene products of Escherichia coli, i.e., 3-methyladenine DNA glycosylases I and II . The tag and alkA gene products were identified radiochemically in maxi- or minicells as polypeptides of 21 and 30 kilodaltons, respectively, which are consistent with the gel filtration molecular weights of the enzyme activities, thus confirming the identity of the cloned genes . High expression of the tag+-coded glycosylase almost completely suppressed the alkylation sensitivity of alkA mutants, indicating that high levels of 3-methyladenine DNA glycosylase I will eliminate the need for 3-methyladenine DNA glycosylase II in repair of alkylated DNA . Furthermore, overproduction of the alkA+-coded glycosylase greatly sensitizes wild-type cells to alkylation, suggesting that only a limited expression of this enzyme will allow efficient DNA repair.

J Bacteriol, 1986 Nov, 168(2), 631 - 5
Purification of the phoU protein, a negative regulator of the pho regulon of Escherichia coli K-12; Surin BP et al.; Thermally induced transcription of the phoU gene under control of the major leftward promoter, pL, of phage lambda resulted in production of the PhoU protein to compose approximately 5% of the total cell protein . The PhoU protein was present in the cytoplasm in the form of an aggregate . The amino acid composition and N-terminal amino acid sequence of the purified protein confirmed the reading frame established earlier for the phoU gene.

J Bacteriol, 1986 Nov, 168(2), 613 - 8
Increase in cell mass during the division cycle of Escherichia coli B/rA; Kubitschek HE; Increase in the mean cell mass of undivided cells was determined during the division cycle of Escherichia coli B/rA . Cell buoyant densities during the division cycle were determined after cells from an exponentially growing culture were separated by size . The buoyant densities of these cells were essentially independent of cell age, with a mean value of 1.094 g ml-1 . Mean cell volume and buoyant density were also determined during synchronous growth in two different media, which provided doubling times of 40 and 25 min . Cell volume and mass increased linearly at both growth rates, as buoyant density did not vary significantly . The results are consistent with only one of the three major models of cell growth, linear growth, which specifies that the rate of increase in cell mass is constant throughout the division cycle.

J Bacteriol, 1986 Nov, 168(2), 553 - 62
Poly-beta-hydroxybutyrate membrane structure and its relationship to genetic transformability in Escherichia coli; Reusch RN et al.; The effects of competence-inducing treatments on the composition and organization of membrane lipids in Escherichia coli K-12, DH1, DH5, HB101, and RR1 were investigated for two widely used protocols in which transformability is developed at low temperatures in Ca2+ buffers . At stages during each procedure, the lipid compositions of the cells were determined, and the thermotropic lipid phase transitions were observed in whole cell culture by fluorescence assay with the hydrophobic probe N-phenyl-1-naphthylamine . Competence was evaluated by determining transformation efficiencies with plasmid pBR322 DNA . The competence-inducing procedures effected only slight changes in phospholipid compositions which did not correlate with transformability . However, the induction of competence was coincident with de novo synthesis and incorporation of poly-beta-hydroxybutyrate into the cytoplasmic membranes and with the appearance of a sharp lipid phase transition above physiological temperatures . Transformation efficiencies correlated with poly-beta-hydroxybutyrate concentrations and with the intensity of the new phase transition . Transformability, poly-beta-hydroxybutyrate synthesis and the new phase transition were not significantly affected by inhibition of protein synthesis with chloramphenicol or inhibition of respiration or ATP synthesis with azide, cyanide, arsenate, or 2,4-dinitrophenol; however, when poly-beta-hydroxybutyrate synthesis was inhibited with acetaldehyde, the new phase transition was not observed, and competence failed to develop . These studies suggest that genetic transformability in E . coli may be physiologically regulated.

J Bacteriol, 1986 Nov, 168(2), 494 - 504
DNA replication termination in Escherichia coli parB (a dnaG allele), parA, and gyrB mutants affected in DNA distribution; Norris V et al.; We investigated the Escherichia coli mutants carrying the parB, parA, and gyrB mutations, all of which display faulty chromosome partitioning at the nonpermissive temperature, to see whether their phenotype reflected a defect in the termination of DNA replication . In the parB strain DNA synthesis slowed down at 42 degrees C and the SOS response was induced, whereas in the parA strain DNA synthesis continued normally for 120 min and there was no SOS induction . To see whether replication forks accumulated in the vicinity of terC at the nonpermissive temperature, the mutants were incubated for 60 min at 42 degrees C and then returned to low temperature and pulse-labeled with {3H}thymidine . In all cases the restriction pattern of the labeled DNA was incompatible with that of the terC region, suggesting that replication termination was normal . In the parA mutant no DNA sequences were preferentially labeled, whereas in the parB and gyrB strains there was specific labeling of sequences whose restriction pattern resembled that of oriC . In the case of parB this was confirmed by DNA-DNA hybridization with appropriate probes . This test further revealed that the parB mutant over initiates at oriC after the return to the permissive temperature . Like dna(Ts) strains, the parB mutant formed filaments at 42 degrees C in the absence of SOS-associated division inhibition, accompanied by the appearance of anucleate cells of nearly normal size (28% of the population after 3 h), as revealed by autoradiography . The DNA in the filaments was either centrally located or distributed throughout . The parB mutation lies at 67 min, and the ParB- phenotype is corrected by a cloned dnaG gene or by a plasmid primase, strongly suggesting that parB is an allele of dnaG, the structural gene of the E . coli primase . It is thus likely that the parB mutant possesses an altered primase which does not affect replication termination but causes a partial defect in replication initiation and elongation and in chromosome distribution.

J Bacteriol, 1986 Nov, 168(2), 1026 - 9
Glutathione in Escherichia coli is dispensable for resistance to H2O2 and gamma radiation; Greenberg JT et al.; Escherichia coli devoid of glutathione (because of transposon insertions in the gshA gene) has normal resistance to H2O2, cumene hydroperoxide, heat, or ionizing radiation . Intracellular glutathione thus does not protect E . coli from such lethal oxidative damage . The use of gshA::Tn10 mutants also revealed a glutathione-independent, H2O2-inducible resistance to N-ethylmaleimide.

J Bacteriol, 1986 Nov, 168(2), 1010 - 3
Partition functions of unit-copy plasmids can stabilize the maintenance of plasmid pBR322 at low copy number; Austin S et al.; The maintenance of plasmid pBR322 is highly unstable in a polA12 strain of Escherichia coli at 29 degrees C due to severely reduced copy number . Under these conditions, introduction of the par (partition) locus of plasmid P1 or the par (sop) region of F into pBR322 stabilizes it . A region with similar activity was detected in the P7 plasmid . The activity of the P1 par locus was dependent on the P1 parA gene product and was sensitive to par-specified incompatibility.

J Bacteriol, 1986 Nov, 168(2), 1002 - 4
Purification of nitrogen regulator II, the product of the glnL (ntrB) gene of Escherichia coli; Ninfa AJ et al.; We purified the product of glnL (ntrB), NRII, and the product of a mutant glnL allele, NRII2302 . In vitro transcription of the nitrogen-regulated promoter glnAp2 by purified components of Escherichia coli required NRII or NRII2302 when the template DNA was linear.

Biochem Pharmacol, 1986 Nov 1, 35(21), 3853 - 5
Inhibition of uridine phosphorylase from Escherichia coli by benzylacyclouridines; Park KS et al.; The benzylacyclouridines, potent and specific inhibitors of mammalian uridine phosphorylase, were also found to be inhibitors of uridine phosphorylase but not thymidine phosphorylase from Escherichia coli . Competitive inhibition was observed in all cases and the most potent of these compounds was HM-BBAU (5-(3-benzyloxybenzyl)-1-{(2'-hydroxy-1'-hydroxymethyl)methyl}urac il) with a Ki value of 0.15 microM . The inhibitory potencies of these compounds parallel those obtained with enzymes from mammalian sources {Niedzwicki et al., Biochem . Pharmac . 31, 1857 (1982) and Naguib et al., manuscript in preparation} indicating that the structure of the active site of uridine phosphorylase from E . coli may resemble that of the mammalian enzyme.

Am Rev Respir Dis, 1986 Nov, 134(5), 885 - 90
The effects of prostaglandin E1 on the adult respiratory distress syndrome in septic primates; Brockmann DC et al.; The effects of prostaglandin E1 (PGE1) on the adult respiratory distress syndrome were studied in the septic primate (Macaca fascicularis) . A 30-min infusion of Escherichia coli (1 X 10(10)/kg) resulted in severe septic shock and adult respiratory distress syndrome . Primates, if living, were killed 4 h after completion of the E . coli infusion . Three groups of primates were studied (n = 4 in each group) . The control group (Group 1) received PGE1 at 100 ng/kg/min throughout the experiment . The septic group (Group 2) received a 30-min infusion of E . coli . The treatment group (Group 3) received a continuous PGE1 infusion (100 ng/kg/min) along with the E . coli infusion which was begun 30 min after the PGE1 infusion was started . Control primates had hemodynamic changes consistent with the vasodilatory effect of PGE1 (heart rate and cardiac output increased; blood pressure and systemic vascular resistance (SVR) decreased) . All control animals survived the experiment and had no evidence of pulmonary damage . Primates given E . coli developed severe hypotension, decreased SVR, and lung injury evidenced by pulmonary edema, decreased oxygenation, and increased extravascular lung water . Primates treated with both PGE1 and E . coli developed similar cardiovascular and pulmonary changes as the septic group . There was no statistically significant difference between Group 2 and Group 3 animals with regard to mean arterial blood pressure, SVR, extravascular lung water, alveolar-arterial oxygen difference, or survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Virology, 1986 Nov, 155(1), 89 - 96
Precore sequence of hepatitis B virus inducing e antigen and membrane association of the viral core protein; Uy A et al.; Hepatitis B virus (HBV) DNA contains a precore (pre-c) sequence of 29 codons with unknown function upstream of its gene for the major core protein . Its significance was studied by expression of core proteins with and without pre-c in Escherichia coli . Core protein without pre-c, P22c, assembled spontaneously to core particles and formed core antigen . It had the same size and antigenicity as core particles from infected liver . Core protein with pre-c, P25e, instead formed membrane-associated e antigen (HBeAg) . The data suggest that pre-c functions as a signal peptide for the attachment of core protein P25e to cellular membranes . This hypothesis can explain the not yet understood relation between viremia and HbeAg and the protective role of anti-HBe antibody.

Urol Clin North Am, 1986 Nov, 13(4), 637 - 45
Pyelonephritis, cortical abscess, and perinephric abscess; Roberts JA; The causative agent in nonobstructive pyelonephritis has been shown most often to be P-fimbriated Escherichia coli, mainly because receptors for these fimbriae are found in the bladder, ureter, and the kidney tubules . Age and sex are factors leading to differences in the presentation of the disease, and early diagnosis followed by intensive therapy lessens the chance of renal damage . Renal abscess, perinephric abscess, and pyonephrosis all follow pyelonephritis in the host who is compromised by the presence of stone, obstruction, diabetes, or immunosuppression.

Surgery, 1986 Nov, 100(5), 876 - 83
The pulmonary and systemic response to recurrent endotoxemia in the adult sheep; Demling RH et al.; The pulmonary and systemic hemodynamic effects of recurrent endotoxemia were studied in the adult sheep with lung lymph fistulas . Six sheep were given 1 mu/kg Escherichia coli endotoxin every 12 hours for 5 days, after which animals were monitored for another 3 days . The pulmonary response to the first three injections was characterized by an initial severe pulmonary hypertension, hypoxia, and a two- to threefold increase in lymph flow, QL . Lymph and plasma thromboxane A2 (TxB2) and prostacyclin (6-keto-PGF1 alpha) levels increased from baseline values of nearly 200 pg/ml to values exceeding 2000 pg/ml . The systemic response to initial doses was characterized by an increase in systemic vascular resistance, a decrease in cardiac index, and a transient 20% increase in oxygen consumption . With later endotoxin doses, the pulmonary response was markedly attenuated, with only modest changes in pulmonary artery pressure, lymph flow, and arterial oxygen tension noted . TxB2 increases were less than 800 pg/ml, and 6-keto-PGF1 alpha levels remained unchanged . However, we noted the progressive onset of a hyperdynamic state characterized by a sustained increase in cardiac index and body temperature, and a 50% increase in oxygen consumption, whereas systemic vascular resistance decreased by 45% . Three days after endotoxin injections were discontinued, the hyperdynamic state (including leukocytosis) was still present, whereas pulmonary variables returned to baseline levels . We conclude that a hyperdynamic state can be produced by repeated doses of endotoxin that will present even after the endotoxin insult is discontinued, which is a characteristic of the multisystem organ failure syndrome.

Proc Natl Acad Sci U S A, 1986 Nov, 83(22), 8506 - 10
Translation of a synthetic two-cistron mRNA in Escherichia coli; Schoner BE et al.; A synthetic two-cistron expression system was constructed for the high-level expression of eukaryotic genes in Escherichia coli . This system was designed to overcome translational inhibition of mRNAs containing eukaryotic sequences . The first cistron in this system is a 31-base A + T-rich synthetic sequence that provides for efficient translation initiation . The second cistron contains the protein coding sequence for the eukaryotic gene . Insertion of the first cistron between the 5' untranslated region of the mRNA and the protein coding region separates the two and thereby potentially minimizes the formation of local secondary structures that might prevent ribosomes from binding and initiating translation . The 31-base cistron contains three nonsense codons (TAA), one in each of the three translational reading frames, and an 8-base Shine-Dalgarno sequence that is complementary to the 3' end of the 16S rRNA . The effects of translation of the first cistron in all three reading frames on the expression of the second cistron was examined . The most efficient expression of the second cistron seemed to occur when the stop codon that terminates translation of the first cistron is located 3' to the Shine-Dalgarno sequence and close to the AUG start codon for the second cistron . When the Shine-Dalgarno sequence was deleted from the first cistron, no detectable expression of the second cistron was observed . This two-cistron system has been used to express the gene encoding methionylalanyl bovine growth hormone with its native codons and the gene encoding methionyl human growth hormone at a level greater than 20% of total cell protein . In the case of human growth hormone, we show that the amount of gene product is not significantly diminished by placing a "functional" first cistron in front of a gene that can be expressed without a cistron.

Proc Natl Acad Sci U S A, 1986 Nov, 83(22), 8447 - 51
beta-Glucuronidase from Escherichia coli as a gene-fusion marker; Jefferson RA et al.; We have developed a gene-fusion system based on the Escherichia coli beta-glucuronidase gene (uidA) . The uidA gene has been cloned from E . coli K-12 and its entire nucleotide sequence has been determined . beta-Glucuronidase has been purified to homogeneity and characterized . The enzyme has a subunit molecular weight of 68,200, is very stable, and is easily and sensitively assayed using commercially available substrates . We have constructed gene fusions of the E . coli lacZ promoter and coding region with the coding region of the uidA gene that show beta-glucuronidase activity under lac control . Plasmid vectors have been constructed to facilitate the transfer of the beta-glucuronidase coding region to heterologous control regions, using many different restriction endonuclease cleavage sites . There are several biological systems in which uidA-encoded beta-glucuronidase may be an attractive alternative or complement to previously described gene-fusion markers such as beta-galactosidase or chloramphenicol acetyltransferase.

Proc Natl Acad Sci U S A, 1986 Nov, 83(21), 8077 - 81
Action mechanism of ABC excision nuclease on a DNA substrate containing a psoralen crosslink at a defined position; Van Houten B et al.; Many carcinogenic as well as chemotherapeutic agents cause covalent linkages between complementary strands of DNA . If unrepaired, DNA crosslinks are blocks to DNA replication and transcription and therefore represent potentially lethal lesions to the cell . Genetic studies of Escherichia coli have demonstrated that the repair enzyme ABC excision nuclease, coded for by the three unlinked genes, uvrA, uvrB, and uvrC, plays a crucial role in DNA crosslink repair . To study the molecular events of ABC excision nuclease-mediated crosslink repair, we have engineered a DNA fragment with a psoralen-DNA interstrand crosslink at a defined position, digested this substrate with pure enzyme, and analyzed the reaction products on DNA sequencing gels . We find that the excision nuclease cuts only one of the two strands involved in the crosslink, incises the crosslink by hydrolyzing the ninth phosphodiester bond 5' and the third phosphodiester bond 3' to the furan-side thymine of the crosslink, and does not produce double-strand breaks at any significant level . Based on these data, we present a model by which ABC excision nuclease initiates crosslink repair in vivo.

Lab Invest, 1986 Nov, 55(5), 580 - 7
Relationship between increased vascular permeability and extravascular albumin clearance in rabbit inflammatory responses induced with Escherichia coli; Hamilton SM et al.; Intradermal injections of killed Escherichia coli are known to cause a variety of pathophysiological changes in the microcirculation that facilitate the extravasation of plasma constituents into the interstitium . In an attempt to learn more of the factors that regulate the magnitude and duration of inflammatory edema, we have focused on the relationship between the extravasation of protein into the interstitium and the removal of extravascular protein from the lesion sites . Vascular permeability changes have been assessed by the local accumulation of systemically administered {131I} or {125I}-albumin and extravascular protein clearance measured by monitoring the disappearance of {125I}-albumin from the same sites . Radioactivity was quantitated with an external gamma-scintillation probe or by punching out the lesion sites in sacrificed animals and counting in a gamma-spectrometer . Scintillation probe measurements of the net accumulation of intravenously administered {125I}-albumin in E . coli-induced skin lesions revealed that the extravasation of albumin was greater than the clearance of protein from the same sites . Comparisons of the removal rates of albumin injected directly into the E . coli sites revealed that, despite increases in vascular permeability amounting to 170 to 700% of control values, the mobilization of deposited albumin was no greater than that from control tissues that received saline; in fact with high concentrations of E . coli (10(8) injected/site) the mobilization of protein from the lesions was significantly reduced . The systemic administration of 055:B5 endotoxin (0.3, 1.6, or 3.3 micrograms/kg) also suppressed the clearance of albumin from skin . In contrast to these results, 300 to 1500% increases in vascular permeability induced with other inflammatory stimuli including thermal injury, high concentrations of bovine serum albumin, or bradykinin, resulted in enhanced clearance of extravascular protein from lesion or injection sites . These experiments suggest that an inability to effectively mobilize extravascular protein from the inflammatory focus could be a major contributing factor in regulating edema in inflammatory reactions induced with E . coli and may possibly contribute to the edema associated with septicemia.

J Clin Microbiol, 1986 Nov, 24(5), 753 - 8
Blinded, two-laboratory comparative analysis of Escherichia coli heat-stable enterotoxin production by using monoclonal antibody enzyme-linked immunosorbent assay, radioimmunoassay, suckling mouse assay, and gene probes; Thompson MR et al.; Heat-stable enterotoxin (ST)-producing enterotoxigenic Escherichia coli (ETEC) can be identified by a variety of assays, including the suckling mouse assay (SMA), radioimmunoassay (RIA), polyclonal or monoclonal antibody enzyme-linked immunosorbent assay (ELISA), and DNA hybridization with STh and STp gene probes . To compare the sensitivity and reliability of these assays, 100 coded ETEC and non-ETEC isolates were blindly tested in two independent laboratories . SMA, RIA, and monoclonal ELISA were performed in Cincinnati, Ohio, while gene probe analysis was performed in Baltimore, Md . The method of storage of organisms had a profound effect on the stability of plasmids in certain strains . Hybridization experiments to determine the presence or absence of the enterotoxin gene showed that strains stored on Dorset egg medium at room temperature better retained their plasmids than strains stored frozen in skim milk . Forty-four of the 100 organisms obtained from the skim milk stock were found to produce STa in liquid culture by the RIA, SMA, and monoclonal ELISA (100% agreement) . However, 50 of 54 of the strains stored on Dorset egg medium which were originally classified as STa+ or ST+ LT+ (positive for both heat-stable and heat-labile {LT} enterotoxins) were found to produce STa and retain the plasmid by each of these assays . Three additional strains were found which harbored the plasmid but did not elaborate STa by any of the assays (3% discrepancy) . The monoclonal antibody ELISA appears to be highly reliable for determination of STa production by ETEC and can be easily scored visually even by untrained personnel . Furthermore, when this STa assay is coupled with a polyclonal antibody assay, it is possible to predict the genotype of STh- and STp-producing organisms.

Infect Immun, 1986 Nov, 54(2), 587 - 9
Production of type II heat-labile enterotoxin by Escherichia coli isolated from food and human feces; Guth BE et al.; Escherichia coli strains isolated in Sao Paulo, Brazil, from feces of patients with diarrhea and from food samples produced toxin(s) that was shown to be related both immunologically and genetically to the recently characterized type II heat-labile enterotoxin of E . coli . The new isolates of type II heat-labile enterotoxin-producing E . coli belonged to five different serotypes and did not represent a single clone.

EMBO J, 1986 Nov, 5(11), 3051 - 6
Subregions of a conserved part of the HIV gp41 transmembrane protein are differentially recognized by antibodies of infected individuals; Certa U et al.; A 240-bp DNA fragment encoding a peptide, designated ENV(80), homologous to a conserved part of the gp41 transmembrane glycoprotein of human immunodeficiency virus (HIV) was chemically synthesized and inserted into different plasmid expression vectors . Escherichia coli transformants containing these plasmid constructs produced upon induction high amounts of either an ENV(80) peptide of relative molecular mass (Mr) of 10,000 or the same ENV(80) peptide N-terminally fused to E . coli chloramphenicol acetyltransferase (CAT) or to mouse dihydrofolate reductase (DHFR) having Mr of 36,000 and 31,000 respectively . All polypeptides containing the ENV(80) sequences were strongly reactive with antibodies present in sera from AIDS virus-infected individuals, but not with control sera . The strategy of gene assembly allowed the expression of ENV(80) subfragments fused to DHFR . The serodiagnosis of 15 positive sera by Western blot analysis using these bacterially synthesized ENV(80) subfragments revealed the presence of several immunoreactive epitopes on the 80-amino acid polypeptide which were recognized differently by the various patients.

Proc Natl Acad Sci U S A, 1986 Nov, 83(22), 8516 - 20
GCN4 protein, a positive transcription factor in yeast, binds general control promoters at all 5' TGACTC 3' sequences; Arndt K et al.; The GCN4 gene is required for the general amino acid control derepression response in yeast . GCN4 protein protects a repeated sequence motif in the 5'-untranslated region of HIS4, HIS3, ILV1, and ILV2 genes subject to general control . At low concentrations of GCN4, only certain repeats in these genes are bound . The repeats differ slightly from the 5' TGACTC 3' consensus core sequence, and the selective binding of some sites at low GCN4 concentrations is related to the relative affinity of these sites to GCN4 . Using purified GCN4 protein obtained from an overproducing strain of Escherichia coli, we were able to obtain complete protection of all of the repeat elements in these four genes at high GCN4 concentrations . Analysis of the relative binding constant to the 15 repeated sequences protected by GCN4 shows that the optimal binding site for GCN4 is 5' RRTGACTC 3' followed by a short stretch of thymidines . Another protein, present mostly in yeast nuclear extracts, binds to the HIS4 promoter at a site overlapping one of the GCN4 binding sites . This protein is displaced from its binding site at high GCN4 concentrations.

Proc Natl Acad Sci U S A, 1986 Nov, 83(21), 8273 - 7
Restricted ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum cells; Bredberg A et al.; A shuttle vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in cultured skin cells from a patient with the skin-cancer-prone, DNA repair-deficient disease xeroderma pigmentosum and in repair-proficient cells . After replication in the human cells, progeny plasmids were purified . Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of Escherichia coli carrying a suppressible amber mutation in the beta-galactosidase gene . Plasmid survival in the xeroderma pigmentosum cells was less than that of pZ189 harvested from repair-proficient human cells . The point-mutation frequency in the 150-base-pair tRNA marker gene increased up to 100-fold with ultraviolet dose . Sequence analysis of 150 mutant plasmids revealed that mutations were infrequent at potential thymine-thymine dimer sites . Ninety-three percent of the mutant plasmids from the xeroderma pigmentosum cells showed G X C----A X T transitions, compared to 73% in the normal cells (P less than 0.002) . There were significantly fewer transversions (P less than 0.002) (especially G X C----T X A) and multiple base substitutions (P less than 0.00001) than when pZ189 was passaged in repair-proficient cells . The subset of mutational changes that are common to ultraviolet-treated plasmids propagated in both repair-proficient and xeroderma pigmentosum skin cells may be associated with the development of ultraviolet-induced skin cancer in humans.

Proteins, 1986 Nov, 1(3), 230 - 8
The DNA replication inhibitor microcin B17 is a forty-three-amino-acid protein containing sixty percent glycine; Davagnino J et al.; Microcin B17 is a low-molecular-weight protein that inhibits DNA replication in a number of enteric bacteria . It is produced by bacterial strains which harbor a 70-kilobase plasmid called pMccB17 . Four plasmid genes (named mcbABCD) are required for its production . The product of the mcbA gene was identified by labelling minicells . The mcbA gene product was slightly larger when a mutation in any of the other three production genes was present . This indicates that these genes are involved in processing the primary mcbA product to yield the active molecule . The mcbA gene product predicted from the nucleotide sequence has 69 amino acids including 28 glycine residues . Microcin B17 was extracted from the cells by boiling in 100 mM acetic acid, 1 mM EDTA, and purified to homogeneity in a single step by high-performance liquid chromatography through a C18 column . The N-terminal amino acid sequence and amino acid composition demonstrated that mcbA is the structural gene for microcin B17 . The active molecule is a processed product lacking the first 26 N-terminal residues . The 43 remaining residues include 26 glycines . While microcin B17 is an exported protein, the cleaved N-terminal peptide does not have the characteristic properties of a "signal sequence", which suggests that it is secreted by a mechanism different from that used by most secreted proteins of E . coli.

J Gen Microbiol, 1986 Nov, 132 ( Pt 11), 3231 - 4
Nutritional variation in Escherichia coli; Olukoya DK; Nutritional tests were carried out on 62 strains of Escherichia coli as part of a study on the genetic basis of natural nutritional variation . The ability of these strains to utilize 84 compounds as carbon, nitrogen and carbon plus nitrogen sources was tested using an auxanographic method . The tests revealed polymorphic characters which are suitable for genetic analysis . Very few of these strains grew on the amino acids classified as 'essential' for humans.

J Gen Microbiol, 1986 Nov, 132 ( Pt 11), 3187 - 93
The phosphonium ion efflux system of Escherichia coli: relationship to the ethidium efflux system and energetic studies; Midgley M; The extent of accumulation of methyltriphenylphosphonium ion by Escherichia coli was shown to be dependent on the permeability of the outer membrane and the activity of an efflux system for this compound . Evidence consistent with the operation of a single efflux system for compounds such as phosphonium ions, phenanthridiniums and flavines is presented . Studies on the energy coupling mechanism for this efflux system indicated that it was driven by the transmembrane proton electrochemical gradient.

Can J Microbiol, 1986 Nov, 32(11), 842 - 6
An estimate of the extent of deamination of L-serine in auxotrophs of Escherichia coli K-12; Ramotar D et al.; We have shown that serine-glycine auxotrophs of Escherichia coli K-12 use exogenous L-serine inefficiently as a source of biosynthetic intermediates . Much of the L-serine supplied in the medium is not used to satisfy the auxotrophic requirement, owing to its diversion by L-serine deaminase, presumably to pyruvate . This is the first proof that the activity known as L-serine deaminase actually deaminates L-serine in vivo.

Mol Gen Genet, 1986 Nov, 205(2), 285 - 90
Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives; Lopilato J et al.; We describe mutants of Escherichia coli that decrease the plasmid copy number of pBR322 derivatives . One mutant was partially characterized genetically and its mutation, designated pcnB for plasmid copy number, was mapped to approximately 3 min on the E . coli chromosome . This locus is distinct from other genes whose products are known to affect plasmid replication or stable plasmid maintenance . The pcnB mutant strain should be useful for cloning genes into pBR322 that have aberrant or deleterious effects on the cell when present in high copy number.

Genetika, 1986 Nov, 22(11), 2649 - 57
{Inversion of the metE-oriC-rpsE chromosome segment in Escherichia coli K12}; Kulakauskas ST et al.; We have described recently a large inversion of the Escherichia coli chromosome (designated udpPf1), including region of the chromosomal replication region (oriC) . The udpPf1 inversion was induced by Tn10 transposon (metE::Tn10) . It results in increased expression of the uridine phosphorylase gene (udp) which is closely linked to the metE gene . The data of conjugational and transductional experiments presented in this report demonstrate that the udpPf1 inversion covers a chromosomal segment extending over 12 min of the E . coli genetic map and including the rpsE, crp and metE::Tn5 markers . The results are presented indicating that the increased uridine phosphorylase activity is due to fusion of the udp gene to a more strong promoter located, probably, in the operon for ribosomal proteins cluster, near 73 min on the E . coli chromosome.

Mol Biochem Parasitol, 1986 Nov, 21(2), 179 - 88
Sequence and expression of a major egg antigen from Schistosoma mansoni . Homologies to heat shock proteins and alpha-crystallins; Nene V et al.; One of the major proteins of eggs and miracidia (p40) of Schistosoma mansoni has an apparent molecular weight of 40,000 and elicits a strong immune response in over 90% of patients . The antigen consists of a family of at least four near identical proteins, probably encoded by a multi-gene family, and expression of the p40 polypeptides is differentially regulated around the parasite's life cycle . We have isolated and sequenced cDNA clones encoding two variants of the antigen and expressed one p40 clone in Escherichia coli . The fusion protein elicits antibodies which immunoprecipitate p40 and recognise antigens of identical sizes in S . haematobium and S . bovis . The open reading frame encoding this antigen specifies a protein which shares a block of sequence homology with alpha-crystallins and Drosophila small heat shock proteins.

J Bacteriol, 1986 Nov, 168(2), 936 - 9
Induction of SOS functions by alkaline intracellular pH in Escherichia coli; Schuldiner S et al.; Alkalinization of intracellular pH (pHi) causes an increase in UV resistance in wild-type and pH-sensitive mutant (DZ3) cells of Escherichia coli . Utilizing cells transformed with a plasmid (pA7) which bears the uvrA promoter fused to galK galactokinase structural gene, it was shown that alkaline pHi leads to an increase in the specific activity of galactokinase . This effect was not displayed in a mutant bearing a recA-insensitive lexA gene, nor in cells harboring a plasmid (pA8) in which the galK is fused to a lexA-insensitive uvrA promoter . Hence, the effects of pHi on cells functions may involve the lexA product of the SOS system.

Proc Natl Acad Sci U S A, 1986 Nov, 83(22), 8703 - 7
Mr 26,000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase; Smith DB et al.; Mice of the inbred strain 129/J bred at this Institute (WEHI 129/J) are relatively resistant to chronic infection with the parasitic helminth Schistosoma japonicum . In contrast to more permissive mouse strains such as BALB/c, the WEHI 129/J mice are high responders to a Mr 26,000 adult worm antigen designated Sj26 . Cloned cDNAs corresponding to Sj26 have been identified in a S . japonicum phage lambda gt11 amp3 expression library, and their nucleotide sequences have been deduced . The predicted amino acid sequence of the antigen specified by these cDNAs shows striking homology with class mu isozymes of mammalian glutathione S-transferases (RX:glutathione R-transferase, EC 2.5.1.18) . Extracts of adult worms contain glutathione S-transferase activity, and affinity chromatography of enzyme activity on glutathione columns leads to the purification of a Mr 26,000 molecule that comigrates with Sj26 . Although vaccination studies in mice with a beta-galactosidase-Sj26 fusion protein from Escherichia coli are encouraging, more immunogenic preparations of the antigen are likely to be required to establish the utility of Sj26 as a model vaccine.

Proc Natl Acad Sci U S A, 1986 Nov, 83(22), 8604 - 8
Photocrosslinking of the signal sequence of nascent preprolactin to the 54-kilodalton polypeptide of the signal recognition particle; Krieg UC et al.; Photoreactive moieties were incorporated into nascent polypeptides in a wheat germ protein-synthesizing system by using a plasmid-derived preprolactin mRNA and a Lys-tRNA analog, N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA (epsilon ANB-Lys-tRNA) . The presence of the abnormally large amino acid side chains in the nascent chains did not impair function: complete preprolactin chains were synthesized in the absence of the signal recognition particle (SRP), elongation was arrested in the presence of SRP, and SRP-dependent translocation across the membrane of the endoplasmic reticulum and signal peptidase cleavage were observed in the presence of salt-extracted microsomes . Photolysis of elongation-arrested ribosomes resulted in several light- and epsilon ANB-Lys-tRNA-dependent crosslinks . By using antibodies specific for each of the proteins, one covalent complex was shown to be a photocrosslink between the preprolactin nascent chain and the 54-kDa protein subunit of SRP . This demonstrates that the N-terminal end of a secretory protein is located adjacent to the SRP in elongation-arrested ribosomes and strongly suggests that the signal sequence is recognized by and binds to the 54-kDa subunit of SRP . The other photocrosslinks involve as-yet-unidentified proteins in the large ribosomal subunit, indicating that this method of incorporating probes provides a powerful approach to examining the environment and interactions of the nascent chain during translation and translocation across the membrane of the endoplasmic reticulum . The Lys-tRNA analog also successfully photoaffinity-labeled the Escherichia coli elongation factor Tu (EF-Tu) in the epsilon ANB-Lys-tRNA.EF-Tu.GTP ternary complex.

Proc Natl Acad Sci U S A, 1986 Nov, 83(21), 8258 - 62
Genetic construction, expression, and melanoma-selective cytotoxicity of a diphtheria toxin-related alpha-melanocyte-stimulating hormone fusion protein; Murphy JR et al.; The structural gene for diphtheria toxin, tox, has been modified at its Sph I site by the introduction of an oligonucleotide linker encoding a unique Pst I restriction endonuclease site and a synthetic oligonucleotide encoding alpha-melanocyte-stimulating hormone (alpha-MSH) . The resulting fusion gene directs the expression of a diphtheria toxin-related alpha-MSH hybrid protein in which the diphtheria toxin receptor-binding domain has been replaced with alpha-MSH sequences . The chimeric toxin has been partially purified from periplasmic extracts of recombinant Escherichia coli K-12 and has been found to be selectively toxic for alpha-MSH receptor-positive human malignant melanoma NEL-M1 cells in vitro.

J Gen Microbiol, 1986 Nov, 132 ( Pt 11), 3209 - 19
Mutations affecting gluconate catabolism in Escherichia coli . Genetic mapping of the locus for the thermosensitive gluconokinase; Isturiz T et al.; An Escherichia coli strain unable to use gluconate was isolated by spontaneous curing of lambda cI857 s7 xis6 b515 b519, lambda cI857 s7 delta(A-att) dargI valS lysogens . Two lesions, linked to asd and pyrB markers, respectively, were necessary to produce this phenotype . The asd-linked mutation gnt-17, of regulatory type, seems to affect the expression of the major system of gluconate utilization (min 75) as well as that of 6-phosphogluconate dehydratase (gene edd, min 41), the first enzyme of the Entner-Doudoroff pathway . A closely linked suppressor of gnt-17 causes constitutivity of these activities; this suppressor resembles gntR, which is also in the asd region . Hence, it is possible that gnt-17 is a super-repressing allele of gntR, rather than a positive controlling element . Lesion gnt-17 alone does not prevent the utilization of gluconate; for this, the mutation gnt-18 at 96.9 min is also necessary . This mutation abolishes the thermosensitive gluconokinase activity and thus eliminates the subsidiary ability to catabolize gluconate . Accordingly, gnt-18 seems to be allelic with gntV, the locus postulated as being in the pyrB region specifying the thermosensitive gluconokinase.

Mol Gen Genet, 1986 Nov, 205(2), 380 - 2
Revised sequence of the nusA gene of Escherichia coli and identification of nusA11 (ts) and nusA1 mutations which cause changes in a hydrophobic amino acid cluster; Saito M et al.; The mutant nusA DNAs (nusA11 and nusA1) were sequenced . Single base substitutions caused by these mutations were found in the coding region of nusA . The nusA11 mutation, which is conditionally lethal, substituted Thr for the 181st Ala . Also, nusA1, which restricts lambda growth, substituted Ala for the 183rd Ser . These two positions were located in the same hydrophobic amino acid cluster . This cluster seemed to be an essential region in the functional domain of NusA . In the course of these experiments, several mistakes in the published nusA nucleotide sequence were found . These errors are revised in this article . The molecular weight of NusA is accordingly revised to 54,430.

Mol Gen Genet, 1986 Nov, 205(2), 358 - 65
Regulation of expression of the gene for vitamin B12 receptor cloned on a multicopy plasmid in Escherichia coli; Aufrere R et al.; The btuB gene of Escherichia coli codes for a protein (BtuB) located in the outer membrane . BtuB is the receptor for vitamin B12 (cyanocobalamin) . We have cloned the btuB gene into pUC8 using transposon Tn5 as the marker to first isolate several parts of the relevant DNA fragment from the specialized transducing phage lambda darg13 . After reconstitution of the gene, Tn5 was removed by selecting for spontaneous excision . The partial nucleotide sequence and transcriptional start of the btuB gene were determined . The BtuB+ plasmid allowed a large amplification of the synthesis of BtuB, resulting in a 65-fold increased level of vitamin B12 binding . The level of vitamin B12 binding was reduced by a factor of 22 when cells were grown in the presence of high concentrations of vitamin B12 . The regulation of the gene was studied in more detail by the use of a protein fusion between the extreme amino-terminus of BtuB and beta-galactosidase of E . coli . The kinetics of repression and derepression were consistent with the presence in the cells of a large amount of a regulatory molecule exhibiting an apparent Km for vitamin B12 of 3 microM.

Mol Gen Genet, 1986 Nov, 205(2), 339 - 48
Mutagenesis by random linker insertion into the lamB gene of Escherichia coli K12; Boulain JC et al.; Gene lamB encodes an outer membrane protein involved in maltose and maltodextrin transport as well as phage adsorption . The active form is a trimer . We characterized 11 mutations in lamB, obtained after random insertion of a BamH1 linker and screening for stable immunodetectable mutant proteins . Six mutations resulted in the loss of the distal part of the LamB protein either by deletion (five cases) or frameshift (one case) . The six corresponding proteins had all lost the ability to confer phage sensitivity and the capacity to grow on dextrins, and to yield immunodetectable oligomers . Induction of a high level of the four longest of these proteins was toxic to the cell . Five other mutations were due to in-frame insertions . In four cases, the corresponding proteins still had the ability to yield immunodetectable oligomers, to confer phage sensitivity and the capacity to grow on dextrins and were not toxic on induction . In one case (AJC73), the mutant protein had lost the first three properties and was toxic on induction . Deletions and duplications between some of the inserts were also constructed and studied . To account for our results we present a hypothetical scheme in which trimerization would not only be needed for phage sensitivity and growth on dextrins but also for proper insertion into the outer membrane . The C-terminus of the protein, as well as other regions such as the site of mutation AJC73, would be required for the formation of stable trimers . We tentatively interpret toxicity as due to improper insertion into the outer membrane . Our results also show that it is possible to insert several amino acids (up to 11 in one case) at a number of positions in LamB without appreciably affecting its export and activities.

Mol Gen Genet, 1986 Nov, 205(2), 298 - 304
The recQ gene of Escherichia coli K12: primary structure and evidence for SOS regulation; Irino N et al.; A 2,695 bp chromosomal segment of Escherichia coli K12 containing the recQ gene was sequenced . Analysis of the sequence revealed an open reading frame thought to represent recQ, with a clockwise direction of transcription relative to the standard genetic map of E . coli K12 and having a coding capacity for a protein of Mr 68,350 . The -10 region of the presumptive recQ promoter overlapped the putative terminator for the upstream gene pldA, and was immediately followed by a 15 bp stretch of DNA bearing a strong resemblance to the reported sequences of LexA repressor binding sites . This latter finding suggested the possibility of SOS regulation of recQ gene expression, which was substantiated by experiments with recQ-lacZ fusions.

Mol Gen Genet, 1986 Nov, 205(2), 260 - 9
Cloning and complete nucleotide sequence of the Escherichia coli glutamine permease operon (glnHPQ); Nohno T et al.; The glutamine permease operon encoding the high-affinity transport system of glutamine in Escherichia coli could be cloned in one of the mini F plasmids, but not in pBR322 or pACYC184, by selection for restoration of the Gln+ phenotype, the ability to utilize glutamine as a sole carbon source . We determined the nucleotide sequence of the glutamine permease operon, which contains the structural gene of the periplasmic glutamine-binding protein (glnH), and indispensable component of the permease activity . The N-terminal amino acid sequence and the overall amino acid composition of the purified glutamine-binding protein were in good agreement with those predicted from the nucleotide sequence, if the N-terminal 22 amino acid residues were discounted . The latter comprised two Lys residues (nos . 2 and 6) followed by 16 hydrophobic amino acid residues and was assumed to be a signal peptide for transport into the periplasmic space . There were two additional reading frames (glnP and glnQ) downstream of glnH sharing a common promoter . It was concluded that the glnP and glnQ proteins as well as the glnH protein are essential for glutamine permease activity.

Mol Gen Genet, 1986 Nov, 205(2), 234 - 9
Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli; Blanco M et al.; Two multicopy plasmids carrying either the umuDC or the mucAB operon were used to compare the efficiency of UmuDC and MucAB proteins in UV mutagenesis of Escherichia coli K12 . It was found that in recA+ uvr+ bacteria, plasmid pIC80, mucAB+ mediated UV mutagenesis more efficiently than did plasmid pSE117, umuDC+ . A similar result was obtained in lexA41 (Def) cells, excluding the possibility that this was due to a differential regulation by LexA of the umuDC and mucAB operons . We conclude that some structural characteristic of the UmuDC and MucAB proteins determines their different efficiency in UV mutagenesis . This characteristic could be also responsible for the observation that in the recA430 mutant, pIC80 but no pSE117 can mediate UV mutagenesis . In the recA142 mutant, pIC80 also promoted UV mutagenesis more efficiently than pSE117 . In this mutant, the recombination proficiency, the protease activity toward LexA and the mutation frequency were increased by the presence of adenine in the medium . In recA+ uvrB5 bacteria, plasmid pSE117, umuDC caused both an increase in UV sensitivity as well as a reduction in the mutation frequency . These negative effects resulting from the overproduction of UmuDC proteins were higher in recA142 uvrB5 than in recA+ uvrB5 cells . In contrast, overproduction of MucAB protein in excision-deficient bacteria containing pIC80 led to a large increase in the mutation frequency . We suggest that the functional differences between UmuDC and MucAB proteins might be due to their different dependence on the direct role of RecA protease in UV mutagenesis.

J Biochem Biophys Methods, 1986 Nov, 13(4-5), 251 - 7
New type of linker useful for cloning experiments; Koster H et al.; A new class of linker oligodeoxynucleotide sequences is described which allows the original sequence of a foreign DNA to be restored after cloning . In the standard linker approach the terminal base pairs of the linker-sequences are irreversibly attached to the cloned DNA, thus altering the genetic information . The use of the new type of linker is demonstrated by the transformation of the unique Pvu II site in pBR322 into Bam HI site using the ISO ('in-site-out') linker d(GATCCGGATC) . Possible further applications of these linker sequences are described.

Genetika, 1986 Nov, 22(11), 2713 - 20
{Cloning of genes for proline biosynthesis in Escherichia coli}; Nersisian AA et al.; Restriction map of Escherichia coli chromosome fragment (7.4 MD) carrying proAB genes was constructed . Localization of proA and proB genes on the cloned chromosome fragment was determined by complementation test and the measuring of glutamylkinase activity (proB gene product) . ProA and proB genes were cloned separately on multicopy plasmids of alternative orientation and their expression being, probably, under the control of their own regulatory regions, studied.

Virus Res, 1986 Nov, 6(2), 141 - 54
Duplication of a viral enhancer sequence improves the stability of a vector based on BPV-1 DNA; Allshire RC et al.; Various recombinant constructions involving bovine papillomavirus type 1 (BPV-1) DNA and bacterial plasmids have been tested for their ability to transform mouse C127 cells and replicate as intact extrachromosomal monomeric structures . When BPV-1 DNA was linked to pBR328, pAT153 or derivatives of these plasmids lacking the 344 bp HindIII-BamHI fragment or another small segment, the resulting vectors replicated in C127 cells as high molecular weight structures and, in some cases, deleted extrachromosomal forms . The sequences which became deleted were generally the non-BPV-1 sequences . Duplication of the 3' distal enhancer sequence of BPV-1 DNA in one of the vectors increased its stability upon introduction into C127 cells, but some deleted and high molecular weight forms were still observed.

EMBO J, 1986 Nov, 5(11), 2825 - 30
Mapping the collagen-binding site of human fibronectin by expression in Escherichia coli; Owens RJ et al.; The collagen-binding domain of human fibronectin has been expressed as a cro/beta-galactosidase fusion protein in Escherichia coli . The hybrid polypeptide was recognized by an anti-(human plasma fibronectin) serum and bound specifically to gelatin-Sepharose . The collagen-binding region was subdivided by constructing a series of overlapping bacterial expression plasmids . The fusion proteins produced by these constructs were analysed for gelatin-binding activity . The results indicate that the binding site lies within an approximately 12.5 kd fragment of fibronectin, and show that the following 14 amino acid sequence is critical for gelatin-binding activity: Ala-Ala-His-Glu-Glu-Ile-Cys-Thr-Thr-Asn-Glu-Gly-Val-Met . This sequence links the second type II homology unit with the adjacent type I repeat in the amino-terminal third of the fibronectin molecule.

Carbohydr Res, 1986 Nov 1, 155, 119 - 29
Synthesis of 5-amino-5-deoxy-D-galactopyranose and 1,5-dideoxy-1,5-imino-D-galactitol, and their inhibition of alpha- and beta-D-galactosidases; Legler G et al.; A 12-step route is presented starting from 1,2:5,6-di-O-isopropylidene-alpha-D-glucofuranose for the preparation of the title compounds and their L-altro analogues . Their synthesis is based on the reduction with Raney nickel of a protected 5-hydroxyimino derivative of L-arabino-hexofuranos-5-ulose, with the following improvements for the preparation of a D-galactofuranose derivative: oxidation at C-3 with pyridinium dichromate-acetic anhydride, stereospecific reduction of a 3-O-acetyl-hex-3-enofuranose intermediate to the D-gulo derivative, and inversion at C-3 of its 3-tosylate with tetrabutylammonium acetate in chlorobenzene . alpha-D-Galactosidase from coffee beans and from Escherichia coli and beta-D-galactosidase from E . coli and Aspergillus wentii were inhibited with Ki values that ranged from 0.0007 to 8.2 microM . Formation of the enzyme-inhibitor complexes with the D-galactose analogue was on the time-scale of minutes, whereas the D-galactitol analogue showed a slow approach to the inhibition only with alpha-D-galactosidase from coffee beans and beta-D-galactosidase from A . wentii . N-Alkylation of the D-galactitol analogue was detrimental to the inhibition except for beta-D-galactosidase from E . coli and beta-D-glucosidase from almonds, but, even with these enzymes, the observed affinity enhancements were 10(2) to 10(3)-times smaller than those of N-alkylated D-galactosylamine and D-glucosylamine.

Am J Vet Res, 1986 Nov, 47(11), 2441 - 4
Detection of transmissible gastroenteritis coronavirus antigens by a sandwich enzyme-linked immunosorbent assay technique; Bernard S et al.; A new sandwich enzyme-linked immunosorbent assay, using monoclonal and polyclonal antibodies, was developed to detect transmissible gastroenteritis virus antigens from cell culture and from intestinal wash or feces obtained from experimentally infected pigs . This technique was shown to be suitable for the detection of virulent field strain unadapted to cell culture . Cross reactions had not been observed with other enteric pathogens, rotavirus, porcine epizootic diarrhea virus, and Escherichia coli.

Radiat Res, 1986 Nov, 108(2), 189 - 95
Induction of L-arabinose isomerase in gamma-irradiated Escherichia coli; Chatterjee A et al.; Gamma irradiation of Escherichia coli B/r caused a dose-dependent inhibition of the capacity of the cells to synthesize L-arabinose isomerase in response to the inducer . At higher doses (18 krad and above), postirradiation incubation led to further inhibition of the capacity to synthesize L-arabinose isomerase, whereas cells receiving lower doses recovered from the damage to the enzyme synthesizing system following incubation . Cyclic AMP partially reversed the inhibitory effect on L-arabinose isomerase induction produced immediately after irradiation by all gamma-ray doses (up to 30 krad), but the enhanced inhibitory effect caused by induction in cells irradiated at higher doses could not be reversed by the nucleotide . It is suggested that although catabolite repression is partly responsible for causing the inhibition of the enzyme synthesizing capacity of the cells observed immediately after gamma irradiation, the enhanced inhibition caused by incubating cells irradiated at higher doses is not due to interference with the control mechanism regulated by catabolite repression.

J Bacteriol, 1986 Nov, 168(2), 999 - 1001
Dominance relationships among mutant alleles of regulatory gene araC in the Escherichia coli B/R L-arabinose operon; Sheppard DE; The araBAD operon of Escherichia coli B/r is positively and negatively regulated by the araC+ regulatory protein . Mutations in gene araC can result in a variety of different regulatory phenotypes: araC null mutants (those carrying a null allele exhibiting no repressor or activator activity) are unable to achieve operon induction; araC-constitutive (araCc) mutants are partially constitutive, inducible by D-fucose, and resistant to catabolite repression; araCh mutants are hypersensitive to catabolite repression; and araCi mutants are resistant to catabolite repression . Various mutant alleles of gene araC were cloned into a derivative of plasmid pBR322 by in vivo recombination . Various heterozygous araC allelic combinations were constructed by transformation . Analysis of isomerase (araA) specific activity levels under various growth conditions indicated the following dominance relationships with regard to sensitivity to catabolite repression: araCh greater than araC+ greater than (araCc and araCi) greater than araC . It was concluded that the araCh protein may form a repressor complex that is refractory to removal by cyclic AMP receptor protein-cyclic AMP complex . This was interpreted in terms of the known nucleoprotein interactions between ara regulatory proteins and ara regulatory DNA.

J Bacteriol, 1986 Nov, 168(2), 940 - 6
recA (Srf) suppression of recF deficiency in the postreplication repair of UV-irradiated Escherichia coli K-12; Wang TC et al.; The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells . The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter-strand gaps . In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells . The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis . These results are consistent with the suggestion (M . R . Volkert and M . A . Hartke, J . Bacteriol . 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein.

J Bacteriol, 1986 Nov, 168(2), 931 - 5
Escherichia coli DnaK protein possesses a 5'-nucleotidase activity that is inhibited by AppppA; Bochner BR et al.; AppppA and the DnaK protein have both been hypothesized to function in regulating the heat shock response of Escherichia coli . The proposals are that AppppA serves as a signal (alarmone) to turn on the heat shock response, whereas the DnaK protein is necessary to turn off the heat shock response . A simple model would be that the DnaK protein turns off the response by degrading AppppA . We disproved this model by demonstrating that the DnaK protein possesses a 5'-nucleotidase activity capable of degrading many cellular nucleotides but not AppppA . Although AppppA was not a substrate, it did inhibit the 5'-nucleotidase activity of the DnaK protein . This inhibition may be specific and have biological function since the mutant DnaK756 protein, which is defective in turning off the heat shock response, is partially desensitized to AppppA inhibition . These findings led us to consider other possible mechanisms for AppppA and the DnaK protein in heat shock regulation.

J Bacteriol, 1986 Nov, 168(2), 795 - 8
Paraquat-mediated selection for mutations in the manganese-superoxide dismutase gene sodA; Bloch CA et al.; We report the unexpected result that Escherichia coli isolates containing a multicopy plasmid (pDT1.5) carrying the manganese-superoxide dismutase gene sodA were more sensitive than the wild type to paraquat-mediated growth inhibition . The pDT1.5 locus responsible for the paraquat-sensitive phenotype was delimited to a 0.6-kilobase segment by transposon Tn5 mutagenesis . Moreover, superoxide dismutase activity was the same as in the wild type in strains carrying pDT1.5::Tn5 insertions mapping to the 0.6-kilobase locus . These data identify the 0.6-kilobase segment as the locus of sodA and establish an association between growth inhibition by paraquat and the function of the plasmid-borne sodA gene.

Proc Natl Acad Sci U S A, 1986 Nov, 83(21), 8303 - 7
Structure-function analysis of murine interleukin 1: biologically active polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of a 270-amino acid precursor; DeChiara TM et al.; Murine interleukin 1 (IL-1) is initially synthesized as a 270-amino acid precursor protein . Guided by amino-terminal end sequence analyses of mouse macrophage-derived IL-1, it was shown that expression of the carboxyl-terminal 156 amino acids (i.e., amino acids 115-270) of this precursor in Escherichia coli yields biologically active recombinant IL-1 (rIL-1) protein . To answer questions about precursor processing and the size of the smallest biologically active IL-1 fragment, we have engineered deletions of the rIL-1 (115-270) gene to encode two amino-terminal deletion analogs, rIL-1 (131-270) and rIL-1 (144-270), and a carboxyl-terminal deletion analog, rIL-1 (131-257, 270) . The analogs were produced in E . coli, purified to homogeneity, and assayed for biological activity on murine thymocytes, human rheumatoid synovial cells, and human dermal fibroblasts and for their ability to bind to IL-1 receptors on murine EL-4 thymoma cells . The amino-terminal deletion analog rIL-1 (131-270) possessed a specific activity in the murine thymocyte proliferation assay equivalent to that of the 115-270 parent protein and exhibited significant biological activity in stimulating the production of collagenase and prostaglandin E2 by synovial cells and fibroblasts . The more extensive amino-terminal deletion analog rIL-1 (144-270) was inactive in all biological assays and failed to compete in the receptor binding assay . The carboxyl-terminal deletion analog rIL-1 (131-257, 270) competed less efficiently (by a factor of 100) in the receptor binding assay, retained weak biological activity on synovial cells and fibroblasts, and only demonstrated full intrinsic activity in the thymocyte proliferation assay when 100-200 times more protein was assayed . These results suggest that biologically active murine IL-1 polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of the 270-amino acid precursor . Furthermore, it appears that the integrity of the carboxyl terminus of the 270-amino acid precursor is important for activity but that different amino termini can be utilized to generate molecules with equivalent specific activities . This amino-terminal end flexibility supports a processing model for IL-1 maturation that partially explains IL-1 polypeptide heterogeneity.

Proc Natl Acad Sci U S A, 1986 Nov, 83(21), 8268 - 72
Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase; Farr SB et al.; Escherichia coli double mutants (sodA sodB) completely lacking superoxide dismutase (SOD) have greatly enhanced mutation rates during aerobic growth . Single mutants lacking manganese SOD (MnSOD) but possessing iron SOD (FeSOD) have a smaller increase, and single mutants lacking FeSOD but possessing MnSOD do not show such an increase . The enhancement of mutagenesis is completely dependent on the presence of oxygen, and treatments that increase the flux of superoxide radicals produce even higher levels of mutagenesis . The presence of a plasmid overproducing either form of SOD reduces the level of mutagenesis to that of wild type, showing that the O2-dependent enhancement results from a lack of SOD . The enhancement of mutagenesis is RecA-independent, and a complete lack of SOD does not induce the SOS response during aerobic growth . However, the enhanced mutagenesis in aerobically grown sodA sodB mutants is largely dependent on functional exonuclease III, suggesting that the increased flux of superoxide radicals results in DNA lesions that can be acted on by this enzyme, leading to mutations.

J Virol, 1986 Nov, 60(2), 782 - 6
Molecular cloning of the temperature-sensitive 371 Kirsten murine sarcoma virus and expression in Escherichia coli of the mutant and wild-type viral Kirsten ras p21 proteins; Stein RB et al.; Rodent fibroblasts infected with the ts371 Kirsten murine sarcoma virus (KiMuSV) are temperature sensitive for the maintenance of transformation because of the production of an abnormal p21 protein . We cloned the ts371 KiMuSV provirus from the genome of a conditionally transformed nonproducer cell line, ts371 KiMuSV NRK clone 5 (T . Y . Shih, M . O . Weeks, H . A . Young, and E . M . Scolnick, J . Virol . 31:546-556, 1979) . The molecularly cloned virus had 1,000-fold lower transformed focus-forming activity at 39 degrees C than at 34 degrees C . The ts371-v-Ki-ras gene differed from the wild type (wt) by a single point mutation, resulting in the substitution of arginine for glutamine at amino acid residue 43 of the encoded p21 . A second difference from the published sequence for wt v-Ki-ras (N . Tsuchida, T . Ryder, and E . Ohtsubo, Science 217:937-939, 1982) at amino acid residue 37 was found . However, on sequencing the wt v-Ki-ras in this region, we found that it also contained a glutamate at residue 37 . Preliminary characterization of bacterially expressed wt and ts371-v-Ki-ras p21 proteins is discussed.

J Infect Dis, 1986 Nov, 154(5), 778 - 83
Changes in the structural and functional properties of human eosinophils during experimental hookworm infection; White CJ et al.; Normal volunteers were infected with hookworm larvae Necator americanus . Peripheral blood counts showed a mean of 524 +/- 29 eosinophils/mm3 of blood before infection and a mean of 3,008 +/- 456 eosinophils/mm3 of blood during infection (P less than .01) . Absolute numbers of neutrophils did not change . Eosinophils and neutrophils from the infected period were compared with the noninfected state in each subject . The percentage of hypodense eosinophils increased from a mean of 34% +/- 13% to 80% +/- 7% during infection (P less than .05) . Superoxide production of eosinophils increased from a mean of 56 +/- 9 to 97 +/- 12 nmol of O2-./10(6) cells per 60 min (P less than .05) during infection . Chemotaxis of eosinophils to Escherichia coli endotoxin-activated serum increased from a mean average distance migrated of 19 +/- 2 micron (P less than .05), whereas neutrophil responsiveness did not change . This is the first report of changes in eosinophil density and stimulation of eosinophil function in normal hosts experimentally infected with hookworm . The data indicate that hookworm infection preferentially increases eosinophil production and activity with little effect on neutrophils.

Biochem J, 1986 Nov 1, 239(3), 699 - 704
Purification and characterization of 3-dehydroquinase from Escherichia coli; Chaudhuri S et al.; A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli . Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield . The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000 . The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric . The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.

Mol Gen Genet, 1986 Nov, 205(2), 225 - 33
Binding protein dependent transport of glycine betaine and its osmotic regulation in Escherichia coli K12; May G et al.; Glycine betaine, which functions as an osmoprotectant, is accumulated to high intracellular concentrations in Escherichia coli at high osmolarity . We demonstrate the presence of a high-affinity, binding protein dependent transport system for glycine betaine, which is encoded by the proU region . We show the osmotically regulated synthesis of a 32 kDa periplasmic protein that is a glycine betaine binding protein with a KD of 1.4 microM . ProU-mediated glycine betaine transport is osmotically stimulated at the level of gene expression . The osmolarity of the medium also regulates the activity of the transport system, while binding of glycine betaine to its binding protein is independent of the osmolarity . We also find a second glycine betaine transport system that is dependent on proP and exhibits a lower substrate affinity . Like ProU, this system is regulated at two levels: both gene expression and the activity of the transport system are osmotically stimulated . Using lambda plac Mu-generated lacZ operon and gene fusions, we find that expression of the proU region is osmotically regulated at the level of transcription . We cloned a part of the proU region together with the phi(proU-lacZ)hyb2 gene fusion into a multicopy plasmid and show that the DNA sequences required in cis for osmotic regulation are present on the plasmid.

J Med Virol, 1986 Nov, 20(3), 229 - 46
Expression of the X gene of hepatitis B virus; Pugh JC et al.; The hepatitis B virus (HBV) genome carries an open reading frame of 462 bases, the X region, but the corresponding protein has yet to be identified as a natural product . In rodent cells cotransformed with the thymidine kinase gene of herpes simplex virus and HBV DNA, however, Gough {1983} identified a mRNA that hybridises uniquely with the X region of the HBV genome . A large fragment of the X region was inserted into plasmid pCL19 delta Y-T in order to produce, in Escherichia coli, the X gene product, HBxAg, as a polypeptide fused to the N-terminal part of the phage lambda cro gene product . Antisera raised against this fused polypeptide gave positive immunofluorescence reactions with the transformed rodent cells . This provides direct evidence for the expression of the HBxAg gene in eukaryotic cells transformed with HBV DNA . The approach used here should be generally applicable.

J Bacteriol, 1986 Nov, 168(2), 648 - 54
Expression of the cloned ColE1 kil gene in normal and Kilr Escherichia coli; Altieri M et al.; The kil gene of the ColE1 plasmid was cloned under control of the lac promoter . Its expression under this promoter gave rise to the same pattern of bacterial cell damage and lethality as that which accompanies induction of the kil gene in the colicin operon by mitomycin C . This confirms that cell damage after induction is solely due to expression of kil and is independent of the cea or imm gene products . Escherichia coli derivatives resistant to the lethal effects of kil gene expression under either the normal or the lac promoter were isolated and found to fall into several classes, some of which were altered in sensitivity to agents that affect the bacterial envelope.

J Bacteriol, 1986 Nov, 168(2), 990 - 8
Nucleotide sequence of the tra YALE region from IncFV plasmid pED208; Finlay BB et al.; The pED208 plasmid is a 90-kilobase conjugative plasmid which is the derepressed form of Fo lac plasmid (IncFV) . A 3.3-kilobase HindIII-PstI fragment from the pED208 plasmid was cloned and sequenced and was found to contain four open reading frames which were highly homologous to the traA, traL, traE, and traY gene products of the F plasmid . The pED208 traA propilin protein was 119 amino acids in length, consisting of a leader sequence of 55 amino acids and a mature pilin subunit of 64 residues . The leader sequence contained a hydrophobic region followed by a classic signal peptidase cleavage site (Ala-Ser-Ala-55) . F and pED208 pilin proteins shared 27 conserved residues and had similar predicted secondary structures . The pED208 traA and traL genes were separated by a single base pair, and no ribosome binding site preceded the traL gene . The pED208 traY gene contained an IS2 insertion element in orientation II 180 nucleotides (60 residues) upstream of the traY stop codon . This insertion of IS2 resulted in a predicted fusion peptide of 69 residues for traY which may provide the observed traY activity . Since IS2 is absent in the wild-type plasmid, Fo lac, derepression and concomitant multipiliation may be due to the insertion of IS2 providing constitutive expression of the pED208 tra operon.

Infect Immun, 1986 Nov, 54(2), 549 - 54
Change in degree of type 1 piliation of Escherichia coli during experimental peritonitis in the mouse; Alkan ML et al.; To determine whether expression of type 1 pili varies during the course of Escherichia coli infection in vivo, mice were injected intraperitoneally with 5 X 10(7) CFU of piliated or nonpiliated phase variants per ml, and the degree of piliation was measured in peritoneal exudate by an enzyme-linked immunosorbent assay inhibition method . In the animals challenged with the piliated bacteria, the numbers of organisms increased a log over 9 h and the amount of pilus antigen decreased from 3 to 0.075 micrograms/10 bacteria . After a 4-h delay, nonpiliated bacteria also increased by one log over 9 h; however, the amount of piliation remained virtually undetectable . Piliated E . coli were more virulent than nonpiliated variants in this model (50% lethal dose of 7.5 X 10(6) versus 3 X 10(7), respectively) . The difference was significantly reduced by prior passive immunization with rabbit serum containing high titers of antipili antibody . Piliated bacteria adhered in significantly greater numbers to isolated mouse peritoneal membranes than did nonpiliated variants (15,400 +/- 2,700 versus 1,300 +/- 700 bacteria/mm2, respectively; P = 0.05) . Adherence was inhibited by the presence of 0.1 M alpha methyl mannose (1,500 +/- 1,800 bacteria/mm2, P = 0.01) . These results confirm the results of previous qualitative studies showing that phase variation of type 1 pili occurs in vivo and suggest that these pili may confer an initial advantage for growth of E . coli in the peritoneal cavity, presumably by fostering colonization of the peritoneal serosal surface.

Infect Immun, 1986 Nov, 54(2), 328 - 32
Localization of binding sites for purified Escherichia coli P fimbriae in the human kidney; Korhonen TK et al.; Binding sites in the human kidney for purified P fimbriae of pyelonephritogenic Escherichia coli were determined . The purified KS71A (F7(1)) fimbriae bound only to epithelial elements of the kidney, i.e., to the apical aspect of proximal and distal tubular cells, as well as to the apical and cytoplasmic sites of collecting ducts . In addition, binding was seen at the vascular endothelium throughout the kidney and at the parietal epithelium of the glomeruli . The binding was specifically inhibited by the receptor analog of E . coli P fimbriae, globotriose . The binding sites identified suggested a possible pathogenetic mechanism for the invasion of P-fimbriated bacteria into the renal parenchyma, as well as for their subsequent spread into the circulatory system.

Infect Immun, 1986 Nov, 54(2), 322 - 7
Binding of Escherichia coli S fimbriae to human kidney epithelium; Korhonen TK et al.; Purified S fimbriae and an Escherichia coli strain carrying the recombinant plasmid pANN801-4 that encodes S fimbriae were tested for adhesion to frozen sections of human kidney . The fimbriae and the bacteria bound to the same tissue domains, and in both cases the binding was specifically inhibited by the receptor analog of S fimbria, sialyl(alpha 2-3)lactose . S fimbriae bound specifically to the epithelial elements in the kidneys; to the epithelial cells of proximal and distal tubules as well as of the collecting ducts and to the visceral and parietal glomerular epithelium . In addition, they bound to the vascular endothelium of glomeruli and of the renal interstitium . No binding to connective tissue elements was observed . The results suggest that the biological function of S fimbriae is to mediate the adhesion of E . coli to human epithelial and vascular endothelial cells.

Proteins, 1986 Nov, 1(3), 263 - 6
Preliminary X-ray diffraction analysis of HhaII endonuclease-DNA cocrystals; Chandrasegaran S et al.; HhaII restriction endonuclease purified from an overproducing recombinant E . coli clone has been cocrystallized with a heptanucleotide duplex, d-GGAGTCC:GGACTCC . The cocrystals are monoclonic and belong to the space group C2 . The unit cell dimensions are a = 199.0 +/- 1.0 A, b = 100.0 +/- 0.5 A, c = 80.3 +/- 0.4 A, and beta = 101.0 +/- 1.0 degrees . There appear to be two dimers per asymmetric unit and the crystals diffract to 4-A resolution.

Acta Chem Scand B, 1986 Nov, 40(10), 817 - 25
Synthesis of an mRNA fragment of alanyl-tRNA synthetase gene in Escherichia coli using the 6-methyl-3-pyridyl group for protection of the imide functions of uridine and guanosine; Welch CJ et al.; The synthesis of 5'-GpGpUpGpU-3' is reported to demonstrate the synthetic use of the 6-methyl-3-pyridyl group for the protection of the O-4 and O-6 imide functions of uridine and guanosine, respectively . The 2'- and 5'-hydroxyl functions of the key intermediates were protected with two acid-labile groups: 3-methoxy-1,5-dicarbomethoxypentane-3-yl (MDMP) and 9-(4-octadecyloxyphenyl)xanthen-9-yl (C18Px), respectively . The internucleotide phosphotriesters were protected with 2-chlorophenyl and the 9-fluorenylmethyl group was employed for 3'-terminal phosphate protection.

Plasmid, 1986 Nov, 16(3), 204 - 12
Mathematical model for the control of ColE1 type plasmid replication; Ataai MM et al.; A mathematical model for the molecular events controlling replication of ColE1 type plasmids is described . All the model parameters can be evaluated independently . The model simulates plasmid replication and accurately predicts the copy-number of ColE1 plasmids carrying a variety of regulatory mutations . The model is used to test the plausibility of hypotheses concerning the interactions of regulatory elements involved in the replication apparatus . The model favorably supports the mechanism proposed by Tomizawa and co-workers concerning the nature of RNA-RNA interactions and that the Rom protein increases the binding between the two RNA species . The hypothesis that the interactions of RNA I-II increases the susceptibility of RNA II to the action of endonucleases is not a plausible mechanism.

Mol Gen Genet, 1986 Nov, 205(2), 248 - 52
Molecular cloning and characterization of the trpC gene from Penicillium chrysogenum; Sanchez F et al.; We cloned the Penicillium chrysogenum trpC gene from a genomic library by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity . The gene encodes a 2.7 kb poly(A)+ RNA . We localized the gene by sequence analysis in a 2.9 kb DNA insert found in the smallest plasmid selected from the library . Sequence data strongly suggest that the organization of the gene is similar to that described in other Ascomycetes . We found that a DNA fragment which codes only for the carboxy-terminal portion of the polypeptide is sufficient for complementation of the E . coli trpC9830 mutation.

J Bacteriol, 1986 Nov, 168(2), 728 - 33
Methylation-dependent transcription controls plasmid replication of the CloDF13 cop-1(Ts) mutant; van Putten AJ et al.; The CloDF13 cop-1(Ts) mutant expresses a temperature-dependent plasmid copy number . At 42 degrees C the mutant shows a "runaway" behavior, and cells harboring this plasmid are killed . The cop-1(Ts) mutation is a G-to-A transition that disturbs one of the two methylation sites which are located opposite in the stem-loop structure within a region involved in both the initiation of primer synthesis for DNA replication and the termination of the cloacin operon transcript . We demonstrate that the mutation results in an increased primer (RNA II) synthesis resulting from nonconditional enhanced RNA II promoter activity, which at 42 degrees C causes a decrease in the amount of active replication repressor molecules (RNA I) synthesized from the opposite strand . We found that the absence of Dam methylation abolishes the mutant phenotype and that under this condition the high mutant level of RNA II synthesis is reduced, which is accompanied by a restoration of the regulation by RNA I . The role of methylation in the regulation of plasmid replication is discussed.

J Virol, 1986 Nov, 60(2), 813 - 6
The L2 open reading frame of human papillomavirus type 1a encodes a minor structural protein carrying type-specific antigens; Komly CA et al.; The proteins encoded by the open reading frames of papillomavirus genomes and the minor polypeptides detected in purified virions are still poorly defined . We show here by its expression in Escherichia coli that the open reading frame L2 of human papillomavirus type 1a codes for a minor structural protein of Mr 76,000 . Antisera raised against a truncated L2-beta-galactosidase fusion protein in which the conserved N-terminal region of L2 is missing are type specific for human papillomavirus type 1 virions and are reactive at high dilutions . Expression of the L2-encoded type-specific antigens thus provides a powerful new tool for the identification of papillomaviruses.

Infect Immun, 1986 Nov, 54(2), 529 - 36
Variation in chemical properties and antigenic determinants among type II heat-labile enterotoxins of Escherichia coli; Guth BE et al.; Type II heat-labile enterotoxin (LT-II) from Escherichia coli 41 was purified and compared with prototype LT-II encoded by genes from E . coli SA53 . Both toxins were oligomeric proteins consisting of polypeptides A (Mr, 28,000) and B (Mr, 11,800) . The A polypeptides were cleaved by trypsin into fragments A1 (Mr, 21,000) and A2 (Mr, about 7,000) . These two toxins were shown to belong to two different subclasses of LT-II . We propose to designate the prototype toxin LT-IIa and the new variant LT-IIb . The pI of LT-IIb was between 5.2 and 5.6, significantly lower than the pI of 6.8 for LT-IIa, and the behavior of LT-IIb during purification differed significantly from that of LT-IIa . The toxic dose of unnicked LT-IIb in the Y1 adrenal-cell assay was 94 pg, but trypsin-treated, nicked LT-IIb was toxic at about 3 pg . In contrast, the toxic dose of LT-IIa was previously shown to be 0.5 to 1 pg for several preparations that varied from unnicked to partially nicked, and treatment with trypsin was not required for full toxicity . The titer of LT-II antiserum in neutralization tests was 100-fold greater against LT-IIa than against LT-IIb . In immunodiffusion tests, LT-IIa and LT-IIb gave a reaction of partial identity . In a radioimmunobinding assay, the titer of LT-IIa antiserum against homologous LT-IIa was approximately 10-fold greater than against LT-IIb . The cholera-E . coli family of heat-labile enterotoxins has been divided into serogroup I, which includes cholera toxin and the antigenic variants of E . coli heat-labile toxin designated LTh-I and LTp-I, and serogroup II, which includes LT-IIa and LT-IIb . The type I and type II toxins do not cross-react in neutralization or immunodiffusion tests . By using very sensitive radioimmunobinding assays, it was possible to demonstrate common antigenic determinants between the type I and type II toxins . However, the titers of antibodies in hyperimmune sera that recognized these common determinants were very low.

Circulation, 1986 Nov, 74(5), 1066 - 70
Coronary thrombolysis with recombinant single-chain urokinase-type plasminogen activator in patients with acute myocardial infarction; Van de Werf F et al.; Seventeen patients with acute transmural myocardial infarction and angiographically confirmed complete coronary occlusion were treated with heparin combined with intravenous single-chain urokinase-type plasminogen activator (scu-PA), obtained by expression of the cDNA encoding mature human scu-PA in Escherichia coli . In eight patients, recombinant scu-PA (rscu-PA) was given as a 10 mg bolus followed by 30 mg over 1 hr . Recanalization was obtained in six patients, but with persistent delayed opacification of the vessel in four of these patients . During infusion, a plateau level of rscu-PA antigen in plasma of 3.4 micrograms/ml (median value, range 1.4 to 5.5) was reached . At the end of the infusion the alpha 2-antiplasmin level had decreased to 54% (median, range 22% to 82%) of the preinfusion level, the fibrinogen level to 89% (median, range 26% to 101%), and fibrinogen degradation products (FDPs) to 20 micrograms/ml (median, range 8 to 387) . In nine patients, rscu-PA was administered as a 10 mg bolus followed by 60 mg over 1 hr . This resulted in recanalization with normal distal filling of the vessel in seven patients, within 46 +/- 17 min (mean +/- SD) . During infusion the concentration of rscu-PA in plasma increased to a median value of 7.4 micrograms/ml (range 4.0 to 13.3) . At the end of the infusion the alpha 2-antiplasmin level was 22% of baseline (range 5% to 47%), the fibrinogen level 45% (range 4% to 94%), and the concentration of FDPs 87 micrograms/ml (range 6 to 1034) . No significant bleeding or short-term side effects were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1986 Oct 30, 140(2), 602 - 8
Effect of vector type, host strains and transcription terminator on heterologous gene expression in yeast; Choo KB et al.; Using the surface antigen gene of the hepatitis B virus, and the promoter and terminator sequences of the yeast pho5 gene as a model system, a series of closely related expression plasmids were constructed to investigate the effect of vector type, genetic background of host strains and the presence of transcription terminator on the expression of heterologous gene in yeast . Plasmids carrying the replication origin of the 2 mu plasmids were found to be much more stable than those either independently or simultaneously carrying ars1 sequences . Gene expression was also higher with 2 micron-based plasmids . Yeast selection marker (trp1 or leu2) and therefore the host strains used did not have significant effects on gene expression . Addition of transcription terminator sequences downstream to the HBsAg gene also contributed only limited increases in gene expression levels.

Biochem Biophys Res Commun, 1986 Oct 30, 140(2), 508 - 14
Biosynthesis of reovirus-specified polypeptides . Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain bicistronic s1 mRNA which encodes the minor capsid polypeptide sigma 1a and the nonstructural polypeptide sigma 1bNS; Munemitsu SM et al.; Human reovirus serotype 1 Lang strain s1 mRNA, which encodes the minor capsid cell attachment protein sigma 1a and the nonstructural protein sigma 1bNS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13 . The Lang strain s1 mRNA is 1462 nucleotides in length and possesses two open reading frames . The first begins at nt 14 and has a coding capacity of 418 amino acids, sufficient to account for sigma 1a; the second begins at nt 75 and has a coding capacity of 119 amino acids, sufficient to account for sigma 1bNS . Comparison of the Lang serotype s1 sequence derived from cDNA clones of s1 mRNA with the Lang S1 sequence derived from cDNA clones of the S1 dsRNA genome segment definitively establishes that reovirus plus-strand mRNA is structurally equivalent to the plus-strand of the dsRNA genome segment.

FEBS Lett, 1986 Oct 27, 207(2), 205 - 12
3'-Hydroxymethyl 2'-deoxynucleoside 5'-triphosphates are inhibitors highly specific for reverse transcriptase; Kutateladze TV et al.; dNTP(3'-OCH3), a 3'-O-methyl derivative of dNTP, is a chain terminator substrate for DNA synthesis catalyzed by AMV reverse transcriptase . The enzyme seems to be the only DNA polymerase susceptible to the inhibitor while all the other DNA polymerases tested are fully resistant to the nucleotide analog . The resistant polymerases are: E . coli DNA polymerase I, Klenow's fragment of DNA polymerase I, phage T4 DNA polymerase, calf thymus DNA polymerase alpha, rat liver DNA polymerase beta and calf thymus terminal deoxyribonucleotidyl transferase.

J Biol Chem, 1986 Oct 25, 261(30), 14135 - 41
Construction of DNA substrates modified with psoralen at a unique site and study of the action mechanism of ABC excinuclease on these uniformly modified substrates; Van Houten B et al.; Psoralens bind to DNA noncovalently and upon exposure to near UV (320-400 nm) light produce covalent adducts . Thymidine residues in DNA, especially those at 5'-TpA-3' sequences, are most susceptible to the photochemical reaction . This property of the reaction and the recent advances in oligonucleotide synthesis and separation has enabled us to construct DNA fragments containing psoralen adducts at a specific site . The octanucleotide 5'-TCGTAGCT-3' was photoreacted (in the presence of the complementary strand) with the synthetic psoralen 4'-hydroxymethyl-4,5',8-trimethylpsoralen to obtain oligonucleotides adducted via the furan or pyrone ring at the internal thymine . These modified octanucleotides were ligated to nonmodified oligonucleotides to obtain a 40-base pair DNA fragment containing a psoralen adduct at a central location . The modified fragment having the thymine-furan side 4'-hydroxymethyl-4,5',8-trimethylpsoralen adduct was irradiated with 360 nm of light to produce an interstrand cross-link, and this cross-linked DNA was purified to homogeneity . These uniquely modified DNAs were used as substrates for Escherichia coli ABC excinuclease to determine its incision mechanism unambiguously and to determine the contact sites of the enzyme . ABC excinuclease mediates the cleavage of the 8th and 5th phosphodiester bonds 5' and 3', respectively, to psoralen monoadducts, and the 9th (5') and 3rd (3') phosphodiester bonds to the furan-side thymine of the cross-link . Preliminary DNaseI footprinting studies show that ABC excinuclease protects the whole 40-base pair fragment from DNaseI, and binding of the A and B subunits to the furan side-monoadducted substrate produces two hypersensitive phosphodiester bonds in the vicinity of the 5' incision site of ABC excinuclease.

J Biol Chem, 1986 Oct 25, 261(30), 13892 - 7
Two monoclonal antibodies against Escherichia coli ribosomal protein L2 distinguish different locations for their respective epitopes in intact ribosomes; Nag B et al.; Two monoclonal antibodies raised against intact Escherichia coli ribosomal protein L2 were isolated, affinity-purified, and characterized . One of the antibodies (Ab 5-186) recognizes an epitope within residues 5-186, and the other (Ab 187-272) recognizes an epitope within residues 182-272 . Both antibodies strongly inhibit in vitro polyphenylalanine synthesis when they are first allowed to bind to 50 S subunits prior addition of 30 S subunits . However, only Ab 187-272 is inhibitory when added to preformed 70 S ribosomes . Ab 5-186 binds to 50 S subunits but not to 70 S ribosomes . Ab 187-272 does not cause dissociation of 70 S ribosomes under the ionic conditions of the assay for polyphenylalanine synthesis (15 mM magnesium), although at 10 mM magnesium it does cause dissociation . Both antibodies inhibit the reassociation of 50 S with 30 S subunits . Both antibodies strongly inhibit peptidyltransferase activity . The two antibodies differ in their effects on interactions with elongation factors Tu (EF-Tu) and G (EF-G) . Neither antibody significantly inhibits EF-G-dependent GTPase activity, nor the binding of EF-G when the antibodies are incubated with 50 S subunits; however, Ab 187-272 causes a decrease in the binding of EF-Tu X aminoacyl-tRNA X GTP ternary complex and of EF-Tu-dependent GTPase when it is incubated with 70 S ribosomes . The Fab fragments of both antibodies had effects similar to the intact antibodies . The results show that monoclonal antibodies can be used to discriminate different regions of L2 and that EF-Tu and EF-G do not have identical ribosomal binding sites.

Nucleic Acids Res, 1986 Oct 24, 14(20), 8061 - 71
Cloning and sequencing of chloroperoxidase cDNA; Fang GH et al.; An oligod-d(T) 12-18 primed cDNA library has been prepared from Caldariomyces fumago mRNA . A clone containing a full-length insert was sequenced on the supercoiled plasmid, pBR322 . The complete primary sequence of chloroperoxidase has been derived . We have also determined about 73% of the peptide sequence by amino acid sequencing . The DNA sequence data matches all of the available known peptide sequences . The mature polypeptide contains 300 amino acids having a combined molecular weight of 32,974 daltons . A putative signal peptide of 21 amino acids is proposed from DNA sequence data . The chloroperoxidase gene encodes three potential glycosylation sites recognized as Asn-X-Thr/Ser sequences . Three cysteine residues are found in the protein sequence . A small region around Cys87 bears a minimal homology to the active site of cytochrome P450cam . No other heme protein homologues can be detected . We propose that Cys87 serves as a thiolate ligand to the iron of heme prosthetic group . A rare arginine codon, AGG, is used three times out of twelve in contrast to the very infrequent use of this codon in E . coli or yeast.

Nucleic Acids Res, 1986 Oct 24, 14(20), 8091 - 101
The adjacent dnaZ and dnaX genes of Escherichia coli are contained within one continuous open reading frame; Flower AM et al.; The dnaZ and dnaX loci of Escherichia coli have been genetically defined as separate genes, both of which are essential for DNA replication (1) . The 2.1 kb region of DNA that complements mutations in both genes has a maximum coding capacity of approximately 80,000 daltons . Two protein products are produced from this region with molecular weights of 77,000 and 52,000 (2,3) . We have sequenced a 2.7 kb fragment containing the dnaZ and dnaX genes and determined that it contains only one open reading frame of sufficient length to encode either of these proteins . This open reading frame may encode a protein of 71,147 daltons or of 68,451 daltons depending on which potential translational initiation codon is utilized . There are two transcriptional promoters preceding the gene as well as a ribosome binding site preceding the two potential initiation codons . Both the promoters and ribosome binding sites are predicted to be weak, perhaps contributing to the low expression of these genes.

Nucleic Acids Res, 1986 Oct 24, 14(20), 8027 - 46
Copy-number of broad host-range plasmid R1162 is regulated by a small RNA; Kim K et al.; We have shown previously {Kim, K . and Meyer, R.J . (1985) J . Mol . Biol . 185,755-767} that copy-number of the broad host-range plasmid R1162 is controlled by the amounts of two proteins, encoded by cotranscribed genes comprising a region of the plasmid called RepI . We have now demonstrated that expression of RepI is negatively regulated by a 75 base RNA that is complementary to a segment of the RepI message . Increased intracellular amounts of RNA molecules that include this segment relieve the inhibition of RepI gene expression, suggesting that the target for regulation is the mRNA itself . A mutation decreasing the amount of the 75 base RNA results in elevated plasmid copy-number . Thus, consistent with our previous observations, regulation of the expression of the RepI genes is a factor in controlling plasmid copy-number.

Biochemistry, 1986 Oct 21, 25(21), 6391 - 7
Accuracy of natural messenger translation: analysis of codon-anticodon recognition in a simplified cell-free system; Negre D et al.; A simplified plasmid-directed coupled system {Robakis, N., Cenatiempo, Y., Meza-Basso, L., Brot, N., & Weissbach, H . (1983) Methods Enzymol . 101, 690-706} was used to study the accuracy of natural messenger translation in vitro . In this system, protein synthesis is limited to the formation of the N-terminal di- or tripeptide of the gene product . Such a control is obtained by restricting the supply of aminoacyl-tRNAs in the assay medium to those corresponding specifically to the first two or three triplets in the mRNA coding sequence . We analyzed comparatively the interaction of 6 different codons with their cognate tRNAs and 18 noncognate tRNAs able to recognize triplets differing from the legitimate sequences by one base only . Special attention was paid to the single base errors occurring at the first and second codon positions during ribosomal selection of aminoacyl-tRNA molecules . The noncognate tRNAs were assayed either in the absence of the legitimate tRNAs or under competition conditions . They were chosen so that all the possibilities for misreading any particular base as each of the other three bases could be studied . First, it was mainly observed that translation mistakes can be equally detected in the first and second codon positions; there is no compelling evidence for a most or least accurate site . Second, pyrimidines seem to be read more accurately than purines . In particular, U cannot be read as either C or G, and C can hardly be mistaken for any other base.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Oct 21, 25(21), 6361 - 5
Ribosome-catalyzed formation of an abnormal peptide analogue; Roesser JR et al.; The peptidyl-tRNA analogue N-(chloroacetyl)phenylalanyl-tRNAPhe was prepared by chemical aminoacylation and prebound to the P site of Escherichia coli ribosomes in response to poly(uridylic acid) . Admixture of phenylalanyl-tRNAPhe to the A site resulted in the formation of two "dipeptides", one of which was formed by displacement of chloride ion from the peptidyl-tRNA . This constitutes the first example of ribosome-mediated formation of a peptide of altered connectivity and suggests a need for revision of the current model of peptide bond formation . Also suggested by the present finding is the feasibility of utilizing tRNAs to prepare polypeptides of altered connectivity in an in vitro protein biosynthesizing system.

Biochemistry, 1986 Oct 21, 25(21), 6356 - 60
Synergism in folding of a double mutant of the alpha subunit of tryptophan synthase; Hurle MR et al.; The urea-induced unfolding of the inactive single mutants Tyr-175----Cys and Gly-211----Glu and the active double mutant Cys-175/Glu-211 of the alpha subunit of tryptophan synthase from Escherichia coli was examined by using ultraviolet difference spectroscopy . Equilibrium techniques were used to determine the equilibrium free energies of unfolding for the mutant proteins to permit comparison with the wild-type protein . The sum of the changes in stability for the single mutants is not equal to the change seen in the double mutant . This inequality is evidence for a structural interaction between these two residues . Kinetic studies show that this synergism, which destabilizes the native form by 1.5-2.0 kcal/mol at pH 7.8, 25 degrees C, occurs only after the final rate-limiting step of domain association.

Biochemistry, 1986 Oct 21, 25(21), 6593 - 8
Synthesis of in vitro Co1E1 transcripts with 5'-terminal ribonucleotides that exhibit noncomplementarity with the DNA template; Parker RC; A region that forms the S1 nuclease site in Co1E1 DNA is shown to code for an in vitro transcript, called S1 RNA-B, which contains a 5'-terminal GTP residue that exhibits noncomplementarity with the template's DNA sequence . The synthesis of S1 RNA-B initiates four bases upstream from the start point for S1 RNA-C . The initial four bases in S1 RNA-B and S1 RNA-C are identical . The relative synthesis of S1 RNA-B to S1 RNA-C is sensitive to the concentration of GTP, a substrate that is required for elongation past the +4 position in S1 RNA-C . Dinucleotides that are expected to only initiate synthesis of S1 RNA-C yield two transcripts that appear to initiate from the S1 RNA-C and S1 RNA-B start sites . In vitro studies involving other Co1E1 transcripts, RNA-B and RNA-C, provide similar observations concerning the noncomplementary initiation phenomenon . A model involving transcriptional slippage is suggested to explain the noncomplementary initiation phenomenon . The model proposes that the cycling reaction of Escherichia coli RNA polymerase produces tetranucleotides that are transposed to nearby upstream sequences for priming transcription.

Biochemistry, 1986 Oct 21, 25(21), 6397 - 404
Accessibility of the leading end of ribonucleic acid in transcription complexes; Bernhard SL et al.; A photoaffinity-protection technique has been developed to study the accessibility of the leading (5') end of nascent RNA as it passes through the transcription complex formed by Escherichia coli RNA polymerase and phage T7 DNA . The macromolecules contacted by the leading (5') end of the growing RNA chain in the transcription complex have been determined previously by photoaffinity labeling experiments using aryl azides attached to the leading end of nascent RNA {Hanna, M . M., & Meares, C . F . (1983) Proc . Natl . Acad . Sci . U.S.A . 80, 4238-4242} . By using thiols to reduce accessible photoprobes, we have modified the photoaffinity technique so that it tests the accessibility of the leading end of nascent RNA to small molecules in solution, as a function of RNA chain length . We examined in detail RNA molecules containing 11-50 nucleotides, whose 5' ends label the beta and beta' enzyme subunits with good yield . The thiol's accessibility to the leading end of each transcript was determined by comparing the RNAs cross-linked to beta beta' in thiol-treated samples to controls not treated with thiol . Incubation with 1 mM dithiothreitol for 5 min reduced approximately 36% of the 5'-azides on RNAs 11-13 bases long and approximately 43% on RNAs 28-37 bases long but practically none of the 5'-azides on RNAs 40-43 bases long . Also notable was the reduction of 34 +/- 1% of the 5'-azides on RNA 12 bases long but only 14 +/- 2% on the 14-base RNA; on the T7 A1 promoter, the leading end of the transcript diverges from the DNA template when the chain is between 12 and 14 bases long.

Biochim Biophys Acta, 1986 Oct 16, 868(1), 39 - 44
Mitomycin C-induced bidirectional transcription from the colicin E1 promoter region in plasmid ColE1; Parker RC; Treatment of a colicinogenic culture with mitomycin C induces convergent transcription from two adjacent promoters at the beginning of the colicin E1 gene . S1-mapping and primer extension assays indicate that the mitomycin C-inducible transcripts correspond to colicin E1 mRNA (cea mRNA) and to a transcript, designated RNA-C, that may code for an entry exclusion function . Nucleotide sequences that strongly resemble a consensus sequence for LexA protein binding sites span the transcription start points for cea mRNA and RNA-C . These putative operator sequences overlap by one base pair and bind LexA protein (Ebina, Y., Takahara, Y., Kishi, F., Nakazawa, A . and Brent, R . (1983) J . Biol . Chem . 258, 13258-13261) . The data suggest that mitomycin C-induced bidirectional transcription from the cea mRNA and RNA-C promoters is controlled by the SOS regulatory system of Escherichia coli.

Biochem J, 1986 Oct 15, 239(2), 435 - 43
Mannitol-1-phosphate dehydrogenase of Escherichia coli . Chemical properties and binding of substrates; Chase T Jr; Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined . The isoelectric point is 4.19 . Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000 . Thus the enzyme is a dimer . Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively . The zinc content is not significant to activity . The enzyme is inactivated (greater than 99%) by reaction of 5,5'-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification . The pH-dependence indicated a pKa greater than 10.5 for the thiol group . Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5'-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely . These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme . Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate . The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction . Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.

Biochem Biophys Res Commun, 1986 Oct 15, 140(1), 372 - 8
Structural stability of heterologous genes cloned in Streptomyces plasmid pIJ702; Lee YH et al.; A recombinant plasmid pWCL1 containing Streptomyces plasmid pIJ702, E . coli plasmid pUC12, and hepatitis B viral surface antigen (HBsAg) gene was stably maintained in E . coli, but exhibited structural instability in S . lividans 1326 . The deletions were found ranging from 2.75 to 5.65 kilobases (kb) and most of them occurred within the melanin (mel) gene of pIJ702, resulting in the loss of part of the mel gene sequence plus the insert . The removal of the pUC12 sequence from pWCL1 eliminated the instability . However, pUC12 alone inserted in either orientation on pIJ702 also caused the deletion in S . lividans 1326 . The results indicated that the structural instability of hybrid plasmid of pIJ702 depended on the interaction between the mel sequence and the inserted sequence.

Eur J Biochem, 1986 Oct 15, 160(2), 285 - 9
Studies in vitro on the flavinylation of 6-hydroxy-D-nicotine oxidase; Brandsch R et al.; The gene of 6-hydroxy-D-nicotine oxidase (6-HDNO), a flavoenzyme from Arthrobacter oxidans with covalently bound FAD, was expressed with the aid of an expression vector in a cell-free coupled transcription-translation system derived from Escherichia coli MZ9 . Ultraviolet irradiation of the E . coli extract did not affect synthesis of the 6-HDNO polypeptide nor total protein synthesis but enzymatic 6-HDNO activity could not be detected . Addition of FAD to the irradiated cell extract restored the capability of the transcription-translation assays to synthesize enzymatically active 6-HDNO . However, enzymatic activity could not be restored on addition of FAD plus cell-free extract to the ultraviolet-inactivated assays after completion of apo-6-HDNO synthesis (60 min) nor to immunoprecipitates thereof . Under similar conditions, addition of {14C}FAD did not increase the protein-bound radioactivity . These results indicate that under conditions of limited FAD supply in the in vitro system a flavinless apo-6-HDNO-polypeptide was synthesized . It was, however, not possible to bind the cofactor to the completed polypeptide chain . These findings argue for a cotranslational cofactor binding.

J Biol Chem, 1986 Oct 15, 261(29), 13844 - 9
The role of the polar, carboxyl-terminal domain of Escherichia coli leader peptidase in its translocation across the plasma membrane; Dalbey RE et al.; Leader peptidase, an integral membrane protein of Escherichia coli, is made without a cleavable leader sequence . It has 323 amino acid residues and spans the plasma membrane with a small amino-terminal domain exposed to the cytoplasm and a large, carboxyl-terminal domain exposed to the periplasm . We have investigated which regions of leader peptidase are necessary for its assembly across the membrane . Deletions were made in the carboxyl-terminal domain of leader peptidase, removing residues 141-222, 142-323, or 222-323 . Protease accessibility was used to determine whether the polar, carboxyl-terminal domains of these truncated leader peptidases were translocated across the membrane . The removal of either residues 222-323 (the extreme carboxyl terminus) or residues 141-222 does not prevent leader peptidase membrane assembly . However, leader peptidase lacking both regions, i.e . amino acid residues 142-323, cannot translocate the remaining portion of its carboxyl terminus across the membrane . Our data suggest that the polar, periplasmic domain of leader peptidase contains information which is needed for membrane assembly.

J Biol Chem, 1986 Oct 15, 261(29), 13838 - 43
The purification of fully active recombinant transforming growth factor alpha produced in Escherichia coli; Winkler ME et al.; Recombinant human transforming growth factor alpha (TGF alpha), which is active as assessed by competition with epidermal growth factor (EGF) for binding to the EGF receptor, has been produced in Escherichia coli and separated from misfolded and inactive forms of recombinant TGF alpha using reverse-phase high performance liquid chromatography . The purified recombinant TGF alpha was used to produce a monoclonal antibody that binds to active TGF alpha specifically . The antibody was coupled to Sepharose and used as an independent method for purifying active TGF alpha . The EGF receptor binding activity of antibody affinity purified TGF alpha is comparable to that of high performance liquid chromatography-purified active TGF alpha, and is 0.55 mg of EGF eq/mg of TGF alpha . The disulfide arrangement of the active TGF alpha was determined after digestion with thermolysin, and found to be analogous to the disulfide arrangement previously determined for EGF (Savage, C . R., Hash, J . H., and Cohen, S . (1973) J . Biol . Chem . 248, 7666-7672).

J Biol Chem, 1986 Oct 15, 261(29), 13677 - 83
Human adenine phosphoribosyltransferase . Complete amino acid sequence of the erythrocyte enzyme; Wilson JM et al.; We defined the amino acid sequence of adenine phosphoribosyltransferase isolated from human erythrocytes . Peptide fragments formed by cleavage at arginine, lysine, glutamic acid, and methionine were purified by high pressure liquid chromatography and sequenced by manual Edman degradation . The complete primary structure of human adenine phosphoribosyltransferase was established by sequence analysis of 19 peptide fragments . Presumed homology between the human and rodent enzymes was used to order fragments that had inadequate overlapping sequences . The enzyme has 179 residues with a calculated subunit molecular weight of 19,481 . Mass spectrometry indicated that the NH2-terminal residue is acetylated . Human adenine phosphoribosyltransferase has sequence homology with xanthine-guanine phosphoribosyltransferase from Escherichia coli in 110-amino acid region encompassing the NH2-terminal section of the enzyme.

J Biol Chem, 1986 Oct 15, 261(29), 13464 - 8
Regulation of the balanced synthesis of membrane phospholipids . Experimental test of models for regulation in Escherichia coli; Jackson BJ et al.; In Escherichia coli, highly effective regulation controls the balanced synthesis of membrane phospholipids, important for optimal growth . Regulation is such that normally about 70% of a common pool of cytosine liponucleotide precursor is utilized by phosphatidylserine synthase and eventually converted to phosphatidylethanolamine, while about 30% is utilized by the competing enzyme phosphatidylglycerophosphate synthase and converted to phosphatidylglycerol (25%) plus cardiolipin (5%) . Although the ratio of phosphatidylglycerol to cardiolipin may vary with conditions of growth, the sum of these two lipids remains relatively constant at about 30% of the total . Alternative models, postulating coordinate regulation of the two competing enzymes, or independent feedback regulation are proposed . These models were tested in experiments in which phosphatidylglycerol was continuously removed from growing cells treated with arbutin (4-hydroxyphenyl-O-beta-D-glucoside), causing its conversion to arbutinphosphoglycerol (Bohin, J.-P., and Kennedy, E.P . (1984) J . Biol . Chem . 259, 8388-8393.) The synthesis of phosphatidylglycerol was increased by a factor of 7 in cells treated with arbutin, with only small changes in phospholipid composition and with no significant change in the level of phosphatidylglycerophosphate synthase . The synthesis of phosphatidylethanolamine was not significantly increased, decisively eliminating the model that requires coordinate regulation of phosphatidylserine synthase and phosphatidylglycerophosphate synthase, and supporting the model of independent feedback inhibition, sensitive to very small changes in composition of cellular phospholipids.

Int J Cancer, 1986 Oct 15, 38(4), 587 - 95
Development of quantitative liquid competition radioimmunoassays for the ras oncogene and proto-oncogene p21 products; Caruso A et al.; The ras gene family of rodents and humans is highly conserved and consists of several distinct genes, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma . This gene family mediates transformation via (1) a point-mutation resulting in the change of one amino acid in the 21 kDA ras gene product (p21) or (2) increased expression of ras p21 . Group-specific, type-selective and interspecies indirect binding liquid competition radioimmunoassays (RIAs), capable of providing truly quantitative analyses of the 21 ras oncogene and proto-oncogene products, have been developed . Using purified recombinant ras p21 from Escherichia coli expressing the full-length T24 mutant human Harvey-ras gene protein product as a standard in these RIAs, we have defined the absolute numbers of pg, fM and molecules of ras p21 in: (1) E . coli expressing the point-mutated or proto-ras p21 and (2) mammalian cell lines of human and murine origin . Two of the RIAs developed can be termed group-specific in that they have the ability to detect the point-mutated and proto forms of all 3 human ras genes (Harvey, Kirsten, and neuroblastoma), while the third RIA is type-selective, since it detects an antigenic determinant located primarily on the Harvey ras p21 . All 3 RIAs are interspecies-specific since they are able to detect ras p21 in rodent as well as human cells . The adaptability of the RIAs to various assay conditions and ease of methodology make these immunoassays applicable to the study of several parameters associated with ras p21 expression . These assays, used in conjunction with specific cDNA probes to identify specific ras proto-oncogenes or point-mutated oncogenes being expressed, now provide truly quantitative analysis of ras p21 in mammalian cells to further the study of the association between ras p21 expression and transformation.

Cell Immunol, 1986 Oct 15, 102(2), 307 - 14
LPS-stimulated release of prostaglandin E and thromboxane B2 from the U937 cell line; Schenkein HA; Human monocytes are known to metabolize arachidonic acid (AA) and to release prostaglandins upon stimulation . Previous data indicate that in vitro maturation and differentiation of monocytes result in alteration of this property with greatly diminished response to stimulators of release of prostaglandin E (PGE) and thromboxane B2 (TxB2) occurring after cells have been cultured . To further study the effects of differentiation on human monocyte AA metabolism, a model system was established based upon the human histiocytic cell line U937 . Among tested stimulants, which included opsonized zymosan, complement fragment C3b, phorbol myristate acetate (PMA), calcium ionophore A23187, and concanavalin A, it was found that Escherichia coli lipopolysaccharide (LPS) was unique in that it stimulated increased release of TxB2 from U937 cells . The effect of the phorbol ester PMA, a compound commonly used to induce differentiation of U937, on the ability of U937 to respond to LPS was examined . Following 48 hr of treatment with PMA, U937 became capable of releasing both PGE and TxB2 in response to small doses of LPS . As previously observed for human monocytes, the release of PGE was delayed for several hours following stimulation and failed to reach maximal cumulative levels in culture until 24-48 hr following stimulation . In contrast to human monocytes, PMA-induced U937 were capable of maintaining their responsiveness to LPS for several days . Thus, the U937 cell line provides a useful model for study of the effects of differentiation of human mononuclear phagocytes on their ability to metabolize AA, and for the effects of LPS on histiocytic tumor cell prostaglandin release.

J Biol Chem, 1986 Oct 15, 261(29), 13610 - 6
On the fidelity of DNA synthesis . Pyrophosphate-induced misincorporation allows detection of two proofreading mechanisms; Kunkel TA et al.; The effect of pyrophosphate on the fidelity of in vitro DNA synthesis has been examined . Pyrophosphate enhances misincorporation by Escherichia coli DNA polymerase I in copying phi X174 DNA . The increased misincorporation is directly proportional to the extent of inhibition of the rate of polymerization . In contrast, pyrophosphate is not detectably mutagenic with avian myeloblastosis virus DNA polymerase or DNA polymerases alpha and beta from animal cells, which lack associated proofreading activities . This suggests that increased misincorporation by pyrophosphate is not due to an increase in misinsertions by DNA polymerase, but rather due to inhibition of proofreading by pyrophosphate . However, the pyrophosphate-induced infidelity has a different specificity from, and is not competitive with, two experimental markers of 3'----5' exonuclease proofreading; i.e . the effects of the next nucleotide or the addition of deoxynucleoside monophosphates . These distinctive features suggest a second mode of proofreading susceptible to inhibition by pyrophosphate . This concept is discussed in relation to models for proofreading described in the literature.

J Biol Chem, 1986 Oct 15, 261(29), 13446 - 50
Cloning and expression of an intron-deleted phage T4 td gene; West DK et al.; The 1017-bp intron within the cloned phage T4 td gene was deleted by oligonucleotide-directed mutagenesis . Induction of thymidylate synthase activity and mature td mRNA from this intronless construct (pKTd delta I) was compared both in vivo and in vitro with expression from plasmids bearing td genes in which the introns contain either no change (pKTd2), an XbaI linker inserted about 200 nucleotides from the 3'-end (pKTdX-1), or a deletion of two-thirds of the central portion (pKTd delta 1-3) . Slightly more synthase accumulated in cells carrying pKTd delta I as compared to the other td genes when induction was performed at 30, 37, or 42 degrees C . Dramatically different results were observed in vitro, where enzyme activity synthesized from pKTd delta I DNA appeared earlier and reached severalfold higher levels than with pKTd2 DNA . In addition, thymidylate synthase expression from pKTdX-1 was impaired relative to pKTd2, while pKTd delta 1-3 accumulated enzyme at levels intermediate between those of pKTd2 and pKTd delta I . Under both in vivo and in vitro conditions, increasing levels of mature td mRNA preceded and paralleled those in enzyme activity for all four plasmids, demonstrating comparable translation of the mRNAs produced . From these results it would appear that the splicing of td RNA is much more efficient in vivo than in vitro, suggesting that other cellular components may facilitate in vivo processing of this intron-containing transcript.

Nucleic Acids Res, 1986 Oct 10, 14(19), 7737 - 49
Codon usage in regulatory genes in Escherichia coli does not reflect selection for 'rare' codons; Sharp PM et al.; It has often been suggested that differential usage of codons recognized by rare tRNA species, i.e . "rare codons", represents an evolutionary strategy to modulate gene expression . In particular, regulatory genes are reported to have an extraordinarily high frequency of rare codons . From E . coli we have compiled codon usage data for highly expressed genes, moderately/lowly expressed genes, and regulatory genes . We have identified a clear and general trend in codon usage bias, from the very high bias seen in very highly expressed genes and attributed to selection, to a rather low bias in other genes which seems to be more influenced by mutation than by selection . There is no clear tendency for an increased frequency of rare codons in the regulatory genes, compared to a large group of other moderately/lowly expressed genes with low codon bias . From this, as well as a consideration of evolutionary rates of regulatory genes, and of experimental data on translation rates, we conclude that the pattern of synonymous codon usage in regulatory genes reflects primarily the relaxation of natural selection.

Nucleic Acids Res, 1986 Oct 10, 14(19), 7695 - 703
Complete nucleotide sequence of the Escherichia coli ptr gene encoding protease III; Finch PW et al.; The nucleotide sequence of a 3120 bp region of the E . coli chromosome that includes the entire ptr gene has been determined . The proposed coding region for Protease III is 2889 nucleotides long, which would encode a protein consisting of 962 amino acids with a calculated molecular mass of 107,719 daltons . The predicted primary structure of the protein includes a 23-residue signal sequence, cleavage of which would give rise to a mature protein of molecular mass 105,124 daltons . At its 3' end, the ptr gene overlaps the start of the recB coding sequence by 8 bases, suggesting that these genes may form part of an operon.

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