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-dia