J Biol Chem, 1988 Oct 5, 263(28), 14176 - 83 In vitro transcription of the origin region of replication of the Escherichia coli chromosome; Nozaki N et al.; Transcription was carried out in vitro by RNA polymerase on supercoiled minichromosome DNA containing the replication origin of the Escherichia coli chromosome (oriC) and the products were analyzed . Leftward transcription starting from the 16-kDa gene promoter located adjacent to the right of oriC was inhibited by the DnaA protein, a protein essential for initiation of replication . This inhibition is due to binding of DnaA protein to the 9-base pair sequence (dnaA box) located just upstream of the 16-kDa gene promoter . The inhibition was observed at levels of DnaA protein and RNA polymerase comparable to those required for replication of oriC plasmids in vitro and was abolished at high levels of RNA polymerase . These results suggest that a balance of DnaA protein and RNA polymerase is important in regulating transcription and that this regulation operates under conditions that allow initiation of replication . The transcription traversed oriC with very few transcripts terminating before or within it . DnaA protein did not affect the chain length of these transcripts . Furthermore, we identified leftward transcripts which start close to position 177 in the oriC sequence. J Mol Biol, 1988 Oct 5, 203(3), 699 - 705 Specific recognition of the 3'-terminal adenosine of tRNAPhe in the exit site of Escherichia coli ribosomes; Lill R et al.; Ribosomes from Escherichia coli possess, in addition to A and P sites, a third tRNA binding site, which according to its presumed function in tRNA release during translocation has been termed the exit site . The exit site exhibits a remarkable specificity for deacylated tRNA; charged tRNA, e.g . N-AcPhe-tRNAPhe, is not bound significantly . To determine the molecular basis of this discrimination, we have measured the exit site binding affinities of a number of derivatives of tRNAPhe from E . coli, modified at the 3' end . Binding to the exit site of the tRNAPhe derivatives was measured fluorimetrically by competition with a fluorescent tRNAPhe derivative . We show here that removal of the 2' and 3' hydroxyl groups of the 3'-terminal adenosine decreases the affinity of tRNAPhe for the exit site 15 and 40-fold, respectively . Substitutions at the 3' hydroxyl group (aminoacylation, phosphorylation, cytidylation) as well as removal of the 3'-terminal adenosine (or adenylate) of tRNAPhe lower the affinity below the detection limit of 2 x 10(5) M-1, i.e . more than 100-fold . Modification of the adenine moiety (1,N6-etheno adenine) or replacement of it with other bases (cytosine, guanine) has the same dramatic effect . In contrast, the binding to both P and A sites is virtually unaffected by all of the modifications tested . These results suggest that a major fraction (at least -12 kJ/mol, probably about -17 kJ/mol) of the free energy of exit site binding of tRNAPhe (-42 kJ/mol at 20 mM-Mg2+) is contributed by the binding of the 3'-terminal adenine to the ribosome . The binding most likely entails the formation of hydrogen bonds. Biochemistry, 1988 Oct 4, 27(20), 7658 - 64 Recombinant rat liver guanidinoacetate methyltransferase: reactivity and function of sulfhydryl groups; Fujioka M et al.; Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule . No disulfide bond is present . Analysis of the chymotryptic peptides derived from the iodo{14C}acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity . Incubation of the enzyme with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine {2,2'-dithiobis(ethylamine)} causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively . Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity . The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics . Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity . The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates . Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine . Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity. Biochemistry, 1988 Oct 4, 27(20), 7951 - 9 Mischarging Escherichia coli tRNAPhe with L-4'-{3-(trifluoromethyl)-3H-diazirin-3-yl}phenylalanine, a photoactivatable analogue of phenylalanine; Baldini G et al.; The Boc-protected derivative of a photoactivatable, carbene-generating analogue of phenylalanine, L-4'-{3-(trifluoromethyl)-3H-diazirin-3-yl}phenylalanine {(Tmd)Phe}, was used to acylate 5'-O-phosphorylcytidylyl(3'-5')adenosine (pCpA) . A diacyl species was isolated which upon successive treatments with trifluoroacetic acid and 0.01 M HCl yielded a 1:1 mixture of 2'(3')-O-(Tmd)phenylalanyl-pCpA and of its 2'-5'-phosphodiester isomeric form . Adapting a procedure introduced by Hecht's group {Heckler, T.G., Chang, L.H., Zama, Y., Naka, T., Chorghade, M.S., & Hecht, S.M . (1984) Biochemistry 23, 1468-1473}, brief incubation of a 15 molar excess of this material with Escherichia coli tRNAPhe, missing at the acceptor stem the last two nucleotides (pCpA), in the presence of T4 RNA ligase and ATP afforded "chemically misaminoacylated" tRNAPhe in approximately 50% yield . Following chromatographic purification on DEAE-Sephadex A-25, benzoylated DEAE-cellulose, and Bio-Gel P-6, the misaminoacylated tRNAPhe was characterized by (i) urea-polyacrylamide gel electrophoresis, (ii) enzymatic reaminoacylation under homologous conditions following chemical deacylation, and (iii) its ability to stimulate protein synthesis in an in vitro translation system which, through the addition of the phenylalanyl-tRNA synthetase inhibitor phenylalaninyl-AMP, was unable to charge its endogenous tRNAPhe . The data demonstrate that we have prepared a biologically active misaminoacylated tRNAPhe. Biochemistry, 1988 Oct 4, 27(20), 7931 - 9 Purification and NMR studies of {methyl-13C}methionine-labeled truncated methionyl-tRNA synthetase; Rosevear PR; A procedure for the rapid purification of a truncated form of the Escherichia coli methionyl-tRNA synthetase has been developed . With this procedure, final yields of approximately 3 mg of truncated methionyl-tRNA synthetase per gram of cells, carrying the plasmid encoding the gene for the truncated synthetase {Barker, D.G., Ebel, J.-P., Jakes, R., & Bruton, C.J . (1982) Eur . J . Biochem . 127, 449}, can be obtained . The catalytic properties of the purified truncated synthetase were found to be identical with those of the native dimeric and trypsin-modified methionyl-tRNA synthetases . A rapid procedure for obtaining milligram quantities of the enzyme is necessary before the efficient incorporation of stable isotopes into the synthetase becomes practical for physical studies . With this procedure, truncated methionyl-tRNA synthetase labeled with {methyl-13C}methionine was purified from an Escherichia coli strain auxotrophic for methionine and containing the plasmid encoding the gene for the truncated methionyl-tRNA synthetase . Both carbon-13 and proton observe-heteronuclear detect NMR experiments were used to observe the 13C-enriched methyl resonances of the 17 methionine residues in the truncated synthetase . In the absence of ligands, 13 of the 17 methionine residues could be resolved by carbon-13 NMR . Titration of the synthetase, monitoring the chemical shifts of resonances B and M (Figure 3), with a number of amino acid ligands and ATP yielded dissociation constants consistent with those derived from binding and kinetic data, indicating active site binding of the ligands under the conditions of the NMR experiment.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1988 Oct 4, 27(20), 7886 - 94 Homology-dependent underwinding of duplex DNA in recA protein generated paranemic complexes; Schutte BC et al.; RecA protein promoted formation of paranemic joints, in which a recA-ssDNA complex and a nicked circular dsDNA molecule are homologously aligned without net cross-strand interwinding, is accompanied by extensive underwinding of the dsDNA molecule . When the nick is sealed by DNA ligase, a highly negatively superhelical DNA molecule is formed . This underwinding has the following properties: (a) it occurs within 2 min; (b) it is completely homology dependent; (c) it does not require a homologous free DNA end . The resulting underwound DNA species is comprised of a heterogeneous population of topoisomers . The degree of unwinding exhibits a strong dependence on the fractional length of homology in the dsDNA molecule and indicates that paranemic joints can extend for at least 2900 base pairs . The rapid underwinding associated with paranemic joint formation is followed by a longer phase in which the dsDNA molecule is more extensively underwound. Biochemistry, 1988 Oct 4, 27(20), 7633 - 40 Kinetics of appearance of an early immunoreactive species during the refolding of acid-denatured Escherichia coli tryptophan synthase beta 2 subunit; Murry-Brelier A et al.; A reversible acid-denaturation process of the beta 2 subunit of Escherichia coli tryptophan synthase has been set up . The acid-denatured state has been physically characterized: though not in a random-coiled conformation, it is extensively denatured . The renaturation of this denatured state of beta 2 has been observed in a stopped-flow system, in the presence of a monoclonal antibody directed against native beta 2 . It is shown that the association occurs very early in the folding of beta 2 . The association rate constants of the antibody with the immunoreactive folding intermediate and with native beta 2 are the same (3 X 10(5) M-1.s-1) . But at high antibody concentrations the formation of the antigen/antibody complex is rate limited by a rapid (5.4 X 10(-2) s-1) isomerization of refolding beta chains . This isomerization appears to reflect the formation of at least part of the epitope recognized by the antibody during the folding of beta 2 . Further conformational adjustments occurring later in the folding pathway would then allow the ultimate structuring of the epitope. J Virol, 1988 Oct, 62(10), 3772 - 8 Vaccinia virus gene D8 encodes a virion transmembrane protein; Niles EG et al.; Transcription mapping studies and DNA sequence analysis of the vaccinia virus HindIII D fragment predict that gene D8 encodes a protein 304 amino acids in length, with a molecular mass of 35,426 daltons, that is expressed at late times in infection . In order to determine whether the native D8 protein is required for virus propagation, we constructed a frameshift mutation in the D8 coding sequence . Virus containing this mutation were isolated and shown to replicate in a single-step growth experiment with wild type virus growth kinetics, demonstrating that the normal-length D8 protein is not essential for virus propagation in tissue culture . In order to investigate the synthesis of the wild-type and the mutant D8 proteins in virus-infected cells, we raised polyclonal antisera to a fusion protein consisting of a portion of the D8 coding sequence linked to the Escherichia coli trpE gene . Western blot (immunoblot) analysis of the time course of D8 protein synthesis in cells infected with either wild-type or mutant virus demonstrated that D8 protein was synthesized late in infection in each case and accumulated throughout the experiment . To determine whether the D8 protein was incorporated into the mutant or wild-type virus, purified virions were fractionated into Nonidet P-40-soluble, deoxycholate-soluble, and detergent-insoluble fractions . In both the wild-type and the mutant viruses, the D8 protein was an integral viral protein . The wild-type protein partitioned into the Nonidet P-40-soluble fraction, suggesting that it was a viral membrane protein . The mutant protein fractionated into the detergent-insoluble component, demonstrating that although the altered protein was incorporated into the virus, it was found in a abnormal location . In order to determine whether the D8 protein was present on the virion surface, the susceptibility of the D8 protein to proteolysis was tested by analyzing the products of incubation of the wild-type and mutant viruses with either chymotrypsin or trypsin . These studies demonstrated that the wild-type D8 protein was a transmembrane protein with a major extraviral domain that was released largely intact from the virus by trypsin . The mutant D8 protein was relatively refractory to proteolysis, confirming the hypothesis that although it is associated with the virus, it is in a conformation different from that of the wild-type protein . Tryptic digestion of the wild-type virus increased plaque formation severalfold, concomitant with the removal of the extraviral domain of the D8 protein.(ABSTRACT TRUNCATED AT 400 WORDS) Am J Pathol, 1988 Oct, 133(1), 47 - 53 Ocular inflammatory effects of intravitreally-injected tumor necrosis factor; Rosenbaum JT et al.; Many of the pathophysiologic effects of bacterial endotoxin have recently been attributed to a monokine, tumor necrosis factor (TNF) . The rabbit eye is extremely sensitive to locally injected endotoxin . The authors have investigated the possible contribution of TNF to ocular inflammation in a rabbit model . The intravitreal injection of 10(5) to 5 X 10(5) units of recombinant human TNF produced a sustained disruption of the blood-aqueous barrier as manifested by elevated aqueous humor protein levels . In addition, 83% of rabbits receiving this dose of TNF developed hyperemia of limbal vessels and early neovascularization of the cornea . Many developed posterior synechiae (fibrous adhesions between the iris and the lens) . TNF induced only a slight cellular response in the anterior chamber . Histologic studies confirmed the presence of new vessels and demonstrated a marked mononuclear infiltrate within and beneath the epithelium of the iris and ciliary body . Lower doses of TNF produced inconsistent results . Heating TNF completely destroyed its inflammatory effects . The time course of the ocular response to TNF and the quantity of recombinant protein needed to produce consistent effects were vastly different from effects observed with interleukin-1 . For example, 24 hours after an intravitreal injection, 2.2 X 10(4) ng of TNF (5 X 10(5) units) produced significantly less protein extravasation and polymorphonuclear leukocyte infiltration than 4 ng of recombinant interleukin-1 . Similarly, 24 hours after intravitreal injection, 1 ng of Escherichia coli endotoxin tended to be a more potent inflammatory stimulus than this quantity of TNF . These observations indicate that the ocular pathophysiologic effects of TNF can be readily distinguished from changes induced by either endotoxin or another endotoxin induced monokine, interleukin-1. J Biochem (Tokyo), 1988 Oct, 104(4), 638 - 42 Physicochemical properties of charge isomers of recombinant human superoxide dismutase; Kajihara J et al.; Recombinant human Cu2Zn2SOD expressed in Escherichia coli consisted of mainly three isomers with isoelectric points of 5.14 (A), 5.06 (B), and 4.99 (C) . Each isomer was isolated by DEAE-Toyopearl chromatography and the physiochemical properties were investigated . No significant differences in chemical and spectrophotometric properties, such as specific activity, metal contents, amino acid composition, and UV and ESR spectra, were found . The result of labeling of free cysteine residues with ABD-F showed the disulfide bond to be formed between 57Cys and 146Cys in every isomer . A few differences were found in the CD spectrum around 260 nm and in the elution patterns on reverse-phase HPLC . The isoelectric points of the three isomers became the same after treatment by reduction and carboxymethylation and even after reduction only, pI of isomers tended to be at the value of component (A) . These results suggest that the three isomers are identical in primary structure but slightly different in secondary or tertiary structure . These differences are probably derived from structural alterations around 111Cys. Bioorg Khim, 1988 Oct, 14(10), 1372 - 86 {Effectiveness of distal gene translation in polycistrons depends upon the arrangement of regulatory signals on a template}; Kravchenko VV et al.; The role of the translational terminator and initiator signals arrangement for two adjacent genes in polycistronic mRNA has been studied . Semisynthetic beta-galactosidase gene (lacZ) of E . coli and fragment of phage M13 DNA (with promoter PVIII, gene IX, and part of gene VIII) were used for constructing of the IX-VIII-lacZ artificial polycistronic operon . Cloning of the constructs into pBR322 vector resulted in a number of pLZ381N plasmids differing by the mutual arrangement of gene VIII translation terminator codon and SD site and initiator codon (SD-ATG-region) of lacZ gene . The mutual arrangement of gene VIII terminator codon and SDlacZ-ATG region has been altered by means of deletions and insertions that have not affected lacZ translation initiation signals . The beta-galactosidase (beta-Gal) synthesis in E . coli harbouring different types of pLZ381N plasmids has been found to depend on type of cistron coupling (gene VIII and lacZ) . The overlapping of terminator and initiator codons (ATGA) for genes VIII and lacZ (type I of polycistrons) provide approximately equal translational level for both cistrons . On the other side, levels of beta-Gal synthesis in case of polycistrons type II (gene VIII stop-codon position at the beginning of SDlacZ or 10 nucleotides upstream) were 20-30 times as high as for type I . Differences in beta-Gal levels have also been found for variants of VIII-lacZ coupling in types IV and III polycistrons (the SDlacZ-ATG region in 27-50 nucleotides downstream from the proximal cistron VIII stop-codon, which, in turn, is 41 nucleotides upstream this terminator) . These data cannot be explained on the basis of possible secondary structure including the SDlacZ-ATG region and other parts of polycistronic mRNA . In all these cases similarly stable stem-loop structures have been found . Therefore, the arrangement of the translation termination and initiation signals for two adjacent genes in essential for distal gene translation efficiency . One can imagine that ribosome or its 30S subpartical, stalling on the proximal gene terminator codon, affects the distal gene translation initiation. Circ Shock, 1988 Oct, 26(2), 169 - 83 Increased microvascular permeability in canine endotoxic shock: protective effects of ibuprofen; Hubbard JD et al.; The ability of sodium ibuprofen to prevent endotoxin-induced changes in vascular permeability was examined in an anesthetized canine model . Ibuprofen was administered i.v . (3.75 mg/kg) in two pretreatment doses before the administration of Escherichia coli endotoxin (0.5 mg/kg) . Serum and left thoracic duct lymph samples were collected for measurement of total protein and separation by polyacrylamide gel electrophoresis . Four protein fractions with molecular weights (MW) ranging from 60,000 to 1,000,000 were consistently analyzed . Administration of endotoxin alone resulted in hypotension and was accompanied by an increase in microvascular permeability as evidenced by increases in lymph flow rate, protein flux, lymph/plasma protein ratio (L/P), and permeability-surface area product (PS) . Pretreatment with ibuprofen attenuated the increase in permeability as reflected by significantly lower lymph flow rate, protein flux, L/P, and PS . Electrophoretic data illustrate partial to complete protection for all four MW fractions . These results suggest that endotoxin damages microvascular integrity and increases extravasation of macromolecules as great as 1,000,000 MW . This damage is attenuated significantly by pretreatment with ibuprofen. Vaccine, 1988 Oct, 6(5), 440 - 4 Regression of line-10 hepatocellular carcinoma by a less toxic cord factor analogue combined with L18-MDP or synthetic lipid A analogues; Ishida H et al.; A transplantable hepatocarcinoma of strain 2 guinea pigs was used as an experimental model for immunotherapy of cancer . 6,6'-Dideoxy-6,6'-bis-mycoloylamino-alpha,alpha- trehalose (TDNM) was found to be more effective in producing regression of transplantable line-10 tumours than 6,6'-di-O-mycoloyl-alpha,alpha-trehalose (TDM) when combined with 6-O-stearoyl muramyldipeptide (L18-MDP) . TDNM showed potent antitumour activity in combination with synthetic lipid A of Escherichia coli (compound 506), but not with the lipid A analogues (GLA-59 and 60) . As with the combination of MDP derivative and lipid A analogue, MDP derivatives conjugated with GLA-60 (GMD compounds) showed no tumour regression activity of line-10 cells in guinea-pigs. Int J Radiat Oncol Biol Phys, 1988 Oct, 15(4), 823 - 9 High level expression of fms proto-oncogene mRNA is observed in clinically aggressive human endometrial adenocarcinomas; Kacinski BM et al.; Six micron paraffin sections of paraformaldehyde-fixed endometrial currettings of 21 benign and neoplastic endometrial specimens were assayed for tumor cell-specific oncogene expression by in situ hybridization with probes for six oncogenes, beta-actin, and the E . coli plasmid pBR322 . In the benign hyperplasias and invasive adenocarcinomas, multiple oncogenes, including erbB, fms, c-myc, and Ki-ras were expressed at significant levels . For the adenocarcinomas, statistical analysis demonstrated that high levels of expression of fms-complementary mRNA correlated strongly with clinicopathologic features (high FIGO histologic grade, high FIGO clinical stage, deep myometrial penetration) predictive of aggressive clinical behavior and poor outcome . The authors discuss the role which M-CSF receptor (the fms gene product) and locally-produced M-CSF may play in the development of the observed aggressively-malignant phenotypes . They also propose that pre-hysterectomy assay of fms gene expression in endometrial currettings in FIGO Stage I patients might be clinically useful to help identify preoperatively those patients with deep myometrial penetration or other locoregional spread. Proc Natl Acad Sci U S A, 1988 Oct, 85(19), 7317 - 21 Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: dihydrolipoamide acetyltransferase; Coppel RL et al.; Primary biliary cirrhosis is a chronic, destructive autoimmune liver disease of humans . Patient sera are characterized by a high frequency (greater than 95%) of autoantibodies to a Mr 70,000 mitochondrial antigen, a component of the M2 antigen complex . We have identified a human cDNA clone encoding the complete amino acid sequence of this autoantigen . The predicted structure has significant similarity with the dihydrolipoamide acetyltransferase (EC 2.3.1.12) of the Escherichia coli pyruvate dehydrogenase multienzyme complex . The human sequence preserves the Glu-Thr-Asp-Lys-Ala motif of the lipoyl-binding site and has two potential binding sites . Expressed fragments of the cDNA react strongly with sera from patients with primary biliary cirrhosis but not with sera from patients with autoimmune chronic active hepatitis or sera from healthy subjects. Proc Natl Acad Sci U S A, 1988 Oct, 85(19), 7049 - 53 Human uroporphyrinogen III synthase: molecular cloning, nucleotide sequence, and expression of a full-length cDNA; Tsai SF et al.; Uroporphyrinogen III synthase {URO-synthase; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75}, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III . The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria . To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides . Two synthetic oligonucleotide mixtures were used to screen 1.2 x 10(6) recombinants from a human adult liver cDNA library . Eight clones were positive with both oligonucleotide mixtures . Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da . The authenticity of this clone was established by colinearity of the predicted amino acid sequence with 81 microsequenced residues from the purified enzyme . In addition, high levels of enzymatic activity and immunoreactive protein were expressed when a blunt-ended 971-base-pair Ava II cDNA fragment containing the entire coding region was inserted into vectors for expression in Escherichia coli . The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria. Eur J Biochem, 1988 Oct 1, 176(3), 661 - 7 Primary structures and catalytic properties of isoenzymes encoded by the two 4-coumarate: CoA ligase genes in parsley; Lozoya E et al.; We have determined the primary structures of two 4-coumarate: CoA ligase (4CL) isoenzymes in parsley (Petroselinum crispum) by sequencing near full-length cDNAs corresponding to the two 4CL genes, Pc4CL-1 and Pc4CL-2, present in this plant . Comparison of the cDNA and genomic nucleotide sequences showed that each 4CL gene is organized in five exons separated by introns of varying lengths . The positions of introns are the same in both genes and 97-99% of the corresponding nucleotide sequences are identical . The two isoenzymes, which are nearly identical in their primary structures, were separated by ion-exchange chromatography, and were found to be indistinguishable with regard to substrate specificity . Assignment to Pc4CL-1 and Pc4CL-2 was achieved by comparison with catalytically active 4CL proteins, isolated from Escherichia coli cells which had been transformed with plasmids harboring the corresponding cDNAs. Protein Eng, 1988 Oct, 2(4), 293 - 6 Site-directed mutagenesis to fine-tune enzyme specificity; Uemura H et al.; We have used a combination of a genetic selection and oligonucleotide-directed mutagenesis to introduce a series of amino acid replacements for a single residue into Escherichia coli glutaminyl-tRNA synthetase . The mutant enzymes mischarge supF tRNA(Tyr), with glutamine, to varying degrees depending on the polarity of the side chain introduced but apparently not depending on the size or shape of the side chain . These results indicate that repulsive charge-charge interactions may be important for specific recognition of nucleic acids by proteins and illustrate how a mutant, derived from genetic selection, may be further modified in activity by oligonucleotide-directed mutagenesis. Protein Eng, 1988 Oct, 2(4), 283 - 91 Improved peptide function from random mutagenesis over short 'windows'; Dunn IS et al.; We have applied random mutagenesis over short contiguous residue tracts ('windows') within an active peptide (the alpha-peptide of beta-galactosidase) such that all window residues are replaced simultaneously . A novel technique using mixed synthetic oligonucleotides and selection against an EcoK restriction site has allowed the construction of libraries of mutants for two separate windows, sites A and B . Mutant phenotypes can be easily assessed in vivo by a complementation test, and panels of mutants have been quantitatively tested in vitro . This allowed the rapid probing of structural requirements for each site . The two windows yielded markedly disparate results . Site B was much less stringent in its sequence requirements for significant function than Site A, and mutants with improved function were isolated at Site B alone . In addition, one Site B mutant with wild-type levels of activity showed enhanced stability to heat or a protein denaturant . We propose that short tracts with the characteristics of Site B constitute 'secondary' interaction sites which are more tolerant of sequence diversity . Random manipulation of such secondary sites is thus more likely to yield upmutations for standard or altered environments . Window mutagenesis can in principle be applied to any protein--protein or protein--ligand interaction. Biochimie, 1988 Oct, 70(10), 1343 - 52 Evidence for a translation-mediated attenuation of a spinach chloroplast rDNA operon; Laboure AM et al.; The presence of potential hairpin structures H1, H2, H3 in the leader region of a spinach rDNA operon led us to postulate that this operon is regulated by premature termination . The mechanism would be controlled by the presence or absence of ribosomes translating a leader peptide . In vitro synchronized transcription by E . coli RNA polymerase shows that pauses do occur in the leader region . By their sizes, the transient transcripts could correspond to pauses on H1 and H2 as predicted by the model in the absence of ribosomes . The complete leader sequence (pKOPH) and the leader sequence with the hairpin structures deleted (pKOP) have been used to the GalK gene in the pK01 plasmid . The resulting plasmids have been used to transform a GalK- E . coli strain . Measurements of GalK expression show that the promoter region of spinach chloroplast rDNA is neither subjected to the growth rate nor to the stringent control . However, under growth conditions leading to an excess of free ribosomes, the expression of GalK gene appears systematically to be reduced in pKOPH when compared with that of pKOP . These results are consistent with a role of the leader region in a translation-mediated attenuation of the chloroplast rDNA expression. Eur J Clin Invest, 1988 Oct, 18(5), 512 - 6 Asparaginase-induced derangements of glutamine metabolism: the pathogenetic basis for some drug-related side-effects; Ollenschlager G et al.; Several side-effects of asparaginase therapy have been said to be a consequence of the glutaminase activity of Escherichia coli asparaginase, especially the deleterious influence on the liver function . We report here the drug-induced impairments of asparagine and glutamine metabolism in correlation to concentrations changes of plasma proteins, synthesized in the liver, in patients with acute lymphatic leukaemia . One hour after asparaginase application, plasma glutamine decreased to 5% (0-39%: median, range) of the initial values, with a subsequent rise to concentrations slightly lower than those prior to therapy . During the 14 days of drug application the fasting plasma concentrations of glutamine fell to a median of 63% of the pre-therapeutic levels, indicating a depletion of the glutamine pools . Two days after the end of asparaginase application, in one patient the glutamine concentrations increased to the pre-therapeutic range . Plasma concentrations of fibrinogen and antithrombin III decreased to 46% and 56%, respectively, of the initial values, with a slight increase 2 days after the end of therapy . The changes of plasma protein concentrations followed the course of plasma glutamine and asparagine . From that we deduce that the hepatic synthesis of the plasma proteins might be influenced by asparagine and glutamine depletion as a consequence of the therapy with E . coli asparaginase. Mol Cell Biol, 1988 Oct, 8(10), 4552 - 6 The yeast regulatory protein ADR1 binds in a zinc-dependent manner to the upstream activating sequence of ADH2; Eisen A et al.; The yeast ADR1 protein contains two zinc finger domains that are essential for its role in transcriptional activation of alcohol dehydrogenase (ADH2) . These domains are thought to function as DNA-binding structures . An ADR1-beta-galactosidase fusion protein made in Escherichia coli and containing the finger domains of ADR1 binds in vitro in a zinc-dependent manner to DNA fragments containing the two ADH2 upstream activation sequences . The strongest binding is to upstream activation sequence 1, a 22-base-pair palindrome. J Bacteriol, 1988 Oct, 170(10), 4963 - 6 Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death; Suit JL et al.; Expression of the kil gene of the ColE1 plasmid in certain classes of Escherichia coli mutants (Kilr) resistant to kil-caused cell death brought about release of periplasmic enzymes and of colicin . Phospholipase A was present but was not activated by kil expression in any of the mutants . This indicates that in these mutants the various effects of kil gene expression have become dissociated. J Bacteriol, 1988 Oct, 170(10), 4769 - 74 Thermosensing properties of Escherichia coli tsr mutants defective in serine chemoreception; Lee L et al.; Tsr, a chemoreceptor for serine and repellents in Escherichia coli, also functions as a thermoreceptor . The relationship between the chemoreceptor and thermoreceptor functions of Tsr was examined in five tsr mutants with altered serine detection thresholds . The thermosensing abilities of the mutant Tsr proteins were not affected by the alterations in their affinities to serine . In contrast, the ability of serine to inactivate thermoreceptor function was altered in these mutants . The minimal serine concentration required for thermoreceptor inactivation was directly related to the decreased affinity of the mutant Tsr for serine . The amino acid replacements in the mutant receptors were deduced from DNA sequence analyses and occurred at two different locations in the presumed periplasmic domain of Tsr . Two mutations caused histidine or cysteine replacements at arginine 64, whereas three others caused isoleucine or proline replacements at threonine 156. J Bacteriol, 1988 Oct, 170(10), 4466 - 76 Cloning, nucleotide sequence, and mutagenesis of a gene (irpA) involved in iron-deficient growth of the cyanobacterium Synechococcus sp . strain PCC7942; Reddy KJ et al.; We describe the cloning and sequencing of a gene from the cyanobacterium Synechococcus sp . strain PCC7942, designated irpA (iron-regulated protein A), that encodes for a protein involved in iron acquisition or storage . Polyclonal antibodies raised against proteins which accumulate during iron-deficient growth were used as probes to isolate immunopositive clones from a lambda gt11 genomic expression library . The clone, designated lambda gtAN26, carried a 1.7-kilobase (kb) chromosomal DNA insert and was detected by cross-reactivity with antibody against a 36-kilodalton protein . It was possible to map a 20-kb portion of the chromosome with various DNA probes from lambda gt11 and lambda EMBL-3 clones, and Southern blot analysis revealed that the irpA gene was present in a single copy and localized within a 1.7-kb PstI fragment . DNA sequencing revealed an open reading frame of 1,068 nucleotides capable of encoding 356 amino acids which yields a protein with a molecular weight of 38,584 . The hydropathy profile of the polypeptide indicated a putative N-terminal signal sequence of 44 amino acid residues . IrpA is a cytoplasmic membrane protein as determined by biochemistry and electron microscopy immunocytochemistry . The upstream region of the irpA gene contained a consensus sequence similar to the aerobactin operator in Escherichia coli . This fact, plus a mutant with a mutation in irpA that is unable to grow under iron-deficient conditions, led us to suggest that irpA is regulated by iron and that the gene product is involved in iron acquisition or storage. J Virol, 1988 Oct, 62(10), 3855 - 61 Vaccinia virus recombinants expressing an 11-kilodalton beta-galactosidase fusion protein incorporate active beta-galactosidase in virus particles; Huang C et al.; Recombinant plasmids in which vaccinia virus transcriptional regulatory sequences were fused to the Escherichia coli lacZ gene were constructed for insertion of the lacZ gene into the vaccinia virus genome . beta-Galactosidase (beta-gal) was found in some purified recombinant vaccinia virions . By enzyme activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and microscopic techniques, the evidence suggested that beta-gal accounted for 5% of the total protein in the virion . These recombinant viruses were constructed so that a portion of the coding sequences of a late vaccinia virus structural polypeptide was fused to the amino terminus of beta-gal to produce the fusion protein . Removal of the coding sequences resulted in the complete loss of beta-gal activity . This demonstrated that a vaccinia virus DNA segment from a late structural gene is responsible for the incorporation of beta-gal into the virion. Biotechniques, 1988 Oct, 6(9), 834, 837 - 8 A simple method for the preparation of the covalently closed circular form of plasmid DNA; Le Brun JJ et al.; We describe a simple, rapid, inexpensive method for isolation of covalently closed circular plasmid DNA . The method involves the electrophoresis of crude DNA preparations in an agarose gel, electrotransfer onto a dialysis membrane and elution of the highly purified circular covalently closed plasmid DNA . Native and recombinant plasmid DNA have been purified by this method and shown to be suitable for restriction enzyme digestion and transformation of bacteria . The yield of this rapid purification procedure makes it a good alternative method to standard centrifugation in cesium chloride ethidium bromide gradients. J Gen Microbiol, 1988 Oct, 134 ( Pt 10), 2757 - 68 Nucleotide sequence of fruA, the gene specifying enzyme IIfru of the phosphoenolpyruvate-dependent sugar phosphotransferase system in Escherichia coli K12; Prior TI et al.; The Enzyme IIfru of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system (PTS), which catalyses the uptake of fructose and its concomitant phosphorylation to fructose 1-phosphate by Escherichia coli, is specified by a gene designated fruA . The nucleotide sequence of a 2.5 kb PvuII restriction fragment spanning fruA+, cloned on a plasmid, was determined . This fragment contained three open reading frames (ORFs) but only one complete ORF, 1689 base pairs long, which was preceded by a well-defined Shine-Dalgarno sequence and ended with a rho-independent transcription terminator . The amino acid sequence deduced from this DNA corresponds to that of a protein of 563 amino acids (57.5 kDa), which has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0.40) and which is characterized by a number of well-marked hydrophobic loops that may correspond to membrane-spanning regions . There is relatively little overall homology between this protein and those of other Enzymes II of the PTS but there is considerable correspondence between the region surrounding one of the six histidine residues (His381) of Enzyme IIfru and those surrounding the particular histidines of other Enzymes II, and of HPr, known to be involved in phosphorylation . A plasmid carrying the complete fruA+ nucleotide sequence, but not that of any other functional protein, fully restored the ability of fruA mutants to grow on fructose and of extracts of fruA mutants to phosphorylate fructose, which confirms that the nucleotide sequence determined species Enzyme IIfru. Protein Eng, 1988 Oct, 2(4), 307 - 11 Expression of human calmodulin cDNA in Escherichia coli and characterization of the protein; West CA et al.; A cDNA clone of human calmodulin, isolated from liver, was subcloned into the expression vector pKK233-2 . The resulting expression plasmid, designated pCWCaM1, produced human calmodulin in Escherichia coli SG5 . The cDNA was sequenced using novel primers designed for use in plasmid-sequencing protocols with pKK233-2 and pKK223-3 . The expressed calmodulin was purified and subjected to NMR analysis which revealed a structure essentially the same as natural calmodulin isolated from human tissue . The activation of myosin light chain kinase by the genetically engineered human calmodulin and bovine brain calmodulin was studied and found to be comparable to a high degree . The expressed calmodulin appears to be comparable to normal calmodulin and can be used for site-directed mutagenesis and structure/function investigations. Protein Eng, 1988 Oct, 2(4), 277 - 82 Crystal structure of T4-lysozyme generated from synthetic coding DNA expressed in Escherichia coli; Rose DR et al.; The polypeptide produced by expressing a chemically synthesized gene coding for the amino-acid sequence of T4-lysozyme has been crystallized and subjected to X-ray diffraction . The crystal structure has been refined to a standard R-factor of 0.191 for data between 8 and 2 A resolution . The refined model is essentially the same as the well-known structure of wild-type T4-lysozyme determined previously by Matthews et al . (1987) . Some small changes in the C-terminal region, which is important in maintaining the folded structure, have been noted . In addition to confirming that the synthetic gene product is very close to the wild type, this structure provides a benchmark for protein engineering experiments on the folding and the catalytic activity of this molecule by the method of gene synthesis. J Clin Lab Immunol, 1988 Oct, 27(2), 83 - 6 Modulation of acute endotoxin pulmonary inflammation by a corticosteroid; Rylander R et al.; The effect of corticosteroid on an acute inflammation in the lungs was studied in guinea pigs exposed to an aerosol of bacterial endotoxin, the subsequent inflammatory response was evaluated counting the number of cells obtained from airway lavage and in the lung interstitium as well as the chemotactic effect of alveolar macrophages . Pretreatment with corticosteroid decreased the number of neutrophils in the airways at 24 hours after exposure but did not influence neutrophils in airways or lung interstitium at 4 hours after exposure, not the secretion of chemotactic factor(s) from alveolar macrophages at that time . It is suggested that the antiinflammatory effect of corticosteroids is a protective effect on the epithelium, preventing the late influx of serum fluid and neutrophils into the airways. Biochem Int, 1988 Oct, 17(4), 617 - 27 Resistance of both the 5'- and 3'-domain of isolated Escherichia coli 23S rRNA against digestion with alpha-sarcin; Hausner TP et al.; The ribonuclease alpha-sarcin exclusively cleaves the phosphodiester bond after G2661 in the 23S rRNA within 50S subunits, thus inactivating the ribosomes . The resulting alpha-fragment is 243 nucleotides long and contains the 3'-end of the 23S rRNA . The specificity is changed dramatically if isolated 23S rRNA is used as substrate . We have shown previously that 23S rRNA is digested completely except for two fragments, one of which is identical to the alpha-fragment . Here we show that the other fragment comprises the 5'-end of 23S rRNA and contains 385 nucleotides . A similar fragment was obtained when isolated 23S rRNA was digested with RNase A (specific for pyrimidines in single strands) . It appears that the 5'-domain (equivalent to 5.8S rRNA of eukaryotic ribosomes) as well as the 3'-domain (equivalent to 4.5S rRNA of chloroplast ribosomes) have a compact and defined tertiary structure in isolated 23S rRNA in contrast to the rRNA region in between . Thus, alpha-sarcin is a convenient tool for detecting compact domains in isolated RNA. Tohoku J Exp Med, 1988 Oct, 156(2), 109 - 20 Airway hyperresponsiveness after endotoxin inhalation depends on leukocyte infiltration; Horie T et al.; We have investigated the relationship between acute airway hyperresponsiveness and polymorphonuclear cell (PMN) migration in airways following saline or endotoxin (ET) aerosol inhalation in 63 guinea pigs . In 20 of these animals, granulocytopenia was induced by prior treatment with hydroxyurea (HU) . Airway responsiveness (AR) to inhaled methacholine, together with leukocyte counts in bronchoalveolar lavage fluid (BALF) and in tracheal epithelium, was also examined before and at 30 min, 1, 3 and 6 hr after saline or ET inhalation . In saline inhalation groups, AR and PMN counts in BALF and in tracheal epithelium did not differ from control animals at any time points . However, in ET inhalation groups AR increased significantly at 1 and 3 hr and returned to the pre-exposure level at 6 hr . This period of hyperresponsiveness was associated with an increment of PMN migration into tracheal epithelium . However, the increment of PMN counts in BALF was delayed to 3 and 6 hr after ET . In HU treated animals, AR and PMN counts in BALF and in tracheal epithelium did not change during 6 hr after ET inhalation . These results suggest that the hyperresponsiveness induced by ET inhalation either depends upon PMN migration into the tracheal epithelium. Mol Gen Genet, 1988 Oct, 214(2), 361 - 4 Anti-mutagenic effect of ultraviolet light on spontaneous tyrosine tRNA ochre suppressor mutations in Escherichia coli; Bockrath R et al.; The numbers of tyrosine tRNA ochre suppressor mutations arising spontaneously or after UV irradiation in different strains of Escherichia coli K12 are considered . The DNA sequence change requisite for this type of mutation would be a transversion at a cytosine between two purines, where pyrimidine-pyrimidine photoproducts could not form . We find that UV mutagenesis does not produce these tyrosine tRNA ochre suppressor mutations . With lexA51 recA441 defective cells, the spontaneous yield of these mutations is elevated and UV irradiation produces a significant decrease in the numbers of this particular mutation . As explanation we suggest that the spontaneous appearance of these mutations reflects mutation at apurinic sites, the efficiency of which is elevated in lexA51 recA441 cells (with derepressed SOS functions and an activated form of RecA protein) . The addition of UV damage in the DNA of these cells cannot further stimulate the positive functions that are required for the production of these mutations and are typically associated with UV mutagenesis (induction of SOS functions, activation of RecA protein and introduction of a targeting photoproduct) but apparently can have a negative effect on mutagenesis, hitherto not realized. Mol Gen Genet, 1988 Oct, 214(2), 353 - 7 The F factor of Escherichia coli carries a locus of stable plasmid inheritance stm, similar to the parB locus of plasmid RI; Golub EI et al.; We found that a 1.4 kb fragment of the F factor of Escherichia coli (coordinates 62.8-64.2) considerably increased the stable inheritance of different plasmids which carried it . The fragment has a 589 bp DNA sequence (coordinates 63.3-63.9) with extensive homology to the parB locus of plasmid RI and, probably like the parB region, ensures the presence of plasmids in bacterial populations by killing those cells which have lost the plasmid. Mol Gen Genet, 1988 Oct, 214(2), 286 - 94 "Strong incompatibility" between derivatives of the Streptomyces multi-copy plasmid pIJ101; Deng ZX et al.; Some derivatives of pIJ101, a 8.9 kb Streptomyces multi-copy plasmid, can co-exist with each other at similar copy numbers but others are strongly incompatible . The DNA sequence, sti, which causes this "strong incompatibility" was localised on a DNA segment of about 200 bp which is not part of the essential replication region of pIJ101 . The sti function is active only when the DNA fragment carrying it is present in the natural orientation with respect to the basic replicon region of pIJ101 . Pairs of plasmids which either both possess sti in the correct orientation (Sti+) or both lack sti or carry it in reverse orientation (Sti-) can co-exist, but Sti+ and Sti- plasmids cannot; in this case the Sti+ plasmid is retained and the Sti- plasmid is lost . This phenomenon is called strong incompatibility to distinguish it from classical incompatibility where identical or related plasmids are incompatible and dissimilar plasmids are compatible . pIJ101 probably replicates via a single-stranded intermediate; sti would be a site where the synthesis of the second (lagging) DNA strand is initiated because Sti- plasmids accumulate more single-stranded plasmid DNA than Sti+ plasmids . The copy number of pIJ101 and its derivatives is influenced by sti and by an additional trans-acting function (cop). Mol Gen Genet, 1988 Oct, 214(2), 198 - 203 Temperature dependent survival of UV-irradiated Escherichia coli K12; Ganesan AK et al.; We have found that several excision deficient derivatives of Escherichia coli K12 survive better after UV irradiation if incubated at 42 degrees C than if incubated at 30 degrees C . The highest survival was observed when incubation at 42 degrees C followed UV irradiation and was maintained for at least 16 h . Our results indicate that this temperature dependent resistance (TDR) requires a functional recA gene, but not uvrA, uvrB, recF, or recB genes, or the recA441 (tif-1) mutation which allows thermoinduction of the recA-lexA regulon . Our data are consistent with the idea that the increase in survival observed at 42 degrees C reflects enhanced daughter-strand gap repair by DNA strand exchange . Although the conditions used to elicit TDR can induce heat shock proteins and thermotolerance in E . coli, the relationship between the two responses remains to be elucidated. J Biomed Eng, 1988 Oct, 10(5), 448 - 52 Low-cost digital impedance meter for the detection of micro-organisms; Felice CJ et al.; The digital impedance meter is a microprocessor-based instrument able to detect, quantify and identify micro-organisms . The equipment makes use of the bipolar technique of measuring the impedance modulus of six cells containing inoculated culture broth . It performs temperature compensation automatically . Growth curves are stored in memory as time course events and can be displayed on any suitable device. Genetika, 1988 Oct, 24(10), 1894 - 6 {Mutations in the Escherichia coli RNA-polymerase beta-subunit gene cloned in a multicopy plasmid}; Ogryz'ko EP et al.; A multicopy plasmid pLMN1 expressing a wild type rpoB gene encoding Escherichia coli RNA polymerase beta subunit gene was constructed . Introduction of this plasmid into rifampicin-resistant RpoB mutants makes them rifampicin-sensitive . Rifampicin-resistant clones appear in such strains with frequencies up to 10(-3), due to recombinational (recA-dependent) transfer of rif-r mutations from chromosome to pLMN1 . This provides a simple selection procedure for transfer of any rpoB mutation, together with a rif-r mutation from a chromosome to pLMN1 . In this way, we transferred rpoB22 amber mutation to pLMN1 for localization of the mutant codon by DNA sequencing. Anal Biochem, 1988 Oct, 174(1), 235 - 8 Specific protein-DNA complexes: immunodetection of the protein component after gel electrophoresis and Western blotting; Granger-Schnarr M et al.; A method is described to determine the presence and the relative amount of proteins within specific protein-DNA complexes . The system studied is the LexA repressor from Escherichia coli and its interaction with the operator of the caa gene encoding the bacterial toxin colicin A . After separation of the free and the complexed 32P-labeled DNA on a native polyacrylamide gel, the bound proteins are transferred on a polyvinylidine difluoride (PVDF) membrane after sodium dodecyl sulfate denaturation . Development of the protein on the membrane was achieved on reaction with an anti-LexA antibody and the use of a second anti-antibody crosslinked with alkaline phosphatase . The phosphatase activity is monitored using 5-bromo-4-chloro-3-indolyl phosphate as a substrate and 4-nitroblue tetrazolium salt . A quantitation by densitometry of both the stained protein bands on the PVDF membrane and the DNA on autoradiograms allowed us to assign the relative stoichiometry of the two different complexes formed between LexA and the caa operator . The method should allow unraveling of complicated band shift patterns arising from the presence of several binding sites for a same protein, as in our case, or from the presence of different proteins binding to a same DNA fragment. J Dairy Sci, 1988 Oct, 71(10), 2782 - 9 Effect of dexamethasone on experimental Escherichia coli mastitis in the cow; Lohuis JA et al.; The effect of intramuscular administration of dexamethasone on milk production and on several clinical and hematologic indicators in cows with experimental Escherichia coli mastitis was studied . Two groups of three cows each were intramammarily inoculated with 20 x 10(5) cfu E . coli 0101 K99 F41 in rear quarters . Immediately after inoculation one groups received 30 mg dexamethasone intramuscularly, whereas the other group was injected similarly with pyrogen-free saline . Fourteen days later milk production losses were markedly less in the dexamethasone-treated group . Dexamethasone treatment reduced local clinical signs of inflammation, significantly increased rectal temperatures, and diminished inhibition of rumen amplitude during the acute phase of infection . Moreover, cows treated with dexamethasone showed diminished neutropenia, more pronounced hypozincemia, and delayed onset in the decrease in plasma iron concentrations . Possible mechanisms by which these changes are accomplished are discussed. J Dairy Sci, 1988 Oct, 71(10), 2772 - 81 Growth of Escherichia coli in whole and skim milk from endotoxin-induced mastitic quarters: in vitro effects of deferoxamine, zinc, and iron supplementation; Lohuis JA et al.; A marked growth inhibition of Escherichia coli 0101 K99 F41 was observed in whole and skim milk collected from inflamed quarters 18 and 36 h after intramammary administration of .1 mg E . coli lipopolysaccharide . Individual cow variation in the ability of milk from endotoxin-infused quarters to inhibit growth of E . coli was found . Growth inhibition of E . coli was observed in milk from endotoxin-infused quarters and was most pronounced in skim milk sampled at postinfusion h 18, and incubated at 38 degrees C . The mechanism by which bacterial growth was depressed was probably of noncellular origin . Addition of Fe (45.5 micrograms/ml) and Zn (2.7 micrograms/ml) to whole and skim milk sampled from inflamed quarters at 18 h after endotoxin infusion resulted in a growth-promoting effect . Addition of deferoxamine (6 mg/ml) depressed bacterial growth . Effects of Fe, Zn, and deferoxamine on bacterial growth did not differ in whole and skim milk . No clear relationship was observed between reduction in Zn concentrations in skim milk from inflamed quarters at 18 h after endotoxin infusion and growth inhibition of E . coli in the same samples. J Antimicrob Chemother, 1988 Oct, 22(4), 521 - 8 Neurotoxicity of benzylpenicillin in experimental Escherichia coli meningitis; Schliamser SE et al.; The neurotoxic potential of benzylpenicillin administered intravenously as a continuous infusion was studied in rabbits with experimental Escherichia coli meningitis . As controls a group of rabbits was injected with saline into the cisterna magna . The concentrations of benzylpenicillin in serum, CSF and brain tissue fluid were studied at onset of epileptogenic electroencephalographic activity (thirteen rabbits) or convulsions (ten rabbits), with a previously developed method for neurotoxicity studies . E . coli meningitis did not increase the neurotoxicity of benzylpenicillin, despite high concentrations of the drug in both CSF and brain tissue fluid . The intracisternal injection of saline in the control group produced slight pleocytosis in some rabbits indicating some degree of damage of the blood-CSF barrier. Avian Dis, 1988 Oct-Dec, 32(4), 779 - 86 Effects of Escherichia coli on iron, copper, and zinc metabolism in chicks; Tufft LS et al.; The present report describes the effects of Escherichia coli endotoxin and infection on kinetic changes of copper (Cu), iron (Fe), and zinc (Zn) levels in the transport (serum), storage (liver), and immune organs (spleen and bursa of Fabricius) of the chicken . During infection and endotoxin challenge, increased serum and bursal Cu were noted . Infection and endotoxin both led to a redistribution of Fe with a decrease in serum and an increase in the spleen . Infection decreased serum Zn and concomitantly increased hepatic and splenic Zn . Seven days postinfection, when recovery was well underway, hepatic and splenic Cu and splenic Zn were elevated . Hepatic Fe decreased with recovery, whereas splenic Fe increased . Endotoxin and infection changed trace element kinetics . The endotoxin produced tissue elemental alterations similar to the early stages of infection . This indicated that in early infection, some of the disease responses may be due to endotoxin, whereas the later responses may be due to other aspects of infection such as stress. Appl Environ Microbiol, 1988 Oct, 54(10), 2464 - 71 Product inhibition of immobilized Escherichia coli arising from mass transfer limitation; Stewart PS et al.; Mass transfer-limited removal of metabolic products led to product-inhibited growth of Escherichia coli that was immobilized in a model system . Comparison of the growth kinetics of immobilized and free-living cells revealed no further physiological differences between cells in these two modes of existence beyond those manifested in the local concentrations of substrate and product . Bacteria were retained on a microporous membrane in a dense, planar aggregate and were grown anaerobically on a glucose-based minimal medium . Radioisotope labeling of the immobilized cell mass with 35S was used to determine growth kinetic parameters . Growth rates in the immobilized cell layer were measured by an autoradiographic technique which allowed comparison of the size of the growing region with the rate of cell convection caused by growth . Immobilized cell growth rates and growth yields ranged from near maximal (0.56 h-1 and 39 g of dry cell weight/mol of glucose, respectively) to substantially reduced (0.15 h-1 and 15 g/mol) . The depression of these kinetic parameters was attributed to product inhibition arising from mass transfer-limited removal of acidic waste products from the cell mass . A simple one-dimensional reaction-diffusion model, which incorporated data on the product-inhibited growth kinetics of free-living cells collected in a product-limited chemostat, satisfactorily predicted product inhibition of immobilized cell growth. Inflammation, 1988 Oct, 12(5), 491 - 501 Kinetics of endotoxin-induced inflammation in ovine mammary gland; Colditz IG et al.; The inflammatory response to stimulation with endotoxin of lactating and nonlactating mammary glands of sheep was examined . Similar numbers of neutrophils and mononuclear cells were recovered from nonlactating glands sampled every 2 h for 8 h and in glands sampled once at 8 h . Thus the inflammatory response was initiated by 2 h and repeated sampling did not modify the time course of the response . In contrast, in lactating ewes, fewer cells were recovered from glands sampled every 2 h than from glands sampled once at 8 h . Fewer neutrophils were also recovered when glands were serially sampled from 4 h to 8 h . Thus, removal of milk and inflammatory exudate modified the time course of the leukocyte influx into lactating glands . Significant accumulation of neutrophils occurred by 2 h in dose-response experiments in nonlactating glands . Peak accumulation of neutrophils occurred between 2 and 6 h, and a marked decline occurred after 8 h . In lactating glands, a slower onset and longer duration of neutrophil accumulation occurred . Twenty- to 30-fold more neutrophils were recovered by 8 h in lactating than nonlactating glands . This difference was not due to a lower threshold of sensitivity to endotoxin . Infusion of milk into nonlactating glands did not modify the intensity or time course of the inflammatory response to endotoxin . Thus, the physiological state of resident cells within the lactating gland, rather than the interaction of inflammatory exudate with milk, can account for the different reaction pattern in lactating glands . Inflammation in the nonlactating gland closely resembles inflammatory responses in skin and provides a convenient model for investigating the initiation and regulation of inflammatory processes. Genetics, 1988 Oct, 120(2), 359 - 66 Molecular evolution of the Escherichia coli chromosome . II . Clonal segments; Milkman R et al.; Remarkable sequence similarities in the trp region among Escherichia coli strains of diverse natural origins imply the existence of worldwide clones of very recent origin . This in turn implies a low rate of fixation of new universally favorable alleles, which carry adjacent stretches of chromosome to high frequency . These clonal segments begin as entire chromosomes; recombination shortens them progressively by substituting less closely related homologous DNA . The rate of this recombination, comprising the introduction of a homologous chromosomal fragment to a cell and the replacement of part of the original chromosome, is estimated from observations. Genetics, 1988 Oct, 120(2), 345 - 58 Molecular evolution of the Escherichia coli chromosome . I . Analysis of structure and natural variation in a previously uncharacterized region between trp and tonB; Stoltzfus A et al.; We present the sequence of a 3500-bp region of the Escherichia coli strain K12 chromosome lying between the tryptophan operon and the tonB gene . Analysis of the sequence yields six open reading frames that have properties characteristic of genes for proteins . The reading frames are closely spaced, and putative transcription units and control sites compose over 95% of the DNA . The sequences of several wild strains of E . coli have been determined for a large segment of the region described . Comparison of these sequences reveals the effects of base substitutions, DNA rearrangements, and recombination . In the regions presumably expressed as polypeptides, most of the natural variation results from synonymous substitutions . However, the DNA rearrangements identified have end points within the open reading frames and disrupt them in a variety of ways . The effects of genetic recombination between strains, recently found to be significant on a large scale in E . coli, are also apparent in the region between trp and tonB. Vaccine, 1988 Oct, 6(5), 389 - 92 Development and protective efficacy of a recombinant-DNA derived fimbrial vaccine against enterotoxic colibacillosis in neonatal piglets; Greenwood PE et al.; A vaccine containing Escherichia coli fimbriae produced by recombinant-DNA techniques was tested in controlled challenge trials and in commercial piggeries experiencing neonatal enterotoxic colibacillocis . In the challenge trials, piglets suckling vaccinated sows were significantly protected from diarrhoea and colonization of the small intestine by enterotoxigenic E . coli . High titres of anti-fimbrial IgG antibody were detected in the serum and colostrum of vaccinated sows and in serum of their piglets . In the field trials, prevalence of diarrhoea and preweaning mortality due to diarrhoea were greatly reduced in piglets suckling vaccinated sows. J Antibiot (Tokyo), 1988 Oct, 41(10), 1462 - 70 Cloning of macromomycin apoprotein gene from Streptomyces macromomyceticus by use of 50-mer deoxynucleotide probes; Hori M et al.; A mixed probe consisting of two synthetic deoxynucleotides (52 and 54 mers referred to as 50-mer) with arbitrarily chosen C or G for the third letters was prepared based on the amino acid sequences No . 31-48 and No . 72-90 of macromomycin (MCM) apoprotein and successfully used to clone the MCM apoprotein gene . Digestion with Sph I of total DNA of MCM-producing Streptomyces macromomyceticus M480-M1 yielded a 2.6-kb fragment that hybridized strongly to the probes . The hybridized probe was stable to washing with 3 x SSC at 75 degrees C . Radioactivity derived from the hybridized probe was comparable to that expected theoretically from hybridization between the probe and the true target sequence . The 2.6-kb fragment was cloned into Escherichia coli RR1 with pBR322 and subsequently subcloned into Streptomyces lividans TK21 with pIJ702 . Nucleotide sequence analysis of the cloned fragment verified the existence of the sequence corresponding to the amino acid sequence of MCM apoprotein and about 90% homologies with the probes . Thus, the use of relatively long deoxynucleotide probes with arbitrarily chosen C or G for the third letters will be advantageous in cloning Streptomyces protein genes where more than 90% of the third letters have been known to be C or G . In addition, theoretical diagnosis of hybridization should be a great help to distinguish true positives from false ones. Am J Vet Res, 1988 Oct, 49(10), 1695 - 8 In vitro effects of a mixture of Escherichia coli heat-stable enterotoxins on chloride flux in everted jejunal sacs of male pigs; Panichkriangkrai W et al.; In vitro effects of a mixture of Escherichia coli heat-stable enterotoxins (STa and STb) on isolated jejunum of 3-week-old male pigs were studied, using everted intestinal sac techniques . Heat-stable enterotoxins increased chloride secretion and chloride absorption in everted intestinal sacs . The increase of secretory flux was greater than that for absorptive flux . Vasoactive intestinal peptide (6 x 10(-9) M) increased chloride secretion, but had no effect on chloride absorption . Neither vasoactive intestinal peptide nor pilocarpine (10(-5) M) had additive effect to ST . Secretory effects of ST were not blocked by atropine (2 x 10(-5) M), clonidine (10(-6) M), or morphine (4.2 x 10(-6) M). Mol Biochem Parasitol, 1988 Oct, 31(1), 11 - 7 Mitochondrial DNA of the human malarial parasite Plasmodium falciparum; Gardner MJ et al.; Covalently closed circular DNA molecules were isolated from Plasmodium falciparum total DNA by isopycnic centrifugation in CsCl gradients containing either ethidium bromide or 2',6-diamidino-2-phenylindole . The circular molecules had an average contour length of 11.1 +/- 0.5 micron, similar to the analogous molecules previously isolated from the simian malaria parasite P . knowlesi . Both circular molecules shared considerable sequence homology and conserved restriction sites . The nucleotide sequence of one 936 bp fragment of the P . falciparum molecule was determined and identified, by a data base homology search, as part of a mitochondrial small rRNA subunit, thus confirming the mitochondrial origin of the circular DNAs of both malarial species. J Clin Microbiol, 1988 Oct, 26(10), 2231 - 2 Improved detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli by using a commercial coagglutination test; Rudensky B et al.; Escherichia coli strains grown on lincomycin-supplemented Mundell agar and on blood agar were compared for their ability to produce heat-labile enterotoxin, as detected by a commercial coagglutination kit . The special agar allowed more strains to be detected, and the results were much more clear-cut. J Clin Microbiol, 1988 Oct, 26(10), 2177 - 9 Lack of Shiga-like cytotoxin production by enteroinvasive Escherichia coli; Cleary TG et al.; Enteroinvasive Escherichia coli has not been extensively studied for cytotoxin production . We evaluated 30 well-characterized enteroinvasive E . coli strains of all the known invasive serogroups from several geographic regions for their ability to produce Shiga-like cytotoxic activity assayed in a HeLa cell system . None of these strains produced cytotoxic activity that was neutralizable with antibody to Shiga-like toxin I or II. J Clin Microbiol, 1988 Oct, 26(10), 2173 - 6 Identification of enterotoxigenic Escherichia coli isolates with enzyme-labeled synthetic oligonucleotide probes; Medon PP et al.; Commercially available kits containing alkaline phosphatase-labeled oligonucleotide probes for Escherichia coli heat-stable enterotoxins (STI-H, STI-P, and STII) and the heat-labile enterotoxin were compared with bioassays and radiolabeled recombinant DNA probes to identify enterotoxigenic E . coli from 100 clinical isolates . There was very good agreement between the three methods. J Clin Microbiol, 1988 Oct, 26(10), 2127 - 31 Isolation and characterization of monoclonal antibodies to Shiga-like toxin II of enterohemorrhagic Escherichia coli and use of the monoclonal antibodies in a colony enzyme-linked immunosorbent assay; Perera LP et al.; The major obstacle in large-scale epidemiological investigations of the incidence of Shiga-like toxin (SLT)-producing Escherichia coli in diarrheal stools is the lack of a rapid, specific test to detect toxin . Enterohemorrhagic E . coli produces elevated levels of SLT-I, SLT-II, or both cytotoxins (also called Verotoxins) . SLT-I but not SLT-II can be neutralized by antiserum to purified Shiga toxin and by monoclonal antibodies to the B subunit of SLT-I . In this study, monoclonal antibodies were generated against a crude preparation of SLT-II produced by an E . coli K-12 strain lysogenized with the 933W toxin-converting phage of enterohemorrhagic E . coli 933 . Hybridoma culture supernatants were screened for anti-SLT-II antibodies by a cytotoxicity neutralization assay and by an enzyme-linked immunosorbent assay (ELISA) . Of 53 ELISA-positive lines, 5 were capable of neutralizing the cytotoxicity of SLT-II but not of SLT-I, Shiga toxin, or a variant of SLT-II produced by E . coli that causes edema disease of swine . All five monoclonal antibodies immunoprecipitated the isolated A subunit of SLT-II but not the B subunit . Of these five neutralizing monoclonal antibodies, four were of the immunoglobulin M class and one belonged to the immunoglobulin G1 subclass . All five lines had kappa light chains . These neutralizing monoclonal antibodies have been used as probes in a colony ELISA to detect SLT-II-positive bacterial colonies . The colony ELISA with these monoclonal antibodies is a specific, sensitive test with potential diagnostic value. J Appl Physiol, 1988 Oct, 65(4), 1586 - 91 Effect of endotoxin pretreatment on the pulmonary vascular response to hypoxia in O2-exposed lambs; Hazinski TA et al.; We recently reported that endotoxin infusion before O2 exposure significantly reduced or delayed the onset of pulmonary edema formation and respiratory failure by reducing the oxidant stress of O2 exposure . Despite these beneficial effects of endotoxin treatment, lung microvascular permeability eventually increased, but postmortem lung water content was less than expected . Prolonged O2 breathing blunts or abolishes the pulmonary constrictor response to alveolar hypoxia in some species, and it is possible that the loss of this response could contribute further to edema formation . To determine whether the reduction in lung edema observed in endotoxin-treated, O2-exposed lambs was linked to the preservation of hypoxic pulmonary vasoconstriction (HPV), we measured pulmonary vascular resistance before and after 8 min of isocarbic hypoxia (inspired O2 fraction 0.12) during each day of O2 exposure . In six control lambs, the pressor response to hypoxia was abolished after 72 h in O2, and the lambs developed respiratory failure shortly thereafter . In six endotoxin-treated lambs, HPV was preserved for as long as 144 h of O2 exposure . In two control O2-exposed lambs in whom HPV was abolished, the infusion of either angiotensin or prostaglandin H2 analogue increased pulmonary vascular resistance by greater than 75% . We conclude that in lambs 1) hyperoxia abolishes the pulmonary vascular response to hypoxia, 2) endotoxin pretreatment reduces acute O2-induced lung injury and preserves the pulmonary constrictor response to hypoxia, and 3) the loss of HPV during O2 exposure may be the result of oxidant-mediated injury to the hypoxia response itself and not the result of diffuse damage to the vasoconstrictor effector mechanism. J Appl Physiol, 1988 Oct, 65(4), 1579 - 85 Pulmonary O2 toxicity in lambs: physiological and biochemical effects of endotoxin infusion; Hazinski TA et al.; Hyperoxic adult rats have prolonged survival and reduced morphological evidence of lung injury when treated with a single dose of bacterial endotoxin; this effect is mediated by an augmentation of antioxidant enzyme activity in lung homogenate . To determine whether endotoxin would prolong survival and influence antioxidant enzyme levels in lambs whose physiological response to O2 breathing can be serially measured, we administered a single intravenous dose of endotoxin (0.75 microgram/kg body wt) to 13 lambs before exposing them to greater than 95% O2 (n = 11) or air (n = 2) . Seven additional lambs were placed in O2 after receiving only saline vehicle . All lambs had been instrumented to measure pulmonary vascular pressures and cardiac output, and 10 lambs had lung lymph fistulas . O2-exposed control lambs developed noncardiogenic pulmonary edema and respiratory failure within 85 +/- 10 h (range 76-110 h); antioxidant enzymes were not increased, but reduced glutathione (GSH) levels fell and oxidized glutathione (GSSG) increased, reflecting the oxidant stress of O2 exposure . By contrast, endotoxin-treated O2-exposed lambs had a delayed increase in microvascular permeability to protein, a reduced rate of lung edema formation, normal gas exchange after 72 h in O2, and prolonged survival (136 +/- 15 h; range 90-160 h; all variables P less than 0.05) . Despite prolonged survival, postmortem lung water content was no greater in the lambs that received endotoxin . Treatment with endotoxin did not increase antioxidant enzyme levels in lung homogenate, but levels of GSH relative to GSSG were significantly elevated.(ABSTRACT TRUNCATED AT 250 WORDS) Epidemiol Infect, 1988 Oct, 101(2), 239 - 47 A two-year survey of the incidence of heat-labile enterotoxin-producing Escherichia coli and other enteric pathogens in travellers returning to the Sheffield area; Chapman PA et al.; A case-controlled study of the incidence of heat-labile enterotoxin-producing Escherichia coli (LT+ETEC) and other enteric pathogens in travellers returning to the Sheffield area was conducted from May 1984 to April 1986 . LT+ETEC were found in 35 (5.8%) of 600 travellers to developed countries (mainly popular Mediterranean holiday resorts), 36 (11.3%) of 320 travellers to less-developed countries, and 11 (0.9%) of 1282 control patients whose illness was not associated with recent travel abroad . A seasonal peak of LT+ETEC infection was observed only in travellers to developed countries, with infections being significantly commoner in August to October . There was no significant deviation from expected age/sex distribution of LT+ETEC infection . Strains of LT+ETEC from travellers produced more toxin than strains from control patients, strains from travellers to less-developed countries producing most of all. Proc Natl Acad Sci U S A, 1988 Oct, 85(20), 7709 - 13 Identification of specific residues of human interleukin 2 that affect binding to the 70-kDa subunit (p70) of the interleukin 2 receptor; Collins L et al.; Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70) . Substitution of Asp20 or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high-affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor . These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit . The presence of the carboxylic acid in the side chain of Asp20 was necessary for effective binding to the p70 protein . In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays . The effects of these mutations on protein conformation were assessed by circular dichroism . These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity. Proc Natl Acad Sci U S A, 1988 Oct, 85(20), 7685 - 9 Alteration of the amino terminus of the mature sequence of a periplasmic protein can severely affect protein export in Escherichia coli; Li P et al.; Escherichia coli alkaline phosphatase, coded for by the phoA gene, is normally translocated across the cytoplasmic membrane into the periplasm with high efficiency . We have constructed a series of derivatives of the phoA gene that code for a wild-type signal sequence but result in altered amino acid sequences at the amino terminus of the mature alkaline phosphatase . Our results suggest that the presence of two positively charged amino acids very early in the mature sequence interferes significantly with protein export . In one case, phoA2AB, the presence of the sequence Arg-Ile-Arg at the amino terminus of alkaline phosphatase results in a 50-times reduction in the export of the protein . By using oligonucleotide-directed mutagenesis, we have constructed mutant derivatives of phoA2AB that are greatly enhanced for export . In all cases, these derivatives reduce the net positive charge in the region . Our results may explain the failure of E . coli to export a number of proteins coded for by artificial constructs and suggest a way to improve export in these cases. Proc Natl Acad Sci U S A, 1988 Oct, 85(19), 7270 - 3 Coexpression and assembly of myosin heavy chain and myosin light chain in Escherichia coli; McNally EM et al.; A fragment of the Dictyostelium discoideum myosin heavy chain gene representing heavy meromyosin was coexpressed in Escherichia coli with the entire essential myosin light chain from the scallop . The expressed myosin heavy chain and essential myosin light chain copurify through ammonium sulfate fractionation, anion exchange, and gel filtration chromatography . The purified complex consists of about 1 mol of light chain per mol of heavy chain . This stoichiometry, which is that of native myosin, suggests that no special eukaryotic machinery is required for coassembly of these two proteins . By coexpressing different myosin heavy chain and myosin light chain combinations, it should be possible to study various isoforms of these two proteins, which are both products of multigene families in mammals . E . coli is thus an ideal system in which to study expression and multimeric assembly of individual components of the eukaryotic contractile apparatus. Proc Natl Acad Sci U S A, 1988 Oct, 85(19), 7144 - 8 Coregulation of processing and translation: mature 5' termini of Escherichia coli 23S ribosomal RNA form in polysomes; Srivastava AK et al.; In Escherichia coli, the final maturation of rRNA occurs in precursor particles, and recent experiments have suggested that ongoing protein synthesis may somehow be required for maturation to occur . The protein synthesis requirement for the formation of the 5' terminus of 23S rRNA has been clarified in vitro by varying the substrate of the reaction . In cell extracts, pre-23S rRNA in free ribosomes was not matured, but that in polysomes was efficiently processed . The reaction occurred in polysomes without the need for an energy source or other additives required for protein synthesis . Furthermore, when polysomes were dissociated into ribosomal subunits, they were no longer substrates for maturation; but the ribosomes became substrates again when they once more were incubated in the conditions for protein synthesis . All of these results are consistent with the notion that protein synthesis serves to form a polysomal complex that is the true substrate for maturation . Ribosomes in polysomes, possibly in the form of 70S initiation complexes, may more easily adopt a conformation that facilitates maturation cleavage . As a result, the rates of ribosome formation and protein synthesis could be coregulated. Proc Natl Acad Sci U S A, 1988 Oct, 85(19), 7069 - 73 The B- to Z-DNA equilibrium in vivo is perturbed by biological processes; Zacharias W et al.; Right-handed B and left-handed Z conformations coexist in equilibrium in portions of plasmids in Escherichia coli . The equilibria are influenced by the length of the sequences that undergo the structural transitions and are perturbed by biological processes . The composite results of three types of determinations indicate a supercoil density of -0.025 in vivo . The coexistence of alternative DNA conformations in living cells implies the potential of these structures or their transitions for important functions in genetic regulatory processes. Mutat Res, 1988 Oct, 201(2), 423 - 30 A genetic assay for aneuploidy: quantitation of chromosome loss using a mouse/human monochromosomal hybrid cell line; Sandhu SS et al.; A genetic assay is described in which a mouse/human hybrid cell line R3-5 containing a single human chromosome (a monochromosomal hybrid) is used to detect chemically induced aneuploidy . In this assay the frequency of chromosome loss determined by the cloning efficiency of the cells in a selection medium is used as an index for the potential of a chemical to induce aneuploidy . The hybrid cells are deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT) and contain human chromosome 2, marked with Ecogpt, an E . coli gene for xanthine guanine phosphoribosyltransferase . These cells with a genotype of hgprt-/Ecogpt+ can grow in medium containing mycophenolic acid and xanthine (MX medium) but not in medium containing 6-thioguanine (6-TG) . The loss of the human chromosome from R3-5 cells as a result of chemical treatment produces cells with a genotype of hgprt-/Ecogpt- which are capable of growth in the medium containing 6-TG . Thus, the cloning efficiency of cells treated with a test chemical in 6-TG provides a method to determine the frequency of cells that have lost the human chromosome . Two chemicals, colcemid and nocodazole, previously known to induce aneuploidy in mammalian cells were used for a preliminary evaluation of this test system . Both of these compounds at concentrations ranging from 0.002 to 0.032 micrograms/ml showed a concentration-related positive response in this assay. J Med Microbiol, 1988 Oct, 27(2), 99 - 103 Effects of chlorpromazine, berberine and verapamil on Escherichia coli heat-labile enterotoxin-induced intestinal hypersecretion in rabbit ileal loops; Askri H et al.; The effects of chlorpromazine (CPZ), berberine and verapamil on intestinal hypersecretion in the rabbit ileal loop model by the heat-labile enterotoxin (LT) of Escherichia coli were studied in relation to their ability to inhibit the stimulation of intestinal adenylate cyclase (AC) by LT . CPZ 5 mg by the intraluminal (i.1.) route and 4 mg/kg by the intramuscular (i.m.) route significantly reduced LT-induced intestinal hypersecretion . Berberine (10 mg) exerted an inhibitory effect, but only after i.1 . administration, whereas verapamil did not exert any significant inhibitory effect when administered either i.1 . (2.5 mg) or i.m . (4 mg/kg) . At concentrations of (0.17-1.34) X 10(-3) M CPZ, the anti-secretory effect of CPZ correlated with its inhibitory effect on rabbit LT-stimulated intestinal AC . Inhibition of cAMP synthesis was probably not involved in the mechanism of action of the two other substances . These results indicate that CPZ and phenothiazines in general are efficient drugs for reducing LT-induced intestinal hypersecretion and could represent a model for synthesis of new anti-secretory drugs with no tranquiliser side effects. J Bacteriol, 1988 Oct, 170(10), 4969 - 71 Availability of porphobilinogen controls appearance of porphobilinogen deaminase activity in Escherichia coli K-12; Umanoff H et al.; A hemin-permeable hemB mutant had no 5-aminolevulinate dehydratase (ALA D) and extremely low porphobilinogen deaminase (PBG D) activity . When the structural gene for hemB was introduced into this strain on a single-copy plasmid, both activities were observed . When the mutant was grown on PBG, normal PBG D activity was observed . Moreover, a hemA mutant had little or no PBG D activity unless it was grown on ALA or PBG . Neither hemin nor PBG affected the level of PBG D protein produced from in vitro transcription and translation of a plasmid harboring the hemC gene as an insert . We conclude that, in Escherichia coli, PBG availability controls the activity of PBG D at some posttranscriptional level. J Bacteriol, 1988 Oct, 170(10), 4967 - 8 Very short patch mismatch repair activity associated with gene dcm is not conferred by a plasmid coding for EcoRII methylase; Lieb M et al.; The only cytosine methylase in Escherichia coli K-12 methylates the second cytosine in the sequence CC (A/T)GG and is encoded by gene dcm . Methylation and very short patch mismatch repair activities lacking in a dcm mutant of E . coli were restored by a plasmid containing the cloned dcm gene . In contrast, plasmids with the gene for EcoRII methylase, which is a homolog of dcm, restored only cytosine methylase activity and not mismatch repair. J Bacteriol, 1988 Oct, 170(10), 4958 - 9 Direction of conjugative transfer of IncI1 plasmid ColIb-P9; Howland CJ et al.; The origin-of-transfer region of ColIb-P9 was inserted into a lambda prophage to give a bacterial chromosome mobilizable by the parental conjugative plasmid . The polarity of mobilization of chromosomal genes indicated that ColIb-P9 transfer is unidirectional, such that the transfer genes adjacent to oriT enter the recipient cell last. J Bacteriol, 1988 Oct, 170(10), 4946 - 9 DNA sequence of the gene (tyrP) encoding the tyrosine-specific transport system of Escherichia coli; Wookey PJ et al.; The nucleotide sequence of 1,947 bases of DNA containing the tyrP structural gene was determined, and an open reading frame of 1,260 nucleotides was identified . The putative structural gene encodes an extremely hydrophobic protein which comprises 404 amino acids, 70% of which are nonpolar, and which has a molecular weight of 43,261. J Bacteriol, 1988 Oct, 170(10), 4798 - 807 Translational coupling between the ilvD and ilvA genes of Escherichia coli; Harms E et al.; The hypothesis that translation of the ilvD and ilvA genes of Escherichia coli may be linked has been examined in strains in which lacZ-ilvD protein fusions are translated in all three reading frames with respect to ilvD . In these strains, the nucleotide sequence was altered to obtain premature termination of ilvD translation, and in one strain translation termination of ilvD DNA occurred two bases downstream of the ilvA initiation codon . In the wild-type strain, the ilvD translation termination site was located two bases upstream of the ilvA start codon . In each of the mutant strains, expression of ilvA, as determined by the level of threonine deaminase activity, was strikingly lower than in the wild-type strain . The data suggest that expression of ilvD and ilvA is translationally coupled . By inserting a promoterless cat gene downstream of ilvA, it was shown that the differences in enzyme activity were not the result of differences in the amount of ilvA mRNA produced. J Bacteriol, 1988 Oct, 170(10), 4714 - 7 Effects of release factor context at UAA codons in Escherichia coli; Martin R et al.; In Escherichia coli, nonsense suppression at UAA codons is governed by the competition between a suppressor tRNA and the translational release factors RF1 and RF2 . We have employed plasmids carrying the genes for RF1 and RF2 to measure release factor preference at UAA codons at 13 different sites in the lacI gene . We show here that the activity of RF1 and RF2 varies according to messenger context . RF1 is favored at UAA codons which are efficiently suppressed . RF2 is preferred at poorly suppressed sites. J Bacteriol, 1988 Oct, 170(10), 4706 - 13 Transcriptional analysis of the major surface array gene of Caulobacter crescentus; Fisher JA et al.; The major component of the paracrystalline surface array of Caulobacter crescentus CB15 and one of the most abundant cellular proteins is a protein designated 130K . We have determined the DNA sequence of the 5' portion of the 130K gene, including the N-terminal one-third of the protein coding region, and analyzed the transcription of the gene . The site of transcription initiation was determined by S1 mapping of Caulobacter RNA . Although the DNA sequence upstream from the transcription start site showed significant homology to the consensus promoter sequences of Escherichia coli, S1 analysis of RNA from E . coli carrying the 130K gene on a plasmid indicated that the 130K promoter was not transcribed by E . coli RNA polymerase in vivo . Quantitative S1 analysis of RNA isolated from synchronously growing Caulobacter cells suggested that this promoter was not under developmental regulation; the amount of 130K transcript varied no more than 1.5-fold during the cell cycle . The length of the 130K mRNA was determined to be 3.3 kilobases by Northern (RNA blot) analysis, indicating that the 130K mRNA is not part of a polycistron . The amino acid sequence predicted from the DNA sequence agreed well with the N-terminal amino acid sequence determined by sequencing of the 130K protein . The 130K protein appears to be synthesized without an N-terminal leader sequence, but the N-terminal 20 amino acids are relatively hydrophobic and may function like a signal sequence during transmembrane translocation. J Bacteriol, 1988 Oct, 170(10), 4619 - 24 Determinations of the DNA sequence of the mreB gene and of the gene products of the mre region that function in formation of the rod shape of Escherichia coli cells; Doi M et al.; The 6.5-kilobase mre region at 71 min in the Escherichia coli chromosome map, where genes involved in formation of a rod-shaped cell form a gene cluster, was analyzed by in vivo protein synthesis in a maxicell system and by base sequencing of DNA . An open reading frame that may code for a protein with an Mr of about 37,000 on sodium dodecyl sulfate-polyacrylamide gels was found and was correlated with the mreB gene . N-terminal amino acid sequencing of the hybrid mreB-lacZ protein confirmed the production by mreB of a protein of 347 amino acid residues with a molecular weight of 36,958 . The amino acid sequence of this protein deduced from the DNA sequence showed close similarity with that of a protein of the ftsA gene which is involved in cell division of E . coli . Three other contiguous genes that formed three proteins with Mrs of about 40,000, 22,000, and 51,000, respectively, were detected downstream of the mreB gene by in vivo protein synthesis . The mreB protein and some of these three proteins may function together in determination of cell shape. J Bacteriol, 1988 Oct, 170(10), 4598 - 602 Overproduction of MalK protein prevents expression of the Escherichia coli mal regulon; Reyes M et al.; The mal regulon of Escherichia coli comprises a large family of genes whose function is the metabolism of linear maltooligosaccharides . Five gene products are required for the active accumulation of maltodextrins as large as maltoheptaose . Two cytoplasmic gene products are necessary and sufficient for the intracellular catabolism of these sugars . Two newly discovered enzymes have the capacity to metabolize these sugars but are not essential for their catabolism in wild-type cells . A single regulatory protein, MalT, positively regulates the expression of all of these genes in response to intracellular inducers, one of which has been identified as maltotriose . In the course of studying the mechanism of the transport system, we have placed the structural gene for one of the transport proteins, MalK, under the control of the Ptrc promoter to produce large amounts of this protein . We found that although high-level expression of MalK was not detrimental to E . coli, the increased amount of MalK decreased the basal-level expression of the mal regulon and prevented induction of the mal system even in the presence of external maltooligosaccharides . Constitutive mutants in which MalT does not depend on the presence of the internal inducer(s) were unaffected by the increased levels of the MalK protein . These results are consistent with the idea that MalK protein somehow interferes with the activity of the MalT protein . Different models for the regulatory function of MalK are discussed. J Bacteriol, 1988 Oct, 170(10), 4542 - 7 Nucleotide sequence of the xth gene of Escherichia coli K-12; Saporito SM et al.; The xth gene of Escherichia coli K-12, which encodes exonuclease III, has been sequenced . Exonuclease III from a cloned copy of the E . coli K-12 gene has been purified and characterized . The molecular weight (30,921), the amino-terminal amino acid sequence, and the amino acid composition of the polypeptide predicted from the nucleotide sequence are in excellent agreement with those properties determined for the purified enzyme . The xth promoter was mapped by primer extension of in vivo transcripts . Inspection of the nucleotide sequence reveals that a region of dyad symmetry which could form a hairpin stem-loop structure in RNA characteristic of a rho-dependent terminator lies immediately downstream from the xth gene. J Bacteriol, 1988 Oct, 170(10), 4528 - 36 Isolation, hyperexpression, and sequencing of the aceA gene encoding isocitrate lyase in Escherichia coli; Matsuoka M et al.; A structural gene for isocitrate lyase was isolated from a cosmid containing an ace locus of the Escherichia coli chromosome . Cloning and expression under control of the tac promoter in a multicopy plasmid showed that a 1.7-kilobase-pair DNA segment was sufficient for complementation of an aceA deletion mutation and overproduction of isocitrate lyase . DNA sequence analysis of the cloned gene and N-terminal protein sequencing of the cloned and wild-type enzymes revealed an entire aceA gene which encodes a 429-amino-acid residue polypeptide whose C-terminus is histidine . The deduced amino acid sequence for the 47.2-kilodalton subunit of E . coli isocitrate lyase could be aligned with that for the 64.8-kilodalton subunit of the castor bean enzyme with 39% identity except for limited N- and C-terminal regions and a 103-residue stretch that was unique for the plant enzyme and started approximately in the middle of that peptide. J Bacteriol, 1988 Oct, 170(10), 4516 - 21 Maltose chemoreceptor of Escherichia coli: interaction of maltose-binding protein and the tar signal transducer; Kossmann M et al.; The maltose chemoreceptor in Escherichia coli consists of the periplasmic maltose-binding protein (MBP) and the Tar signal transducer, which is localized in the cytoplasmic membrane . We previously isolated strains containing malE mutations that cause specific defects in the chemotactic function of MBP . Four of these mutations have now been characterized by DNA sequence analysis . Two of them replace threonine at residue 53 of MBP with isoleucine (MBP-TI53), one replaces an aspartate at residue 55 with asparagine (MBP-DN55), and the fourth replaces threonine at residue 345 with isoleucine (MBP-TI345) . The chemotactic defects of MBP-TI53 and MBP-DN55, but not of MBP-TI345, are suppressed by mutations in the tar gene . Of the tar mutations, the most effective suppressor (isolated independently three times) replaces Arg-73 of Tar with tryptophan . Two other tar mutations that disrupt the aspartate chemoreceptor function of Tar also suppress the maltose taxis defects associated with MBP-TI53 and MBP-DN55 . One of these mutations introduces glutamine at residue 73 of Tar, the other replaces arginine at residue 69 of Tar with cysteine . These results suggest that regions of MBP that include residues 53 to 55 and residue 345 are important for the interaction with Tar . In turn, arginines at residues 69 and 73 of Tar must be involved in the recognition of maltose-bound MBP and/or in the production of the attractant signal generated by Tar in response to maltose-bound MBP. J Bacteriol, 1988 Oct, 170(10), 4509 - 15 Aspartate taxis mutants of the Escherichia coli tar chemoreceptor; Wolff C et al.; The Tar protein of Escherichia coli belongs to a family of methyl-accepting inner membrane proteins that mediate chemotactic responses to a variety of compounds . These transmembrane signalers monitor the chemical environment by means of specific ligand-binding sites arrayed on the periplasmic side of the membrane, and in turn control cytoplasmic signals that modulate the flagellar rotational machinery . The periplasmic receptor domain of Tar senses two quite different chemoeffectors, aspartate and maltose . Aspartate is detected through direct binding to Tar molecules, whereas maltose is detected indirectly when complexed with the periplasmic maltose-binding protein . Saturating levels of either aspartate or maltose do not block behavioral responses to the other compound, indicating that the detection sites for these two attractants are not identical . We initiated structure-function studies of these chemoreceptor sites by isolating tar mutants which eliminate aspartate or maltose taxis, while retaining the ability to respond to the other chemoeffector . Mutants with greatly reduced aspartate taxis are described and characterized in this report . When present in single copy in the chromosome, these tar mutations generally eliminated chemotactic responses to aspartate and structurally related compounds, such as glutamate and methionine . Residual responses to these compounds were shifted to higher concentrations, indicating a reduced affinity of the aspartate-binding site in the mutant receptors . Maltose responses in the mutants ranged from 10 to 80% of normal, but had no detectable threshold shifts, indicating that these receptor alterations may have little effect on maltose detection sensitivity . The mutational changes in 17 mutants were determined by DNA sequence analysis . Each mutant exhibited a single amino acid replacement at residue 64, 69, or 73 in the Tar molecule . The wild-type Tar transducer contains arginines at all three of these positions, implying that electrostatic forces may play an important role in aspartate detection. J Bacteriol, 1988 Oct, 170(10), 4484 - 92 Feedback regulation of the spc operon in Escherichia coli: translational coupling and mRNA processing; Mattheakis LC et al.; The spc operon of Escherichia coli encodes 10 ribosomal proteins in the order L14, L24, L5, S14, S8, L6, L18, S5, L30, and L15 . This operon is feedback regulated by S8, which binds near the translation start site of L5 and inhibits translation of L5 directly and that of the distal genes indirectly . We constructed plasmids carrying a major portion of the spc operon genes under lac transcriptional control . The plasmids carried a point mutation in the S8 target site which abolished regulation and resulted in overproduction of plasmid-encoded ribosomal proteins upon induction . We showed that alteration of the AUG start codon of L5 to UAG decreased the synthesis rates of plasmid-encoded distal proteins, as well as L5, by approximately 20-fold, with a much smaller (if any) effect on mRNA synthesis rates, indicating coupling of the distal cistrons' translation with the translation of L5 . This conclusion was also supported by experiments in which S8 was overproduced in trans . In this case, there was a threefold reduction in the synthesis rates of chromosome-encoded L5 and the distal spc operon proteins, but no decrease in the mRNA synthesis rate . These observations also suggest that transcription from ribosomal protein promoters may be special, perhaps able to overcome transcription termination signals . We also analyzed the state of ribosomal protein mRNA after overproduction of S8 in these experiments and found that repression of ribosomal protein synthesis was accompanied by stimulation of processing (and degradation) of spc operon mRNA . The possible role of mRNA degradation in tightening the regulation is discussed. J Bacteriol, 1988 Oct, 170(10), 4445 - 50 Two regions of mature periplasmic maltose-binding protein of Escherichia coli involved in secretion; Duplay P et al.; Six mutations in malE, the structural gene for the periplasmic maltose-binding protein (MBP) from Escherichia coli, prevent growth on maltose as a carbon source, as well as release of the mutant proteins by the cold osmotic-shock procedure . These mutations correspond to insertion of an oligonucleotide linker, concomitant with a deletion . One of the mutations (malE127) affects the N-terminal extension (the signal peptide), whereas the five others lie within the mature protein . As expected, the export of protein MalE127 is blocked at an early stage . This protein is neither processed to maturity nor sensitive to proteinase K in spheroplasts . In contrast, in the five other mutants, the signal peptide is cleaved and the protein is accessible to proteinase K added to spheroplasts . This indicates that the five mutant proteins are, at least in part, exported through the inner membrane . We propose that the corresponding mutations define two regions of the mature protein (between residues 18 and 42 and between residues 280 and 306), which are important for release of the protein from the inner membrane into the periplasm . We discuss the results in terms of possible conformational changes at this late step of export to the periplasm. Infect Immun, 1988 Oct, 56(10), 2723 - 30 Quantitative relationship between capsular content and killing of K1-encapsulated Escherichia coli; Vermeulen C et al.; Since there are conflicting reports in the literature on a possible relationship between the K1 capsular polysaccharide (CP) content of Escherichia coli and its susceptibility to killing, we reexamined this issue in a strain that had a smooth lipopolysaccharide (LPS) phenotype (E . coli O18:K1:H7 Bort) and in a strain with a deep rough LPS phenotype (E412, spontaneously agglutinable: K1:H-) . When cell-associated K1 capsular content was greater than 90 micrograms of K1 polysaccharide per 10(10) CFU, neither strain was lysed by 20% normal human serum . In contrast, at equivalent but lower levels of K1 CP content, E412 but not strain Bort was lysed by normal human serum . Thus, LPS phenotype is an additional surface determinant that affects bacterial susceptibility to killing . Organisms obtained from very early log phase, when cell-associated K1 CP is greatest, were significantly more virulent for mice than were bacteria harvested in stationary phase, when cell-associated K1 polysaccharide is lowest . We conclude that (i) there is a threshold level of K1 CP needed to confer protection from lysis by serum, and this is usually exceeded under standard growth conditions; (ii) at a given level of K1 CP the LPS phenotype is an important determinant of bacterial killing; and (iii) the loss of capsule at low pH may be an additional mechanism by which hosts defend against invasive infection by K1-encapsulated E . coli. Endocrinology, 1988 Oct, 123(4), 1848 - 53 Inhibition of parathyroid hormone bioactivity by human parathyroid hormone (PTH)-(3-84) and PTH-(8-84) synthesized in Escherichia coli; Born W et al.; Human PTH (hPTH)-(3-84) and hPTH-(8-84) were synthesized in Escherichia (E.) coli when the cells were transformed with a multicopy plasmid in which the transcription of human preproPTH cDNA is directed by the E . coli lac promoter . PTH fragments were extracted from cells and purified by reverse phase HPLC . PTH bioactivity and PTH antagonist activity were estimated in a renal cytochemical bioassay . hPTH-(3-84) and hPTH-(8-84) exhibited less than 1% and less than 0.1%, respectively, of the biological activity of synthetic hPTH-(1-84) . hPTH-(8-84) had 1% of the PTH inhibitory activity of synthetic {Nle8,18,Tyr34}bovine PTH-(3-34)amide, whereas hPTH-(3-84) was 100 times more active as a PTH inhibitor than the synthetic bovine PTH-(3-34) analog . The latter has so far been recognized as the most potent PTH antagonist in vitro . A 5-fold molar excess of hPTH-(3-84) over hPTH-(1-84) completely blocked the biological action of intact hPTH-(1-84) in the renal cytochemical bioassay . These findings suggest that the carboxyl-terminal portion of the intact hPTH-(1-84) molecule contributes importantly to inhibitor potency. Cancer Res, 1988 Oct 1, 48(19), 5503 - 9 Preparation of anti-ras Mr 21,000 protein monoclonal antibodies and immunohistochemical analyses on expression of ras genes in human stomach and thyroid cancers; Yoshida K et al.; Sixteen clones (RASK-1 to -16) of murine monoclonal antibodies were raised against ras Mr 21,000 protein (p21) . The p21 produced by Escherichia coli with inserted v-Ki-ras genes was used as immunogen . RASK-1 was found to be specific for Ki-ras p21, whereas RASK-2 to -16 reacted with the p21s of Ki-, N-, and Ha-ras genes in both enzyme-linked immunosorbent and immunoblotting assays . Binding inhibition assays with biotinylated monoclonal antibodies by enzyme-linked immunosorbent assay showed that the monoclonal antibodies of the 16 clones included those binding to several mutually distinct sites on p21 . The expressions of ras p21 in human stomach and thyroid tissues were examined with RASK-3, which reacted with all the Ki-, N-, and Ha-ras p21s immunohistochemically by the avidin-biotin peroxidase complex method . Formalin-fixed, paraffin-embedded tissues of 101 cases of stomach cancer, 53 cases of noncancerous stomach, 74 cases of cancer of the thyroid, and 59 cases of noncancerous thyroid were analyzed . In both the stomach and thyroid, cancer cells expressed p21 predominantly . Cells of cases with various noncancerous disorders as well as certain types of normal cells were also p21 positive . These findings suggest that precaution is required in use of p21 as a cancer marker . Expression of p21 was noted in moderately to well-differentiated stomach cancer, intestinal metaplasia, and atypical hyperplasia . This finding suggests that the appearance of p21 in stomach cancer may be initiated before cytological transformation. J Biochem (Tokyo), 1988 Oct, 104(4), 643 - 7 Synthesis of recombinant human single-chain urokinase-type plasminogen activator variants resistant to plasmin and thrombin; Miyake T et al.; Single-chain urokinase-type plasminogen activator (scu-PA), a potential therapeutic reagent for thrombosis, is activated in plasma by plasmin . The activated enzyme is further digested by plasmin to generate low-molecular-weight urokinase (LMW-UK), which has no affinity for fibrin . To circumvent this dual effect of plasmin, we synthesized in Escherichia coli a variant of scu-PA, which is not converted to LMW-UK on treatment with plasmin . In another variant, the activation cleavage site was modified such that activation by plasmin was slowed down and that inactivation by thrombin was greatly diminished . The combination of these variants may be applicable as an effective thrombolytic reagent for clinical use. Mol Gen Genet, 1988 Oct, 214(2), 317 - 20 2-Aminopurine and 5-bromouracil induce alleviation of type I restriction in Escherichia coli: mismatches function as inducing signals? Efimova EP, Delver EP, Belogurov AA. The EcoK restriction of unmodified phage lambda is 1000-fold alleviated in Escherichia coli grown in the presence of base analogs 2-aminopurine (2AP) and 5-bromouracil (5BU) . 2AP treatment of bacteria affects specifically the type I restriction systems (EcoA, EcoB, EcoD and EcoK) and does not influence type II (EcoRI) and type III (EcoP1) restriction . 2AP-induced alleviation of restriction occurs in bacteria which are deficient in the SOS response (recA and lexA) and mismatch repair (mutH, mutL and mutS) and can be distinguished from the alleviation of restriction observed in dam- strains . We suggest that mismatches induced by 2AP and 5BU may function as an inducing signal for the alleviation of restriction observed in the presence of base analogs. Mol Gen Genet, 1988 Oct, 214(2), 313 - 6 Alleviation of type I restriction in adenine methylase (dam) mutants of Escherichia coli; Efimova EP et al.; The host-controlled EcoK-restriction of unmodified phage lambda.O is alleviated in dam mutants of Escherichia coli by 100- to 300-fold . In addition, the EcoK modification activity is substantially decreased in dam- strains . We show that type I restriction (EcoB, EcoD and EcoK) is detectably alleviated in dam mutants . However, no relief of EcoRI restriction (Type II) occurs in dam- strains and only a slight effect of dam mutation on EcoP1 restriction (Type III) is observed . We interpret the alleviation of the type I restriction in dam- strains to be a consequence of induction of the function which interferes with type I restriction systems. Genetika, 1988 Oct, 24(10), 1739 - 51 {Rpo pathway as a possible recombination mechanism of the interaction between DNA of transducing phages and the Escherichia coli genome}; Kholodii GIa et al.; The conditions affecting recombination of DNAs of transducing lambdoid rifd phages with the chromosome of Escherichia coli K-12 recA in the region of homology were studied . In support to the previously obtained data, the Int system was shown to take no part in the process . The homologous character of recombination interactions and their dependence on efficiency of transcription were demonstrated . It is therefore suggested that recombination takes the Rpo pathway . Similar peculiarities were revealed in the processes of interaction between DNAs of rifd phages as well as of lambdoid phages carrying trp genes and the host genome . A hypothesis is put forward that the Rpo pathway operates, depending on the density of DNA supercoiling. J Antibiot (Tokyo), 1988 Oct, 41(10), 1452 - 61 Adenylylation of trospectomycin by crude enzyme preparations from Escherichia coli; Yagi Y et al.; Employing osmotically shocked lysate of a spectinomycin resistant strain of Escherichia coli, trospectomycin, a new alkylspectinomycin, was adenylylated in the presence of adenosine 5'-triphosphate and magnesium ion . A highly resistant strain of E . coli was obtained by transforming a laboratory strain with a newly constructed plasmid consisting of pBR322 and a determinant for spectinomycin resistance originally found on a low copy number plasmid in E . coli strain NR79 . The biologically inactive adenylylated trospectomycin was found to be trospectomycin 6-(5'-adenylate). EMBO J, 1988 Oct, 7(10), 3171 - 80 Rapid turnover of adenovirus E1A is determined through a co-translational mechanism that requires an aminoterminal domain; Slavicek JM et al.; The product of the adenovirus E1A 13S mRNA can both stimulate and repress the expression of certain viral and cellular genes . As with several other regulatory proteins, E1A has a short half-life, approximately 40 min . Although this short half-life is observed in cells expressing the E1A gene, it is not the case with cells injected with E1A protein, where its half-life is