Escherichia coli

Biochem J, 1983 Jul 1, 213(1), 187 - 91
Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli; Lewendon A et al.; A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described . Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield . The key purification step involves substrate elution of the enzyme from a cellulose phosphate column . The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate . The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.

Appl Environ Microbiol, 1983 Jul, 46(1), 37 - 43
Amplification of the aroA gene from Escherichia coli results in tolerance to the herbicide glyphosate; Rogers SG et al.; The predominant cellular target of the herbicide glyphosate is thought to be the enzyme 5-enolpyruvylshikimate-3-phosphoric acid synthase (EPSP synthase) . As a means of biologically testing this finding, we cloned a segment of DNA from Escherichia coli that encodes this enzyme . Clones carrying the gene for EPSP synthase were identified by genetic complementation . Cells that contain a multicopy plasmid carrying the EPSP synthase gene overproduce the enzyme 5- to 17-fold and exhibit at least an 8-fold increased tolerance to glyphosate . These experiments provide direct biological evidence that EPSP synthase is a major site of glyphosate action in E . coli and that, in an amplified form, it can serve as a selectable glyphosate resistance marker.

Appl Environ Microbiol, 1983 Jul, 46(1), 291 - 2
Effect of pH on conjugal transfer at low temperatures; Singleton P et al.; The inhibitory effects of nonoptimal temperature and nonoptimal pH on F-type conjugal transfer were found to be synergistic . This finding is briefly discussed in an environmental context.

J Clin Microbiol, 1983 Jul, 18(1), 23 - 8
Rapid test for identification of heat-labile enterotoxin-producing Escherichia coli colonies; Finkelstein RA et al.; A latex particle agglutination test is described which is suitable for the recognition of heat-labile enterotoxin-producing colonies of Escherichia coli immediately after primary culture on a variety of commonly used enteric diagnostic media . The test is simple and economical to perform, and the results are available in minutes.

J Clin Microbiol, 1983 Jul, 18(1), 199 - 202
Genetic probes for enterotoxigenic Escherichia coli isolated from childhood diarrhea in the Central African Republic; Georges MC et al.; Escherichia coli strains were isolated from 778 children with diarrhea and 151 well children in the Central African Republic over a period of 1 year . These 929 strains were assayed for heat-labile and heat-stable enterotoxin production and were hybridized (probed) with structural genes for these enterotoxins . Twenty-four isolates from diarrheal patients and one isolate from a well child were found to be toxigenic by assay and probe . Minor discrepancies were encountered with both assays and probes during initial screening procedures, but the two methodologies were ultimately comparable.

In Pract, 1983 Jul, 5(4), 135 - 46
Coliform mastitis; Jackson E et al.; The majority of coliform mastitis cases are relatively mild and self-limiting . Teat dipping and dry cow therapy will not prevent coliform problems, neither do they cause them . Bad housing and poor milking time hygiene lead to coliform mastitis . Efforts to control coliform mastitis must be directed at reducing exposure to the bacteria . In acute cases fluid therapy is indicated to counteract endotoxic shock.

Drugs, 1983 Jul, 26(1), 80 - 90
Acute secretory diarrheas . Current concepts in pathogenesis and treatment; Hughes S; Acute secretory diarrheas constitute a major source of mortality and morbidity world-wide . Our current understanding of the underlying mechanisms involved is reviewed with particular reference to cholera and enterotoxigenic E . coli infections . From the physiological principles involved, a unified concept for the treatment of acute secretory diarrheas is presented . The importance of rehydration is highlighted and practical instructions for the use of oral glucose-electrolyte solutions in the treatment of acute secretory diarrhoeas are given, along with some discussion of the rationale behind their use and optimum composition . The important role of nutritional factors during acute diarrhoea is underlined and the place of various drugs, some established, some experimental, are briefly discussed.

Radiat Res, 1983 Jul, 95(1), 158 - 64
The effects of radioprotectors on DNA polymerase I-directed repair synthesis and DNA strand breaks in toluene-treated and X-irradiated Escherichia coli; Billen D; In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is induced by exposure to X rays . This repair synthesis may be amplified and easily measured through inhibition of DNA ligase action . In an effort to learn more of the relationship between X-ray-induced strand breaks in cellular DNA and the extent of this repair synthesis, experiments designed to compare the influence of radioprotectors on both strand-break production and repair synthesis have been carried out . The results show that cysteamine, sodium formate, and glycerol not only protect against strand breaks but also reduce DNA polymerase I-directed repair synthesis . However, I-, an efficient hydroxyl radical scavenger, is not as effective a protective agent against strand breaks and does not measurably affect repair synthesis in our system.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4446 - 9
Targeted mutation at cytosine-containing pyrimidine dimers: studies of Escherichia coli B/r with acetophenone and 313-nm light; Fix D et al.; We have tested the two-event model for UV mutagenesis producing class 2 suppressor mutations at glutamine tRNA genes in Escherichia coli . In the model used, the induction/indexing lesion is any type of pyrimidine dimer and the premutational photoproduct at the target site is a cytosine-containing dimer . Specific mutation-frequency responses were analyzed under conditions in which the ratio of thymine-thymine dimers to cytosine-containing dimers was modified by using 313-nm light and 0.0%, 0.1%, or 0.2% acetophenone . Changes observed in the production of class 2 suppressor mutations were consistent with the model and suggested that the G X C leads to A X T transitions responsible for class 2 suppressor mutations are targeted by cytosine-containing pyrimidine dimers at the mutational sites.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4213 - 7
Testing an alternative model for the ribosomal peptide elongation cycle; Rheinberger HJ et al.; A kinetic analysis of poly(U)-dependent poly(Phe) synthesis with {14C}tRNAPhe and {3H}phenylalanine demonstrated that, in the course of efficient poly(Phe) synthesis, two tRNAs are present per 70S ribosome at all times, although at least 70% of the poly(Phe)-tRNAPhe is found at the peptidyl-tRNA (P) site . Together with our recent observation of a third tRNA-binding site on Escherichia coli ribosomes, these findings suggest a model for the peptide elongation cycle in which two tRNA molecules are present on the ribosome at both the pre- and the post-translocational state . This model predicts that deacylated tRNA is not released from the P site but translocated to the exit (E) site before release occurs . A series of translocation experiments with deacylated {14C}tRNAPhe at the P site and oligo {( 3H}Phe)-tRNA at the aminoacyl-tRNA (A) site proved that efficient elongation factor G-dependent translocation is not accompanied by a corresponding {14C}tRNAPhe release . However, significant {14C}tRNAPhe release was observed after translocation when an aminoacyl-tRNA was bound to the A site . Thus, deacylated tRNA is not released from the P site but is translocated to the E site, which therefore must be located "upstream" adjacent to the P site . Furthermore, the trigger for the release of deacylated tRNA from the E site is the binding of aminoacyl-tRNA to the A site.

Proc Natl Acad Sci U S A, 1983 Jul, 80(13), 3884 - 8
Iron superoxide dismutase from Escherichia coli at 3.1-A resolution: a structure unlike that of copper/zinc protein at both monomer and dimer levels; Stallings WC et al.; The structure of iron superoxide dismutase (EC 1.15.1.1) from Escherichia coli has been determined at 3.1-A resolution . The dimeric molecule is constructed from identical subunits, which are two-domain polypeptides . The NH2-terminal domain is composed of two antiparallel crossing helices and the COOH-terminal domain is a three-layered structure characterized by mixed alpha/beta secondary structural features . The active center iron atoms, separated by 18 A and located near the monomer-monomer interface, are coordinated by two amino acid residues from each domain . Azide binding has been investigated by using difference Fourier techniques . Consistent with the notion of the independent evolution of the copper/zinc dismutase gene, the iron dismutase structure resembles the copper/zinc protein at neither the monomer nor the dimer level.

J Virol, 1983 Jul, 47(1), 227 - 32
Construction of an infectious molecular clone of the autonomous parvovirus minute virus of mice; Merchlinsky MJ et al.; The linear single-stranded DNA genome of minute virus of mice, an autonomous parvovirus, was cloned in duplex form into the bacterial plasmid pBR322 . The recombinant clones of minute virus of mice were infectious when transfected into monolayers of human 324K cells and produced virus plaques with an efficiency of about 6% that obtained with duplex replicative-form DNA purified from cells infected with minute virus of mice . Southern blot analysis of transfected cells indicated that the cloned minute virus of mice genome requires both termini to be intact for excision and replication as a linear duplex molecule.

J Med Chem, 1983 Jul, 26(7), 990 - 6
Theory and application of molecular potential energy fields in molecular shape analysis: a quantitative structure--activity relationship study of 2,4-diamino-5-benzylpyrimidines as dihydrofolate reductase inhibitors; Hopfinger AJ; A general formalism, based upon molecular mechanics pairwise potential functions, has been developed to compute the molecular potential energy fields inherent to a given molecule in a given conformation . Molecular descriptors are derived from the potential energy fields, which can be used in QSAR studies based upon molecular shape analysis . These descriptors have been computed for a set of 2,4-diamino-5-benzylpyrimidines that are dihydrofolate reductase (DHFR) inhibitors . A QSAR is derived in which DHFR inhibition activity can be explained in terms of molecular shape, as represented by differences in molecular potential energy fields between pairs of superimposed molecules, and the sum of the pi constants of substituents on the 3- and 4-position of the benzyl ring . This QSAR is superior to one developed earlier (Hopfinger, A . J . J . Med . Chem . 1981, 24, 818) in which molecular shape is described by common overlap steric volume . Ancillary information defining the "active" conformation and electrostatic nature of the binding site are realized in the construction of the QSAR.

J Bacteriol, 1983 Jul, 155(1), 420 - 3
Inhibition of RNA synthesis by oxolinic acid is unrelated to average DNA supercoiling; Manes SH et al.; Oxolinic acid reduced RNA synthesis rates whether chromosome supercoiling decreased, increased, or remained unchanged . Thus, inhibition of RNA synthesis by oxolinic acid appears to involve factors other than average DNA supercoiling level . Coumermycin A1 caused RNA synthesis rates to increase or decrease roughly in parallel with DNA supercoiling.

J Bacteriol, 1983 Jul, 155(1), 412 - 6
Expression of the Rickettsia prowazekii citrate synthase gene in Escherichia coli; Wood DO et al.; Recombinant DNA techniques were used to isolate the Rickettsia prowazekii citrate synthase gene on the plasmid vector pBR322 by functional complementation of a gltA mutation of Escherichia coli K-12 . Analysis of citrate synthase activity in crude extracts revealed that the enzyme expressed in E . coli retains the regulatory control mechanisms characteristic of the rickettsial enzyme.

J Bacteriol, 1983 Jul, 155(1), 402 - 6
Involvement of a plasmid in Escherichia coli envelope alterations; Rosas SB et al.; A plasmid-containing wild-type Escherichia coli strain was treated with two plasmid-curing agents, sodium dodecyl sulfate and ethidium bromide . Plasmid elimination was accompanied by drastic changes in the morphology of the colonies . Analysis of the cured strain by scanning and transmission electron microscopy showed important alterations in size and morphology of the cells . Metabolic differences were also found between the wild-type and cured cells.

J Bacteriol, 1983 Jul, 155(1), 391 - 7
Structure of fumarate reductase on the cytoplasmic membrane of Escherichia coli; Lemire BD et al.; The terminal electron transfer enzyme fumarate reductase has been shown to be composed of a membrane-extrinsic catalytic dimer of 69- and 27-kilodalton (kd) subunits and a membrane-intrinsic anchor portion of 15- and 13-kd subunits . We prepared inverted membrane vesicles from a strain carrying the frd operon on a multicopy plasmid . When grown anaerobically on fumarate-containing medium, the membranes of this strain are highly enriched in fumarate reductase . When negatively stained preparations of these vesicles were examined with an electron microscope, they appeared to be covered with knob-like structures about 4 nm in diameter attached to the membrane by short stalks . Treatment of the membranes with chymotrypsin destroyed the 69-kd subunit, leaving the 27-, 15-, and 13-kd subunits bound to the membrane; these membranes appeared to retain remnants of the structure . Treatment of the membranes with 6 M urea removed the 69- and 27-kd subunits, leaving the anchor polypeptides intact . These vesicles appeared smooth and structureless . A functional four-subunit enzyme and the knob-like structure could be reconstituted by the addition of soluble catalytic subunits to the urea-stripped membranes . In addition to the vesicular structures, we observed unusual tubular structures which were covered with a helical array of fumarate reductase knobs.

J Bacteriol, 1983 Jul, 155(1), 275 - 80
Effects of inserting eight amino acid residues into the major lipoprotein on its assembly in the outer membrane of Escherichia coli; Inukai M et al.; A DNA sequence consisting of 24 base pairs was inserted into the structural gene (lpp) coding for the major lipoprotein of the Escherichia coli outer membrane which was carried on a high-copy-number plasmid in which expression was regulated through a lac promoter-operator region . This modification resulted in the insertion of eight amino acid residues, Glu-Glu-Phe-Leu-Glu-Glu-Phe-Leu, between the glutamine residue at position 9 and the leucine residue at position 10 of the wild-type lipoprotein sequence . When production of the mutant lipoprotein was induced by a lac inducer, the cells became swollen, showed unusual morphology, and eventually lysed . When the membrane fraction was analyzed after the induction, the mutant lipoprotein was found to have been normally secreted across the cytoplasmic membrane and assembled in the outer membrane . This lipoprotein was modified with glycerol and palmitic acid and even formed the bound form, which was linked covalently to peptidoglycan . The major difference between the membrane-associated mutant lipoprotein and the wild-type lipoprotein was that the mutant lipoprotein became sensitive to trypsin treatment . These results indicate that the substantial alteration in mutant lipoprotein structure near the amino-terminal end does not interfere with modification of the amino-terminal cysteine residue or cleavage of the signal peptide by the prolipoprotein-specific signal peptidase . However, this mutant lipoprotein assembled in the outer membrane appears to have deleterious effects with respect to envelope structure and cellular morphology and viability.

J Bacteriol, 1983 Jul, 155(1), 228 - 37
Coordination of flagella on filamentous cells of Escherichia coli; Ishihara A et al.; Video techniques were used to study the coordination of different flagella on single filamentous cells of Escherichia coli . Filamentous, nonseptate cells were produced by introducing a cell division mutation into a strain that was polyhook but otherwise wild type for chemotaxis . Markers for its flagellar motors (ordinary polyhook cells that had been fixed with glutaraldehyde) were attached with antihook antibodies . The markers were driven alternately clockwise and counterclockwise, at angular velocities comparable to those observed when wild-type cells are tethered to glass . The directions of rotation of different markers on the same cell were not correlated; reversals of the flagellar motors occurred asynchronously . The bias of the motors (the fraction of time spent spinning counterclockwise) changed with time . Variations in bias were correlated, provided that the motors were within a few micrometers of one another . Thus, although the directions of rotation of flagellar motors are not controlled by a common intracellular signal, their biases are . This signal appears to have a limited range.

J Bacteriol, 1983 Jul, 155(1), 113 - 21
Catabolism of phenylpropionic acid and its 3-hydroxy derivative by Escherichia coli; Burlingame R et al.; A number of laboratory strains and clinical isolates of Escherichia coli utilized several aromatic acids as sole sources of carbon for growth . E . coli K-12 used separate reactions to convert 3-phenylpropionic and 3-(3-hydroxyphenyl)propionic acids into 3-(2,3-dihydroxyphenyl)propionic acid which, after meta-fission of the benzene nucleus, gave succinate, pyruvate, and acetaldehyde as products . Enzyme assays and respirometry showed that all enzymes of this branched pathway were inducible and that syntheses of enzymes required to convert the two initial growth substrates into 3-(2,3-dihydroxyphenyl)propionate are under separate control . E . coli K-12 also grew with 3-hydroxycinnamic acid as sole source of carbon; the ability of cells to oxidize cinnamic and 3-phenylpropionic acids, and hydroxylated derivatives, was investigated . The lactone of 4-hydroxy-2-ketovaleric acid was isolated from enzymatic reaction mixtures and its properties, including optical activity, were recorded.

Infect Immun, 1983 Jul, 41(1), 383 - 90
Evidence that a new enterotoxin of Escherichia coli which activates adenylate cyclase in eucaryotic target cells is not plasmid mediated; Green BA et al.; Escherichia coli SA53 produces a new enterotoxin that has a biological activity similar to that of E . coli heat-labile enterotoxin (LT) but is not neutralized by antiserum against LT or cholera enterotoxin . Strain SA53 contained two plasmids, pRB1 (69.2 +/- 4.3 megadaltons) and pRB2 (57.6 +/- 5.3 megadaltons) . Studies were undertaken to determine whether either plasmid was required for production of the LT-like toxin . We isolated a derivative of SA53 lacking both plasmids and confirmed that radioactively labeled pRB1 and pRB2 DNAs failed to hybridize to total DNA digests of the cured strain . The new enterotoxin was still produced by the cured strain, demonstrating that the gene(s) encoding the toxin was not located on pRB1 or pRB2 and was most likely on the bacterial chromosome . Although sonic extracts from SA53 contained no detectable LT antigen, plasmid pRB1 DNA did contain sequences with partial homology to the LT-A and LT-B genes . No sequence homology with LT genes was detected with pRB2 DNA . When the enterotoxin plasmid pCG86 was introduced into a rifampin-resistant derivative of SA53, LT was produced . Thus, plasmid-coded LT could be produced in the E . coli SA53 host, and the sequences homologous to LT in pRB1 were cryptic.

Infect Immun, 1983 Jul, 41(1), 190 - 6
Physical and biological properties of U.S . standard endotoxin EC after exposure to ionizing radiation; Csako G et al.; Techniques that reduce the toxicity of bacterial endotoxins are useful for studying the relationship between structure and biological activity . We used ionizing radiation to detoxify a highly refined endotoxin preparation . U.S . standard endotoxin EC . Dose-dependent changes occurred by exposure to 60Co-radiation in the physical properties and biological activities of the endotoxin . Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis showed gradual loss of the polysaccharide components (O-side chain and R-core) from the endotoxin molecules . In contrast, although endotoxin revealed a complex absorption pattern in the UV range, radiation treatment failed to modify that pattern . Dose-related destruction of the primary toxic component, lipid A, was suggested by the results of activity tests: both the pyrogenicity and limulus reactivity of the endotoxin were destroyed by increasing doses of radiation . The results indicate that the detoxification is probably due to multiple effects of the ionizing radiation on bacterial lipopolysaccharides, and the action involves (i) the destruction of polysaccharide moieties and possibly (ii) the alteration of lipid A component of the endotoxin molecule.

Infect Immun, 1983 Jul, 41(1), 137 - 44
Biphasic effect of pertussis vaccine on serum insulin in mice; Hewlett EL et al.; Administration of pertussis vaccine, consisting of whole, killed Bordetella pertussis organisms, causes hyperinsulinemia and enhanced secretion of insulin in response to a variety of secretagogues in rats and mice . In examining the time course and properties of this phenomenon, we discovered two distinct and separate effects of the bacteria on glucose and insulin levels in mice . First, a heat-stable (80 degrees C for 30 min) component causes a brief hyperinsulinemia which is +measureable by 1 h, maximal at 8 h, and ends in less than 48 h . This effect appears to be due to B . pertussis endotoxin, is mimicked by Escherichia coli endotoxin, and is associated with a transient, mild hypoglycemia . Second, there is a heat-labile component of the B . pertussis organism which induces a sustained (greater than 14 days), dose-dependent hyperinsulinemia which reaches a maximum at 5 to 7 days and has no associated hypoglycemia . The two effects are further distinguishable in that the early, endotoxin-induced hyperinsulinemia exhibits the normal suppressibility by exogenous epinephrine, whereas epinephrine markedly enhances the hyperinsulinemia occurring at 7 days . These two effects of B . pertussis appear to be mediated by different mechanisms and may be important in the well-recognized reactogenicity of pertussis vaccine in humans.

Eur J Biochem, 1983 Jul 1, 133(3), 531 - 8
The accessibility of the active site and conformation states of the beta 2 subunit of tryptophan synthase studied by fluorescence quenching; Lane AN; The rate of quenching of the fluorescence of pyridoxal 5'-phosphate in the active site of the beta 2 subunit of tryptophan synthase from Escherichia coli was measured to estimate the accessibility of the coenzyme to the small molecules iodide and acrylamide . The alpha subunit and the substrate L-serine substantially reduced the quenching rate . For iodide, the order of decreasing quenching was: Schiff's base of N alpha-acetyl-lysine with pyridoxal 5'-phosphate greater than holo beta 2 subunit greater than holo alpha 2 beta 2 complex approximately equal to holo beta 2 subunit + L-serine greater than holo alpha 2 beta 2 complex + L-serine . The coenzyme in the beta 2 subunit is apparently freely accessible to both iodide and acrylamide (kappa approximately equal to 2 X 10(9) M-1 s-1), but the alpha subunit and L-serine decrease the rate by factors of 2-5 . Quenching of the fluorescence of the single tryptophan residue of the beta 2 subunit revealed that the apo and holo forms exist in different states, whereas the alpha subunit stabilizes a third conformation . As the alpha subunit binds to the beta 2 subunit, the tryptophan residue, which is within 2.2 nm of the active site of the beta 2 subunit, probably rotates with respect to the plane of the ring of the coenzyme, such that fluorescence energy transfer from tryptophan to pyridoxal phosphate is greatly reduced . The alpha subunit strongly protects the active-site ligand indole propanol phosphate from quenching with acrylamide, consistent with the active site being deep in a cleft in the protein . Iodide induces dissociation of the holo alpha 2 beta 2 complex {E . W . Miles & M . Moriguchi (1977) J . Biol . Chem . 252, 6594-6599} . The effect of iodide on the fluorescence properties of holo alpha 2 beta 2 complex allows us to estimate an upper limit for the dissociation constant for the alpha 2 beta 2 complex of 10(-8) M, in the absence of iodide.

Eur J Biochem, 1983 Jul 1, 133(3), 481 - 9
The pyruvate dehydrogenase complex of Escherichia coli K12 . Nucleotide sequence encoding the dihydrolipoamide acetyltransferase component; Stephens PE et al.; The nucleotide sequence of the aceF gene, which encodes the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase complex of Escherichia coli K12, has been determined using the dideoxy chain-termination method . The aceF gene comprises 1887 base pairs (629 codons excluding the initiation codon AUG); it is preceded by a short intercistronic segment of 14 base pairs containing a good ribosomal binding site, and it is followed closely by a potential rho-independent terminator . The results extend by 1980 base pairs the previously sequenced segment of 3780 base pairs containing the structural gene (aceE) of the pyruvate dehydrogenase component (E1) and they confirm that aceE and aceF are the proximal and distal genes of the ace operon . The amino terminus, carboxy-terminal sequence and amino acid composition of the acetyltransferase subunit predicted from the nucleotide sequence are in excellent agreement with previous studies with the purified protein . The predicted molecular weight (Mr = 65959) confirms experimental values derived from sedimentation equilibrium analysis and indicates that the higher values (78000-89000) that have been reported are due to unusual features of the protein that lead to anomalous mobilities during sodium dodecyl sulphate/polyacrylamide gel electrophoresis and in gel filtration . The primary structure fully supports conclusions, based on limited tryptic proteolysis, that the acetyltransferase subunit possesses two heterologous domains: the lipoyl domain and the subunit binding and catalytic domain . The lipoyl domain corresponds to the amino-terminal segment of the protein . It is acidic and contains three remarkably homologous repeating units of approximately 100 amino acids, each possessing a potential lipoyl binding site and a region that is characteristically rich in alanine and proline residues . The subunit binding and catalytic domain occupies most of the residual polypeptide in the carboxy-terminal segment.

Arch Pathol Lab Med, 1983 Jul, 107(7), 361 - 3
Dissimilar manifestations of intrauterine infection with echovirus 11 in premature twins; Bose CL et al.; Echoviruses cause neonatal disease following intrauterine and intrapartum acquisition of the organism or by nosocomial infection . Dizygous twins apparently became infected following transplacental transmission of echovirus 11 . At 5 days of age, both twins experienced poor feeding, lethargy and hypothermia, and evidence of coagulopathy and hepatitis . During the sixth week of illness, the convalescence of twin A was complicated by peritonitis and sepsis, and the infant died . Pathologic findings included scattered foci of dystrophic myocardial calcification, distortion of hepatic architecture with fibrous connective tissue surrounding regenerative nodules and large foci of dystrophic calcification, and adrenal hemorrhagic necrosis and calcification . Twin B recovered without sequelae . The disease in twin A was unusual because of the extensive myocardial involvement . Also of interest was the variability of disease in twins who presumably had received a similar inoculum of organism by the same route.

Science, 1983 Jul 1, 221(4605), 59 - 61
Requirement for signal peptide cleavage of Escherichia coli prolipoprotein; Inouye S et al.; Oligonucleotide-directed site-specific mutagenesis was applied to alter the cleavage site in the signal peptide of the major outer membrane lipoprotein of Escherichia coli . Replacing the glycine residue at the cleavage site with an alanine residue did not affect the processing of the signal peptide . However, when the same cleavage site was constructed by the deletion of the glycine residue, the signal peptide was no longer cleaved . These results indicate that stringent structural integrity at the cleavage site in the lipoprotein signal sequence is required for correct processing of prolipoprotein.

Cancer Res, 1983 Jul, 43(7), 3247 - 52
O6-Methylguanine-DNA methyltransferase of human lymphoid cells: structural and kinetic properties and absence in repair-deficient cells; Harris AL et al.; Human lymphoid cell lines contain a DNA repair enzyme which removes the mutagenic alkylation lesion O6-methylguanine from DNA . The enzyme transfers the methyl group to a protein cysteine residue, generating S-methylcysteine, and is inactivated as a consequence of the reaction . Apparently the methylated enzyme represents a dead-end complex . The transfer reaction is very rapid and is completed in less than 1 min at 37 degrees, but methyl group transfer from single-stranded DNA or heavily damaged DNA is less efficient . The active methyltransferase and the methylated protein both have molecular weights of 21,000 to 22,000, as determined by gel filtration . Lymphoid cell lines proficient in repair of O6-methylguanine in vivo, Mex+, contain 10,000 to 25,000 molecules of the methyltransferase per cell . In contrast, repair-deficient cell lines, Mex-, do not contain detectable amounts of the enzyme . The latter point was verified by applying a partial purification procedure for the enzyme to cell-free extracts from two Mex- cell lines.

J Gen Microbiol, 1983 Jul, 129 (Pt 7), 2277 - 83
Incompatibility properties of the IncFIII/FIV haemolytic plasmid pSU316 when integrated in the Escherichia coli chromosome; Navas J et al.; The haemolytic plasmid pSU316 is incompatible with members of the IncFIII and IncFIV incompatibility groups . Plasmid pSU307 (pSU316 hlyC::Tn5) was inserted by integrative suppression into the chromosome of JW112, a temperature-sensitive dnaA mutant of Escherichia coli . The incompatibility properties of this strain (SU51) were studied and it was found that: (1) plasmid pSU306 (pSU316 hlyA::Tn802) was rapidly lost from strain SU51 both at 30 degrees C and 42 degrees C; (2) the IncFIII plasmid pSU397 (ColB-K98::Tn802) was lost from strain SU51 and at 42 degrees C but not at 30 degrees C; and (3) the IncFIV plasmid R124 was stably maintained in strain SU51 at both temperatures . Revertants of pSU307 to the autonomous state could be obtained from SU51 . These revertants exerted incompatibility towards the prototype plasmids pSU306, pSU397 and R124 in the same way as pSU307 itself . Thus, strain SU51 provided a suitable method for distinguishing the three different incompatibility determinants of plasmid pSU316.

Biochimie, 1983 Jul, 65(7), 437 - 40
{Specific association of the CRP-cyclic AMP complex with the control region of the lactose operon of Escherichia coli K 12: a direct fluorimetric study using DNA fragments of different lengths}; Baudras A et al.; Specific-site binding of the cAMP . CRP complex to the control region of the lactose operon of E . coli was measured directly . All of the protein molecules did bind specifically, and the binding constant for the major CRP site was not dependent on the length (62, 219 or 301 base pairs) of the DNA fragments used . Comparing the values of the binding constant measured for the major site and for the weaker "operator" CRP site, and referring to the published "consensus sequence" derived from the known CRP sites in a series of operons, we suggest that two sub-sites support additive contribution to the total binding free energy.

Biochimie, 1983 Jul, 65(7), 417 - 25
CG sites and their nucleotide environment in the human gamma-delta-beta-globin gene cluster: distribution, frequency and possible frame for differential methylation; Poncet D et al.; Available restriction endonucleases including CG dinucleotides in their target sequences (most of them being unable to cut the DNA when the cytosine of the CG sequence is methylated) have been used to map cloned DNA covering the human gamma-delta-beta globin gene cluster . Since the human DNA fragments were cloned in Escherichia coli, only the internal cytosine in the sequence CCAT GG could be methylated . Thus, any recognized "CG enzyme" site can be detected since they are unmethylated . Results show that frequencies of "CG enzyme" sites regularly decrease from the gamma-globin region to the beta-globin region, the latter being very poor in "CG enzyme"' sites . The array of enzymes used here detects 4 times more CG sites than the classical MspI/HpaII system . Examination of previously sequenced parts of the gamma-delta-beta globin gene cluster shows that CG dinucleotides correspond to an average frequency of 1 out of 104 nucleotides in the gamma-globin region and 1 out of 138 nucleotides in the beta-globin region . In the gamma-globin region, 1 CG out of 4 or 5 may be detected by the enzymes used; the detected frequency is less than 1 out of 10 CG in the beta-region . Analysis of nucleotide environment around CG dinucleotides shows occurrence of local differences, the main sequences being CGG in the 5' side flanking the gamma genes and ACG in the corresponding area of the beta gene . The results presented introduce some new considerations about analysis of cytosine methylation which has been previously proposed as playing a role in the control of the activity of gamma, delta and beta genes respectively.

Plasmid, 1983 Jul, 10(1), 31 - 44
Absence of cis-acting transposition immunity with Tn7; Hassan DM et al.; It is possible in two steps to insert into the plasmid RP4 two copies of the transposon Tn7 . This was demonstrated using a wild-type Tn7 in the first step, and a Tn7 derivative (carrying an additional marker), in the second step . The two successive transpositions occurred with the same polarity and frequency . The genetic structures of the resulting plasmids, predicted from the phenotypes of the bacterial host, were confirmed by direct analysis of the plasmid DNAs . Thus, the phenomenon of cis-acting transposition immunity, described with Tn1 or Tn3, does not take place in the case of Tn7.

Plasmid, 1983 Jul, 10(1), 11 - 20
Cloning of an exclusion-determining fragment of the IncI plasmid, R144; Hartskeerl RA et al.; By cloning a distinct 8 MDa fragment of the IncI plasmid, R144, in the vector pACYC184, two recombinant plasmids were isolated . In these plasmids, pRAH303 and pRAH308, the inserted fragment was in opposite orientations . Both plasmids when present in a recipient strain caused a conjugation-specific exclusion in crosses with donor cells carrying the IncI plasmid R144 . Some derivatives of the recombinant plasmids in which parts were deleted, or in which Tn5 transposons were inserted, appeared to be exclusion negative . Analysis in minicells of the gene products of such plasmids together with those of the original recombinant plasmids revealed that the presence of two proteins, with apparent molecular weights of 13,000 and 19,000 Da could be correlated with the exclusion phenomenon.

Mol Biol (Mosk), 1983 Jul-Aug, 17(4), 809 - 17
{Directed fragmentation of tobacco mosaic virus RNA: excision of 3' terminal sections, containing coat protein genes of tobacco mosaic virus}; Rodionova NP et al.; The present work demonstrated that RNA of different Tobacco mosaic virus strains (common; A14; Ni118, U2; wheat strain) are selectively cleaved by RNase H from E . coli in the presence of oligodeoxyribonucleotide d(TTTTTTTCCGG) complementary to the stretch of TMV RNA from the 842-nd to the 852-nd nucleotide from 3'-end, e . g . corresponding to the region adjacent to the 3'-terminus of origin of assemble of TMV . Such cleavage yields two main products . One of them--the short S-fragment--is the 3'-terminal part of TMV RNA . This conclusion is based on its ability to accept histidine and its molecular weight is about 0.28 X 10(6) . The other main product of TMV RNA cleavage by RNase H is the L-fragment . L-fragment is the 5'-terminal moiety of TMV RNA, as follows from its ability to direct the in vitro synthesis of p110-130, which is known to be encoded in the 5'-terminal TMV RNA region.

Gene, 1983 Jul, 23(1), 35 - 40
A method for the generation of small pre-determined deletions in plasmid DNA: deletion analysis of the tetR region of vector pBR322; Heusterspreute M et al.; A general method is described that allows precise deletion of a chosen restriction fragment(s) from a plasmid having many cleavage sites for that restriction enzyme . The DNA to be deleted is first separated from the rest of the plasmid on a larger DNA fragment contained between two different unique restriction sites . This fragment is then subdigested by the restriction endonuclease of interest, which recognises two or more tetranucleotide (cohesive end or blunt end) sequences on the fragment, and is recloned between the two original unique restriction sites . The method is rapid, efficient, and the results are predictable . Examples are given in which predetermined HpaII (9 bp, 147 bp), TaqI (141 bp) and AluI (15 bp, 403 bp) fragments have been selectively removed from the tetR region of plasmid pBR322.

Genetika, 1983 Jul, 19(7), 1210 - 2
{Localization of Kpnl restriction sites in the r-determinant of plasmid NR1}; Perebitiuk AN et al.; Restriction KpnI sites on a molecule of the plasmid NR1 were localized . The position of KpnA (58,0 MD) and KpnB (1,0 MD) fragments on a physical map of the NR1 molecule was shown using endonucleases EcoRI and SaII . Both restriction sites are situated on a r-determinant of the EcoJ fragment of the plasmid NR1.

Biochem J, 1983 Jul 1, 213(1), 137 - 42
Studies by electron-paramagnetic-resonance spectroscopy of the molybdenum centre of spinach (Spinacia oleracea) nitrate reductase; Gutteridge S et al.; The molybdenum centre of spinach (Spinacia oleracea) nitrate reductase has been investigated by e.p.r . spectroscopy of molybdenum(V) in reduced forms of the enzyme . The resting enzyme gives no signals attributable to Mo(V) . However, on reduction with NADH, Mo(V) signals appeared at relatively short reaction times but decreased again on prolonged exposure to excess of the substrate as the enzyme was further reduced . On brief treatment of such samples with nitrate, Mo(V) signals reappeared but disappeared again on longer exposure to excess nitrate as the enzyme became fully reoxidized . Detailed investigation of the signals carried out in both 1H2O and 2H2O revealed the presence of two signal-giving species, referred to as 'signal A' and 'signal B', analogous to corresponding signals from nitrate reductase from Escherichia coli and from liver sulphite oxidase . Signal A has gav . 1.9767 and shows coupling to a single proton, exchangeable with the solvent, with A(1H)av . 1.3mT, whereas signal B shows no more than weak coupling to protons . Investigation of interconversion between the two species indicated that decreasing the pH from 8.0 to 6.7 had little effect, but that signal A was favoured by the presence of Cl- . This suggests, by analogy with recent work on sulphite oxidase by Bray, Gutteridge, Lamy & Wilkinson {Biochem . J . (1983) 211, 227-236} that Cl- is a ligand of molybdenum in the species giving signal A.

Mol Cell Biol, 1983 Jul, 3(7), 1246 - 54
Differential activation of the mouse beta-globin promoter by enhancers; Berg PE et al.; A series of plasmids was constructed to study the effect of two enhancers, the simian virus 40 72-base-pair repeat and the Harvey sarcoma virus 73-base-pair repeat, on the mouse beta maj-globin promoter . These plasmids contain the mouse beta maj-globin promoter linked to the Escherichia coli galK gene, thus allowing galactokinase enzyme activity to be used as a measure of promoter function . In CV-1 (primate) cells, it was found that an enhancer is required for optimal promoter activity and that the simian virus 40 (primate) enhancer increases galactokinase fourfold more than the Harvey sarcoma virus (mouse) enhancer . In L (mouse) cells, however, the Harvey sarcoma virus enhancer is 1.3-fold stronger than the simian virus 40 enhancer . These data support the hypothesis that enhancer activity can be species specific . Furthermore, when both enhancers are present on the same plasmid, their effect is additive on the beta-globin promoter whether the plasmid is in CV-1 cells or L cells.

Am J Trop Med Hyg, 1983 Jul, 32(4), 733 - 7
Migration of Entamoeba histolytica under agarose; Urban T et al.; A procedure for visualizing and quantifying motility of Entamoeba histolytica by migration under agarose is described . Agarose suspended in tissue culture medium 199 supplemented with bovine albumin was poured into plastic dishes and allowed to harden . Six pairs of wells were cut out in a circular configuration . To the inner wells a suspension of E . histolytica in Eagle's Medium (24 X 10(6) cells/ml) was added, and to the outer wells a chemoattractant or the control medium . After overnight incubation at 37 degrees C, the amebae were fixed and stained . The chemotactic and spontaneous migrations were measured in an enlarging projector . Escherichia coli filtrates, suspensions of intact and lysed erythrocytes, and the complement factor C5a acted as good chemoattractants . Both the random and chemotactic motility were correlated to the time of the incubation . Cytochalasin B effected a dose-related inhibition of both chemotactic and random migration, while colchicine caused a decrease of the chemotaxis only . The reproducibility of the method, measured by 10 intra-assay tests, was good . Thus, the described method can be useful for comparative determinations of the motility of different ameba populations . Furthermore, different factors affecting the motility of amebae can be studied.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4432 - 6
Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase; Weinstock GM et al.; We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs) . The 5' end of ompF, an E . coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm . This sequence is coupled to the lacZ gene of E . coli so that expression of beta-galactosidase requires ompF transcription and translation signals . However, this hybrid gene is LacZ- because lacZ is out of frame with respect to ompF . Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments . If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ+ gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and beta-galactosidase . The LacZ+ phenotype thus identifies clones containing an expressed ORF . To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells . We also inserted a fragment from the E . coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein . Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4422 - 6
F sex factor of Escherichia coli K-12 codes for a single-stranded DNA binding protein; Kolodkin AL et al.; In Escherichia coli K-12 strains that carry the mutation ssb-1 in the gene for single-stranded DNA binding protein, the presence of the F sex factor partially reverses the temperature-sensitive growth phenotype caused by the mutation . The region of F (EcoRI fragment 3) responsible for this compensation has been identified and subcloned onto pBR322 . A BamHI cleavage site has been found to intersect the essential coding region for this F function . By using this site, mutational blocks in the function have been constructed and used to identify a protein product (Mr approximately 22,000, slightly larger than the E . coli K-12 single-stranded DNA binding protein) which is correlated with the ssb-1-complementing activity . Labeled extracts from maxicells were used to show that this protein binds tightly to single-stranded DNA . The gene on F that codes for this protein is denoted ssf and is located at approximately 55.2 kilobases on the standard map of F, in the region transferred very early during bacterial conjugation.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4403 - 7
Expression of a beta-galactosidase gene containing the ribosomal protein 51 intron is sensitive to the rna2 mutation of yeast; Teem JL et al.; The temperature-sensitive mutation rna2 causes the accumulation of higher molecular weight transcripts from the ribosomal protein 51 (rp51) gene of yeast and many other yeast ribosomal protein genes . We have determined the DNA sequence of the rp51 gene, confirming that it contains an intron and that the higher molecular weight transcript is an intron-containing precursor RNA . These data and other experiments suggest that the rna2 mutation affects mRNA processing (splicing) and that the presence of an intron is sufficient to render expression of a gene sensitive to the rna2 mutation . To test these hypotheses, we have inserted the rp51 intron into the coding region of a hybrid Escherichia coli beta-galactosidase gene, thereby interrupting the open reading frame subsequent to the initiating methionine codon . Despite the presence of the intron, the beta-galactosidase gene is expressed in yeast . Thus, the rp51 intron is properly excised from the normally intronless gene . The presence of the rp51 intron causes the beta-galactosidase activity to be sensitive to the rna2 mutation, consistent with the notion that this mutation affects gene expression at the level of splicing . The experiments suggest that an intron-containing beta-galactosidase gene can be used in a general way to study mRNA splicing.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4228 - 32
Molecular cloning of a vitamin D-dependent calcium-binding protein mRNA sequence from chick intestine; Hunziker W et al.; We have constructed a recombinant cDNA library to facilitate study of the genomic actions of vitamin D3 and its hormonally active metabolite 1,25-dihydroxyvitamin D3 in initiation of the de novo biosynthesis of a 28,000-dalton vitamin D-dependent calcium binding protein (CaBP) present in chick intestine . The recombinant plasmids were prepared by the homopolymeric tailing and hybridization method using as a starting template poly(A)-enriched mRNA obtained from the intestinal mucosa of vitamin D3-replete (+D) chicks . Screening of 9,516 clones in this library was effected by using a comparative in situ colony hybridization technique with two {32P}cDNA probes; these probes were prepared from total poly(A)-RNA from chick intestinal mucosa of vitamin D-deficient (-D) chicks and a poly(A)-RNA specifically enriched for chick intestinal CaBP mRNA by immunoprecipitation of polysomes derived from vitamin D-replete (+D) chicks . We identified 26 clones that consistently displayed a significantly increased hybridization signal when comparing the -D vs . CaBP-enriched probe . Further evaluation of these clones by hybrid-selected translation showed the presence of CaBP-specific sequences . By "RNA gel" analysis of poly(A)-RNA, three independent mRNA species were found to hybridize to a CaBP clone; none of these RNA species were found in -D poly(A)-RNA . With this comparative colony hybridization procedure, we were able to identify CaBP-specific clones corresponding to a mRNA that is 0.1% of the total poly(A)-mRNA . The differential colony hybridization procedure using an enriched vs . a nonenriched probe should be of value in screening for other cDNA clones complementary to rare mRNA species.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4223 - 7
The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli; Cox MM; The FLP gene of the yeast 2-microns plasmid is involved in a site-specific recombination event that results in the inversion of a set of sequences within the plasmid . This gene has been cloned and expressed in Escherichia coli . Expression of the FLP gene results in efficient recombination within the bacterial cell, which is specific for plasmids containing at least one 2-microns plasmid recombination site . This work demonstrates that (i) FLP protein is actively involved in 2-microns plasmid recombination; (ii) no other factors specific to yeast are required for the reaction; (iii) FLP protein acts efficiently in trans; (iv) FLP protein will promote site-specific insertion and deletion reactions in addition to the inversion reaction; and (v) FLP-promoted recombination is not dependent upon any DNA structural features unique to yeast chromatin.

J Bacteriol, 1983 Jul, 155(1), 69 - 73
Accumulation of cyclic GMP in filaments of Escherichia coli BUG6; Cook WR et al.; Experiments with Escherichia coli BUG6, a temperature-sensitive cell division mutant, have shown that at the restrictive temperature (42 degrees C) the loss of cell division potential (filamentation) was accompanied by an unusual increase in intracellular cyclic GMP (cGMP) . At the permissive temperature (30 degrees C), cell division proceeded normally, and cGMP did not accumulate . Increasing the osmotic strength of the medium with NaCl suppressed filamentation in BUG6 at 42 degrees C and also suppressed the temperature-sensitive accumulation of cGMP . The addition of nalidixic acid to BUG6 at 30 degrees C induced filamentation but failed to cause cGMP accumulation . A similar accumulation of cGMP has not been observed in other E . coli strains.

J Bacteriol, 1983 Jul, 155(1), 407 - 11
Inhibition of secretion of a mutant lipoprotein across the cytoplasmic membrane by the wild-type lipoprotein of the Escherichia coli outer membrane; Lee N et al.; A globomycin-resistant mutant of Escherichia coli was found to produce a precursor of the major outer membrane lipoprotein (prolipoprotein), in which the glycine residue at position 14 within the signal peptide was replaced by an aspartic acid residue . The same mutation has been reported by Lin et al . (Proc . Natl . Acad . Sci . U.S.A . 175:4891-4895, 1978) . The structural gene of the mutant prolipoprotein was inserted into an inducible expression cloning vehicle . When the mutant prolipoprotein was produced in lipoprotein-minus host cells, 82% of the unprocessed protein was found in the membrane fraction, with the remaining 18% localized in the soluble fraction . However, when the production of the mutant prolipoprotein was induced in the wild-type lpp+ host cells, only 31% of the mutant prolipoprotein was found in the membrane fraction, leaving the remaining 69% in the soluble, cytoplasmic fraction . In addition, the assembly of the wild-type lipoprotein in these cells was not affected, whether the mutant prolipoprotein was produced or not . These results suggest that secretions of both mutant and wild-type prolipoproteins utilize the same component(s) responsible for the initial stages of secretion across the cytoplasmic membrane . However, it appears that the wild-type lipoprotein has a higher affinity for these components than does the mutant lipoprotein.

J Bacteriol, 1983 Jul, 155(1), 398 - 401
Identification of a membrane protein induced concurrently with cell filamentation by cyclic AMP in an Escherichia coli K-12 fic mutant; Utsumi R et al.; A membrane protein with a molecular weight of 40,000 (40K protein) was induced concurrently with cell filamentation by cyclic AMP (cAMP) in a fic mutant . In the crp mutant and the wild-type strain, cell filamentation by cAMP was not observed, and the 40K protein was not induced . Induction of the 40K protein is regulated by the cAMP-cAMP receptor protein complex and is closely related to cell filamentation by cAMP in the fic mutant.

J Bacteriol, 1983 Jul, 155(1), 317 - 29
Coliphage P1-mediated transduction of cloned DNA from Escherichia coli to Myxococcus xanthus: use for complementation and recombinational analyses; O'Connor KA et al.; We have found that coliphage P1 can be used to transduce cloned DNA from Escherichia coli to Myxococcus xanthus . Transduction occurred at a high efficiency, and no evidence for DNA restriction was observed . The analysis of the transductants showed that they fall into three general categories: (i) haploid cells which contain portions of the cloned DNA substituted for homologous chromosomal DNA; (ii) heterozygous merodiploids which contain the recombinant plasmid integrated into the chromosome at a region of homology; and (iii) homozygous merodiploids which contain two copies of a portion of the cloned DNA with the loss of the chromosomal copy of the genes . The merodiploids, once formed, are relatively stable . They were used to analyze two genes necessary for aggregation and thus fruiting body formation . P1 transduction also permits the reintroduction and substitution of mutated regions of cloned DNA into M . xanthus for the analysis of the role of the DNA in cellular physiology and development.

J Bacteriol, 1983 Jul, 155(1), 265 - 74
Interactions between chemotaxis genes and flagellar genes in Escherichia coli; Parkinson JS et al.; Escherichia coli mutants defective in cheY and cheZ function are motile but generally nonchemotactic; cheY mutants have an extreme counterclockwise bias in flagellar rotation, whereas cheZ mutants have a clockwise rotational bias . Chemotactic pseudorevertants of cheY and cheZ mutants were isolated on semisolid agar and examined for second-site suppressors in other chemotaxis-related loci . Approximately 15% of the cheZ revertants and over 95% of the cheY revertants contained compensatory mutations in the flaA or flaB locus . When transferred to an otherwise wild-type background, most of these suppressor mutations resulted in a generally nonchemotactic phenotype: suppressors of cheY caused a clockwise rotational bias; suppressors of cheZ produced a counterclockwise rotational bias . Chemotactic double mutants containing a che and a fla mutation invariably exhibited flagellar rotation patterns in between the opposing extremes characteristic of the component mutations . This additive effect on flagellar rotation resulted in essentially wild-type swimming behavior and is probably the major basis of suppressor action . However, suppression effects were also allele specific, suggesting that the cheY and cheZ gene products interact directly with the flaA and flaB products . These interactions may be instrumental in establishing the unstimulated swimming pattern of E . coli.

J Bacteriol, 1983 Jul, 155(1), 254 - 64
Cloning of the pif region of the F sex factor and identification of a pif protein product; Rotman GS et al.; This paper reports a detailed investigation of the pif region of the F factor responsible for inhibition of development of T7 and related "female-specific" phages . We have mapped a series of pif::Tn5 insertions to a region between 39.6 and 42.8 kilobases on the physical map of F . All pif::Tn5 insertions plated T7 at full efficiency; most were clustered in a 1.8-kilobase interval on both sides of the EcoRI site located at F coordinate 40.3 kilobases . A 5.2-kilobase Pst-I fragment with F coordinates 38.9 to 44.1 has been cloned into a pSC101 vector to create the Pif+ plasmid pGS103 . A series of Pif- deletion mutants and nonsense mutants were isolated from pGS103 . Using minicells carrying pGS103 or its derivatives, we have identified a 70,000-dalton pif protein.

J Bacteriol, 1983 Jul, 155(1), 122 - 8
Regulation of expression of the colicin gene of I1 group plasmid TP110; Glazebrook JA et al.; The control of expression of the colicin Ib gene of the I1 group plasmid TP110 has been investigated . The colicin promoter was fused to the structural gene for beta-galactosidase, using the Mu d(Aprlac) phage, and the plasmid carrying this fusion was introduced into a variety of bacterial strains defective in genes involved in the "SOS" response . Colicin Ib belongs to that group of genes directly controlled by the repressor produced by the lexA gene, and expression was inducible by DNA-damaging agents . Mutations in uvrA, -B, and -C reduced the efficiency of induction by mitomycin C, as did mutations in recB . Mutations in recA and recF effectively prevented induction by mitomycin C, whereas mutations in lexA had contrasting effects, depending upon their effect on the properties of lexA protein . The spr-51 mutation (which inactivates lexA protein) led to constitutive expression, whereas the lexA3 mutation (which makes lexA protein refractory to cleavage by recA protein) completely inhibited inducible expression . In addition to lexA control, a TP110-coded function was identified which appeared able to inhibit colicin expression when the gene responsible was present in high copy number.

Infect Immun, 1983 Jul, 41(1), 88 - 96
Acquired chemotactic inhibitors during infection with guinea pig cytomegalovirus; Tannous R et al.; Factors involved in neutrophil and monocyte migrations were serially studied in strain 2 guinea pigs undergoing initial cytomegalovirus infection and sham-inoculated controls . All studies remained unchanged in uninfected animals . Monocyte migrations and neutrophil spontaneous migration remained unchanged in infected animals . However, transient abnormalities occurred early in infection, comprising a decrease in neutrophil-directed migration towards C5-derived chemotactic fractions (C5-fr) and a decrease in the chemotactic activity of zymosan-activated plasma . Consequently, the presence of neutrophil- and chemotaxin-directed inhibitors in plasma was investigated . Normal neutrophils, C5-fr, Escherichia coli-derived bacterial factor, and the synthetic peptide F-met-leu-phe were first incubated with control or infected plasmas and then assayed for directed migration and lysosomal enzyme release . Results indicated the de novo appearance of both neutrophil- and chemotaxin-directed inhibitory activities in plasma during early infection . The neutrophil-directed inhibition was heat stable (56 degrees C for 120 min) and nonspecific (responses to all chemotaxins were inhibited) . The chemotaxin-directed inhibition was heat stable and C5-fr specific . The cytomegalovirus-induced inhibitors may be important in the enhanced susceptibility to concurrent opportunistic infections.

Infect Immun, 1983 Jul, 41(1), 294 - 301
Escherichia coli lipopolysaccharides diminish and enhance cell function of human polymorphonuclear leukocytes; Henricks PA et al.; The effects of the lipopolysaccharide (LPS) of Escherichia coli J5 and 0111B4 on the function of human polymorphonuclear leukocytes (PMN) were tested . E . coli J5 is a UDP-galactose-4-epimerase-deficient mutant of E . coli 0111B4, and its LPS, therefore, contains mainly lipid A, as it lacks the polysaccharide side chains . PMN which had been incubated with J5 LPS showed decreased phagocytic, chemotactic, and metabolic activities as compared with control PMN . In contrast, incubation of PMN with 0111B4 LPS had no effect or even an enhancing effect on PMN function . When lipid A and the polysaccharide fraction were isolated from 0111B4 LPS, it was shown that lipid A had the same deleterious effect on PMN function as did J5 LPS and that the LPS fraction had no effect . When PMN were incubated with J5 LPS or lipid A, it could be shown that these structures were able to induce PMN to generate superoxide and chemiluminescence . 0111B4 LPS and the polysaccharide component were able to generate a metabolic burst by the PMN to a lesser extent . The induced defects in PMN function by J5 LPS could be prevented when polymyxin B or an oxygen-radical scavenger was present . We hypothesize that the lipid A portion of LPS is toxic for PMN due to the induction of toxic oxygen species by the PMN . These toxic oxygen species destroy the phagocytic, chemotactic, and metabolic activities of the PMN.

Mikrobiologiia, 1983 Jul-Aug, 52(4), 563 - 8
{Metabolic limitation of DNA-polymerase I synthesis by Escherichia coli strain CM5199}; Soktoev SA et al.; The work was aimed at studying DNA polymerase I synthesis after induction of the vector prophage lambda polA (NM 964) in lysogenic Escherichia coli CM 5199 in the course of batch cultivation in different media and at various growth phases as well as upon "nutrient shifts" caused by adding organic compounds to the minimal medium . The enzyme activity was highest when the phage was induced at the exponential phase of growth and in media richer in their composition . The enzyme synthesis, the dynamics of protein and RNA content were studied after induction of the phage and enrichment of the medium; the studies have shown that synthesis of DNA polymerase I is influenced by limitation at two levels: (1) biosynthesis of amino acids and (2) biosynthesis of components of the protein-synthesizing apparatus . There is a direct correlation between DNA polymerase I biosynthesis under the control of vector DNA and the growth rate of the culture.

J Antibiot (Tokyo), 1983 Jul, 36(7), 900 - 6
Aclacinomycin A-inhibition of phage phi X174 DNA synthesis in vitro; Tanaka A et al.; Aclacinomycin A inhibited the in vitro conversion of phage phi X174 single-stranded DNA to the replicative form DNA . DNA synthesis was inhibited by 50% in the presence of 15 microM aclacinomycin A . The inhibition was competitive with respect to template DNA (Ki = 13 microM) and was reversed by addition of Escherichia coli cell extracts . Short complementary strands approximately one-third of unit length molecule were synthesized in the presence of 15 microM aclacinomycin A . The data suggest that aclacinomycin A may inhibit the process of phi X174 DNA chain elongation by a direct interaction with the E . coli host enzymes.

J Bacteriol, 1983 Jul, 155(1), 330 - 6
Two fep genes are required for ferrienterochelin uptake in Escherichia coli K-12; Pierce JR et al.; Escherichia coli mutants defective in the assimilation of iron from ferrienterochelin were isolated and characterized . One mutant was able to bind ferrienterochelin to its outer membrane but could not transport it into the cell . Complementation tests with lambda hybrid phage were employed to distinguish the defective gene, which we term fepB, from fepA, the structural gene for the outer membrane ferrienterochelin receptor protein . These same physiological and genetic tests were employed to tentatively classify several previously described fep mutants as carrying either fepA or fepB . The data demonstrate the existence of fepB and provide an explanation for previous difficulties in identifying fepB mutants.

Bioorg Khim, 1983 Jul, 9(7), 900 - 5
{Modification of sulfhydryl groups in ribosomal proteins by a dinitrophenyl hapten}; Kozhukharova MS et al.; A dinitrophenyl hapten capable of protein SH-group modification was synthesized and the specificity of its reaction with SH-groups of the E . coli ribosomal proteins was studied . The possibility of incorporation of the Dnp-modified protein into ribosomal subunits by in vitro reconstitution was demonstrated with the ribosomal protein S12 . The Dnp-hapten attached to the protein S12 was found to be accessible for interaction with the Dnp-specific antibodies and therefore to be exposed on the surface of the reconstituted 30S subunit . Thus, the approach for incorporation of the antigenic groups into the protein components of supramolecular structures was proposed.

J Gen Microbiol, 1983 Jul, 129 (Pt 7), 2079 - 89
Discriminated induction of SOS functions in Escherichia coli by alkylating agents; Barbe J et al.; Treatment of Escherichia coli with the alkylating agents diethyl sulphate, ethyl methane-sulphonate and N-methyl-N'-nitrosoguanidine produces a different pattern of expression of SOS functions . There is a full induction of recA-dependent inhibition of cell respiration, a slight induction of lambda prophage, and no inhibition of cellular division . In a comparative study with bleomycin, an agent which is able to induce these three SOS functions, we have also shown that the differences in expression of SOS functions are not due to any variation in the pattern of DNA synthesis, or DNA degradation after treatment with alkylating agents . These results suggest that the kind of damage induced in the DNA may be important in determining which SOS function is expressed.

Fundam Appl Toxicol, 1983 Jul-Aug, 3(4), 209 - 14
Oxygen and redox-active drugs: shared toxicity sites; Brown OR et al.; Paraquat and nitrofurantoin can accept single electrons and, under appropriate conditions in tissues and cells, can pass these electrons to oxygen, thus participating in redox cycling . Similarities in the response of the target organ (the lung) and in subsequent pathology have also been observed among animals poisoned by oxygen and by these chemicals . We report evidence primarily obtained from Escherichia coli for common biochemical sites of toxicity for these agents . Common sites for oxygen and paraquat involve biosynthesis of specific amino acids, induction of genetic stringency via unloaded tRNAs resulting from amino acid deficiencies, decreased thiamin content, and impaired biosynthesis of pyridine nucleotide coenzyme biosynthesis for paraquat and oxygen . Inhibition of specific amino acid biosynthesis and induction of stringency also have been observed for nitrofurantoin . RNA and DNA biosynthesis are also impaired by oxygen; this has not been examined for paraquat or nitrofurantoin . There is a biochemical basis and preliminary data to support inhibition of NAD biosynthesis as a component of mammalian toxicity for these agents . Niacin may act to circumvent the consequences of the biochemical lesion at quinolinate phosphoribosyl transferase in NAD biosynthesis.

Plasmid, 1983 Jul, 10(1), 66 - 72
Transcription of plasmid DNA in Escherichia coli minicells; Crooks JH et al.; Cellular RNA polymerase in association with plasmid DNA segregates into the minicells of minicell-producing strains . In general, one "plasmid equivalent" of RNA polymerase, reflecting the size of the segregating plasmid DNA and its efficiency of segregation, entered the minicell with the plasmid . The amount of RNA polymerase (measured as the amount of enzyme activity purified from minicells and the rate of RNA synthesis in plasmid-containing minicells), and not the DNA content, appeared to be rate-limiting in plasmid-mediated transcription in minicells . The purified minicell and cellular RNA polymerases showed the same sensitivity to rifampin and streptolydigin; both were associated with sigma factor, although the minicell enzyme appeared to have slightly less than the cellular enzyme . These studies demonstrate that transcription of plasmid DNA in minicells is a function of the efficiency of segregation and the amount of RNA polymerase which enters with the plasmid DNA . Because RNA polymerase is limiting, plasmids with relatively weak promoters for the vector genes should be used when attempting to identify products from inserted foreign DNA.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4450 - 4
Identification of a precursor molecular for the RNA moiety of the processing enzyme RNase P; Gurevitz M et al.; A precursor molecule for 10Sb (M1) RNA, the RNA moiety of the RNA processing enzyme ribonuclease P (EC 3.1.26.5), is accumulated transiently in an Escherichia coli strain containing a plasmid that carries the 10Sb RNA gene . The same RNA precursor molecule is accumulated, in relatively large quantities, in a temperature-sensitive RNase E- mutant at the nonpermissive temperature . The RNA precursor includes 10Sb RNA and an extra 3' fragment that contains a termination stem and loop . It can be processed in vitro to a molecule the size of 10Sb RNA . None of the four endoribonucleases of E . coli--RNase III, RNase E, RNase F, or RNase P--takes part in this cleavage reaction . Therefore, we suggest that the processing of the precursor-10Sb RNA to 10Sb RNA is carried out by a thus-far unidentified endoribonuclease . The accumulation of a RNA molecule in a RNase E- mutant that does not contain a cleavage site for RNase E has been encountered previously and can be explained by assuming the existence of a RNA processing complex in the E . coli cell.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4412 - 6
Selection of functional cDNAs by complementation in yeast; McKnight GL et al.; Yeast cDNA was prepared in a yeast expression plasmid to generate a cDNA plasmid pool composed of approximately 40,000 members . Several yeast mutants were transformed with the cDNA plasmid pool, and the cDNAs for ADC1, HIS3, URA3, and ASP5 were isolated by functional complementation . Restriction enzyme analysis confirmed the genetic identity of the ADC1, HIS3, and URA3 cDNAs and demonstrated that the URA3 cDNA contains 5' noncoding sequences . The relative abundance of the various cDNAs in the cDNA plasmid pool paralleled the abundance of the mRNAs in total poly(A)+ RNA, which ranged from approximately 0.01% to 1% . The utility of this approach to isolate rare cDNAs from higher eukaryotes is discussed.

Cell, 1983 Jul, 33(3), 865 - 76
Expression of rRNA and tRNA genes in Escherichia coli: evidence for feedback regulation by products of rRNA operons; Jinks-Robertson S et al.; We have tested a model for global ribosome biosynthesis by examining the effects of increased gene dosage on the synthesis of rRNA . Increasing gene dosage does not lead to a significant increase in total rRNA transcription; i.e., rRNA synthesis from individual rRNA operons is reduced to keep total rRNA production unchanged . In contrast, when the plasmid-encoded rRNA operons used to increase gene dosage contain deletions within the rRNA coding region, rRNA transcription is gene-dosage-dependent; i.e., rRNA regulation is relieved . We find that the syntheses of most, if not all, tRNAs are also subject to the same controls as rRNA transcription . We conclude that the production of functional rRNA is monitored by the regulatory system that controls rRNA and tRNA transcription . We propose that rRNA and tRNA are negatively controlled by products of rRNA operons and discuss evidence suggesting that ribosomes are the key element involved in the postulated feedback regulation.

Cancer Treat Rep, 1983 Jul-Aug, 67(7-8), 611 - 9
Leukocyte recovery with short-chain RNA fragments in cyclophosphamide-treated rabbits; Beljanski M et al.; Single-stranded short-chain RNA fragments, obtained by mild degradation of purified Escherichia coli ribosomal RNA(s) with pancreatic RNase A, exhibit particular biologic activities in vitro and in vivo . In vitro, these RNA fragments are used by DNA-dependent DNA polymerase I as primers to initiate the replication of DNA(s) isolated from rabbit bone marrow and spleen; they are inactive with DNA isolated from several normal tissues and cancerous cells . Administered iv, RNA fragments restore a normal level of circulating leukocytes in rabbits with high doses of cyclophosphamide (CP) . Granulocyte/lymphocyte balance, upset by daily CP administration, is also restored during the increase of both types of cells . No toxicity is observed, and numerous repeated doses of RNA fragments show no cumulative effect and do not lead to loss of leukopoietic stimulating activity . Tumor-bearing mice can be protected by RNA fragments against the toxic effect of CP without impeding the anticancer activity of this drug.

Arch Biochem Biophys, 1983 Jul 1, 224(1), 196 - 205
Synthesis of polyadenylate-containing RNA in vitro in permeable cells of Escherichia coli B; Gopalakrishna Y et al.; As a starting point for the study of the biosynthesis of polyadenylated RNA in bacteria, the characteristics of RNA synthesis by cells of Escherichia coli B made permeable to small molecules by treatment with toluene were examined . Such cells mediated the incorporation of radiolabeled ribonucleoside triphosphates into RNA in a reaction that was sensitive to inhibitors of RNA polymerase and required the simultaneous presence of the four ribonucleoside triphosphates . Between 10 to 15% of the RNA synthesized under these conditions was polyadenylated as shown by affinity chromatography on oligo(dT)-cellulose . The presence of orthophosphate or dADP, inhibitors of polynucleotide phosphorylase, had no effect on the reaction and the rate of RNA synthesis was indistinguishable in the polynucleotide phosphorylase-deficient strain PR-7 and in its otherwise isogenic parent strain PR-100 . The poly(A) tracts associated with the newly synthesized RNA could be isolated after exhaustive digestion with pancreatic and T1 ribonucleases and accounted for 14% of the poly(A)-RNA . At least 74% of the poly(A) sequences were located at the 3' ends of RNA molecules and their weight-average length was 48 nucleotide residues . The size distribution of total RNA and poly(A)-RNA synthesized in the toluenized cell system was similar to that of the corresponding pulse-labeled fractions derived from growing cultures . The sequence complexity of poly(A)-RNA and unadenylated RNA synthesized in toluenized cells with {alpha-32P}CTP as the labeled substrate was analyzed by hybridization to fragments of Escherichia coli B DNA generated by digestion with EcoRI restriction endonuclease and immobilized on nitrocellulose sheets . Both RNA fractions hybridized with many DNA fractions, the hybridization patterns being similar with poly(A)-RNA and unadenylated RNA . This indicated that many different types of RNA transcripts synthesized in toluenized cells were subject to polyadenylation, but that polyadenylation was incomplete so that each transcript was present in both an adenylated and an unadenylated state.

Arch Biochem Biophys, 1983 Jul 1, 224(1), 134 - 41
DNA sequesters endogenous mRNA during preparation of crude Escherichia coli extracts for protein synthesis; use of an S60 reduces the sequestered mRNA; Goldman E et al.; High backgrounds of endogenous incorporation of amino acids into protein in crude extracts of Escherichia coli are a consequence of endogenous messenger RNA . This RNA survives the preparation by virtue of protection or sequestering by endogenous DNA . Thus, DNase treatment in the crude extract leads to the elimination of this mRNA, while DNase treatment has no effect on the purified RNA . The endogenous mRNA also appears to be physically associated with DNA on CsCl gradients, and can be largely removed along with DNA by centrifugation of extracts at 60,000g . The top layer above a 60,000g centrifugation (S60) appears to be suitable for protein synthesis, and provides for lower background levels of endogenous messenger RNA.

J Virol, 1983 Jul, 47(1), 202 - 16
Characterization of am404, an amber mutation in the simian virus 40 T antigen gene; Rawlins DR et al.; We analyzed the biological activity of an amber mutation, am404, at map position 0.27 in the T antigen gene of simian virus 40 . Immunoprecipitation of extracts from am404-infected cells demonstrated the presence of an amber protein fragment (am T antigen) of the expected molecular weight (67,000) . Differential immunoprecipitation with monoclonal antibody demonstrated that am T antigen was missing the carboxy-terminal antigenic determinants . The amber mutant was shown to be defective for most of the functions associated with wild-type T antigen . The mutant did not replicate autonomously, but this defect could be complemented by a helper virus (D . R . Rawlins and N . Muzyczka, J . Virol . 36:611-616, 1980) . The mutant failed to transform nonpermissive rodent cells and did not relieve the host range restriction of adenovirus 2 in monkey cells . However, stimulation of host cell DNA, whose functional region domain has been mapped within that portion of the protein synthesized by the mutant, could be demonstrated in am404-infected cells . A number of unexpected observations were made . First, the am T antigen was produced in unusually large amounts in a simian virus 40-transformed monkey cell line (COS-1), but overproduction was not seen in nontransformed monkey cells regardless of whether or not a helper virus was present . This feature of the mutant was presumably the result of the inability of am T antigen to autoregulate, the level of wild-type T antigen in COS-1 cells, and the unusually short half-life of am T antigen in vivo . Pulse-chase experiments indicated that am T antigen had an intracellular half-life of approximately 10 min . In addition, although the am T antigen retained the major phosphorylation site found in simian virus 40 T antigen, it was not phosphorylated . Thus, phosphorylation of simian virus 40 T antigen is not required for the stimulation of host cell DNA synthesis . Finally, fusion of am404-infected monkey cells with Escherichia coli protoplasts containing appropriate procaryotic suppressor tRNAs showed that am404 is a suppressible nonsense mutation.

Immunology, 1983 Jul, 49(3), 519 - 28
Alpha-macroglobulin-induced release of anti-Ig-coated particles from a subpopulation of rabbit B lymphocytes; Mackin WM et al.; Approximately half of the rosettes formed by rabbit Ig+ lymphocytes (B cells) and anti-coated erythrocytes or glutaraldehyde-fixed bacteria are dissociated upon the addition of rabbit serum . Rabbit serum was fractionated and the rosette-dissociating activity was found in purified preparations of rabbit alpha 1- and alpha 2-macroglobulins . Studies designed to elucidate the mechanism of rosette dissociation suggested that the alpha-macroglobulins dissociated rosettes by causing the release or proteolytic cleavage of the membrane proteins complexed with the anti-Ig-coated particles . These data suggest that the alpha-macroglobulins may have a role in the interaction of B lymphocytes with particulate antigens.

Zh Mikrobiol Epidemiol Immunobiol, 1983 Jul, (7), 59 - 62
{Electron microscopic study of the interaction of nephritogenic strains of Escherichia coli carrying D-mannose-resistant fimbriae with the cells of a human kidney line}; Gosteva VV et al.; The electron-microscopic study revealed that nephritogenic E . coli having L-mannose-resistant fimbriae contacted with subcultured human renal cells due to the interaction of fimbriae with microvilli and, less frequently, with the cytoplasmic membrane surrounding the main part of the cell, as well as with the electron-opaque fibrillar material in the intercellular space . The possibility of very close interaction was demonstrated; in some cases this interaction was so close that the outlines of bacterial and epitheloid cells followed each other, the invagination of the external membrane of the host cell being sometimes observed . The expediency of using ruthenium red for detecting fimbriae in morphological studies was shown . The cytopathogenic effect observed in this study and developing by the end of the 5-hour period from the moment of the inoculation of the monolayer with E . coli was manifested by the swelling of mitochondria accompanied by the partial ruptures of cristae, the widening of channels in the endoplasmic reticulum, the appearance of the secondary lysosomes and the increase of their number.

Infect Immun, 1983 Jul, 41(1), 174 - 80
Reduction of the secretory response to Escherichia coli heat-stable enterotoxin by thiol and disulfide compounds; Greenberg RN et al.; We examined the effects of disulfide and thiol compounds on Escherichia coli heat-stable enterotoxin (ST) and cyclic GMP-induced secretion . Both cystamine and cystine (disulfide compounds) reduced the secretory responses to submaximal doses of ST in suckling mice (at 0.5 mumol per mouse) and reduced ST activation of guanylate cyclase (by 33 to 73% at 1 mM) . In higher doses, cystamine completely eradicated a maximally effective ST dose as well . In addition, the sulfhydryl (thiol) compounds cysteamine, cysteine, and acetylcysteine strikingly reduced the secretory response and the guanylate cyclase response to ST . Neither the disulfide nor the thiol compounds tested reduced cyclic GMP-induced secretion . These studies suggest that disulfide and thiol compounds both block ST-induced secretion before its activation of guanylate cyclase . Taken with the work of others, these findings suggest that disulfide compounds may alter the oxidation reduction state of a cell or act directly on the guanylate cyclase enzyme, whereas thiol compounds may inactivate ST itself by breaking its disulfide bridges, or it may alter guanylate cyclase activation by ST . Both families of compounds deserve further consideration among potential antisecretory agents for application in the control of ST-induced diarrhea.

Eur J Biochem, 1983 Jul 1, 133(3), 499 - 507
Interaction between the different domains of aminoacyl-tRNA and the elongation-factor-Tu x kirromycin complex; Guesnet J et al.; In this work, we have studied the effect of aa-tRNA and derived 3' aminoacylated fragments on the EF-Tu GTPase in the presence of kirromycin, using two systems: without and with ribosomes . The aa-tRNA fragments were obtained by enzymatic digestion . Procedures for the enzymatic preparation of C-A-Val and Val-tRNA Val1 3' half molecule, as well as a purification method for short 3' aminoacylated fragments based on the amino group charge, were newly developed for this work . Aminoacyl-adenosine was found to be able to stimulate the EF-Tu x kirromycin GTPase, but only to a very small extent . Increasing the length of the aminoacylated fragments increased the stimulatory effect as follow: A-Val much less than C-A-Val less than C-C-A-Val less than 3' valyladenosine dodecanucleotide much less than Val-tRNA Val1 3' half molecule less than Val-tRNA Val1 . The presence of ribosomes did not affect the order of effectiveness, but increased the basic GTPase activity of EF-Tu x kirromycin and the stimulation by aa-tRNA, its 3' half molecule and even more by its 3' short fragments . The effect of aa-tRNA and derived 3' fragments in the absence of ribosomes was not influenced by MgCl2 concentrations of 5-30 mM whereas, in the presence of ribosomes, low concentrations of MgCl2 (5 mM) greatly reduced the stimulation of aa-tRNA and, to a lesser extent, also the effect of the C-C-A-aa as well as the basic activity of the EF-Tu x kirromycin GTPase . The extent of the stimulation by aa-tRNA, and even more by C-C-A-aa, depends on the nature of the amino acid . Among the aminoacyl side chains tested (Arg-, Phe-, Val-, Met-, Leu-, Lys-) arginine was found to be the most active and leucine the least . Our results show that (a) the 3' aminoacylated extremity is of prime importance for the stimulation of the EF-Tu GTPase, (b) in the 3' extremity there are critical sequences for the interaction with EF-Tu and (c) other domains of the aa-tRNA molecule are capable of influencing this reaction: one of the most important is the region including the T psi C loop and stem.

Clin Chim Acta, 1983 Jun 30, 131(1-2), 75 - 85
Immunoassay of human muscle enolase subunit in serum: a novel marker antigen for muscle diseases; Kato K et al.; A sandwich enzyme immunoassay method for measurement of beta subunit of muscle enolase in human serum was developed by use of purified antibodies to enolase beta subunit and beta-D-galactosidase from Escherichia coli as label . The assay was specific to the beta subunit with no cross-reaction with the alpha and gamma subunits of human enolase . The measurable range was from 10 pg to 10 ng per assay tube or 1 to 1000 ng/ml serum . Coefficients of variation within-run and between-run for the assay of serum beta subunit were less than 14% . Normal adult sera contained about 6 ng/ml of the beta subunit, and the levels were significantly elevated in sera of patients with muscular dystrophy and those with myocardial infarction . Serum levels of the beta subunit correlated well with those of creatine phosphokinase, but poorly with those of myoglobin in the same samples . The specific distribution of beta subunit in skeletal muscle and heart was confirmed by measuring the levels in various tissue extracts.

Nature, 1983 Jun 30, 303(5920), 770 - 4
Escherichia coli single-strand binding protein stabilizes specific denatured sites in superhelical DNA; Glikin GC et al.; Escherichia coli single-strand binding protein relaxes supercoiled DNA molecules containing the Drosophila melanogaster histone gene repeat unit, by stabilizing denaturation bubbles that map near the boundaries of the genes, at sites that in native chromatin have been shown to be hypersensitive to nucleases . A similar process may contribute to the propagation of such hypersensitive sites after their induction on the activation of gene expression.

Biochim Biophys Acta, 1983 Jun 29, 745(3), 247 - 58
The pH-dependence of the binding of dihydrofolate and substrate analogues to dihydrofolate reductase from Escherichia coli; Stone SR et al.; The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments . The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as pKa values for groups on the ligand and enzyme that are involved with complex formation . The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit pKa values for their N-1 nitrogens of above 10.0 . These values are about five pH units higher than those for the compounds in free solution . The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand . The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex . A group on the enzyme with a pKa value of about 6.3 is involved with the interactions . However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme . For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form . It appears that dihydrofolate is not protonated in the binary complex.

FEBS Lett, 1983 Jun 27, 157(1), 91 - 4
Quantitative study of interaction of deacylated tRNA with Escherichia coli ribosomes . Role of 50 S subunits in formation of the E site; Kirillov SV et al.; The 30 S subunit contains 2 sites for tRNA binding (Phe-tRNA, AcPhe-tRNA, tRNAPheOH) with the functional properties of D and A sites of the 70 S ribosome after attachment of 50 S subunit . The third (E) site specific for deacylated tRNA is introduced into 70 S ribosome by its 50 S subunit . The E-site binding of tRNAPheOH is not sensitive to either tetracycline and edeine, and practically codon-independent . The affinity constant of tRNAPheOH for the E site is 2-3 orders of magnitude lower than that for the D site.

FEBS Lett, 1983 Jun 27, 157(1), 85 - 90
Physical properties of ribosomal proteins isolated under different conditions from the Escherichia coli 50 S subunit; Tumanova LG et al.; Physical properties of ribosomal proteins obtained with or without denaturating agents were compared . CD measurements and NMR studies have shown that proteins L2, L19, L24 and L30 isolated under denaturing conditions have the same properties as those prepared avoiding denaturating agents . CD and NMR spectra of proteins L1, L6, L11, L23, L25 and L29 obtained by us under denaturating conditions practically coincide with the data for the same proteins reported under 'mild' conditions . These findings suggest that the differences of reported physical properties can be due to different procedures of protein renaturation rather than to the methods of their isolation.

J Mol Biol, 1983 Jun 25, 167(2), 259 - 74
Deletion mutagenesis of the Escherichia coli galactose operon promoter region; Busby S et al.; Using recombinant DNA technology we have created a series of progressively longer deletions both upstream and downstream from the Escherichia coli galactose operon regulatory region . The effects of these lesions on expression of the two overlapping galactose promoters have been quantitated after DNA fragments carrying these deletions were cloned in a plasmid vector, in which the beta-galactosidase gene could be expressed from the truncated galactose regulatory region . The results allow us to determine which sequences are necessary for the activity of the two promoters . Our results show that for the P1 promoter, which is controlled by the cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP), the sequence necessary for full activity starts 56 base-pairs upstream from the transcription initiation point . In contrast, for the P2 promoter, which functions in the absence of cAMP-CRP, the crucial sequence extends to only 39 base-pairs upstream from the transcription start . Deletions that cut into these sequences cause reductions in promoter strength, although some promoter activity is observed even when the "-35 region" of both P2 and P1 are deleted . Analysis of deletions originating downstream from the regulatory region shows that the elimination of the P1 and P2 Pribnow box sequences leads to loss of promoter activity . However, sequences downstream from the P1 start can be replaced without affecting the activity of either promoter . Finally examination of DNA fragments containing total deletions of both galactose promoters allows us to confirm that the flanking sequences contain no significant promoter activity and that the P1 and P2 promoters are principally responsible for galactose operon expression in vivo.

J Biol Chem, 1983 Jun 25, 258(12), 7550 - 5
A novel proteolytic activity apparently initiating degradation of beta-galactosidase nonsense fragments in in vitro extracts of Escherichia coli; McKnight JL et al.; Our previous in vivo studies demonstrated that large premature fragments of beta-galactosidase are degraded in Escherichia coli by a common pathway, and the initial event appears to be a site-specific cleavage (McKnight, J . L., and Fried, V . A . (1981) J . Biol . Chem . 256, 9652-9661) . We now have developed a cell-free system that retains the specificity of this early cleavage event . Immunochemical techniques were used to isolate and quantitate the polypeptide substrate and products in pulse-chase experiments . The in vitro system has an activity that quantitatively converts the prematurely terminated A polypeptide of the lacZ non-sense mutant CSH-10 to the 90-kilodalton common B polypeptide intermediate observed in vivo . The activity is localized in the cytoplasm since the cleavage reaction is not affected by osmotic shock of whole cells or removal of the membrane fraction from cell-free extracts . The lon mutation capR9, which blocks this degradation pathway in vivo, does not affect the initial cleavage event in cell-free extracts of CSH-10 carrying this mutation . The in vitro cleavage event in extracts of lon+ CSH-10 or the isogenic lon- mutant is not stimulated by addition of ATP, not inhibited by depletion of ATP pools by hexokinase-2-deoxyglucose treatment, and not inhibited by EDTA or phenylmethylsulfonyl fluoride . These results suggest that the ATP-dependent proteolytic activity of the lon gene product does not directly catalyze this primary cleavage event.

Nucleic Acids Res, 1983 Jun 25, 11(12), 3873 - 88
Determination of the promoter strength in the mixed transcription system . II . Promoters of ribosomal RNA, ribosomal protein S1 and recA protein operons from Escherichia coli; Kajitani M et al.; Using the in vitro mixed transcription system (Kajitani, M . and Ishihama, A . (1983) Nucleic Acids Res . 11, 671-686), we determined the two parameters of the promoter strength, i.e., the rate of open complex formation between RNA polymerase and promoter, and the saturation level of the open complex formation at equilibrium, for the promoters of ribosomal RNA (rrnE), ribosomal protein S1 (rpsA) and recA protein (recA) operons from Escherichia coli . Taken together with the previous determinations for lactose (lac(UV5)), tryptophan (trp) and ribosomal protein L10 (rp1J) operons, these studies revealed that the relative promoter strengths with respect to the kinetic parameter are 200, 70, 50, 40, 30, 20 and 2% of the reference promoter lacP(UV5) for recAp, rp1Jp, rpsAp3, trpP, rpsAp1, rrnEp1 and rrnEp2, respectively, under our standard reaction conditions (50 mM NaCl and 37 degrees C); and those with respect to the thermodynamic parameter are 70, 35, 20, 10, 10, 10 and 5% the level of lacP(UV5) for rrnEp2, trpP, rpsAp3, rp1Jp, rpsAp1, rrnEp1 and recAp, respectively . The order of the promoter strength, however, changes with variation of the salt concentration or reaction temperature.

J Mol Biol, 1983 Jun 25, 167(2), 411 - 26
Topological arrangement of two transfer RNAs on the ribosome . Fluorescence energy transfer measurements between A and P site-bound tRNAphe; Paulsen H et al.; The relative arrangement of two tRNAPhe molecules bound to the A and P sites of poly(U)-programmed Escherichia coli ribosomes was determined from the spatial separation of various parts of the two molecules . Intermolecular distances were calculated from the fluorescence energy transfer between fluorophores in the anticodon and D loops of yeast tRNAPhe . The energy donors were the natural fluorescent base wybutine in the anticodon loop or proflavine in both anticodon (position 37) and D loops (positions 16 and 17) . The corresponding energy acceptors were proflavine or ethidium, respectively, at the same positions . Four distances were measured: anticodon loop-anticodon loop, 24(+/- 4) A; anticodon loop (A site)-D loop (P site), 46(+/- 12) A: anticodon loop (P site)-D loop (A site), 38(+/- 10) A: D loop-D loop, 35(+/- 9) A . Assuming that both tRNAs adopt the conformation present in the crystal and that the CCA ends are close to each other, the results are consistent with the two anticodons being bound to contiguous codons and suggest an asymmetric arrangement in which the planes of the two L-shaped molecules enclose an angle of 60 degrees +/- 30 degrees.

J Biol Chem, 1983 Jun 25, 258(12), 7676 - 83
Cysteine starvation, isoleucyl-tRNAIle, and the regulation of the ilvGEDA operon of Escherichia coli; Harris CL et al.; The involvement of undermodified tRNA in the regulation of the ilvGEDA operon has been investigated using Escherichia coli C6, a relA-, Cys-, Met- mutant . This strain accumulates thionucleotide-deficient or methyl-deficient tRNA when starved for cysteine or methionine, respectively . The levels of threonine deaminase, the ilvA gene product, and transaminase B, the ilvE gene product, were both lower in cysteine-starved cells, as compared with either growing or methionine-starved cultures . When cysteine was added to cysteine-starved cells, growth ensued promptly and both enzyme activities returned to control levels . Treatment of recovering cultures with valine limited growth by isoleucine limitation, but did not cause a derepression of the ilvGEDA operon . Valine treatment of nonstarved or methionine-starved cells led to the expected increase in threonine deaminase and transaminase B activities . Cysteine-starved cells slowly regained the ability to derepress the operon after 3 h of recovery in complete medium . In contrast, the induction of the lac operon was normal in cysteine-starved cultures, even in the presence of valine . The loss of derepressibility of the ilvGEDA operon was correlated with the presence of a kinetically and chromatographically altered tRNAIle in cysteine-starved cells . No changes in tRNAIle were observed after methionine starvation . Using the periodate method, we found that the charging of tRNAIle increased from the normal level of 60 to 80% or greater after starvation for cysteine . Under conditions where the ilvGEDA operon was fully derepressed in nonstarved cells, the charging of tRNAIle fell to 27% . Unexpectedly, nearly identical results were obtained with cysteine-starved cells after an identical derepression test . These results suggest that factors other than the aminoacylation state of tRNAIle may be important in the regulation of this operon . In particular, modifications to tRNA which involve cysteine may be necessary for controlling the expression of the ilvGEDA operon in E . coli.

J Biol Chem, 1983 Jun 25, 258(12), 7669 - 75
The cycling of Escherichia coli DNA polymerase III holoenzyme in replication; Burgers PM et al.; ATP-activated DNA polymerase III holoenzyme (holoenzyme) forms a stable initiation complex with primed DNA with concomitant hydrolysis of the ATP (Burgers, P . M . J., and Kornberg, A . (1982) J . Biol . Chem . 257, 11468-11478) . Upon replication of primed single-stranded circular DNA to a duplex circle with a small gap (RFII), the holoenzyme remains stably bound . Dissociation requires binding by ATP or the generally nonhydrolyzable analog, adenosine 5'-(3-thiotriphosphate) . Transfer of holoenzyme to another primed DNA absolutely requires ATP (or dATP) and takes about 2 min at 30 degrees C . The rate of cycling of holoenzyme is only slightly dependent on the concentration of primed DNA . However, the transfer time is reduced to only 2 to 5 s when it is intramolecular, as shown by movement to other primers on the same template chain . A rapid transfer of holoenzyme from a completed chain to another primer on the same template molecule is anticipated from the frequency of initiating nascent chains at the replicating fork of the cellular chromosome (about 1 per s at 37 degrees C) and the low cellular abundance of holoenzyme (about 10 to 20 molecules per cell).

J Biol Chem, 1983 Jun 25, 258(12), 7379 - 85
Fatty acyl derivatives of glucosamine 1-phosphate in Escherichia coli and their relation to lipid A . Complete structure of A diacyl GlcN-1-P found in a phosphatidylglycerol-deficient mutant; Takayama K et al.; We have determined the complete structure of a glycolipid (designated lipid X) previously found to accumulate in certain Escherichia coli mutants defective in phosphatidylglycerol synthesis (Nishijima, M., and Raetz, C.R.H . (1979) J . Biol . Chem . 254, 7837-7844) . Based on fast atom bombardment mass spectrometry and proton nuclear magnetic resonance studies, this substance is an acylated metabolite of glucosamine 1-phosphate . Lipid X of E . coli has a Mr = 711.87 as the free acid (C34H66NO12P) and contains two beta-hydroxymyristate moieties, one attached as an amide at the 2 position and the other as an ester at the 3 position of the sugar . It has free hydroxyl groups at the 4 and 6 positions, and the anomeric configuration is alpha . The structure of lipid X from E . coli closely resembles the reducing end subunit of lipid A, and it might represent a very early precursor in the biosynthesis of lipid A . To our knowledge, fatty acyl derivatives of glucosamine 1-phosphate have not been reported previously.

Nucleic Acids Res, 1983 Jun 25, 11(12), 4251 - 6
Blocked 5'-termini in the fragments of chromosomal DNA produced in cells exposed to the antitumor drug 4'-{(9-acridinyl)-amino}methanesulphon-m-anisidide (mAMSA); Marshall B et al.; Comparison of the sensitivity of DNA isolated from untreated and mAMSA-treated PY815 mouse mastocytoma cells to hydrolysis by E.coli 3'-exonuclease III and phage lambda or phage T7 5'-exonucleases show that the fragments of chromosomal DNA produced by mAMSA treatment have free 3'-OH termini and blocked 5'-termini.

Nucleic Acids Res, 1983 Jun 25, 11(12), 4109 - 26
Site-dependent cleavage of pBR322 DNA by restriction endonuclease HinfI; Armstrong KA et al.; Cleavage of pBR322 DNA I by the restriction endonuclease HinfI is preferentially inhibited at specific HinfI cleavage sites . These sites in pBR322 DNA I have been identified and ordered with respect to the frequency with which they are cleaved . The HinfI site most resistant to cleavage in pBR322 DNA I is unique in that runs of G-C base pairs are immediately adjacent on both sites . Two differently permuted linear (DNA III) species were produced by cleavage with two different restriction endonucleases, PstI and AvaI . Only one of these linear molecules, that produced by PstI, exhibits the same preferential cleavage pattern as DNA I . The second linear species, that arising from AvaI digestion, shows pronounced relative inhibition of cleavage at the HinfI sites nearest the ends of the molecule (100 to 120 base pairs away, respectively) . This result suggest that proximity to the termini of a linear DNA molecule might also influence preferential cleavage . The possibility of formation of stem-loop structures does not appear to influence preferential cleavage by HinfI.

J Mol Biol, 1983 Jun 25, 167(2), 227 - 43
Organization of the Escherichia coli chromosome around the genes for translation initiation factor IF2 (infB) and a transcription termination factor (nusA); Plumbridge JA et al.; The genes infB, for translational initiation factor IF2, and nusA for a protein involved in transcription termination are carried on a 4.8 X 10(3) base-pair DNA fragment . This fragment also carries promoters capable of expressing both genes . The order of these genes with respect to the surrounding genes is pnp, rpsO, infB, nusA, argG . Transcription of the two genes is anticlockwise on the standard Escherichia coli map, i.e . directed towards the genes rpsO and pnp . The presence of infB on multicopy plasmids enhances IF2 protein and messenger RNA levels only two to threefold compared to the normal haploid level.

J Biol Chem, 1983 Jun 25, 258(12), 7840 - 6
Tandem promoters in the gene for ribosomal protein S20; Mackie GA et al.; Examination of the nucleotide sequence of the gene for ribosomal protein S20 (rpsT) of Escherichia coli suggested the presence of two promoters ("sites 1 and 2") separated by 90 base pairs (Mackie, G . A . (1981) J . Biol . Chem . 256, 8177-8182) . We have investigated the properties of purified or cloned DNA fragments containing one or other or both these sites for their ability to promote transcription in vivo and in vitro . In reactions in vitro containing DNA and purified RNA polymerase as the sole macromolecular components, both sites 1 and 2 act as promoters directing the synthesis of "runoff" transcripts . The 5' termini of such transcripts have been determined by direct sequencing or by identification of the 5' terminal nucleoside 5'-triphosphate, 3'-monophosphate . In site 1, the major transcript initiates with GTP at residue 141 in the DNA sequence . A minor start occurs at residue 142 and uses CTP as the initiating nucleotide . In site 2, the major transcript (approximately 55% of all initiations in site 2) initiates with CTP at residue 232 while minor transcripts, each comprising approximately 20% of the total, initiate at residues 231 and 233 with GTP and CTP, respectively . In four methods of assay which reflect to varying extents the usage of promoters in vivo, site 1 is responsible for 10-30% of the total transcription of the gene for S20 and site 2 the remainder . Sites 1 and 2 appear to act independently and additively in assays based on the rate of synthesis of S20 in a system for coupled transcription and translation . Together, the two promoters for S20 are from 10-25-fold more active than the fully induced lac operon promoter.

J Biol Chem, 1983 Jun 25, 258(12), 7828 - 39
Carbodiimide inactivation of Escherichia coli RNA polymerase promoters on supercoiled simian virus 40 and ColE1 DNAs occurs by a one-hit process at salt concentrations in the physiological range; Hale P et al.; A previous study (Hale, P., Woodward, R . W., and Lebowitz, J . (1980) Nature 284, 640-644) showed that Escherichia coli RNA polymerase promoters on superhelical SV40 DNA are highly selective targets for chemical modification by the water-soluble carbodiimide, N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethyl carbodiimide (CMC) . To extend the inactivation analysis of supercoiled DNAs, we determined the number and location of RNA polymerase binding sites on the supercoiled and linear forms of ColE1 DNA . We also determined the site distribution of {3H} CMC on the superhelical form . This information, coupled with per cent inhibition of transcription versus CMC-bound curves, allowed a test of the specificity of the CMC inactivation by the Poisson equation . Curves were obtained for supercoiled SV40 DNA modified at 0 and 100 mM NaCl (2 mM NaPi, pH 7.0) and for supercoiled ColE1 DNA modified at 0, 100, and 320 mM NaCl . For supercoiled SV40 DNA, these data, coupled to our knowledge of the number of RNA polymerase binding sites from the study cited above, revealed an excellent fit to a one-hit inactivation by the Poisson equation for DNA modified at 100 mM NaCl . For ColE1 DNA, we obtained an excellent fit to a Poisson distribution when supercoiled DNA was modified at 320 mM NaCl . The Poisson distribution can be applied to {3H} CMC restriction fragment data with equivalent results . These results suggest that promoter sites can be forced into different structural conformations with variable degrees of unpairing.

J Biol Chem, 1983 Jun 25, 258(12), 7469 - 75
Purification and characterization of Escherichia coli guanine-xanthine phosphoribosyltransferase produced by a high efficiency expression plasmid utilizing a lambda PL promoter and CI857 temperature-sensitive repressor; Liu SW et al.; The gene for Escherichia coli guanine-xanthine phosphoribosyltransferase was placed after the high efficiency lambda phage leftward promoter in plasmid pHEGPT also containing the lambda CI857 temperature-sensitive repressor . Guanine-xanthine phosphoribosyltransferase increases 780-fold when cells containing pHEGPT are shifted from 30 to 42 degrees C . Guanine-xanthine phosphoribosyltransferase represents approximately 5% of the protein in a crude extract of induced cells . Guanine-xanthine phosphoribosyltransferase may be purified to apparent homogeneity by ammonium sulfate fractionation, Sephadex G-100, and DEAE-cellulose column chromatography . The enzyme has a subunit molecular weight of 18,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaves as a trimer during Sephadex G-100 column chromatography . Guanine-xanthine phosphoribosyltransferase is active from pH 7.5 to 10.5 with maximum activity at pH 9.5 . The enzyme is protected from heat inactivation by phosphoribosylpyrophosphate (PRPP) . At 65 degrees C, the enzyme has a half-life of 2 min in the absence of PRPP and 90 min in the presence of PRPP . The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for guanine, xanthine, hypoxanthine, and PRPP of 2.6, 39, 167, and 95 microM, respectively . The activity of the enzyme with guanine is 2-fold greater than that with xanthine and 3-fold greater than that with hypoxanthine.

J Biol Chem, 1983 Jun 25, 258(12), 7395 - 401
The nucleotide sequence of the Mr = 28,500 flagellin gene of Caulobacter crescentus; Gill PR et al.; The DNA sequences which encode the Mr = 28,500 flagellin polypeptide of Caulobacter crescentus CB15 have been determined . The size of the protein, deduced from its DNA sequence (276 amino acids), is in agreement with its apparent molecular weight as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The distribution of arginine residues within the protein sequence encoded by the gene correlates with their relative location as predicted by peptide alignment analysis (Gill, P.R., and Agabian, N . (1982) J . Bacteriol . 150, 925-933) . DNA sequences 5' and 3' to the coding sequence were also determined . In the 5' region, DNA sequences homologous to consensus sequences associated with RNA polymerase recognition and transcription initiation sites in Escherichia coli (Pribnow box) are found . These are centered around 60, 90, and 120 base pairs upstream from the ATG codon at the beginning of the structural gene . Sequences 3' to the coding region were identified which might signal transcription termination . A typical E . coli 16 S ribosomal binding site (Shine-Dalgarno sequence) is located just 5' to the coding sequence, and for most of the amino acids there is a strong codon usage preference . Although this protein is exported from the cell (Gill, P.R., and Agabian, N . (1982) J . Bacteriol . 150, 925-933), the encoded NH2-terminal amino acid sequence is not different from the mature product.

J Biol Chem, 1983 Jun 25, 258(12), 7345 - 51
Human beta-glucuronidase pinocytosis and binding to the immobilized phosphomannosyl receptor . Effects of treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase; Talkad V et al.; beta-D-Glucuronidase (EC 3.2.1.31) was purified to homogeneity from human spleen, and enzyme fractions from CM-Sephadex were examined for uptake by fibroblasts and retention by a column of immobilized phosphomannosyl receptor . Uptake and binding were enhanced by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase, greatly reduced by prior treatment with alkaline phosphatase, and restored by subsequent treatment with alpha-N-acetylglucosaminyl phosphodiesterase . Immobilized phosphomannosyl receptor was used to separate high and low uptake enzyme forms . About 25% of the total beta-glucuronidase was retained by the receptor column and eluted with mannose 6-phosphate . The rate of uptake of retained enzyme was 2.5-3.0-fold greater than that of the enzyme applied to the receptor column . The fraction retained by the column was reduced to 5-10% by prior treatment of the enzyme with alkaline phosphatase . This phosphatase-resistant, receptor-retained fraction was taken up at only 24% the rate of non-phosphatase-treated, receptor-retained enzyme . However, its uptake was increased 7-fold by treatment with alpha-N-acetylglucosaminyl phosphodiesterase . The enhanced rate of pinocytosis conferred by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase was destroyed by a subsequent treatment with alkaline phosphatase . These studies demonstrate that although most of the "high uptake" enzyme in beta-glucuronidase from human spleen binds to receptors through phosphomonoesters of mannose, a significant fraction can interact with immobilized phosphomannosyl receptor and be taken up by fibroblasts through interactions involving mannose 6-phosphate in diester linkage with N-acetyl-D-glucosamine.

J Chromatogr, 1983 Jun 24, 262, 193 - 8
Characteristics of immobilized histamine for pyrogen adsorption; Minobe S et al.; The characteristics of immobilized histamine for pyrogen adsorption were investigated . The adsorbent showed a high affinity for pyrogen at low ionic strength, at around neutral pH, at high temperature and at low flow-rates of a solution containing pyrogen . The adsorption capacity per millilitre of the adsorbent was 0.9 mg pyrogen . Immobilized histamine could be completely regenerated by washing with 0.2 M sodium hydroxide solution containing 10--30% ethanol followed by 1.5 M sodium chloride solution, or 0.2 M sodium hydroxide solution followed by 0.5% sodium deoxycholate solution, 0.2 M sodium hydroxide solution and 1.5 M sodium chloride solution.

Biochemistry, 1983 Jun 21, 22(13), 3285 - 92
CTP synthetase from Escherichia coli: an improved purification procedure and characterization of hysteretic and enzyme concentration effects on kinetic properties; Anderson PM; Previous studies have shown that CTP synthetase exists as a dimer which aggregates to a tetramer in the presence of the substrates ATP and UTP {Long, C . W., Levitzki, A., & Koshland, D . E., Jr . (1970) J . Biol . Chem . 245, 80} . A new, relatively simple purification procedure resulting in enzyme of high purity and in good yield has been established by using two successive hydrophobic chromatography steps, the first in the absence of ATP and UTP (dimer binds) and the second in the presence of ATP and UTP (tetramer does not bind) . Several previously unreported properties of CTP synthetase are described which suggest that alterations in the state of association and dissociation of the enzyme have a controlling influence on the observed kinetic properties of the enzyme . The specific activity of CTP synthetase decreases with decreasing enzyme concentration, particularly when the concentrations of ATP and UTP in the reaction mixture are nonsaturating . The concentration of ATP or UTP required for half-maximal activity is significantly increased as the concentration of enzyme in the reaction mixture is decreased . CTP synthetase displays reversible cold lability and hysteretic properties (lags or bursts in the time course of product formation), both of which are influenced by the concentration of enzyme and/or the presence of ATP and UTP in the preincubation mixture and/or assay mixture . Gel filtration studies have shown that CTP synthetase can dissociate to an apparently inactive monomer . The dissociation is reversible, and the rate of association is slow.

Biochemistry, 1983 Jun 21, 22(13), 3162 - 70
Direct cross-links between initiation factors 1, 2, and 3 and ribosomal proteins promoted by 2-iminothiolane; Boileau G et al.; Complexes were prepared containing 30S ribosomal subunits from Escherichia coli and the three initiation factors IF1, IF2, and IF3 . In different experiments, each of the factors was radiolabeled with the others unlabeled . The complexes were allowed to react with 2-iminothiolane and then oxidized to promote the formation of intermolecular disulfide bonds, some of which were between factors and ribosomal proteins . Each of the labeled factors becomes covalently cross-linked to the complex as judged by its failure to dissociate when centrifuged in a sucrose gradient containing a high salt concentration . Proteins from the complexes were extracted and analyzed on two-dimensional polyacrylamide gels by nonequilibrium isoelectric focusing and sodium dodecyl sulfate gel electrophoresis . Spots corresponding to cross-linked dimers that contained initiation factors, as indicated on autoradiographs, were cut out and analyzed further . The following cross-linked dimers between factors and ribosomal proteins were identified: IF1-S12, IF1-S18, IF2-S13, IF3-S7, IF3-S11, IF3-S13, and IF3-S19 . Cross-links between factors IF1-IF2 and IF3-IF2 were also identified . A model integrating these findings with others on the protein topography of the ribosome is presented.

Biochemistry, 1983 Jun 21, 22(13), 3157 - 62
Identification of tyrosine residues that are susceptible to lactoperoxidase-catalyzed iodination on the surface of Escherichia coli 50s ribosomal subunits or 70s ribosomes; Maly P et al.; Further to our studies on the Escherichia coli 30S ribosomal subunit, the detailed surface topography of both 50S subunits and 70S ribosomes has been investigated by using iodination catalyzed by immobilized lactoperoxidase as the surface probe . In the 50S subunit, only proteins L2, L5, L10, and L11 were iodinated to a significant and reproducible extent . The targets of iodination were identified, after isolation of the individual iodinated proteins, and were as follows: in protein L2 (271 amino acids), tyrosine-102 and -160; in protein L5 (178 amino acids), tyrosine-142; in protein L10 (165 amino acids), tyrosine-132; in protein L11 (142 amino acids), tyrosine-7 and -61 . In the 70S ribosome, only protein L5 was still iodinated to a significant extent from the 50S subunit, whereas in the 30S subunit the same spectrum of iodinated proteins was observed as that from iodinated isolated 30S subunits, with the exception that S21 was no longer present.

Biochemistry, 1983 Jun 21, 22(13), 3082 - 90
Rotational diffusion of Escherichia coli RNA polymerase free and bound to deoxyribonucleic acid in nonspecific complexes; Austin RH et al.; We have studied the rotational diffusion of Escherichia coli RNA polymerase free in solution and bound nonspecifically to DNA fragments . The rotational motion was measured by the decay in anisotropy of the triplet-triplet absorption by using as probes either the liganded enzyme inhibitor Rose Bengal or eosin 5'-isothiocyanate conjugated to the protein . The time resolution extended from 10 ns to 1 ms . Free RNA polymerase (holoenzyme) at high salt concentration (1 M NaCl) is monomeric and diffuses at 5 degrees C with a rotational correlation time of 0.66 microseconds, corresponding to an equivalent hydrodynamic sphere with a radius of 7.4 nm . These values and the known molecular weight are most compatible with a nonspherical shape, e.g., an oblate ellipsoid with an axial ratio of about 3 . In 0.1 M NaCl, the holoenzyme is dimeric and has a rotational correlation time of 2 microseconds . The decay of anisotropy is at least biexponential upon binding RNA polymerase to calf thymus DNA or to poly{d(A-T)} . The fast component with half of the amplitude has decay kinetics comparable to those seen with the free monomeric enzyme . The slow component has a rotational correlation time of about 14 microseconds and is independent of DNA chain length in the range greater than 180 base pairs . Both rotational correlation times decrease with temperature, and the relative amplitudes change such that the faster component dominates at higher temperature . The rotational relaxation of the enzyme-DNA complexes is discussed in terms of alternative models involving rigid rod-sphere diffusion, conformational changes in the enzyme and/or DNA, sliding motions of the protein along the DNA, and torsional-bending motions of DNA envisioned as a deformable rod.

Brain Res, 1983 Jun 20, 269(2), 251 - 7
Bilateral lesions of the fastigial nucleus prevent the recovery of blood pressure following hypotension induced by hemorrhage or administration of endotoxin; Lutherer LO et al.; The present studies were undertaken to determine if bilateral lesions of the fastigial nuclei of the cerebellum would impair the recovery and maintenance of mean arterial blood pressure during hypotension caused by hemorrhage or administration of endotoxin . We had shown previously that cerebellectomy would produce such an impairment, and the fastigial nuclei were implicated as the specific area involved due to the known pressor response observed when they are stimulated electrically . Chloralose-anesthetized dogs were made hypotensive by administration of E . coli endotoxin or hemorrhage to 50 mm Hg and observed over the subsequent 3 h . Dogs with fastigial nucleus lesions had a significantly lower mean arterial pressure during both the recovery and maintenance phases when compared with intact animals under both hypotensive protocols . In the hemorrhage study, a significant number of lesioned animals died whereas none of the controls died . Lesion of the fastigial nuclei produced an impairment similar to that seen with cerebellectomy . It is concluded that the fastigial nuclei play an important role in the recovery of blood pressure following a hypotensive episode.

J Mol Biol, 1983 Jun 15, 167(1), 103 - 17
Binding of yeast tRNAPhe anticodon arm to Escherichia coli 30 S ribosomes; Rose SJ 3rd et al.; A 15-nucleotide fragment of RNA having the sequence of the anticodon arm of yeast tRNAPhe was constructed using T4 RNA ligase . The stoichiometry and binding constant of this oligomer to poly(U)-programmed 30 S ribosomes was found to be identical to that of deacylated tRNAPhe . The anticodon arm and tRNAPhe also compete for the same binding site on the ribosome . These data indicate that the interaction of tRNAPhe with poly(U)-programmed 30 S ribosomes is primarily a result of contacts in the anticodon arm region and not with other parts of the transfer RNA . Since similar oligomers which cannot form a stable helical stem do not bind ribosomes, a clear requirement for the entire anticodon arm structure is demonstrated.

J Mol Biol, 1983 Jun 15, 167(1), 205 - 9
Codon-induced transfer RNA association . A property of transfer RNA involved in its adaptor function?
Labuda D, Porschke D.
It is shown by equilibrium sedimentation that the binding of cognate codons to tRNAPhe (yeast), tRNAPhe (Escherichia coli), tRNALys, tRNAfMet and of the wobble codon UUU to tRNAPhe (yeast) induces dimerization of codon transfer RNA complexes . Analysis of the sedimentation profiles with a quantitative evaluation of the coupling between sedimentation and association equilibrium provides dimerization constants in the range from 1 X 10(4) to 6 X 10(4) M-1 . These results on various tRNAs from different organisms suggest that the codon-induced tRNA association is a general phenomenon . Probably the codon-induced tRNA association facilitates the aminoacyl transfer reaction.

Eur J Biochem, 1983 Jun 15, 133(2), 371 - 7
N-acetylmuramoyl-L-alanine amidase of Escherichia coli K12 . Possible physiological functions; Parquet C et al.; Various experiments were carried out in an attempt to determine the possible physiological function of the N-acetylmuramoyl-L-alanine amidase purified from Escherichia coli K12 on the basis of its activity on N-acetylmuramoyl-L-alanyl-D-gamma-glutamyl-meso-diaminopimelic acid {MurNAc-LAla-DGlu(msA2pm)} . A Km value of 0.04 mM was determined with this substrate . Specificity studies revealed that compounds with a MurNAc-LAla linkage are the most probable substrates of this enzyme in vivo . Purified amidase had no effect on purified peptidoglycan and only low levels (1-2.5%) of cleaved MurNAc-LAla linkages were detected in peptidoglycan isolated from normally growing cells . However, the action of the amidase in vivo on peptidoglycan was clearly detectable during autolysis . The amidase activity of cells treated by osmotic shock, ether or toluene, as well as that of mutants with altered outer membrane composition was investigated . Attempts to reveal a transfer reaction catalysed by amidase were unsuccessful . Furthermore, by its location and specificity, amidase was clearly not involved in the formation of UDP-MurNAc . The possibility that it might be functioning in vivo as a hydrolase degrading exogeneous peptidoglycan fragments in the periplasma was substantiated by the fact that MurNAc itself and MurNAc-peptides could sustain growth of E . coli as sole carbon and nitrogen sources . Finally, out of 200 thermosensitive mutants examined for altered amidase activity, only two strains had less than 50% of the normal level of activity, whereas ten strains were found to possess more than 50% . In fact, two of the overproducers encountered presented a 4-5-fold increase in activity.

FEBS Lett, 1983 Jun 13, 156(2), 366 - 70
Nucleotide sequence of the promoter region of the citrate synthase gene (gltA) of Escherichia coli; Hull EP et al.; The gltA gene, specifying the citrate synthase (EC 4.1.3.7) of Escherichia coli, has been isolated and the nucleotide sequence of a 752 basepair segment containing the gltA promoter and encoding 96 aminoterminal residues of the protein has been defined using the dideoxy/M13 method . The results confirm the location and transcriptional polarity of the gltA gene and indicate that the gltA transcript may contain a long leader sequence of 302-306 nucleotides upstream from the coding region.

FEBS Lett, 1983 Jun 13, 156(2), 307 - 10
The TolC protein of Escherichia coli K12 is synthesised in a precursor form; Hackett J et al.; We examined the biosynthesis of the TolC protein of Escherichia coli K12 in a pulse-chase experiment, followed by immunoprecipitation with anti-TolC antibody and SDS-PAGE of the immunoprecipitate . This showed that TolC protein was originally synthesised in a precursor form (Mr 54 500) which could be chased into the mature form (Mr 52 000) . DNA sequencing of a portion of the cloned tolC gene showed that the N-terminus of the mature rotein was preceded by a typical signal sequence of 22 residues (Mr 2542) . The initiator Met was preceded by a Shine-Dalgarno sequence, with the correct spacing.

Nucleic Acids Res, 1983 Jun 11, 11(11), 3823 - 32
A nucleotide change in the anticodon of an Escherichia coli serine transfer RNA results in supD-amber suppression; Steege DA; The tRNAs specified by the wild type and amber suppressor alleles of the Escherichia coli supD gene have been identified, and their primary structures determined . The sequences differ by a single nucleotide in the middle of the anticodon . A CUA anticodon allows the suppressor tRNA to read the UAG stop codon; the CGA anticodon in the minor serine tRNA species from which the suppressor is derived is specific for the serine codon UCG.

Nucleic Acids Res, 1983 Jun 11, 11(11), 3547 - 57
Nucleotide sequence for the catalytic domain of colicin E3 and its immunity protein . Evidence for a third gene overlapping colicin; Mock M et al.; We have determined the nucleotide sequence of a segment of Co1E3 DNA coding for the carboxyl-terminal, catalytic peptide of colicin E3 and for the immunity protein . The end of the colicin E3 gene is separated from the beginning of the immunity gene by a nine-basepair intercistronic region, suggesting the two genes are expressed as a single transcriptional unit . The immunity gene is expressed, however, in E . coli strains containing Co1E3-pBR322 hybrid plasmids deleted for the 5'-end of the colicin gene . The DNA sequence also contains an unexpected open reading frame (ORF) . This ORF is contained within the colicin gene and is in the +1 reading frame with respect to that gene . Plasmids containing the ORF, directed the synthesis of an 11 kilodalton protein in a cell-free, transcription-translation system.

Nucleic Acids Res, 1983 Jun 11, 11(11), 3451 - 65
Cloning and promoter analysis of the Escherichia coli adenylate cyclase gene; Aiba H et al.; The gene for adenylate cyclase of E . coli has been cloned in the plasmid pBR322 . The Cya- strain transformed with the isolated plasmids produces significant amounts of adenylate cyclase and cAMP . Some of the Cya+ plasmids were shown to direct the synthesis of a 85,000 dalton polypeptide in a cell-free system . The direction of transcription and the location of the cya promoter including the transcriptional start site were determined by an S1 digestion method . DNA sequence around the promoter region indicates that a putative coding region for adenylate cyclase begins at +233 . The 233 bp leader region could encode a potential small polypeptide containing 30 amino acids . Two probable CRP binding sites were found in the leader region, suggesting a negative control at the transcriptional level by CRP-cAMP.

Nucleic Acids Res, 1983 Jun 11, 11(11), 3679 - 86
The pR UV+ plasmid, transfected into mammalian cells, enhances their UV survival; Elli R et al.; It has been recently reported that the pR plasmid enhances the UV survival in E.coli c600 . In order to test whether this function may be expressed also in mammalian cells, LTA (tk- aprt-) mouse cells were cotransformed with pR plasmid DNA and ptk1 plasmid as selectable marker . Tk+ transformants were analyzed for their UV survival and for the presence of pR DNA sequences by blot-hybridization . The results show a correlation between the enhanced UV survival and presence of pR DNA sequences in cotransformed LTA mouse cells.

J Biol Chem, 1983 Jun 10, 258(11), 7141 - 8
Effects of the complete removal of basic amino acid residues from the signal peptide on secretion of lipoprotein in Escherichia coli; Vlasuk GP et al.; We have examined the importance of the positively charged NH2 terminus of the major outer membrane lipoprotein precursor, prolipoprotein, in the early steps of secretion in Escherichia coli . For this purpose, we have generated three mutants using oligonucleotide-directed mutagenesis in which the charge at the NH2-terminal region was changed from +2 to +1, 0, and -2 . The results indicate that the synthesis of prolipoprotein is facilitated by the presence of a positively charged NH2 terminus . In addition, the translocation of prolipoprotein across the cytoplasmic membrane does not absolutely require any basic amino acids at its NH2 terminus . However, the presence of a net negatively charged NH2 terminus causes an initial cytoplasmic accumulation of prolipoprotein which is slowly, post-translationally translocated across the cytoplasmic membrane at a rate which is dependent on the number of positive charges present in this region . The analysis of these mutants clearly demonstrates the importance of the NH2 terminus of the lipoprotein signal peptide in initiating the secretion of this protein in E . coli.

J Biol Chem, 1983 Jun 10, 258(11), 6963 - 4
S-adenosylmethionine synthetase from Escherichia coli . Crystallization and preliminary X-ray diffraction studies; Gilliland GL et al.; S-Adenosylmethionine synthetase from Escherichia coli can be crystallized by the method of vapor diffusion using ammonium sulfate as the precipitant . Two of several crystal forms of the tetrameric (Mr = 180,000) enzyme have been examined by x-ray diffraction methods . One form, a hexagonal bipyramid, develops when Mg2+ and PPi are added to the protein solution . The space group is P62(22) or P64(22) with a = b = 128.8 A, c = 140.0 A, and one subunit in the asymmetric unit . The other, a regular octrahedron, forms with the addition of 5% (v/v) 2-methyl-2,4-pentanediol . It is of space group P41(2)1(2) or P4(3)2(1)2, with a = b = 121.8 A, c = 172.6 A, and two subunits in the asymmetric unit.

J Biol Chem, 1983 Jun 10, 258(11), 7072 - 8
Cyclic AMP-dependent initiation and rho-dependent termination of colicin E1 gene transcription; Ebina Y et al.; We have analyzed the initiation and termination sites of transcription in vivo of the colicin E1 gene in Escherichia coli cells by S1-mapping assay and RNA blot hybridization . According to the S1-mapping assay, the transcription was initiated at about 75 base pairs upstream from the NH2-terminal codon of the gene . The initiation site corresponded with one of the two promoters which were previously determined by in vitro transcription experiments (Ebina, Y., Kishi, F., Miki, T., Kagamiyama, H., Nakazawa, T., and Nakazawa, A . (1981) Gene 15, 119-126) . Transcription in vivo of the colicin E1 gene was stimulated by cyclic AMP in the adenylate cyclase-defective mutant cells . Two transcripts of the colicin E1 gene, approximately 1700 and 2200 nucleotides, were detected by the blot hybridization . Since initiation of the transcription started at one site in vivo, these results indicated two termination sites . The location of the termination sites were approximately 60 and 560 base pairs downstream from the COOH-terminal codon of the gene as judged by S1-mapping assay . In vitro transcription experiments with rho-factor strongly suggested that the termination in the proximal terminator was rho-dependent . In the terminator structure, there is the sequence CAAACAAA which is homologous to a common sequence CAATCAA found in other rho-dependent terminators.

J Biol Chem, 1983 Jun 10, 258(11), 6932 - 40
A comparative study on the genes for three porins of the Escherichia coli outer membrane . DNA sequence of the osmoregulated ompC gene; Mizuno T et al.; The DNA sequence of the ompC gene which encodes one of the outer membrane porins has been determined . The gene appears to encode a secretory precursor of OmpC protein consisting of a total of 367 amino acid residues with a signal peptide of 21 amino acid residues at its NH2-terminal end . The 5' end noncoding region including the promoter of the ompC gene is extremely {A-T}-rich, and the codon usage in the ompC gene is unusual as are those in genes for other abundant outer membrane proteins . The promoter sequence of the ompC gene was compared with that of the ompF gene, both of which are controlled by the osmoregulatory operon, ompB . The deduced amino acid sequence of the OmpC protein showed extensive homology with that of the other porins (OmpF and PhoE proteins) . The homology in the primary amino acid sequences, as well as the coding DNA sequences among the porins, indicates that the structural genes for the three porins evolved from a common ancestral gene . Comparison of the amino acid sequences among the OmpC, OmpF, and PhoE porins will be discussed with regard to structure and function.

J Biol Chem, 1983 Jun 10, 258(11), 6912 - 9
Regulatory properties of the pyruvate dehydrogenase complex from Escherichia coli . Studies on the thiamin diphosphate-dependent lag phase; Horn F et al.; The pyruvate dehydrogenase complex from Escherichia coli shows an appreciable lag phase (tau) of some minutes when its overall reaction rate was tested with very limiting amounts of thiamin diphosphate . tau depends on the concentration of thiamin diphosphate in a nonlinear fashion . Sodium diphosphate, a competitive inhibitor with respect to thiamin diphosphate (Ki = 5.2 . 10(-4) M) prolongs the lag, while the strongly binding transition state analog thiamin thiazolone diphosphate has no effect . tau is independent of the enzyme concentration, thus no dissociation-association step is involved . Incubation of the pyruvate dehydrogenase complex with thiamin diphosphate, Mg2+, and pyruvate leads to a shortening of the lag phase, as well as to a decrease of the intrinsic tryptophan fluorescence in a time-dependent process, which evinces the same characteristics as tau . Dependence of pyruvate, as well as of the substrate analog methylacetylphosphonate, can be established by measurements of fluorescence quenching, thus ruling out an essential role of hydroxyethyl thiamin diphosphate in the process reflected by the lag phase . The results demonstrate that the lag phase is induced after the binding of both thiamin diphosphate . Mg2+ and pyruvate to the catalytic site to form a ternary enzyme complex, which undergoes subsequently a slow conformational change to an active enzyme form . This change is confined to single subunits, and no interactions between neighboring monomers could be observed . A model is proposed to describe the mechanism represented by the lag phase.

J Immunol Methods, 1983 Jun 10, 60(3), 351 - 8
A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site . Application to hybridomas specific for the beta 2-subunit of Escherichia coli tryptophan synthase; Friguet B et al.; Seven hybridoma clones, producing antibodies directed against the beta 2-subunit of Escherichia coli tryptophan synthase, have been obtained from mouse cells . To test whether the corresponding monoclonal antibodies recognize different epitopes on beta 2, an ELISA double antibody binding system has been developed and is reported here . The antigen is first coated onto a microtitration plate . Two monoclonal antibodies are then added either separately or simultaneously, and the amount of bound antibody is quantitatively measured by use of immunoglobulin (rabbit anti-mouse IgG) linked to beta-galactosidase . Additivity of the bound enzymatic activity is observed when the monoclonal antibodies bind to distinct epitopes . Using this test, it is shown that, of the 7 anti-beta 2 monoclonal antibodies obtained, 5 recognize the same antigenic site and the 2 others recognize a second site.

Biochemistry, 1983 Jun 7, 22(12), 2832 - 8
Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: stereospecificity of proton transfer in the phosphorylation of enzyme I from (Z)-phosphoenolbutyrate; Hoving H et al.; The stereochemistry of the proton transfer in the reaction of phosphoenolbutyrate with enzyme I has been established . During the reaction of the pure Z isomer of this analogue of phosphoenolpyruvate with enzyme I, to yield phosphoenzyme I and 2-oxobutyrate, the substrate is protonated at C-3 from the 2re,3si face . This stereospecificity was established for the transfer of a proton to (Z)-phospho{3-D}enolbutyrate and for the transfer of a deuteron to (Z)-phospho{3-H}enolbutyrate . The E isomer of phosphoenolbutyrate is not a substrate for enzyme I . Accordingly, the reaction of phosphoenzyme I with 2-oxobutyrate yields exclusively the Z isomer of phosphoenolbutyrate, and only the pro-S proton at C-3 of 2-oxobutyrate is abstracted . A kinetic H/D isotope effect of 6.8 in this reaction demonstrates the rate-limiting nature of the proton-transfer step . The stereochemical analysis of 2-oxo{3(R)-H,D}butyrate and of 2-oxo-{3(S)-H,D}butyrate was carried out by using the pyruvate kinase catalyzed enolization of this compound . This enzymatic enolization, with phosphate as a cofactor, is rapid at neutral pH and is a highly stereospecific reaction: only the pro-R proton at C-3 of 2-oxobutyrate is exchanged with solvent . This reaction was also used to generate the pure 3R and 3S enantiomers of 2-oxo{3-H,D}butyrate . The degree of protonation/deuteration at C-3 of 2-oxobutyrate was detected from the fine structure of the methyl proton nuclear magnetic resonance signal.

J Mol Biol, 1983 Jun 5, 166(4), 557 - 80
Studies on transformation of Escherichia coli with plasmids; Hanahan D; Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated . A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell . These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III) . Transformation efficiency declines linearly with increasing plasmid size . Relaxed and supercoiled plasmids transform with similar probabilities . Non-transforming DNAs compete consistent with mass . No significant variation is observed between competing DNAs of different source, complexity, length or form . Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.

J Appl Physiol, 1983 Jun, 54(6), 1463 - 8
Effect of endotoxin on airway responsiveness to aerosol histamine in sheep; Hutchison AA et al.; This study tested the hypothesis that in the awake sheep, airway responsiveness to aerosol histamine would be increased acutely by endotoxemia . Eleven sheep were chronically instrumented to allow for measurements of lung lymph flow, vascular pressures, and lung mechanics . Awake sheep were studied in a whole-body plethysmograph designed to measure dynamic compliance (Cdyn), resistance of the lung (RL), and functional residual capacity (FRC) . Pulmonary responsiveness to aerosol histamine was assessed by giving five breaths of increasing concentrations of histamine (0.1-50 mg/ml) until Cdyn decreased to 65% (of control) or until 50 mg/ml of histamine had been given . Escherichia coli endotoxin (0.2-0.5 microgram/kg) was then infused, and at 5 h after endotoxemia pulmonary responsiveness to aerosol histamine was remeasured . After endotoxin, 9 of the 11 sheep exhibited decreased Cdyn at a lower concentration of histamine compared with the preendotoxin level (P less than 0.05) . The mean of the log dose of histamine necessary to reduce Cdyn to 65% of control was 1.00 +/- 0.16 (SE) before endotoxin and 0.027 +/- 0.29 5 h after endotoxin; i.e., histamine responsiveness was increased . In the last 3 sheep studied, atropine (0.1 mg/kg iv) was given after the second aerosol histamine challenge, and a third dose-response curve was performed . Atropine did not return the endotoxin-induced increase in histamine responsiveness to base line . There was no correlation between the change in histamine responsiveness and the endotoxin-induced changes in Cdyn, FRC, RL, alveolar-arterial O2 difference, pulmonary arterial pressure, or lung lymph flow.

Biochem Genet, 1983 Jun, 21(5-6), 579 - 94
Comparisons of liver chromatin proteins and template activities in parental and heterotic rats during postweaned development; Amero S et al.; Electrophoretic profiles of acid-extractable proteins from Holtzman rat liver chromatin display four minor and five major histone bands through certain stages of postweaned development but are qualitatively different from the chromatin protein profiles previously reported during postweaned development for the Fisher 344 rat strain and the F344 X Holtzman heterotic progeny {Tallman, G., et al . (1979) . Biochem, Genet . 17:185} . The protein profiles from the heterotic progeny do not reflect and are not combinations of the profiles from the parental strains . Levels of in vitro transcription with Escherichia coli RNA polymerase of total chromatin and of acid-extracted chromatin from Holtzman rat liver tissue fluctuate in an age-specific manner during postweaned development and are higher than previously published levels determined for the Fisher and F344 X H strains during the same developmental period {Tallman, G., et al . (1978) . J . Hered . 69:282} . The degrees of stimulation in the transcription assays resulting from the acid treatment vary with the age of the animal but are similar for the maternal Holtzman and hybrid strains . These studies suggest that regulation of heterotic growth may involve dominant, or maternal genetic influences.

J Med Chem, 1983 Jun, 26(6), 870 - 6
4'-Methylangelicins: new potential agents for the photochemotherapy of psoriasis; Dall'Acqua F et al.; Three derivatives of angelicin (1) {4'-methyl-, 4,4'-dimethyl-, and 4',5-dimethylangelicin (2a-c)} have been prepared with the aim of obtaining new agents for the photochemotherapy of psoriasis . These compounds form a complex in the dark with DNA that shows an affinity for the macromolecule higher than that of the parent angelicin (1) . A correlation between their octanol/water partition coefficients and the association constants of the complexes has been observed . Compounds 2a-c photobind to DNA to a much higher extent than 1 and also more effectively than 8-methoxypsoralen (8-MOP), taken as reference compound . When activated with UV-A, the three compounds strongly inactivate T2 phage and inhibit epidermal DNA synthesis in mice . Moreover, they show a mutagenic activity markedly lower than that of 8-methoxypsoralen on Escherichia coli wild-type strain . Due to its lack of skin phototoxicity, its low mutagenic activity, and its antiproliferative activity, 2c was chosen for clinical evaluation . It proved to be effective in clearing psoriasis in two patients.

Infect Immun, 1983 Jun, 40(3), 888 - 93
Protection in rabbits immunized with a vaccine of Escherichia coli heat-stable toxin cross-linked to the heat-labile toxin B subunit; Klipstein FA et al.; Rabbits and rats were immunized with a vaccine consisting of synthetically produced Escherichia coli heat-stable toxin cross-linked by the carbodiimide reaction to the B subunit of biologically produced porcine heat-labile toxin . The vaccine contained 50% of each toxin component by weight and antigenicity; the toxicity of the heat-stable enterotoxin component was reduced by greater than 600-fold . Two or three peroral immunizations with vaccine containing 1,000 antigen units of each component raised greater-than-threefold increases in specific mucosal immunoglobulin A antitoxin titers to each component in all animal groups . Protection index values for challenge with either heat-labile or heat-stable toxins in ligated ileal loops were 3.4 to 4.0 in rats immunized by a parenteral primary immunization followed by two peroral booster immunizations, greater than 9 in rabbits immunized by these routes, and greater than 8 in rabbits given just three peroral immunizations . The antigenicity of the B-subunit component of the peroral vaccine was protected equally well against gastric acidity either by pretreatment with cimetidine or by delivery of the vaccine encapsulated in pH-dependent microspheres . The vaccine did not cause diarrhea when given perorally to any of the experimental animals or evoke fluid secretion when instilled into rabbit ligated ileal loops . These observations (i) confirm the effectiveness of this vaccine as an immunogen in a second animal model, (ii) establish that it is effective when given exclusively by the peroral route, and (iii) provide further evidence regarding its lack of toxicity.

Nippon Yakurigaku Zasshi, 1983 Jun, 81(6), 565 - 72
{Immunopharmacological actions of 6-amidino-2-naphthyl-4-guanidinobenzoate (FUT-175) . 1 . Effect of FUT-175 on immune response and host defense in mice}; Yanagihara Y et al.; FUT-175 is a new synthetic protease inhibitor which strongly inhibits complement-mediated hemolysis via the classical and alternative pathways . The present study was undertaken to examine the effects of FUT-175 on antibody formation and host defense in mice since the complement system participates in both immunological responses and host defense against bacterial infection . FUT-175 did not suppress the primary IgM and IgG antibody responses to sheep red blood cells, although FUT-175 was given at 10 to 100 mg/kg/day p.o . for 3 days before or after immunization . On the other hand, the primary anti-DNP IgE antibody response to DNP-conjugated ovalbumin was slightly suppressed only by post-administration of FUT-175 in a dose of 100 mg/kg/day p.o . for 5 days . However, the results of the adoptive transfer experiments indicate that FUT-175 did not affect either T cells or B cells participating in the secondary anti-DNP IgE antibody formation . FUT-175 in a dose of 10(-4)M but not at 10(-6) to 10(-5)M significantly decreased the proliferation of spleen cells caused by concanavalin A, lipopolysaccharide or the one-way mixed lymphocytes culture reaction using 1000 R-irradiated spleen cells from BDF1 mice as stimulator cells and those from C57BL/6 mice as responder cells . FUT-175 had an inhibitory rather than an enhancing effect on host defense to infection with Escherichia coli when administered at 10 to 100 mg/kg/day p.o . for 3 days before or after infection . These results strongly suggest that FUT-175 does not affect antibody formation and host defense in mice.

J Immunol, 1983 Jun, 130(6), 2804 - 8
Conversion of lipopolysaccharides to molecular aggregates with reduced subunit heterogeneity: demonstration of LPS-responsiveness in "endotoxin-unresponsive" C3H/HeJ splenocytes; Vukajlovich SW et al.; Lipopolysaccharides (LPS) of homogeneous monomeric composition were prepared by gel filtration chromatography of detergent-dissociated LPS from the smooth strain of E . coli 055:B5 . Splenocyte mitogenic activity of reassociated column fractions and fraction pools is markedly dependent upon the ratio of O-antigen polysaccharide to lipid A . The activity of the homogeneous LPS in eliciting spleen cell responses varied by approximately three orders of magnitude . One of the fraction pools rich in lipid A (which contains only a trace of O-antigen polysaccharide) induces a spleen cell proliferative response in C3H/HeJ "LPS-nonresponder" spleen cells . This mitogenic activity is not present in either unfractionated LPS or O-antigen-rich fractions . These latter findings indicate LPS macromolecular aggregates of the appropriate physicochemical structure have the capacity to elicit C3H/HeJ spleen cell proliferative responses.

Bull Tokyo Med Dent Univ, 1983 Jun, 30(2), 37 - 46
Thromboxane A2 and hemodynamic-biochemical parameters in canine endotoxin shock; Sakanishi N et al.; Prostaglandins participate in the pathophysiology of endotoxin shock; however, their exact role has not yet been clear . In this study, we investigated the role of the proaggregatory vasoconstrictor, thromboxane A2 (TXA2), an arachidonic acid metabolite, during canine endotoxin shock . The central venous plasma levels of thromboxane B2 (TXB2), the stable metabolite of TXA2, was measured by radioimmunoassay . We also investigated the therapeutic effect of reduced glutathione (GSH), a potential cell-stabilizing sulfhydryl compound, in canine endotoxin shock . Sixty minutes after the intravenous administration of E . coli endotoxin (1 mg/kg), the plasma TXB2 levels were significantly increased from 68.8 +/- 49.0 pg/ml to 318.3 +/- 117.2 pg/ml (N = 5) in the control group and from 67.9 +/- 68,4 pg/ml to 222.6 +/- 133.2 pg/ml (N = 5) in the GSH (300 mg/kg/hr) group . The levels in the GSH group were somewhat lower than in the control group for 60 to 180 minutes after the injection of endotoxin . Thromboxane A2 value appear not to relate to early thrombocytopenia and pulmonary hypertension but to relate to the change of late coagulopathy and of pulmonary vascular resistance . The administration of GSH suppressed the lactic acidemia significantly, however there was a much more decrease in the mean arterial pressure in the GSH group than in the control group . In addition, there was a tendency to inhibit the increase of the serum beta-glucuronidase activity in the GSH group.

Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3223 - 6
Simplified in vitro system for study of eukaryotic mRNA translation by measuring di- and tripeptide formation; Cenatiempo Y et al.; An in vitro system for measurement of rabbit globin mRNA translation has been developed based on the formation of the NH2-terminal dipeptide, fMet-Val . The basic components include a partially purified initiation factor preparation from rabbit reticulocytes supplemented with eukaryotic initiation factor 4A, purified and formylated yeast Met-tRNAi, and rabbit liver or Escherichia coli Val-tRNA1Val . Picomole quantities of fMet-Val are synthesized, dependent on mRNA, and the dipeptide is readily assayed by a simple extraction procedure . In the presence of Leu-tRNA or His-tRNA, the tripeptides fMet-Val-Leu and fMet-Val-His are synthesized, corresponding to the NH2-terminal sequence of alpha- and beta-globin, respectively . Therefore, tripeptide synthesis provides a simple means to distinguish between the expression of the alpha- and beta-globin mRNA species.

Biochimie, 1983 Jun, 65(6), 325 - 38
On the insertion of proteins into membranes; Clement JM; Recent data concerning the primary structure and the interactions of proteins with membranes suggest the existence of two classes of integral membrane proteins . In the first class, the polypeptide chain crosses the membrane only once . The membrane penetrating fragment is markedly hydrophobic and contains several positive charges on its C-terminal border . In the second class, the protein is folded in a complex fashion within the membrane and the knowledge of its amino acid sequence is not sufficient to predict the manner in which the protein interacts with the membrane.

Genetika, 1983 Jun, 19(6), 888 - 96
{Genetic control of the protective effect of spermidine in Escherichia coli cells as affected by mitomycin C}; Tanirbergenov TB et al.; The lethal action of mitomycin C and the effect of mutual treatment with mitomycin C and spermidine on Escherichia coli were studied . DNA repair in cells treated with mitomycin C was shown to have some differences, as compared to that of UV-induced pyrimidine dimers . The presence of the additive sbcB mutation increases the resistance of wild-type bacteria as well as of recBrecC and recF mutants to the lethal action of mytomicin C . Preliminary treatment of bacteria with spermidine increases resistance to the lethal action of the mutagen in wild-type bacteria as well as uvrB, recBrecC and sbcB strains . However, no such effect was observed in recF, recFsbcB and uvrE strains . The data suggest that the protective action of spermidine may be connected with stimulation of RecF-pathway of postreplication repair.

J Appl Biochem, 1983 Jun, 5(3), 210 - 8
Purification and characterization of glutathione synthetase from Escherichia coli B; Gushima H et al.; Glutathione synthetase was purified about 60-fold with 8.5% of activity yield from the cell extracts of Escherichia coli C600 cells transformed with a recombinant plasmid for the glutathione synthetase gene of E . coli B . The purified enzyme had a Mr of 152,000 and was composed of four identical subunits each with a Mr of 38,000 . The Km values of the enzyme for gamma-glutamylcysteine, glycine, and ATP were 2.6, 2.0, and 1.8 mM, respectively . The enzyme was most active at pH 8.5 and at 45 degrees C and required divalent cations such as Mg2+, Mn2+, and Co2+ for activity . The activity was inhibited by oxidized glutathione (Ki = 4.4 mM) . Reduced glutathione showed no effect on glutathione synthetase activity.

Ultrastruct Pathol, 1983 Jun, 4(4), 291 - 304
An ultrastructural study of enteropathogenic Escherichia coli infection in human infants; Rothbaum RJ et al.; Over the past 2 years, we have studied and treated 18 infants with protracted diarrhea due to an enteropathogenic Escherichia coli serogroup 0119 . All patients had persistent stool escretion and jejunal over-growth with this pathogenic E . coli . Jejunal biopsy revealed atrophy of villi with a chronic inflammatory cell infiltrate in the lamina propria . E . coli 0119 adhered to the luminal surface of enterocytes . Electron microscopy showed disappearance of glycocalyx and microvilli at the areas of bacterial adherence . Intracellular damage was indicated by dilatation of rough endoplasmic reticulum, mitochondrial changes, and cytoplasmic pallor . Similar changes in histology and ultrastructure occurred in ileal epithelial cells . Glandular crypt epithelium showed prominent subnuclear vacuolation and separation of lateral intercellular junctions throughout the small intestine . Rectal mucosal biopsy showed mucus depletion and irregular atrophy of the epithelium, with E . coli 0119 adherent to the luminal surface . Ultrastructural damage paralleled that in the small intestine . E . coli 0119 causes damage to epithelial cells throughout the infant intestinal tract . This damage leads to atrophy of villi and a marked reduction in absorptive surface area, resulting in protracted diarrhea.

J Gen Microbiol, 1983 Jun, 129 (Pt 6), 1889 - 97
Citrate synthase activity in Escherichia coli harbouring hybrid plasmids containing the gltA gene; Bloxham DP et al.; A hybrid plasmid, pDB2, was constructed by ligating a 3.24 kb EcoRI/HindIII fragment of the Escherichia coli chromosome into pBR322 . This was used to transform a gltA mutant which was devoid of citrate synthase activity . The resultant strain expressed very high citrate synthase activity and this enabled a simplified purification of the homogeneous enzyme in high yield . The subunit Mr was estimated as 47000-49000 by SDS gel electrophoresis, which closely resembles the eukaryotic form of the enzyme . Evidence for some conservation of sequence between the two proteins was revealed in the acid cleavage pattern at aspartyl-prolyl residues . In addition to coding for the structural gene for citrate synthase, the 3.24 kb EcoRI/HindIII fragment also retained the genetic structure necessary for control of enzyme synthesis since the expression of enzyme activity in the strain harbouring pDB2 was still subject to glucose repression.

Aust J Exp Biol Med Sci, 1983 Jun, 61 (Pt 3), 287 - 99
Host defences in the upper genital tract of the female: studies in a murine system; Sorrell TC et al.; Mechanical and cellular factors which maintain sterility in the fallopian tube were studied in virgin, BALB/c mice . Quantitative cultures of homogenates from ovary/periovarian sac, fallopian tube and uterine horn were obtained at various times after intratubal injection of 6 X 10(7) Escherichia coli (E . coli) by micropuncture . Immediately post-injection, significantly higher bacterial counts were obtained from fallopian tube than from uterine horn, indicating a functional barrier at the uterotubal junction . Viable E . coli were usually absent from the genital tract within 7 days . Reduction in bacterial counts was significant at 24 h post-injection . Ligation of the uterine horn and/or induction of leucopenia prior to injection were associated with decreased bacterial clearance at 24 h . Histology of samples from normal, injected mice revealed marked polymorphonuclear leucocyte (PMNL) infiltration in response to E . coli, confirmed by quantitation of intralumenal PMNL . By 72 h post-injection, viable E . coli and PMNL containing phagocytosed particles were prominent in vaginal washings . No inflammatory response was elicited in leucopenic mice . We conclude that clearance of E . coli from the fallopian tube depends primarily on excretion through the lower genital tract and PMNL-associated bacterial activity.

Zh Mikrobiol Epidemiol Immunobiol, 1983 Jun, (6), 63 - 7
{Invasive Escherichia coli of the O139:K serogroup}; Shurgai MA; The results obtained in the study of the main biological properties of 9 E . coli strains, serogroup O139:K., isolated from monkeys at the Sukhumi monkey breeding colony and yielding the positive result in Sereny's keratoconjunctival test are presented . For the first time E . coli of serovar O139:K.:431 were isolated; these organisms differed from reference strain O139K82:H1 in their enzymatic activity, in the partial composition of O-antigen and in their capacity for inducing experimental keratoconjunctivitis . The isolation of the above-mentioned cultures from monkeys with diagnosed acute intestinal diseases of unclear etiology, both during the life of the animals and in the process of autopsy, from a monkey having had contacts with sick animals in the focus of clinical dysentery, and from monkey subjected to prophylactic examination, as well as the pathogenicity of these strains for guinea pigs, evaluated on the keratoconjunctivitis model, suggest their probable etiological role in E . coli infection in primates and make it possible to regard them as the invasive variants of E . coli, serogroup O139:K..

Vet Microbiol, 1983 Jun, 8(3), 293 - 300
The opsonic activity of bovine milk whey for the phagocytosis and killing by neutrophils of encapsulated and non-encapsulated Escherichia coli; Hill AW et al.; Encapsulated strains of Escherichia coli were found to be more resistant to phagocytosis and killing by bovine neutrophils; requiring in the order of 100 times more serum opsonins than non-encapsulated strains . Mid-lactation pooled whey from cows with no history of mastitis was opsonic for non-encapsulated strains, but had no effect on encapsulated organisms . In contrast, early lactation pooled whey (5-10 days post-partum) was opsonic for all strains of E . coli . It is concluded that since early lactation milk contains sufficient opsonins, severe E . coli mastitis at this stage of lactation is not due to opsonic deficiency.

Tsitologiia, 1983 Jun, 25(6), 703 - 6
{Postreplication DNA repair in Escherichia coli cells . II . The necessity of primase for constitutive repair}; Zhestianikov VD et al.; In a thermosensitive mutant of Escherichia coli--PC3 dnaGts (the dnaG gene controls the synthesis of primase, or rifampycin resistant RNA polymerase in initiation of the Okazaki pieces synthesis in vegetative DNA replication), after UV-irradiation, the postreplication repair of DNA is lower by 5-20% at a non-permissive temperature 43 degrees, than at permissive temperature 30 degrees . A short-term inactivation of the dnaG gene activity before irradiation does not exert influence on the survival of bacteria, which remains the same at 30 and 43 degrees . In the presence of chloramphenicol, the efficiency of postreplication repair of DNA does not change at 30 and 43 degrees, but the survival of bacteria is somewhat higher, than without chloramphenicol treatment, being the same at 30 and 43 degrees . The data obtained indicate, that primase is a necessary constitutive component of postreplication repair of DNA, and that the short-term inactivation of the dnaG gene activity before inactivation does not exert influence on the survival of bacteria . In the PC3 dnaGts strain no inducible component of postreplication repair of DNA was detected.

J Biochem (Tokyo), 1983 Jun, 93(6), 1509 - 15
Temperature-sensitive prolipoprotein signal peptidase in an Escherichia coli mutant: use of the mutant for an efficient and convenient assay system; Yamagata H; Escherichia coli mutant Y815 accumulates the precursor of lipoprotein (prolipoprotein) in its envelope . The accumulated prolipoprotein could be chased to mature lipoprotein at 30 degrees C but not at 60 degrees C (Yamagata, H., Ippolito, C., Inukai, M., & Inouye, M . (1982) J . Bacteriol . 152, 1163) . When the envelope fraction prepared from the mutant was mixed with the envelope fraction prepared from wild-type E . coli cells and incubated at 60 degrees C in the presence of Triton X-100, the prolipoprotein in the mutant envelope fraction was cleaved rapidly to mature lipoprotein . The cleavage was dependent on the addition of wild-type envelope fraction and Triton X-100 to the reaction mixture . This indicated that the prolipoprotein accumulated in the mutant envelope is a good substrate for the signal peptidase which cleaves the signal peptide from the prolipoprotein, and hence the accumulation of prolipoprotein was due to lack of the signal peptidase in the mutant . The optimum concentration of Triton X-100 for the cleavage of the prolipoprotein in the above in vitro system was 0.05 to 0.1% (v/v) at a wild-type envelope concentration of 0.35 mg protein/ml . Prolipoprotein accumulated in wild-type cells on treatment with globomycin, a specific inhibitor of the signal peptidase, was also cleaved to mature lipoprotein under the same conditions . Triton X-100 was shown to solubilize the signal peptidase from the envelope fraction . The cleavage of the prolipoprotein was rapid and complete in the in vitro system described here, which provides an efficient and convenient assay system for the solubilized signal peptidase for prolipoprotein.

Can J Biochem Cell Biol, 1983 Jun, 61(6), 488 - 99
The major heat-shock protein (hsp70) gene family: related sequences in mouse, Drosophila, and yeast; Moran LA et al.; Heat shock induces the synthesis of a 70-kdalton protein in Escherichia coli, Drosophila, yeast, and mouse . We show that the genes for this heat-shock protein in mouse, yeast, and Drosophila share extensive sequence homology as determined by heteroduplex formation at different stringencies . We calculate that the heat-shock gene homology is 74% between yeast and Drosophila and 85% between mouse and Drosophila . The organization of the six copies of the Drosophila gene for the 70-kdalton heat-shock protein at two separate loci is summarized and evidence is presented that the yeast and mouse genomes each contain multiple copies of sequences related to the Drosophila gene for the 70-kdalton heat-shock protein . These results demonstrate that not only the sequence but also the repetitive organization of the major heat-shock genes is conserved.

Biokhimiia, 1983 Jun, 48(6), 959 - 69
{Stoichiometry of GTP breakdown during peptide synthesis on the ribosome . Stoichiometry of GTP hydrolysis during elongation of polyphenylalanine on polyuridylic acid}; Kakhniashvili DG et al.; The stoichiometry of GTP hydrolysis during poly(Phe) elongation by Phe on poly(U) covalently bound to Sepharose was determined . The concentrations of both EF-T and EF-G were saturating . The GTP/Phe stoichiometry was calculated without the usual correction for the uncoupled ribosomal EF-T and EF-G dependent GTP hydrolysis . At the Mg2+ optimum (6 mM) for the poly(Phe) elongation on poly(U) . Sepharose the stoichiometry ratio of GTP/Phe was 1.9/2.1 . This indicates that two (or less) GTP molecules coupled with poly(Phe) elongation by Phe on poly(U) . Sepharose are hydrolyzed.

Biochem J, 1983 Jun 1, 211(3), 631 - 40
Analysis of progress curves . Interaction of pyruvate kinase from Escherichia coli with fructose 1,6-bisphosphate and calcium ions; Boiteux A et al.; The influence of fructose 1,6-bisphosphate and Ca2+ on the kinetics of pyruvate kinase from Escherichia coli K12 was studied (at pH 7.0 and 25 degrees C) by using the pH-stat method for the measurement of the reaction progress as well as initial-rate analysis . The data were analysed on the basis of a concerted model with three conformational states {Markus, Plesser, Boiteux, Hess & Malcovati (1980) Biochem . J . 189, 421-433} by using a novel procedure for a computer-directed treatment of progress curves {Markus & Plesser (1976) Biochem . Soc . Trans . 4, 361-364} . By addition of fructose 1,6-bisphosphate the sigmoid kinetics with respect to phosphoenolpyruvate and Mg2+ is abolished and the activity of the enzyme is described by classical saturation kinetics . This is explained by exclusive binding of fructose 1,6-bisphosphate at an allosteric site of the conformational state that forms the active complex . We observe that Ca2+ is an activator of the enzyme at low Mg2+ and Ca2+ concentrations; otherwise it is an inhibitor . These effects can be understood by assuming that Ca2+ has the same binding properties as Mg2+, although it does not allow a catalytic turnover.

Appl Environ Microbiol, 1983 Jun, 45(6), 1838 - 47
Improved conversion of fumarate to succinate by Escherichia coli strains amplified for fumarate reductase; Goldberg I et al.; Two recombinant plasmid Escherichia coli strains containing amplified fumarate reductase activity converted fumarate to succinate at significantly higher rates and yields than a wild-type E . coli strain . Glucose was required for the conversion of fumarate to succinate, and in the absence of glucose or in cultures with a low cell density, malate accumulated . Two-dimensional gel electrophoretic analysis of proteins from the recombinant DNA and wild-type strains showed that increased quantities of both large and small fumarate reductase subunits were expressed in the recombinant DNA strains.

Mol Biochem Parasitol, 1983 Jun, 8(2), 137 - 43
Preliminary characterization of ribosomes of Entamoeba invadens; Price M et al.; The ribosomes of Entamoeba invadens trophozoites have sedimentation coefficients of 77, 53 and 36 S . Most of the ribosomal proteins are basic and their one- and two-dimensional electrophoretic patterns differ from the corresponding patterns of Escherichia coli and Saccharomyces cerevisiae . Two dozen bands were observed in the 10 000 to 100 000 molecular weight range following sodium dodecylsulfate-gel electrophoresis of amoebal ribosomal proteins . Long, thin pronase-sensitive structures were seen in electron micrographs of E . invadens ribosomal preparations.

J Clin Lab Immunol, 1983 Jun, 11(2), 101 - 4
The effect of interleukin 1(IL-1) containing supernatants on murine thymocyte maturation; Phillips R et al.; IL-1 containing supernatants were produced by E . coli lipopolysaccharide (LPS) stimulated adherent human mononuclear cells . Thymocytes from 4--6-week-old BALB/c mice were pretreated with IL-1 containing supernatants or control supernatants . IL-1 containing supernatant pretreated thymocytes when added to enriched splenic B cells increased the proliferative response of these cells after PWM activation . IL-1 pretreated thymocytes furthermore, increased the number of immunoglobulin secreting cells in the mouse splenocyte population, as compared to control supernatant pretreated thymocytes . These results indicate that IL-1 matures thymocytes into predominantly helper cells which augment B cell proliferation and antibody production.

J Clin Microbiol, 1983 Jun, 17(6), 965 - 9
Ganglioside GM1 enzyme-linked immunospot assay for simple identification of heat-labile enterotoxin-producing Escherichia coli; Czerkinsky CC et al.; A new method has been developed for demonstration of heat-labile (LT) enterotoxin produced by Escherichia coli . This method is based upon the release of LT from bacteria grown directly onto agar plates which have been coated with ganglioside GM1 . Toxin bound to the GM1 solid state is subsequently demonstrated by means of a three-step immunoenzymatic procedure in which enzyme-substrate reactions are visualized as dark spots in agarose . When analyzing LT production from 105 E . coli strains, results obtained by this procedure (GM1-ELISPOT) correlated well with those of the GM1 enzyme-linked immunosorbent assay (GM1-ELISA); in no instance were any false-positive reactions observed when highly specific monoclonal antibodies against LT were used . Easy to perform, the GM1-ELISPOT allows demonstration of LT within 24 h after inoculation of the plates, and large numbers of specimens can be screened at the same time without the need of any special equipment . Thus, this new method should meet the requirements of any diagnostic laboratory.

J Appl Bacteriol, 1983 Jun, 54(3), 437 - 9
A note on the repair of the Escherichia coli nucleoid structure after heat shock; Pellon JR; The repair of the Escherichia coli nucleoid structure after heat shock (50 degrees C, 5 min) was studied . After heat shock the repair process did not include the association of the nucleoid to protein structures as is the case after more severe heat treatments resulting in cell death or inactivation.

Immunopharmacology, 1983 Jun, 6(1), 1 - 5
Effect of radio-detoxified endotoxin on the liver microsomal drug metabolizing enzyme system in rats; Bertok L et al.; E . coli endotoxin (LPS) depresses the hepatic microsomal mono-oxygenase activity . Radio-detoxified LPS (TOLERIN: 60 Co irradiated endotoxin preparation) decreases this biotransforming activity to a smaller extent . Phenobarbital, an inducer of this mono-oxygenase system, failed to induce in LPS-treated animals . In radio-detoxified LPS-treated rats, phenobarbital induced the mono-oxygenase and almost fully restored the biotransformation.

Clin Exp Immunol, 1983 Jun, 52(3), 586 - 94
Cyclosporin A inhibits acute serum sickness nephritis in rabbits; Neild GH et al.; The effect of the immunosuppressive agent cyclosporin A (Cy A) on the renal injury in acute serum sickness was examined in rabbits . Serum sickness was induced in 23 untreated NZW rabbits by a single intravenous injection of bovine serum albumin (BSA) 250 mg/kg with E . coli endotoxin (5 micrograms/kg): BSA was eliminated after 8.6 +/- 0.16 days (mean +/- s.e . (mean}; proteinuria occurred in 19 (84%) and glomerular proliferation in 20 (87%) rabbits . When Cy A (15 or 25 mg/kg) was given daily by intramuscular injection, starting either 2 days before or at the time of induction of acute serum sickness, proteinuria was profoundly reduced and glomerular proliferation was inhibited . Even when rabbits were first treated with Cy A (25 mg/kg) 5 days after the induction of disease proteinuria and glomerular proliferation were similarly inhibited . When the treated animals were compared with controls there were no differences in the following: time to elimination of BSA, amount or size of circulating immune complexes, fall in serum C3 at immune elimination, or deposition of immune reactants in the glomeruli . These results show that Cy A inhibits the renal injury of acute serum sickness and indicate that T cells may play a role in mediating the nephritis in this condition.

Am J Vet Res, 1983 Jun, 44(6), 949 - 54
Susceptibility of sows to experimentally induced Escherichia coli mastitis; Ross RF et al.; A study was conducted to compare susceptibility of sows from 2 herds to experimentally induced Escherichia coli mastitis . Four sows each from herds R and S were inoculated intramammarily at postpartum hour 8 with a strain of E coli shown previously to be capable of producing mastitis . After inoculation with E coli, sows from herd S had higher temperatures, lower WBC counts, and lower plasma protein:fibrinogen ratios than did sows from herd R . Inoculated sows from herd S lost 83% of newborn pigs due to starvation by 14 days after inoculation, whereas sows from herd R lost none . Control, noninoculated sows from both herds had normal temperatures, normal hematologic values, and minimal mortality of piglets . Levels of antibodies (complement fixing, enzyme-linked immunosorbent assay, and agglutinating) to E coli in preinoculation sera from the 2 populations of sows did not differ . Assay of lactoferrin by radial immunoassay revealed comparable concentrations in milk of sows from both herds during the first 24 hours after sows had delivered, but significantly higher values were detected in milk from sows of herd S at postpartum days 2 and 3 . The basis for the marked difference in susceptibility to E coli-induced mastitis was not determined except that "susceptible" sows (herd S) were from a conventional herd and "resistant" sows (herd R) were from a specific-pathogen-free herd.

Scand J Immunol, 1983 Jun, 17(6), 569 - 74
IgA antibodies in rat bile are not solely derived from thoracic duct lymph; Dahlgren U et al.; The IgA level in rat bile was significantly decreased by drainage of the thoracic duct, and passively administered IgA antibodies to Escherichia coli O antigen decreased similarly . In contrast, specific IgA antibodies against E . coli O antigen raised by immunization in the Peyer's patches did not diminish significantly in the bile . Rats immunized in the Peyer's patches with sheep erythrocytes had IgA-forming cells in the thoracic lymph nodes, in the mesenteric lymph nodes, and in the spleen . Perfusion of the liver of immunized animals significantly decreased the bile levels of the IgA antibodies . It seems that IgA antibodies reach the bile not only via the thoracic duct but also via lymph ducts originating from thoracic lymph glands and the spleen.

Cell, 1983 Jun, 33(2), 629 - 37
RecA-independent recombination between direct repeats of IS50; Zupancic TJ et al.; Recombination between the IS50 sequences that flank Tn5 has been found to occur readily after the transformation of E . coli recA strains with a plasmid that contains direct repeats of these sequences . Analysis of mutants of IS50 indicates that the polypeptide encoded by IS50 that is required for transposition is also required for the recombination . Surprisingly, the mechanism of recombination appears to be similar to general homologous recombination rather than site-specific recombination . This IS50 recombination system may be responsible for resolving transient cointegrate structures containing direct repeats of IS50 or Tn5 during the transposition of IS50 and Tn5.

Am J Physiol, 1983 Jun, 244(6), H763 - 8
Effect of Escherichia coli endotoxin on dog lung fluid balance; Scott RL et al.; Endotoxin may cause an increase in pulmonary capillary permeability and thus promote edema formation . We used a gravimetric technique to estimate the pulmonary capillary filtration coefficient (KF) and the maximum capillary pressure at which the lung could maintain a constant weight (Pccritical) in dogs after intravenous administration of Escherichia coli (E . coli) endotoxin . KF should be increased and Pccritical should be decreased by an increase in permeability . Four groups of three to four dogs were given 1, 10, 1,000, or 3,000 micrograms/kg of endotoxin . A fifth group of five dogs, which served as controls, was given no endotoxin . KF was significantly (P less than 0.05) greater than control {0.049 +/- 0.031 (SD) ml . min-1 . mmHg-1} in only the 1-micrograms/kg group (0.100 +/- 0.027), indicating a possible increase in permeability . However, changes in capillary surface area may have affected KF . Pccritical was not significantly different from control (20.7 +/- 2.4 mmHg) in any of the E . coli groups . We conclude from these results that E . coli endotoxin may have caused a slight increase in permeability; however, the lung retained its ability to resist edema formation.

Proc Natl Acad Sci U S A, 1983 Jun, 80(12), 3845 - 9
cDNA clone for the alpha subunit of the acetylcholine receptor from the mouse muscle cell line BC3H-1; Merlie JP et al.; Sequences from a gene coding for mouse acetylcholine receptor alpha subunit have been inserted into a recombinant plasmid and cloned in Escherichia coli . mRNAs for acetylcholine receptors occur in low abundance in vertebrate muscle . To clone the mouse alpha-subunit cDNA, we made use of (i) a cell line, BC3H-1, that overproduces the alpha-subunit mRNA and (ii) a polysome fractionation procedure that results in enrichment of alpha-subunit mRNA . Polyadenylylated RNA was used to construct a cDNA library of 750 recombinant clones . Acetylcholine receptor-specific sequences were identified by hybrid-selected translation, followed by monoclonal antibody precipitation and peptide mapping of the translation product . One clone (pA59) that fit these criteria was found in the first 80 isolates . It had a 700-base-pair insert that was excised with Pst I . Blot-hybridization experiments with nick-translated pA59 DNA showed that BC3H-1 cells contain 100-1,000 times more alpha-subunit mRNA than does newborn or adult mouse muscle . Blot hybridization of restriction digests of mouse liver DNA revealed that pA59 is homologous to a very small number (probably one) of genomic sequences.

Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3322 - 6
Topology of the lac carrier protein in the membrane of Escherichia coli; Goldkorn T et al.; Proteolysis of topologically sealed right-side-out and inside-out membrane vesicles from Escherichia coli with chymotrypsin, trypsin, or papain inactivates lac carrier function in a symmetrical manner . Concomitantly, the electrophoretic mobility of lac carrier protein photoaffinity labeled in situ with p-nitro{2-3H}phenyl-alpha-D-galactopyranoside is altered from a relative Mr of 33,000 to 20,000, and the time course of proteolysis is almost identical in vesicles of opposite polarities . In contrast, solubilization of the vesicles in NaDodSO4 followed by proteolysis causes fragmentation of the Mr 33,000 band into material that electrophoreses at the solvent front . Notably, proteolysis has no effect whatsoever on the ability of the lac carrier protein to bind substrate, as judged by photoaffinity-labeling experiments . Furthermore, the electrophoretic patterns of samples proteolyzed prior to photoaffinity labeling are the same as those observed when the procedures are reversed . These results show that the lac carrier protein spans the membrane and indicate that the binding site resides within a segment that is embedded in the bilayer.

Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3301 - 4
Ribonuclease BN: identification and partial characterization of a new tRNA processing enzyme; Asha PK et al.; A new ribonuclease, RNase BN, has been identified and partially purified from a strain of Escherichia coli lacking RNase II and RNase D by using the artificial tRNA precursor tRNA-C-{14C}U as substrate . This enzyme is present in E . coli B but absent from the tRNA processing mutant strain BN which is unable to process extraneous 3' residues on certain phage T4-specified tRNA precursors . The properties of RNase BN clearly distinguish this enzyme from other known E . coli exoribonucleases . It is optimally active at pH 6.5 with 0.2 mM divalent cation and 0.2 M monovalent cation . It is most active against tRNA substrates containing nucleotide substitutions within the -C-C-A sequence and relatively inactive against other types of RNAs . This substrate specificity in vitro is consistent with a processing function in vivo . However, in contrast to the other processing enzymes whose function has been confirmed by mutation, RNase BN is an exoribonuclease . The presence of multiple RNases in E . coli and a strategy for their identification and separation are discussed.

Lab Invest, 1983 Jun, 48(6), 705 - 10
Prevention of the generalized Shwartzman reaction in pregnant rats by prostacyclin infusion; Campos A et al.; The effects of prostacyclin infusion on endotoxin-induced generalized Shwartzman reaction were studied in pregnant rats . Four hours after administration of Escherichia coli endotoxin (0.4 mg) to pregnant rats on the 20th day of gestation, fibrin and platelet thrombi were observed in 72% of the animals . These findings were associated with a decrease in renal blood flow and glomerular filtration rate from 7.0 to 0.7 and 0.8 +/- 0.1 to 2.6 +/- 0.4 and 0.2 +/- 0.8 ml/minute, respectively (p less than 0.0001 and less than 0.05) and a decrease in platelet count (p less than 0.05) . The infusion of prostacyclin, at a mean dose of 57 ng/kg/minute prior to and after administration of endotoxin strikingly inhibited the development of fibrin and platelet thrombi in glomerular capillaries (p less than 0.001) . Despite hemodynamic changes which were similar to those in control animals, renal blood flow and glomerular filtration rate decreased from 7.2 +/- 0.2 and 0.9 to 2.9 +/- 0.03 and 0.3 +/- 0.1 ml/minute, respectively (p less than 0.001 and less than 0.025) . In addition, the mean blood pressure decreased from 118 +/- 3.8 to 86 +/- 3.7 mm Hg (p less than 0.025), whereas the platelet count remained normal . We conclude that prostacyclin infusion prior to endotoxin administration significantly inhibits endotoxin-induced generalized Shwartzman reaction in pregnant rats.

J Bacteriol, 1983 Jun, 154(3), 1508 - 12
DNA sequences of the D-serine deaminase control region and N-terminal portion of the structural gene; McFall E et al.; We determined the DNA sequence of the D-serine deaminase promoter region and of the N-terminal region of the structural gene . There are possibilities in the promoter for secondary structure and for initiation recognition sequences, and there is an open reading frame . The N-terminal sequence for the structural gene confirms that part of the amino acid sequence previously determined by E . Schlitz and W . Schmitt (FEBS Lett . 134:57-62, 1981), including the active site of the enzyme, and spans the two regions unresolved by their work.

J Bacteriol, 1983 Jun, 154(3), 1505 - 7
Identification of the heat-inducible protein C15.4 as the groES gene product in Escherichia coli; Tilly K et al.; The product of the Escherichia coli morphogenetic gene groES (mopB) was identified as the heat-inducible protein C15.4 by two-dimensional gel analysis of the products of wild-type and mutant alleles carried on the bacterial chromosome, on a hybrid plasmid, and on a transducing phage.

J Bacteriol, 1983 Jun, 154(3), 1339 - 46
Coupling of DNA replication and cell division: sulB is an allele of ftsZ; Lutkenhaus JF; Treatments that damage DNA in Escherichia coli result in the inhibition of cell division . This inhibition is controlled by the lexA-recA regulatory circuit and can be specifically uncoupled by the mutations sulA (sfiA) and sulB (sfiB), which map at 21 and 2 min, respectively . Presently it is thought that sulA codes for an inducible inhibitor of cell division, the expression of which is controlled directly by the lexA repressor . In this report, it is shown that sulB is an allele of ftsZ, an essential cell division gene . A sulB mutation leads to an altered ftsZ gene product which is slightly thermosensitive and has an altered mobility on polyacrylamide gels . It is suggested that the altered ftsZ gene product is resistant to the sulA inhibitor, thus permitting cell division after induction of the SOS response . It is also shown that an increase in the gene dosage of ftsZ delays the onset of filamentation after SOS induction.

J Bacteriol, 1983 Jun, 154(3), 1329 - 38
Suppressed nonsense mutations in the araC gene of Escherichia coli provide three novel variant proteins; Schecter JB; A total of 400 suppressible mutations have been isolated in the araC gene of Escherichia coli . Based on deletion mapping, growth patterns when suppressed, and intragenic recombination, 37 mutants have been determined to contain unique mutations . Rapid plate assays were developed to test for each of the three AraC protein functions: inducing araBAD, repressing araBAD, and araC self-repression . The 185 mutant proteins, resulting from 37 mutants each suppressed by five different suppressors, were assayed for each of the three AraC functions . These plate assays showed that: (i) for each function, some areas of the gene map are more sensitive to mutation than other areas, and (ii) three of the mutant AraC proteins were unlike previously characterized AraC mutants . Enzyme assays on the mutant proteins confirmed their novel character . The first mutant cannot induce araBAD but retains the capacity to perform both repression functions; and the second and third can each perform one of the two repression functions better than it can perform the other . These characteristics suggest that previously proposed models of ara regulation are incomplete.

J Bacteriol, 1983 Jun, 154(3), 1323 - 8
Levels of ribosomal protein S1 and elongation factor G in the growth cycle of Escherichia coli; Lambert JM et al.; The relative levels of ribosomes, ribosomal protein S1, and elongation factor G in the growth cycle of Escherichia coli were examined with two-dimensional polyacrylamide gel electrophoresis . Nonequilibrium pH gradient polyacrylamide gel electrophoresis was used in the first dimension, and polyacrylamide gradient-sodium dodecyl sulfate gel electrophoresis was used in the second dimension . The identities of protein spots containing S1 and elongation factor G were confirmed by radioiodination of the proteins and peptide mapping of the radiolabeled peptides . The levels of ribosomes and ribosomal protein S1 were coordinately reduced during transition from exponential phase to stationary phase . There was no accumulation of S1 in the stationary phase . In marked contrast, the level of elongation factor G showed no significant change from exponential phase to stationary phase . The relative level of elongation factor G compared with ribosomes or S1 increased by about 2.5-fold during transition from exponential phase to stationary phase . The results show that there are differences between the regulation of the levels of elongation factor G and of ribosomal proteins, including S1, apparent during the transition from exponential to stationary phase.

J Bacteriol, 1983 Jun, 154(3), 1263 - 8
Plasmid-controlled resistance to copper in Escherichia coli; Tetaz TJ et al.; The copper resistance of a strain of Escherichia coli isolated from the effluent of a piggery where pigs were fed a diet supplemented with copper sulfate was controlled by a conjugative 78-megadalton plasmid designated pRJ1004 . Plasmid pRJ1004 exhibited surface exclusion and incompatibility with standard plasmids belonging to incompatibility groups I1 and K . Sensitive strains of E . coli K-12 were unable to form colonies on nutrient agar containing more than 4 mM copper, whereas transconjugants which harbored pRJ1004 were able to form colonies on medium containing up to 20 mM copper.

J Bacteriol, 1983 Jun, 154(3), 1174 - 83
Accumulation of ColE1 early replicative intermediates catalyzed by extracts of Escherichia coli dnaG mutant strains; Fouser L et al.; To investigate the events occurring at the replication forks during DNA synthesis, we studied the replication of plasmid ColE1 DNA in vivo and in vitro, using strains of Escherichia coli carrying either the dnaG3(Ts) or dnaG308(Ts) mutation . Extracts of both mutant strains supported in vitro DNA synthesis, but the amount of {3H}TMP incorporated into DNA was always less for mutant extracts than for extracts of revertant strains, which were able to grow at 42 degrees C . Sucrose gradient analysis, Southern blot analysis, and electron microscopy showed that mutant extracts synthesize a large number of early replicative intermediates containing one or two (one on each template strand) fragments at the origin of replication and some completed molecules, either open circles or covalently closed circles . The revertant extracts synthesized more completed molecules although the fraction of templates used was about the same, 0.27 for mutant extracts and 0.21 for revertant extracts . Our results show that a mutation in dnaG causes a block in the synthesis of both leading and lagging strands after initiation, which results in the accumulation of early replicative intermediates . The average size of the newly replicated region in the early replicative intermediates is 730 bases as measured from electron micrographs of early replicative intermediates . We conclude that the DnaG protein functions in lagging strand synthesis and may be necessary for the continuation of leading strand synthesis as well.

J Bacteriol, 1983 Jun, 154(3), 1145 - 52
Spontaneous deletions and flanking regions of the chromosomally inherited hemolysin determinant of an Escherichia coli O6 strain; Hacker J et al.; The hemolytic Escherichia coli strain 536 (O6) propagates spontaneous hemolysin-negative mutants at relatively high rates (10(-3) to 10(-4)) . One type of mutant (type I) lacks both secreted (external) and periplasmic (internal) hemolysin activity (Hlyex-/Hlyin-) and in addition shows no mannose-resistant hemagglutination (Mrh-), whereas the other type (type II) is Hlyex-/Hlyin+ and Mrh+ . The genetic determinants for hemolysin production (hly) and for mannose-resistant hemagglutination (mrh) of this strain are located on the chromosome . Hybridization experiments with DNA probes specific for various parts of the hly determinant reveal that mutants of type I have lost the total hly determinant, whereas those of type II lack only part of the hlyB that is essential for transport of hemolysin across the outer membrane . Using a probe that contains the end sequence of the plasmid pHly152-encoded hly determinant (adjacent to hlyB), we determined that a related sequence flanks also the hlyB-distal end of the chromosomal hly determinant of E . coli 536 . In addition several other similar or even identical sequences are found in the vicinity of the hlyC- and the hlyB-distal ends of both the chromosomal and the plasmid hly determinants.

J Bacteriol, 1983 Jun, 154(3), 1098 - 103
Trimethoprim-induced DNA polymerase I deficiency in Escherichia coli K-12; Mukhopadhyay P et al.; Curing of the mini-ColE1 plasmid pML21 was observed among cells of Escherichia coli K-12 strain C600(pML21) grown under subinhibitory conditions in the presence of trimethoprim, a specific inhibitor of dihydrofolate reductase . Some of the cured colonies showed (i) a reduction in frequency of transformation with pML21 compared with those of isogenic strains not treated with trimethoprim, (ii) loss of viability after acquisition of a recA mutation, and (iii) UV sensitivity greater than that of the original isogenic strain . These colonies therefore had PolA- phenotypes . Moreover, they were found to be deficient in DNA polymerase I activity in the in vitro assays, indicating the occurrence of a polA mutation in them . Many of the colonies with PolA- phenotypes were also thyA deoC mutants, and these mutations, in addition to the polA mutations, appeared to be involved in the expression of the PolA- phenotypes.

Infect Immun, 1983 Jun, 40(3), 924 - 9
Protection against human and porcine enterotoxigenic strains of Escherichia coli in rats immunized with a cross-linked toxoid vaccine; Klipstein FA et al.; To compare their relative immunogenicities, we used synthetically produced Escherichia coli heat-stable toxin coupled to a protein carrier and the B subunit of porcine heat-labile toxin separately in graded dosages to immunize rats . Equivalent antigen unit dosages of each toxin raised approximately the same level of mucosal immunoglobulin A (IgA) antitoxin response and degree of protection against a challenge with respective heat-stable- or heat-labile-toxin-producing viable bacteria . Conjugation conditions were identified, therefore, which yielded a vaccine of these toxins, cross-linked by the carbodiimide reaction, that consisted of equal antigenic proportions of each toxin component as determined by enzyme-linked immunosorbent assay and expressed in antigen units . The dose-related response to immunization with this vaccine was the same as the response to its components given separately . The toxicity of the heat-stable toxin component was reduced greater than 600-fold . Immunization with optimal antigen unit dosages of the vaccine gave greater than or equal to sixfold increases in mucosal IgA antitoxin titers and provided significant (P less than 0.001) protection against challenge with heterologous serotypes of viable strains, of either human or porcine origin, that produce heat-stable or heat-labile toxin or both.

Infect Immun, 1983 Jun, 40(3), 1236 - 9
Plasmids that code for production of colonization factor antigen II and enterotoxin production in strains of Escherichia coli; Smith HR et al.; Similar plasmids that code for production of colonization factor antigen II and enterotoxins were mobilized into Escherichia coli K-12 from strains of E . coli O6.H16 biotype B or C that expressed colonization factor antigen II components CS2 and CS3 . Further transfer experiments demonstrated that there was production of CS2 and CS3 in an O6.H16 biotype C strain, but there was expression of only CS3 in other serogroups and in E . coli K-12.

Immunology, 1983 Jun, 49(2), 301 - 9
Ontogeny and characterization of mitogen-reactive lymphocytes in the thymus and spleen of the amphibian, Xenopus laevis; Williams NH et al.; The ontogeny of the proliferative response of Xenopus thymocytes to the T-cell mitogens concanavalin A (Con A) and phytohaemagglutinin and to the B-cell mitogen E . coli lipopolysaccharide (LPS) is examined during the first 2 years of life . Responses after metamorphosis are compared with those occurring in the spleen . During much of larval life, mitogen-treated thymocytes display no sign of elevated {3H}TdR uptake . A low level in vitro response to Con A and LPS emerges transiently just prior to metamorphosis, the latter period being associated with a temporary disruption of thymocyte mitogen reactivity . Appreciable stimulation of both thymocytes and splenocytes with all three mitogens emerges within the first few months post-metamorphosis, and, with the exception of the thymocyte response to LPS, these induced proliferative levels are maintained or increased in toadlets aged 7-24 months . Maximal levels of LPS responsiveness in thymocyte cultures are found at 6 months of age . LPS-induced thymocyte proliferation at this time is confirmed morphologically by autoradiography; furthermore, immunofluorescence experiments, using an anti-Xenopus-mu chain antiserum, reveal that LPS induces the differentiation of cytoplasmic IgM cells in both spleen and thymus . The results indicate the presence of substantial populations of mitogen-sensitive cells in B lineage in the anuran thymus.

Eur J Biochem, 1983 Jun 1, 133(1), 155 - 62
The pyruvate dehydrogenase complex of Escherichia coli K12 . Nucleotide sequence encoding the pyruvate dehydrogenase component; Stephens PE et al.; The nucleotide sequence of a 3780-base-pair segment of DNA containing the aceE gene encoding the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method . The aceE structural gene comprises 2655 base pairs (885 codons, excluding the initiation codon AUG), it is preceded by a good ribosome binding site and several potential RNA polymerase binding sites . Its polarity and location in the restriction map of the corresponding segment of DNA are consistent with it being the proximal gene in the ace operon, as defined in previous genetic and post-infection labelling studies . The relative molecular mass (99474), composition (885 amino acids), amino-terminal residue and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with published information obtained from studies with the purified pyruvate dehydrogenase component (E1) . The nucleotide sequence also contains a second gene (gene A) situated upstream of the aceE gene . It appears to be an independent gene containing 708 base pairs (236 codons) and encoding a weakly expressed product (protein A; Mr = 27049) of unknown function.

Gastroenterology, 1983 Jun, 84(6), 1547 - 52
Ability of various adsorbents to bind endotoxins in vitro and to prevent orally induced endotoxemia in mice; Ditter B et al.; The efficacy of various adsorbents for endotoxin was tested in vitro and in vivo using a murine experimental model of gut-derived endotoxemia . A quantitative limulus amebocyte lysate microtiter test and the limulus amebocyte lysate tube test were used to determine intestinal and circulating levels of endotoxin . Kaopectate, kaolin/pectin mixture, kaolin, pectin, bentonite, charcoal particles, and lactulose were tested for their ability to bind endotoxins both in vitro and in vivo . The most effective material in the prevention of endotoxemia provided to be bentonite followed by Kaopectate and charcoal particles . Kaolin least effectively bound endotoxin at similar concentrations, while lactulose and pectin had minimal effects . Good correlation was shown between the ability of these drugs to bind endotoxin in vitro as compared with in vivo action.

Br J Pharmacol, 1983 Jun, 79(2), 421 - 8
Plasma acid phosphatase levels in endotoxaemia: modification by drugs and chemically detoxified endotoxins; Godin DV et al.; The ability of chemically detoxified E . coli endotoxins and membrane-active agents to modify the toxicity of native E . coli endotoxin in vivo was examined . The time- and dose-dependent increase in plasma acid phosphatase activity following toxin administration to rats provided a convenient quantitative measure of in vivo toxicity under various experimental conditions . Treatment of endotoxin with either sodium hydroxide or sodium periodate produced substances which, when injected alone, failed to cause an increase in plasma acid phosphatase activity . When given before native endotoxin, periodate-detoxified toxin produced a dose-dependent reduction in the elevation of plasma enzyme activity caused by unmodified toxin . Pretreatment with pranolium, hydrocortisone or (+)-propranolol also reduced the in vivo toxicity of endotoxin . Mortality studies in mice provided further independent support for the effectiveness of periodate-detoxified endotoxin and membrane-active drugs as endotoxin antagonists . Evidence has been found that under certain conditions gentamicin may act synergistically with bacterial endotoxins in vivo.

J Gen Microbiol, 1983 Jun, 129 (Pt 6), 1651 - 9
The electron transport chain of Escherichia coli grown anaerobically with fumarate as terminal electron acceptor: an electron paramagnetic resonance study; Ingledew WJ; The electron transport chain of Escherichia coli grown anaerobically on glycerol with fumarate as terminal electron acceptor, has been studied using electron paramagnetic resonance (EPR) spectroscopy . The analysis did not include cytochromes, but was confined to the lower potential (dehydrogenase) section of the electron transport chain . Several ferredoxin-type centres were detected and partially characterized, and the possible presence of iron-sulphur centres paramagnetic in their oxidized form ('HiPIP-type') was also noted . An EPR-detectable signal that may have been due to the presence of molybdenum in the electron transport chain is described and assessed . Most of the centres detected were reducible by substrate in the absence of oxidant and some were found to be wholly or partly reduced during steady state oxidation of substrates in the presence of oxygen.

Biochimie, 1983 Jun, 65(6), 339 - 44
Transcription termination factor rho of Escherichia coli K-12: some regulatory aspects of its expression and activity; Biville F et al.; A highly sensitive radioimmunological assay of the transcription termination factor (rho) of Escherichia coli K-12 has been developed . This method allows to measure in crude bacterial extracts quantities as low as 1.8 X 10(-15) moles of rho . We studied, under various conditions of growth and in mutants lacking or overproducing the cyclic AMP receptor protein (CAP) or adenylate cyclase, the relationship between the level of rho and its poly(C)-dependent ATPase activity . We showed that neither growth conditions, nor the presence or absence of a functional cAMP-CAP complex affect the synthesis and the enzymatic activity of the rho protein.

Antimicrob Agents Chemother, 1983 Jun, 23(6), 846 - 51
Safety and tolerance of recombinant leukocyte A interferon in bone marrow transplant recipients; Winston DJ et al.; Five bone marrow transplant recipients with cytomegalovirus infections were treated with pure recombinant leukocyte A interferon produced by recombinant DNA technology from Escherichia coli . All five patients had documented interstitial pneumonia . The daily intramuscular dose of interferon ranged from 18 X 10(6) to 50 X 10(6) U; the mean duration of therapy was 11.0 days (range, 5 to 18 days) . Two patients recovered, one improved, and two died . Clinical side effects (usually fever and chills) occurred in three patients . A 60% or greater reduction in the pretherapy peripheral granulocyte counts occurred in four patients, and four patients had a 37 to 80% reduction in their pretherapy platelet counts . Hematological toxicity was reversible, and there was no loss of marrow graft function . The toxicity of pure recombinant leukocyte A interferon in marrow transplants is similar to that of partially pure leukocyte interferon derived from human cells . Further controlled studies to establish the efficacy of recombinant leukocyte A interferon in marrow transplants are warranted but may be limited by hematological toxicity.

Anal Biochem, 1983 Jun, 131(2), 397 - 403
A rapid two-dimensional fractionation of DNA restriction fragments; Chen CW et al.; Genomic DNA that has been digested with a restriction endonuclease and fractionated by electrophoresis on an agarose gel can be recovered on glass-fiber filters by a new blotting scheme . The DNA fragments in each fraction are then digested with a second restriction nuclease and then separated on a slab gel, resulting in a two-dimensional display of the restriction fragments . This rapid fingerprinting technique is useful in the analysis of complex genomes and in the isolation and cloning of particular sequences.

Genetika, 1983 Jun, 19(6), 903 - 11
{Induction of transposon Tn1 translocations as affected by different mutagens}; Kubaneishvili MG et al.; Transposition of ampicillin Tn1 transposon is repressed under normal conditions and occurs with low frequency (10(-4) per cell) . Treatment of Escherichia coli cells with 22 c+mytomycin C, nitrosoguanidine and UV light induced transposition of Tn1 into different replicons . The degree of induction depended upon the strain of bacteria and the replicon which the transposon was inserted into . Mutation recA did not influence spontaneous translocation of the transposon but fully repressed induction of transposition during the period of mutagen treatment . In the present paper, the connection of inducible transposition process with the recA and lexA inducible functions of E . coli is discussed.

Biochem J, 1983 Jun 1, 211(3), 717 - 26
Mutations in the uncE gene affecting assembly of the c-subunit of the adenosine triphosphatase of Escherichia coli; Jans DA et al.; The amino acid substitutions in the mutant c-subunits of Escherichia coli F1F0-ATPase coded for by the uncE429, uncE408 and uncE463 alleles affect the incorporation of these proteins into the cell membrane . The DNA sequence of the uncE429 allele differed from normal in that a G leads to A base change occurred at nucleotide 68 of the uncE gene, resulting in glycine being replaced by aspartic acid at position 23 in the c-subunit . The uncE408 and uncE463 mutant DNA sequences were identical and differed from normal in that a C leads to T base change occurred at nucleotide 91 of the uncE gene, resulting in leucine being replaced by phenylalanine at position 31 in the c-subunit . An increased gene dosage of the uncE408 or uncE463 alleles resulted in the incorporation into the membranes of the mutant c-subunits . The results are discussed in terms of the 'Helical Hairpin Hypothesis' of Engelman & Steitz {(1981) Cell 23,411-422}.

Virology, 1983 Jun, 127(2), 385 - 96
Comparative studies of the expression of linked Escherichia coli gpt gene and BPV-1 DNAs in transfected cells; Giri I et al.; A series of hybrid plasmids, containing two selective markers that can be expressed in mammalian cells have been constructed . These plasmids are derived from the pSV2gpt recombinant plasmid described by Mulligan and Berg (1980) and contain the entire BPV-1 DNA, or the HindIII-BamHI large transforming fragment (T69) or the early transforming region of polyoma virus DNA . DNA transfers into Fisher rat 3T3 cells were performed either by the calcium phosphate coprecipitation technique, or by protoplast fusion . For all plasmids, the frequency of formation of phenotypically transformed foci or of the expression of the gpt+ marker in selective medium have been scored comparatively . Both series of recombinant plasmids gave similar yield of gpt+ colonies, whereas BPV plasmids (both entire or T69 subgenome) transformed morphologically rat cells 8-50 times less frequently than their polyoma homologues . Although the pSV2gpt BPV-1 plasmids can replicate autonomously to high copy number in monkey COS cells, the rate of transcription of the BPV-1 genome in these cells is 10(2) to 10(3) times lower than that of gpt transcribed from the SV40 early promoter . This low rate of transcription may explain the low frequency of transformation by this viral DNA.

Mutat Res, 1983 Jun, 112(3), 139 - 45
Strand-break formation in DNA modified by benzo{alpha}pyrene diolepoxide . Quantitative cleavage by Escherichia coli uvrABC endonuclease; Seeberg E et al.; Covalently closed circular plasmid DNA was modified by benzo{alpha}pyrene diolepoxide and incubated with partially purified fractions of the Escherichia coli uvr+ gene products . Strand breaks were introduced into the modified DNA by the uvrABC endonuclease; on average, one break was formed for each bound benzo{alpha}pyrene residue in the DNA . These results are direct evidence that benzo{alpha}pyrene adducts in DNA are acted upon by the same repair enzyme as those that handle UV-induced lesions in DNA.

Cell, 1983 Jun, 33(2), 345 - 55
The product of the retroviral transforming gene v-myb is a truncated version of the protein encoded by the cellular oncogene c-myb; Klempnauer KH et al.; Avian myeloblastosis virus (AMV) is an oncogenic retrovirus that rapidly causes myeloblastic leukemia in chickens and transforms myeloid cells in culture . AMV carries an oncogene, v-myb, that is derived from a cellular gene, c-myb, found in the genomes of vertebrate species . We constructed a plasmid vector that allows expression of a portion of the coding region for v-myb in a procaryotic host . We then used the myb-encoded protein produced in bacteria to immunize rabbits . The antisera obtained permitted identification of the proteins encoded by both v-myb and chicken c-myb . The molecular weights of the products of v-myb and c-myb (45,000 and 75,000 respectively) indicate that the v-myb protein is an appreciably truncated version of the c-myb protein.

Arch Biochem Biophys, 1983 Jun, 223(2), 503 - 13
Methylglyoxal-mediated growth inhibition in an Escherichia coli cAMP receptor protein mutant; Puskas R et al.; Under certain growth conditions, some strains of Escherichia coli accumulate toxic levels of methylglyoxal . This report characterizes a strain which synthesizes a mutant cAMP receptor protein in an adenylate cyclase deletion background . When cultured in glucose 6-phosphate minimal medium, this strain (222) was prematurely growth arrested due to methylglyoxal production; growth inhibition did not occur when the strain was grown in glucose minimal medium . A comparison of a variety of enzyme and cofactor levels in the related strains 222 (mutant) and 225 (wild-type) grown on either glucose or glucose 6-phosphate medium was carried out . The only difference found that might explain an increase in methylglyoxal accumulation was an elevated level of phosphofructokinase in strain 222 grown on glucose 6-phosphate . Since this enzyme activity probably limits hexose phosphate metabolism, it is suggested that growth inhibition in strain 222 may be due to increased production of triose phosphate, some of which is converted to methylglyoxal.

Proc Natl Acad Sci U S A, 1983 Jun, 80(12), 3671 - 5
Synthesis of calf prochymosin (prorennin) in Escherichia coli; Emtage JS et al.; A gene for calf prochymosin (prorennin) has been reconstructed from chemically synthesized oligodeoxyribonucleotides and cloned DNA copies of preprochymosin mRNA . This gene has been inserted into a bacterial expression plasmid containing the Escherichia coli tryptophan promoter and a bacterial ribosome binding site . Induction of transcription from the tryptophan promoter results in prochymosin synthesis at a level of up to 5% of total protein . The enzyme has been purified from bacteria by extraction with urea and chromatography on DEAE-cellulose and converted to enzymatically active chymosin by acidification and neutralization . Bacterially produced chymosin is as effective in clotting milk as the natural enzyme isolated from calf stomach.

Proc Natl Acad Sci U S A, 1983 Jun, 80(12), 3599 - 603
Enzymatic deamidation of methyl-accepting chemotaxis proteins in Escherichia coli catalyzed by the cheB gene product; Kehry MR et al.; The methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli undergo reversible methylation that has been correlated with adaptation of cells to environmental stimuli . MCPI, the product of the tsr gene, accepts methyl groups at multiple sites that are located on two tryptic peptides, denoted K1 and R1 . A second modification of the MCPs, which is not methylation, has been designated the CheB-dependent modification . A CheB-dependent modification occurs on methyl-accepting peptide K1 and allows additional methyl groups to be incorporated into this peptide . We have performed partial amino acid sequence analyses on radiolabeled peptides K1 and R1 derived from MCPI and have identified several methyl-accepting sites . We found that, in the absence of CheB-dependent modification, a site in peptide K1 is unable to accept methyl groups . Correlation of this protein sequence data with the nucleotide sequence of the tsr gene {Boyd, A., Kendall, K . & Simon, M.I . (1983) Nature (London) 301, 623-626} suggests that CheB-dependent modification of MCPI is the enzymatic deamidation of glutamine to methyl-accepting glutamic acid . Possible roles for this deamidation in bacterial chemotaxis are discussed.

Proc Natl Acad Sci U S A, 1983 Jun, 80(12), 3563 - 7
Defects in functional expression of an influenza virus hemagglutinin lacking the signal peptide sequences; Sekikawa K et al.; We have investigated the requirement of the signal sequence for expression of influenza virus hemagglutinin (HA) . For this purpose we used a recombinant prepared from a late-region deletion mutant of simian virus 40 (SV40) and cloned influenza HA DNA; the influenza DNA was inserted into the late region of SV40 previously occupied by the deleted sequences coding for SV40 capsid proteins . A simple in-phase deletion was made in the HA DNA, resulting in loss of 11 internal amino acids from the 16 amino acid signal peptide . This deletion HA recombinant was then used to infect African green monkey kidney cells . Mutant HA was not detected on the cell surface but stably accumulated in the cytoplasm at a level similar to that of wild-type HA . NaDodSO4/polyacrylamide gel analysis of lysates from infected cells showed that mutant HA was not glycosylated . Significantly, the amount of mutant HA synthesized was not affected by tunicamycin . In contrast, wild-type HA was decreased more than 90% by tunicamycin . These findings suggest that mutant polypeptide is synthesized on free polyribosomes rather than on membrane-bound polyribosomes . The mutant HA failed to agglutinate erythrocytes, probably due to a defect directly or indirectly associated with the lack of carbohydrate side chains.

Proc Natl Acad Sci U S A, 1983 Jun, 80(12), 3548 - 52
Primary and secondary structure of hamster vimentin predicted from the nucleotide sequence; Quax-Jeuken YE et al.; The nucleotide sequence of two recombinant plasmids containing hamster vimentin cDNA was determined . The sequence comprises 1,640 base pairs and reveals virtually the total primary structure of vimentin and a large part of the 3' noncoding region . Secondary structure prediction methods allow the characterization of two distinct regions of the polypeptide chain, 135 and 145 residues long, which are able to form alpha helices organized in "coiled coils." Three nonhelical domains can be distinguished: a very basic NH2-terminal domain of at least 67 residues, a nonhelical region of 45 amino acids separating the two helix domains, and a COOH-terminal region of 55 residues, which contains an excess of acidic amino acids . The meaning of each of these domains of the vimentin polypeptide for the subunit and filament formation is discussed.

Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3411 - 5
Fine structure analysis and nucleotide sequence of the vaccinia virus thymidine kinase gene; Hruby DE et al.; The thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene of vaccinia virus has previously been mapped near the middle of the viral DNA, within the 4.85-kilobase HindIII J fragment, and shown to encode a Mr 19,000 polypeptide {Hruby, D . E . & Ball, L . A . (1982) J . Virol . 43, 403-409} . To locate the gene more precisely and to determine the structure of the basic transcriptional unit, the positions of cleavage sites for several restriction endonucleases were mapped within the HindIII J DNA fragment . Four appropriate subfragments of HindIII J DNA were inserted into plasmid pBR322 derivatives and cloned in Escherichia coli . These recombinant plasmid DNAs were tested for their ability to inhibit the cell-free synthesis of active thymidine kinase and to retain the mRNA for this enzyme when immobilized on nitrocellulose filters . The data showed that the gene spanned an EcoRI cleavage site that lies 850 base pairs from the left-hand end of the HindIII J fragment (the HindIII L-J boundary) . Because hybridization of vaccinia virus DNA to partially purified thymidine kinase mRNA detected only a single 670-nucleotide RNA species capable of hybridizing to this region of the genome, nuclease S1 mapping experiments were carried out with thymidine kinase mRNA to protect DNA fragments that were terminally labeled at this EcoRI site . The results indicated that the gene extended from about 550 to 1,150 base pairs from the left end of HindIII J, was transcribed in a rightward direction, and contained no intervening sequences . Hence, a 1.04-kilobase Ava II-Hpa II restriction fragment containing this region of DNA was isolated and subjected to nucleotide sequence analysis . An examination of this nucleotide sequence revealed the presence of an open reading frame of 531 nucleotides capable of encoding a protein of 177 amino acids with a Mr of 20,077.

Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3232 - 6
Regulatory regions of ColE1 that are involved in determination of plasmid copy number; Som T et al.; The copy number of plasmid ColE1 has been known to increase when the Hae II-C segment downstream from the replication origin is deleted . The presence of the 306-base-pair (bp) Hpa II region in the segment is sufficient for reduction of the copy number . Plasmids harboring the region express a trans-acting function that is responsible for the copy number reduction and synthesize a unique protein . A protein specified by the region is purified to near homogeneity and identified as the 63-amino-acid protein encoded by the Hpa II segment . The region, which includes segments 19-25 bp and 53-311 bp downstream from the start site of the primer RNA, is involved in determination of sensitivity to the inhibitory function . In vivo transcription of the galK gene, which is directed by the primer promoter in a segment inserted in front of the structural gene, is inhibited by a plasmid carrying the Hpa II region . The inhibition is strong when the promoter segment contains up to 135 bp downstream from the primer RNA start site, whereas it is weak when only the region up to 52 bp downstream is present.

Postgrad Med, 1983 Jun, 73(6), 175 - 82
Infectious diarrhea . Which culprit? What strategy?
Farmer RG.
The patient with diarrhea is generally looking for prompt relief, not a prolonged diagnostic workup . In many cases effective treatment can be initiated after careful review of the clinical symptoms and of the patient's recent travel or other activities . Stool examination, sigmoidoscopy, or other studies may be necessary for definitive diagnosis or in refractory cases . Drugs are useful to combat many of the causative organisms, but in mild, self-limited infections, supportive therapy may suffice . Indeed, antimotility agents are contraindicated in some types of infectious diarrhea.

J Bacteriol, 1983 Jun, 154(3), 1459 - 61
Use of an Escherichia coli mutant strain permits measurement of single-stranded apurinic-apyrimidinic endonuclease in crude extracts: studies with untransformed cells and cells transformed with plasmids containing the uvrC gene; Schultz RA et al.; We have constructed a strain of Escherichia coli that is defective in exonuclease VII and uracil-DNA glycosylase activities . This strain (xse ung) facilitates the quantitation of single-stranded apurinic-apyrimidinic endonuclease activity in crude extracts . Quantitative comparisons of single-stranded apurinic-apyrimidinic endonuclease activity under conditions in which uvrC protein is overexpressed showed no differences, suggesting that single-stranded apurinic-apyrimidinic endonuclease and uvrC protein are probably distinct.

J Bacteriol, 1983 Jun, 154(3), 1446 - 50
Plasmid transformation in Agmenellum quadruplicatum PR-6: construction of biphasic plasmids and characterization of their transformation properties; Buzby JS et al.; Biphasic, chimeric plasmids for the transformation of Agmenellum quadruplicatum PR-6 (Synechococcus sp . strain 7002) were constructed by splicing the 3.0-megadalton cryptic plasmid from strain PR-6 into plasmids pBR322 and pBR325 from Escherichia coli . Transformants of either E . coli or strain PR-6 by these plasmids could be detected on the basis of the drug resistance marker(s) carried by the chimeric plasmids . Plasmid DNA isolated from a PR-6 transformant transformed PR-6 much more efficiently than plasmid DNA prepared from E . coli . Plasmids from which the AvaI recognition site was deleted (AvaI is an isoschizomer of the AquI restriction endonuclease of strain PR-6) also transformed strain PR-6 much more efficiently than did plasmids containing the AvaI recognition site . These and other results suggest that AquI strongly effects plasmid transformation when the donor plasmid contains an unmodified AquI recognition site . Multimeric forms of the chimeric plasmids are also much more efficient at transforming strain PR-6 than are the analogous monomeric forms.

J Bacteriol, 1983 Jun, 154(3), 1371 - 80
Expression of accumulated capacity for initiation of chromosome and minichromosome replication in dnaA mutants of Escherichia coli; LaDuca RJ et al.; Chromosome and minichromosome replication were examined in temperature-sensitive dnaA mutants of Escherichia coli growing at temperatures between permissive and nonpermissive . Periodicities in {14C}thymidine uptake were detected as cultures incubated at intermediate temperatures approached late exponential-early stationary phase of growth . Exposure of the cultures to a nutritional shift-up caused a stimulation of chromosome replication associated with a rapid initiation of new rounds of replication, very similar to that observed after exposure to chloramphenicol . Addition of rifampin also caused a stimulation, but to a much lesser extent . The induced initiations of chromosome replication took place in two waves, as was the case when the cultures were simply shifted to permissive temperature . Minichromosomes were also stimulated to replicate by the addition of chloramphenicol at intermediate temperatures, providing further evidence that the chromosomal region which responded to the chloramphenicol treatment was in the vicinity of oriC . The findings are consistent with the conclusion that the initiations induced by chloramphenicol are consequences of the involvement of the dnaA gene product in a transcriptional step at initiation, as suggested by Orr et al . The results also suggest that the activity of the dnaA gene product is not normally involved in controlling the frequency of initiation of chromosome replication.

J Bacteriol, 1983 Jun, 154(3), 1269 - 75
Isolation and characterization of an Escherichia coli mutant lacking cytochrome d terminal oxidase; Green GN et al.; A screening procedure was devised which permitted the isolation of a cytochrome d-deficient mutant by its failure to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine . Cytochrome a1 and probably cytochrome b558 were also missing in the mutant . Growth and oxygen uptake rates were similar for both parent and mutant strains . However, the strain lacking cytochrome d had an increased sensitivity to cyanide, indicating that cytochrome d confers some resistance to this respiratory inhibitor . The gene responsible for these phenotypes has been named cyd and maps between tolA and sucB.

J Bacteriol, 1983 Jun, 154(3), 1153 - 61
Overproduction of Escherichia coli replication proteins by the use of runaway-replication plasmids; Yasuda S et al.; A derivative of the runaway-replication plasmid was constructed . This plasmid, pSY343, has the gene for kanamycin resistance and single sites for EcoRI, BamHI, HindIII, KpnI, and XhoI that can be used as cloning sites without inactivating the kanamycin resistance gene or the replication genes . Three replication genes of Escherichia coli were cloned on the plasmid . The activity of dnaA, dnaZ, and ssb gene products were 200-, 90-, and 60-fold greater, respectively, in the cells containing these plasmids than in normal cells.

Exp Parasitol, 1983 Jun, 55(3), 265 - 9
Entamoeba invadens and E . histolytica: separation and purification of precysts and cysts by centrifugation on discontinuous density gradients of Percoll; Avron B et al.; The different cell forms in the life cycle of Entamoeba invadens (trophozoites, precysts, and cysts) were rapidly and quantitatively separated on density step gradients of polyvinylpyrolidone-coated colloidal silica particles (Percoll) . With this method, the gradual process of encystation by E . invadens trophozoites could be monitored . Percoll gradients were also efficient in separating trophozoites of Entamoeba histolytica and bacteria . After purification on Percoll, trophozoites display no evidence of damage when examined by light microscopy and no loss in viability as judged by their ability to multiply.

Clin Chem, 1983 Jun, 29(6), 1051 - 6
Simultaneous determination of phenytoin and phenobarbital in serum or plasma by substrate-labeled fluorescent immunoassay; Dean KJ et al.; This assay system for simultaneously determining phenytoin and phenobarbital in serum and plasma is based on the substrate-labeled fluorescent immunoassay technique . A beta-galactosylcoumarin derivative of phenobarbital and a 4-methylcoumarin phosphodiester derivative of phenytoin are used as substrate labels for Escherichia coli beta-galactosidase and Crotalus atrox phosphodiesterase I, respectively . The smallest measurable concentrations are about 1.6 mg/L for phenytoin, 2.7 mg/L for phenobarbital . Within-run coefficients of variation are about 5% for phenytoin and 2% for phenobarbital, about 6% for both between-runs . Results for phenytoin and phenobarbital in serum and plasma correlate well with those determined by the Ames TDA (r = 0.944 and 0.986, respectively) and Syva's EMIT (r = 0.977 and 0.969, respectively) assays.

Can J Microbiol, 1983 Jun, 29(6), 694 - 9
Unusual properties of a new division mutant of Escherichia coli; Drapeau GR et al.; The properties of a division mutant of Escherichia coli were investigated . At 42 degrees C, this mutant forms nonseptate, multinucleate, filamentous cells typical of division mutants, and at the permissive temperature, is sensitive to ultraviolet (UV) irradiation . Temperature and UV sensitivities are probably due to a single mutation . The mutant phenotype is dominant to wild type . The mutant cells make DNA nearly as effectively as control cells at 42 degrees C or following UV irradiation . They exhibit normal host-cell reactivation capacities and can express all manifestations of the SOS response with the exception of Weigle reactivation . The genetic lesion which mediates this pleiotropic effect is located very close to the leu locus of the linkage map.

J Bacteriol, 1983 Jun, 154(3), 1284 - 90
Pool levels of UDP N-acetylglucosamine and UDP N-acetylglucosamine-enolpyruvate in Escherichia coli and correlation with peptidoglycan synthesis; Mengin-Lecreulx D et al.; A high-pressure liquid chromatography procedure was developed for the isolation and quantitation of UDP-N-acetylglucosamine, UDP-N-acetylglucosamine-enolpyruvate, and UDP-N-acetylmuramic acid, which are the early cytoplasmic precursors of bacterial peptidoglycan . In exponential-phase cells of Escherichia coli K-12, the intracellular concentration of UDP-N-acetylglucosamine was about 100 microM, whereas that of UDP-N-acetylglucosamine-enolpyruvate was only 2 microM . The phosphoenolpyruvate: UDP-N-acetylglucosamine transferase and UDP-N-acetylglucosamine-enolpyruvate reductase activities were investigated in extracts from E . coli . These activities appeared to be present in amounts sufficient for the ongoing rate of peptidoglycan synthesis . Certain uridine nucleotide peptidoglycan precursors were found to inhibit phosphoenolpyruvate: UDP-N-acetylglucosamine transferase activity.

Nippon Yakurigaku Zasshi, 1983 Jun, 81(6), 573 - 84
{Immunopharmacological actions of 6-amidino-2-naphthyl-4-guanidinobenzoate (FUT-175) . 2 . Effect of FUT-175 on immunological reactions in man}; Yanagihara Y et al.; FUT-175 is a new anti-complemental drug which strongly inhibits complement-mediated allergic reactions in animals . It was reported that FUT-175 does not affect both antibody formation and host defense to bacterial infection in mice . The present study was undertaken to examine the effects of FUT-175 on various immunological reactions in humans . FUT-175 dose-dependently decreased the antigen-induced anaphylactic histamine release from leukocytes of atopic patients . However, i.d . treatment of FUT-175 neither inhibited antigen- or compound 48/80-induced immediate type skin reactions in atopic patients nor antigen-induced early, late or delayed type skin reactions in Candida-sensitive patients . FUT-175 also did not inhibit the PPD-mediated delayed type skin reaction in healthy subjects . FUT-175 at a dose of 10(-4)M but not at 10(-8) to 10(-5)M significantly decreased the proliferation of human atopic peripheral blood lymphocytes (PBL) caused by mite antigen, concanavalin A or pokeweed mitogen . 51Cr release from human PBL was slightly enhanced by FUT-175 at a dose range of 10(-6) to 10(-4)M . FUT-175 did not change the number of SIg receptors or C3 receptors on human B cells . FUT-175 hardly affected the nitroblue tetrazolium reduction test and Escherichia coli-mediated chemotaxis in human neutrophils . These results strongly suggest that FUT-175 does not affect immunological functions and host defense in humans.

Anal Biochem, 1983 Jun, 131(2), 360 - 4
Purification of isoacceptor transfer RNA by affinity coupling to aldehyde-containing matrix; Wang QS et al.; A method has been developed for the affinity coupling of aminoacyl-tRNA to an aldehyde-containing polymer by means of reduction with sodium cyanoborohydride . Sephadex dialdehyde obtained from periodate oxidation of Sephadex G-50, and Enzacryl polyaldehyde obtained from hydrolysis of Enzacryl polyacetal, were found to provide the requisite polymeric matrix . This coupling reaction permitted the rapid purification of isoacceptor tRNAs of Escherichia coli for the alpha-amino acids glycine, valine, and arginine, as well as the alpha-imino acid proline.

Arch Biochem Biophys, 1983 Jun, 223(2), 521 - 32
Escherichia coli mutants defective in the gamma subunit of proton-translocating ATPase: intracistronic mapping of the defective site and the biochemical properties of the mutants; Kanazawa H et al.; Various hybrid plasmids carrying a portion of the gene for the gamma subunit of the H+-ATPase of Escherichia coli complemented five mutants defective in the enzyme in a genetic test, indicating that the mutants are defective in the gamma subunit . Since the nucleotide sequence of genomic DNA carried on the plasmids is known, the defective site(s) of the mutants could be located within the gene for the gamma subunit as follows: KF10 and NR70, KF1, and KF12 and KF13 have a mutation causing a defect(s) in amino acid residues 1 to 82, 83 to 167, and 168 to 287, respectively, of the gamma subunit . The biochemical properties of all these mutants except NR70 were analyzed in terms of proton permeability of the membranes and assembly of F1 . Results suggested that KF1 and KF10 have defective F1 without at least the alpha and beta subunits on their membranes, whereas KF12 and KF13 have F1's of rather similar structure to that of the wild type . Attempts were made to purify F1 oF KF12 as a single complex . Although the F1 complex dissociated during purification, active alpha and beta subunits of KF12 were partially purified . On the basis of these biochemical and genetic results, it is suggested that structural alterations in the primary sequence of the gamma subunit corresponding to residues 1 to 167 cause more extensive defects in the assembly of F1 than alteration in the sequence of residues 168 to 287.

Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3208 - 12
cDNA cloning and transcriptional mapping of nine polyadenylylated RNAs encoded by the genome of human respiratory syncytial virus; Collins PL et al.; We have isolated cDNA clones representing nine unique poly(A)+ RNAs transcribed from the genome of human respiratory syncytial virus, a paramyxovirus . A cDNA library was constructed by using poly(A)+ RNA from virus-infected cells as template and the Escherichia coli plasmid pBR322 as vector . Viral cDNA clones were identified by hybridization with cDNA probes prepared from viral genomic RNA . The viral clones were grouped into nine different families by hybridization with individual size-selected reverse transcripts representing the major classes of poly(A)+ RNA from virus-infected cells . The largest clone from each family was selected for analysis . These nine clones, molecular sizes ranging from 520 to 2,600 base pairs, were shown to be unrelated on the basis of reciprocal hybridization using dot-blots . These cDNA clones were then used as hybridization probes to analyze intracellular viral RNAs that had been separated by gel electrophoresis and transferred to diazobenzyloxymethyl-paper . All nine clones hybridized with intracellular viral genomic RNA, confirmation of virus specificity . Nine unique intracellular viral poly(A)+ RNAs were identified {molecular sizes ranging from 720 to 7,500 nucleotides, including poly(A)} . Comparison of the sizes of these major RNAs and the cDNA clones indicated that a number of the clones represented nearly complete copies of the corresponding RNAs . Several other intracellular viral poly(A)+ RNAs appeared to be polycistronic by the criteria of molecular weights and homologies to various combinations of cDNA clones . The sizes and sequence contents of these polycistronic RNAs were used to prepare a transcriptional map whose significance is discussed.

J Bacteriol, 1983 Jun, 154(3), 1040 - 5
Regulation of ribonucleoside diphosphate reductase mRNA synthesis in Escherichia coli; Hanke PD et al.; A RNA-DNA hybridization assay for ribonucleoside diphosphate reductase (RDP reductase) mRNA was used to determine whether control of RDP reductase synthesis in Escherichia coli is at the level of RNA transcription . The correlation observed between the level of RDP reductase enzymatic activity and the rate of RDP reductase mRNA synthesis suggested that the control is at the level of RNA transcription . No increase in RDP reductase enzymatic activity or RDP reductase mRNA was observed during the first 15 min after removal of thymine from a thymine-requiring culture . Thereafter, the rate of RDP reductase mRNA synthesis increased linearly for approximately 75 min before beginning to level off . The addition of thymine to a culture starved for thymine resulted in a decreasing rate of RDP reductase mRNA synthesis . However, 30 min of growth in the presence of thymine was required before the rate of RDP reductase mRNA synthesis dropped to the rate observed in an exponentially growing culture . Inhibition of DNA synthesis caused by shifting a culture of a polC mutant or a dnaB mutant to nonpermissive growth conditions resulted in an increase in the rate of RDP reductase mRNA synthesis similar to that observed for a thymine-starved culture.

Infect Immun, 1983 Jun, 40(3), 862 - 8
Fimbria-specific antibodies detach Escherichia coli from human cells; Mett H et al.; Antibodies obtained from rabbits immunized with purified adhesion-mediating fimbriae of Escherichia coli SS142 were specific for the fimbriae of the homologous strain; they did not cross-react with isolated fimbriae of three different E . coli strains or with protein extracts from nine other adhesive E . coli strains . The antibodies inhibited adhesion to Intestine 407 tissue culture cell monolayers and hemagglutinating activity of E . coli SS142 but not of several other E . coli strains . The antibodies were able not only to prevent but also to reverse the adhesion of E . coli SS142 to Intestine 407 cells or human erythrocytes . Analysis of the kinetics of inhibition suggested that the antibodies did not competitively inhibit adhesion . Such antibodies can be useful for distinguishing different mechanistic steps of bacterial adhesion . Their ability to reverse bacterial adhesion in vitro may be of clinical relevance.

J Gen Microbiol, 1983 Jun, 129 (Pt 6), 1975 - 82
A chromatographic method for the purification of K99 pili from enterotoxigenic Escherichia coli; Altmann K et al.; Details of a relatively inexpensive method for the purification of K99 pili in their native conformation are reported . The method involved sequential precipitation of K99 pili with (NH4)2SO4, followed by precipitation in 12% (w/v) mannose or sorbitol solution . The crude pili preparation was adsorbed with Bio-Gel A-5m and subjected to sequential gel filtration on Bio-Gel A-5m equilibrated with Tris/EDTA/NaN3/NaCl buffer and KSCN/KCl solution, respectively . The K99 containing peak was subjected to sequential ion-exchange chromatography on DEAE-Bio-Gel A equilibrated with 0.02 M-Tris/HCl, pH 8.6 containing 0.05 M-NaCl and DEAE-Sephadex A-50 equilibrated with 0.05 M-phosphate buffer, pH 7.2 . The purified pili yielded a single band on SDS-PAGE with an estimated molecular weight of 13000 . Attempts to purify pili by other methods evaluated, viz . MgCl2 precipitation and chromatofocusing were unsuccessful . While the amino acid composition of purified K99 pili was similar to that reported previously the N-terminal amino acid was apparently blocked.

Boll Ist Sieroter Milan, 1983 May 31, 62(2), 157 - 80
{Methods of evaluation of the activity of disinfectants through the use of 2 laboratory technics, one manual and one semi-automated}; Santini GF et al.; We describe Kelsey-Sykes's method for in vitro analysis of the activity of disinfectants and propose some modifications in order to obtain an easier test for routine analysis.

Biochem Biophys Res Commun, 1983 May 31, 113(1), 301 - 8
Toxicity of activated oxygen: lack of dependence on membrane unsaturated fatty acid composition; Ohlrogge JB et al.; Membrane unsaturated fatty acid oxidation has been suggested as a mechanism of toxicity for a variety of activated oxygen species . We have tested this hypothesis by manipulating the fatty acid composition of an Escherichia coli mutant that is unable to synthesize unsaturated fatty acids . To provide a wide range of susceptibility to membrane oxidation we have replaced the naturally occurring monoenoic acyl chains with cyclopropanes to greatly reduce the unsaturation level and with linoleate to increase the membrane unsaturation . These cultures were treated with ozone, hydrogen peroxide, singlet oxygen and paraquat . In no case was there substantial protection from toxicity afforded by cyclopropanes nor was there enhancement of toxicity to cells with the polyunsaturated membranes . We suggest, therefore, that oxidation of membrane unsaturated fatty acids is not an essential component of the toxicity to E . coli of active oxygen species.

FEBS Lett, 1983 May 30, 156(1), 83 - 7
Actinomycin D-binding in vivo: active chromatin preferred; Yu FL; Escherichia coli RNA polymerase and the endogenous engaged RNA polymerase I were used as specific probes to monitor the physiologically inactive and active nucleolar chromatin template function, respectively . Actinomycin D bound preferentially to the physiologically active regions of rat liver nucleolar chromatin in vivo.

J Mol Biol, 1983 May 25, 166(3), 453 - 6
Crystallization and 7 A resolution electron density map of the large fragment of Escherichia coli DNA polymerase I; Brick P et al.; Crystals of the large fragment of Escherichia coli DNA polymerase I have been grown that diffract to better than 2.8 A resolution . They are in tetragonal space group P4(3) with a = b = 104.1 A, c = 86 A . A 7 A resolution map shows the protein to consist of two domains and to be mostly alpha-helical . The active site has been located by binding nucleoside monophosphates.

J Biol Chem, 1983 May 25, 258(10), 6340 - 3
Amplification and purification of exonuclease I from Escherichia coli K12; Prasher DC et al.; Employing the recombinant runaway replication plasmid pDPK13 {sbcB+}, an exonuclease I-overproducing derivative of Escherichia coli K12 has been constructed . The strain SK4258 has exonuclease I activity 140-400-fold higher than wild type control levels . A new purification procedure has been developed such that the protein can be purified to near homogeneity and is free of endonuclease and RNase activities . The specific activity of the purified enzyme is 10-fold higher than reported previously (Ray, R.K., Reuben, R., Molineux, I., and Gefter, M . (1974) J . Biol . Chem . 249, 5379-5381) . Native exonuclease I is a single polypeptide having Mr = 55,000 with a Stokes radius of 3.12 nm.

J Biol Chem, 1983 May 25, 258(10), 6313 - 8
Reverse phase high performance liquid chromatography of Escherichia coli ribosomal proteins: standardization of 70 S, 50 S, and 30 S protein chromatograms; Kerlavage AR et al.; We recently described the use of reverse phase high performance liquid chromatography for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes (Kerlavage, A . R., Kahan, L., and Cooperman, B . S . (1982) Anal . Biochem . 123, 342-348) . In the present studies we report improvements in the technique and its extension to the separation of the proteins of the 50 S subunit and of 70 S ribosomes . Using an octadecasilyl silica column and a trifluoroacetic acid/acetonitrile solvent system, the 21 proteins of the 30 S subunit have been resolved into 17 peaks, the 33 proteins of the 50 S subunit into 22 peaks, and the 53 proteins of the 70 S ribosome into 31 peaks . The proteins present in each peak have been identified by polyacrylamide gel electrophoresis, by comparison with previously standardized chromatograms, and by calibration with authentic samples of purified proteins . All of the known ribosomal proteins have been identified on the chromatograms with the exception of L31 and its variant, L31' . Three protein peaks, not corresponding to known ribosomal proteins, have been observed in preparations from the total protein from 50 S subunits and 70 S ribosomes, but the significance of these peaks is unclear . The reverse phase high performance liquid chromatography technique has the potential for purifying all ribosomal proteins, as demonstrated by the increase in resolution we obtain when a peak isolated under standard gradient conditions and containing several proteins is reapplied to the column and eluted with a shallower gradient . Its utility in preparing proteins for functional studies is demonstrated by a reconstitution of active 30 S particles using 30 S proteins prepared by reverse phase high performance liquid chromatography.

Nucleic Acids Res, 1983 May 25, 11(10), 3113 - 21
Targeted random mutagenesis: the use of ambiguously synthesized oligonucleotides to mutagenize sequences immediately 5' of an ATG initiation codon; Matteucci MD et al.; The nine base pairs immediately 5' of the initiation codon for the bovine growth hormone (BGH) structural gene have been mutagenized . The mutagenesis method employs the ligation of an ambiguously synthesized oligonucleotide duplex into a previously engineered gap in an expression plasmid for BGH . The mutation method, coupled with hybridization screening, is efficient at isolating 1 and 2 base pair changes within the targeted region . The E . coli cultures harboring the mutant plasmids were assayed for relative levels of BGH expression . The most notable result is the varied effect of substitution of G into the mRNA at various positions in this region.

Nucleic Acids Res, 1983 May 25, 11(10), 2999 - 3017
Positively supercoiled plasmid DNA is produced by treatment of Escherichia coli with DNA gyrase inhibitors; Lockshon D et al.; A procedure has been developed whereby the relative amounts of the topoisomers of E . coli plasmid can be determined for cells grown under a variety of conditions . Several applications of the procedure are presented . Addition of either novobiocin or oxolinic acid, two inhibitors of DNA gyrase, gives rise to positively supercoiled plasmid . A likely model for the introduction of positive supercoils, involving DNA gyrase itself, is discussed . Oxolinic acid is also shown to induce linearization of plasmid in vivo . Starvation of cells for ATP is shown to cause relaxation of plasmid . The shift of a gyrB temperature-sensitive strain to the restrictive temperature is also shown to cause plasmid relaxation . Finally, it is noted that polyamine starvation of E . coli has no detectable effect on the distribution of topoisomers.

J Mol Biol, 1983 May 25, 166(3), 283 - 308
Overlapping and separate controls on the phosphate regulon in Escherichia coli K12; Wanner BL; The physiological and genetic controls operating on phosphate-regulated promoters were studied in greater detail . This was done by defining the control for three phosphate-regulated genes: phoA, psiE, and psiO . Each is highly inducible by phosphate starvation . Individually, these phosphate-starvation-inducible, psi, genes at the same time show common and differing features in their molecular control . The phoA gene, encoding alkaline phosphatase, is specifically induced by phosphate starvation . It is negatively controlled by phoR as well as by the phosphate-specific transport (PST) system in Escherichia coli . phoA induction is positively controlled by the phoB, M, and R products; it is unaffected by the cAMP and CAP system . The psiE and psiO genes were studied by using strains with lacZ fused to their respective promoters . psiE-lacZ is induced by phosphate-, carbon- or nitrogen-limited growth . Genetically, psiE-lacZ induction is partially phoB and phoR-dependent . However, its expression is phoM-independent . This implies that phoB/phoR coupled control differs from phoB/phoM coupled control . Repression of psiE-lacZ is substantially altered in only some PST mutants, such as phoT . In addition, psiE-lacZ is negatively controlled by the cAMP and CAP system . psiO-lacZ is induced by phosphate-, carbon- or nitrogen-limited growth or by anaerobiosis . Its expression is unaffected by any pho mutation that has been previously described . A cell density-dependent induction of psiO-lacZ is observed in lon mutants . Also, psiO-lacZ is negatively controlled by the cAMP-CAP system . In summary, these results demonstrate that co-ordinately regulated promoters can have some common regulatory elements while, at the same time, not sharing other controlling factors.

J Mol Biol, 1983 May 25, 166(3), 273 - 82
Isolation and characterization of mutations altering expression of the major outer membrane porin proteins using the local anaesthetic procaine; Taylor RK et al.; Mutations at several different chromosomal locations affect expression of the major outer membrane porin proteins (OmpF and OmpC) of Escherichia coli K12 . Those that map at 21 and 47 minutes define the structural genes for OmpF and OmpC, respectively . A third locus, ompB, is defined by mutations that map at 74 minutes . The ompB locus contains two genes whose products regulate the relative amounts of ompF and ompC expression . One of these genes, ompR, encodes a positive regulatory protein that interacts at the ompF and ompC promoters . Mutations in ompR exhibit an OmpF- OmpC- or an OmpF+ OmpC- phenotype . The product of the second gene, envZ, affects regulation of the porin proteins in an unknown manner . Previously isolated mutations in envZ exhibit an OmpF- OmpC+ phenotype and also have pleiotropic effects on other exported proteins . In the presence of local anaesthetics such as procaine, wild-type strains exhibit properties similar to these envZ mutants, i.e . OmpF- OmpC+ . Using ompF-lac fusion strains, we have exploited this procaine effect to isolate two new classes of envZ mutations . One of these classes exhibits an OmpF+ OmpC- phenotype . The other allows expression of both OmpF and OmpC but alters the relative amounts found under various growth conditions . Like previously isolated envZ mutations, these also affect regulation of other exported proteins, such as lambda receptor . These results permit a more detailed analysis of the omp regulon and they may shed light on one of the mechanisms by which local anaesthetics exert their effect.

J Biol Chem, 1983 May 25, 258(10), 6277 - 81
Catalase and superoxide dismutase in Escherichia coli; Schwartz CE et al.; We assessed the roles of intrabacterial catalase and superoxide dismutase in the resistance of Escherichia coli to killing by neutrophils . E . coli in which the synthesis of superoxide dismutase and catalase were induced by paraquat 10-fold and 5-fold, respectively, did not resist killing by neutrophils . When bacteria were allowed to recover from the toxicity of paraquat for 1 h on ice and for 30 min at 37 degrees C, they still failed to resist killing by neutrophils . Induction of the synthesis of catalase 9-fold by growth in the presence of phenazine methosulfate did not render E . coli resistant to killing by either neutrophils or by H2O2 itself . The lack of protection by intrabacterial catalase from killing by neutrophils could not be attributed to an impermeable bacterial membrane; the evolution of O2 from H2O2 was no less rapid in suspensions of E . coli than in lysates . The failure of intrabacterial catalase or superoxide dismutase to protect bacteria from killing by neutrophils might indicate either that the flux of O-2 and H2O2 in the phagosome is too great for the intrabacterial enzymes to alter or that the site of injury is at the bacterial surface.

J Biol Chem, 1983 May 25, 258(10), 6073 - 7
recA protein promoted DNA strand exchange; Soltis DA et al.; recA protein and circular single-stranded DNA form a stable complex in the presence of single-stranded DNA binding protein (SSB), in which one recA protein monomer is bound per two nucleotides of DNA . These complexes are kinetically significant intermediates in the exchange of strands between the single-stranded DNA and an homologous linear duplex . After completion of strand exchange, the recA protein remains tightly associated with the circular duplex product of the reaction and the SSB is bound to the displaced linear single strand . Upon addition of ADP, the recA protein-duplex DNA complex dissociates . RecA protein also interacts with single-stranded DNA in the absence of SSB; however, the amount of recA protein bound is substantially reduced . These findings provide direct physical evidence for the participation of SSB in the formation of the recA protein-single-stranded DNA complexes inferred earlier from kinetic analysis . Moreover, they confirm the ability of recA protein to equilibrate between bound and free forms in the absence of SSB.

J Mol Biol, 1983 May 25, 166(3), 383 - 405
Formation and properties of a covalent complex between elongation factor Tu and Phe-tRNA bearing a photoaffinity probe on its 3-(3-amino-3-carboxypropyl)uridine residue; Kao T et al.; Escherichia coli Phe-tRNA, modified with the photoaffinity reagent 6-(2-nitro-4-azidophenylamino)caproate on the 3-(3-amino-3-carboxypropyl)uridine residue, was crosslinked to E . coli EFTu Section upon irradiation at 0 degree C with visible light at wavelengths greater than 400 nm . Crosslinking was dependent on irradiation, the photoaffinity probe, and was blocked by pre-photolysis . 1 mM-dithiothreitol completely quenched crosslinking . Binding of the tRNA to EFTu was a prerequisite for crosslinking, because neither EFTu . GDP nor AcPhe-tRNA could substitute; EFTu . GDPCP, however, was almost as active as EFTu . GTP . Crosslinking was complete in less than five minutes and was stable to at least 20 minutes of irradiation with a single 650 W tungsten lamp 4 cm away . The crosslinking yield ranged from 15% to 25% . The crosslinked complex possessed several remarkable properties . At 0.5 mM-Mg2+, the complex protected the AA-tRNA link to chemical hydrolysis, stabilized the bound GTP to dissociation or exchange, and was not adsorbed to cellulose nitrate filters . The purified crosslinked complex could be bound to ribosomes with concomitant hydrolysis of GTP . Extensive peptide bond formation with AcPhe-tRNA in the P site occurred despite the presence of the crosslinked EFTu . We conclude that hydrolysis of GTP is sufficient to release the 3' end of the Phe-tRNA from complexation with EFTu . Translocation of the A site bound complex did not occur . The crosslink site on EFTu is probably near the periphery of the molecule, because shortening the probe from 20 A to 14 A completely blocked crosslinking . A similar but shorter 8 A probe, p-azidophenacyl-4-thiouridine located on the opposite face of the tRNA, did not crosslink.

Biochemistry, 1983 May 24, 22(11), 2622 - 9
Nuclear Overhauser experiments at 500 MHz on the downfield proton spectra of 5S ribonucleic acid and its complex with ribosomal protein L25; Kime MJ et al.; The downfield (9-15 ppm) proton spectrum of Escherichia coli 5S RNA has been examined at 500 MHz by using nuclear Overhauser methods . The data confirm the existence of the terminal and procaryotic loop helices within the molecule {Fox, G . E., & Woese, C . R . (1975) Nature (London) 256, 505-506} . Very little stable, double-helical structure is detectable in the third loop of the molecule, the one comprising bases 12-68 . The downfield spectrum of 5S RNA is perturbed in a highly specific manner upon addition of protein L25 to the system . The changes seen strongly suggest that the binding site for L25 on 5S RNA includes the procaryotic loop helix, but not the terminal stem helix . Similar complexes formed between L25 and the 5S RNA fragment consisting of bases 1-11, 69-87, and 89-120 show exactly the same spectral alterations . A number of downfield resonances appear in the spectra of these complexes which have no counterparts in the free RNA, suggesting the stabilization of new RNA structures by the protein . There are some indications of protein-nucleic acid nuclear Overhauser effects.

Biochemistry, 1983 May 24, 22(11), 2615 - 22
Nuclear Overhauser experiments at 500 MHz on the downfield proton spectrum of a ribonuclease-resistant fragment of 5S ribonucleic acid; Kime MJ et al.; The downfield (9-15 ppm) proton NMR spectrum of a RNase A resistant fragment of E . coli 5S RNA has been studied by nuclear Overhauser methods . The fragment comprises bases 1-11 and 69-120 of the parent molecule {Douthwaite, S., Garrett, R.A., Wagner, R., & Feunteun, J . (1979) Nucleic Acids Res . 6, 2453-2470} . The nuclear Overhauser data identify two double helical structures in the fragment whose sequences are (GC)3(AU)(GC)3 and (GC)2(AU)(GU) . These structures correspond exactly to the central portions of the terminal stem and procaryotic loop helices which should exist in the fragment sequences according to the Fox-Woese model {Fox, G.E., & Woese, C . R . (1975) Nature (London) 256, 505-506} of 5S RNA secondary structure . The significance of these and other nuclear Overhauser effects detected for the structure of 5S RNA and its fragment is discussed.

Biochemistry, 1983 May 24, 22(11), 2610 - 5
Kinetic and thermodynamic characterization of the R17 coat protein-ribonucleic acid interaction; Carey J et al.; A filter retention assay is used to examine the kinetic and equilibrium properties of the interaction between phage R17 coat protein and its 21-nucleotide RNA binding site . The kinetics of the reaction are consistent with the equilibrium association constant and indicate a diffusion-controlled reaction . The temperature dependence of Ka gives delta H = -19 kcal/mol . This large favorable delta H is partially offset by a delta S = -30 cal mol-1 deg-1 to give a delta G = -11 kcal/mol at 2 degrees C in 0.19 M salt . The binding reaction has a pH optimum centered around pH 8.5, but pH has no effect on delta H . While the interaction is insensitive to the type of monovalent cation, the affinity decreases with the lyotropic series among monovalent anions . The ionic strength dependence of Ka reveals that ionic contacts contribute to the interaction . Most of the binding free energy, however, is a result of nonelectrostatic interactions.

Biochemistry, 1983 May 24, 22(11), 2601 - 10
Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site; Carey J et al.; The interaction between phage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA--protein interaction . Nuclease protection and selection experiments define the binding site to about 20 contiguous nucleotides which form a hairpin . A nitrocellulose filter retention assay is used to show that the binding between the coat protein and a synthetic 21-nucleotide RNA fragment conforms to a simple bimolecular reaction . Unit stoichiometry and a Kd of about 1 nM are obtained at 2 degrees C in buffer containing 0.19 M salt . The interaction is highly sequence specific since a variety of RNAs failed to compete with the 21-nucleotide fragment for coat protein binding.

Biochemistry, 1983 May 24, 22(11), 2708 - 14
Immunochemical evidence for conformational flexibility and its modulation by specific ligands in the beta 2 subunit of Escherichia coli tryptophan synthase; Chaffotte AF et al.; The immunochemical reactivity of unfractionated antibodies elicited by denatured beta 2 subunits of Escherichia coli tryptophan synthase {L-serine hydro-lyase (adding indole) EC 4.2.1.20} with the homologous antigen and with the native enzyme is examined . These antibodies recognize the native apoenzyme nearly as well as the denatured protein . On the contrary, after binding of its cofactor, pyridoxal 5'-phosphate, the protein exhibits a much lower immunoreactivity toward these antibodies . This decrease of affinity becomes even more pronounced when the beta 2 protein interacts with the alpha subunit . Similarly, reduction of the Schiff base formed between the cofactor and the protein leads to a strong decrease of immunoreactivity . To account for these results, it is proposed that apo-beta 2 must be a dynamic flexible structure that easily exposes to the solvent regions of its polypeptide chain that normally are buried in its interior . The increase in rigidity of this structure upon binding of the cofactor, reduction of Schiff base, and formation of the alpha 2 beta 2 complex would then account for the decreased immunoreactivity of these various states of the native beta 2 protein.

Eur J Pharmacol, 1983 May 20, 90(1), 143 - 5
Depressed isoprenaline vascular response in endotoxic rats; Auclair MC et al.; The injection of a sublethal dose of E . coli endotoxin (0127 B8) to intact rats (2 mg/kg i.v) or adrenalectomized rats (0.01 mg/kg i.v.) depressed hypotensive responses to isoprenaline (0.2, 0.4, 0.8 microgram/kg i.v.) for at least 4 h following endotoxin injection . These findings evidence an early (under 1 h after administration) and long-lasting (over 4 h) depression of the vascular response to isoprenaline in endotoxic rats . This depression was not related to catecholamine adrenal discharge or to the hypotensive and lethal effects of endotoxin.

Biochim Biophys Acta, 1983 May 20, 740(1), 29 - 37
Partial purification of a ribosomal ribonucleic acid methylase from rat liver nuclei and methylation of undermethylated nuclear ribonucleic acid from regenerating liver of ethionine-treated rat; Long TW et al.; S-Adenosylmethionine-dependent ribosomal RNA (rRNA) methylase has been purified approx . 90-fold from rat liver nuclei . The partially purified methylase catalyzes the methylation of base and ribose in hypomethylated nuclear rRNA prepared from the regenerating rat liver after treatment with ethionine and adenine . The enzyme has an apparent molecular weight of about 3 x 10(4) and a sedimentation coefficient of 3.0 S . The enzyme is optimally active at pH 9.5 and sensitive to p-chloromercuribenzoate . Thiol-protecting reagents, such as dithiothreitol, are necessary for its activity, and the enzyme requires no divalent cations for its full activity . This enzyme did not efficiently transfer the methyl group to nuclear rRNA from normal rat liver, compared with hypomethylated nuclear rRNA . Methyl groups were mainly incorporated into pre-rRNA larger than 28 S, and the extent of 2'-O-methylation of ribose by this enzyme was greater than that of base methylation in the hypomethylated rRNA . No other nucleic acids, including transfer RNA (tRNA) and microsomal RNA from normal as well as ethionine-treated rat livers, tRNA from Escherichia coli, yeast RNA, and DNA from rat liver and calf thymus, were significantly methylated by this methylase . These results suggest that partially purified rRNA methylase from rat liver nuclei incorporates methyl groups into hypomethylated pre-rRNA from S-adenosylmethionine.

Nature, 1983 May 19-25, 303(5914), 256 - 9
Isolation of genetic elements that increase frequencies of plasmid recombinants; James AA et al.; Some plasmid DNAs, when maintained in wild-type Escherichia coli strains, form high levels of oligomeric species while others remain primarily monomers . One explanation of this observation is that the plasmids that do not form circular oligomers lack a DNA sequence necessary for the formation or maintenance of circular oligomeric species . Here we describe the isolation of segments of DNA from the E . coli genome and other sources that through a recA+ -dependent process: (1) stimulate the conversion of monomeric plasmids to different oligomeric forms, (2) stimulate the conversion of an oligomeric plasmid to a mixture of monomeric and different oligomeric forms, and (3) increase the frequency of recovery of figure-8 molecules . Both cis-acting and trans-acting elements were found . These elements seen to act by stimulating either the frequency of the recombination events that lead to the interconversion of different oligomeric plasmid DNA molecules or some process involved in the maintenance of newly-formed recombinant molecules.

Brain Res, 1983 May 16, 267(2), 237 - 40
Reduced hypothalamic thermosensitivity following intraventricular injection of pyrogen in conscious rabbits; Kruk B et al.; Hypothalamic temperature thresholds for vasodilatory and respiratory reactions were determined before and after i.c.v . injection of pyrogen in rabbits . During the rising phase of fever the increases in the hypothalamic thresholds for vasodilatory and respiratory reactions differed from those found in the pre-pyrogen preoptic anterior hypothalamic area (POAH) heating by 2.1 +/- 0.2 degrees C and 1.89 +/- 0.31 degrees C, respectively . During the plateau phase of fever the threshold for vasodilatory reaction was further increased (by 0.7 +/- 0.23 degrees C), whereas that for panting remained at the same level . It is concluded that pyrogen exerts a depressive action on POAH thermosensitivity.

Eur J Biochem, 1983 May 16, 132(3), 645 - 50
Lipid dependence of diacylglycerol kinase from Escherichia coli; Bohnenberger E et al.; Diacylglycerol kinase apoprotein was purified from membranes of Escherichia coli K 12 . The protein was catalytically inactive, but regained activity upon recombination with phospholipids, certain neutral lipids, or fatty acids . Activation proceeded with positive cooperativity and was independent of the exact chemical structure, bilayer arrangement or electrical charge of the lipid . The apoprotein was activated by lysophosphatidylethanolamine but not by lysophosphatidylcholine . 1-Monooleoylglycerol was an effective activator and substrate at the same time . The fluidity and the polarity of lipids appeared to be generally important for activation . Lipid polarity was estimated by a triacylglycerol/phosphatidylcholine-partitioning procedure . All lipids showing preferential association with triacylglycerol failed to activate the kinase apoprotein even in the presence of detergent . It is concluded that a defined hydrophilic/lipophilic balance of the lipid was required for the formation of a functional lipoprotein complex.

J Mol Biol, 1983 May 15, 166(2), 241 - 7
Homology between CAP and Fnr, a regulator of anaerobic respiration in Escherichia coli; Shaw DJ et al.; The fnr gene is essential for the expression of anaerobic respiratory metabolism in Escherichia coli . Genetic and biochemical studies support the view that its product . Fnr, is a transcriptional regulatory protein specific for genes encoding anaerobic respiratory functions (fumarate, nitrate and nitrite reductases, hydrogenase, etc.) . In this respect Fnr may be considered analogous to the well-characterized catabolite gene activator protein (CAP), which mediates the control of catabolite-sensitive gene transcription . With a view to identifying its function, the fnr gene has recently been cloned and the primary structure of the Fnr protein deduced from the nucleotide sequence . This has revealed the presence of three regions of sequence homology with CAP . One corresponds to the DNA-binding site, a region of about 20 highly conserved amino acids that is believed to form a characteristic three-dimensional structure in several transcriptional regulators . The other regions of homology are in the nucleotide binding domain of CAP but the residues that interact with cAMP are not identical in Fnr . These homologies suggest that Fnr and CAP may have similar three-dimensional structures and that the regulation of anaerobic energy metabolism may involve interaction between Fnr and an unidentified effector molecule.

J Mol Biol, 1983 May 15, 166(2), 233 - 40
Deletion analysis of the retroregulatory site for the lambda int gene; Court D et al.; The lambda int gene product, integrase, recombines phage and bacterial DNA at a specific site during the integration step of lysogeny . Regulation of integrase synthesis is complex . (1) Transcription of the gene can occur from either of two promoters . lambda cII protein activates transcription initiation near int at pI . The lambda N protein allows transcription of int from pL . N protein acts by preventing transcription termination at several terminators between pL and int . (2) The expression of integrase is also subject to post-transcriptional regulation by a site, sib, which is located beyond int in the b region of lambda . Expression of int from pL is inhibited by sib, whereas that from pI is not . The negative control of int expression by sib is termed retroregulation . Retroregulation of int is caused, in part, by processing of the pL transcript at the sib site by RNase III of Escherichia coli . The exonuclease, Bal31, was used to generate a set of deletions to define the sib regulatory site . Both sib+ and sib- deletions were sequenced, and it was concluded from this and other work that a dyad symmetry present in the b region, 270 base-pairs from int, was necessary for retroregulation . The RNA structure of this segment is similar to other RNase III-sensitive sites found in E . coli and phage RNAs.

Nucleic Acids Res, 1983 May 11, 11(9), 2927 - 41
Expression of human interferon genes using the recA promoter of Escherichia coli; Feinstein SI et al.; Interferon beta 1 and three alpha-interferon genes were cloned on Eco RI fragments isolated from a human genomic library into the Eco RI site of a plasmid containing the recA promoter of E . coli . Expression of interferon activity from cells carrying these plasmids was nalidixic acid inducible . The alpha-interferon genes were expressed only when in the same transcriptional orientation as the recA promoter while the beta 1 interferon gene was expressed in either orientation . Interferon activity was also inducibly expressed from the recA promoter in cells containing a plasmid carrying a fusion of the recA gene with the beta 1 interferon gene . This interferon activity was thirty-fold less sensitive to neutralization by polyclonal antibodies than authentic interferon, implying that the change near the amino terminus affects either antibody recognition or specific activity or both.

Nucleic Acids Res, 1983 May 11, 11(9), 2745 - 63
Expression of hepatitis B virus surface antigen in yeast; Hitzeman RA et al.; The structural gene of Hepatitis B virus surface protein (HBsAg) was introduced into a plasmid capable of autonomous replication and selection in both the yeast Saccharomyces cerevisiae and E . coli . In this plasmid transcription of the HBsAg is initiated by the 5'-flanking sequence of the yeast 3-phosphoglycerate kinase (PGK) gene and terminated by the 3'-flanking region of the yeast TRP1 gene . Yeast cells containing this plasmid produce a new major species of mRNA of 1200 nucleotides in length coding for HBsAg . Viral surface antigen is made in nonglycosylated form at a level of about 1-2 percent of total yeast protein . A small fraction of this polypeptide (2-5 percent) is found in aggregated form upon yeast cell disruption by glass beads . This material is similar in size, density, and shape to the 22nm particle, isolated from the plasma of human hepatitis carriers, and induced comparable levels of HBsAg antibodies in mice when compared with the natural particle.

Nucleic Acids Res, 1983 May 11, 11(9), 2599 - 616
The spc ribosomal protein operon of Escherichia coli: sequence and cotranscription of the ribosomal protein genes and a protein export gene; Cerretti DP et al.; The genes encoding the 52 ribosomal proteins (r-proteins) of Escherichia coli are organized into approximately 19 operons scattered throughout the chromosome . One of these, the spc operon, contains the genes for ten ribosomal proteins: L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15 (rp1N, rp1X, rp1E, rpsN, rpsH, rp1F, rp1R, rpsE, rpmD, and rp1O) . We now report the entire 5.9 kb nucleotide sequence of the spc operon . DNA sequence analysis has confirmed the genetic organization and refined the amino acid sequence of the ten r-proteins in this operon . It has also revealed the presence of two open reading frames past the last known gene (L15) of the spc operon . One of these corresponds to a gene (pr1A or secY) which recently has been shown by others to be involved in protein export . In addition, S1 mapping experiments indicate that a significant proportion of transcription initiated from the spc operon continues not only into the two putative genes, but also without termination into the downstream alpha r-protein operon.

J Biol Chem, 1983 May 10, 258(9), 5379 - 85
Characterization of the catalytic pathway for D-serine dehydratase . Evidence for variation of the rate-determining step with substrate structure; Federiuk CS et al.; Steady state kinetic parameters (kcat and Km) which were determined for the D-serine dehydratase-catalyzed decomposition of the isomers of serine and threonine at pH 5.7, 7.8, and 8.9 indicated that the enzyme exhibited considerable kinetic specificity . At pH 7.8 and 25 degrees C, the specificity constants (kcat/Km) were as follows: D-serine (3.4 X 10(5) M-1 s-1), D-threonine (2.9 X 10(4) M-1 s-1), D-allothreonine (7.5 X 10(3) M-1 s-1), and L-serine (38 M-1 s-1) . Substrate C-2 deuterium atom isotope effects on the steady state kinetic parameters disclosed that the rate-determining step in the catalytic pathway varied with substrate structure and pH . Removal of the C-2 hydrogen atom of the substrate was shown to be fully rate-determining with L-serine and partially rate-determining with D-allothreonine as substrate at pH 7.8 . Stopped flow measurements of absorbance and fluorescence were used to characterize intermediates in the catalytic pathway . These measurements indicated that D-serine, in addition to being the best substrate, was processed faster than the other substrates through steps in the catalytic pathway which were not rate-controlling . Kinetic evidence also was obtained which indicated that the base which accepts the proton from the C-2 carbon atom of the substrate must be aprotic . Thus, the catalytic site of D-serine dehydratase should contain a basic group in addition to the active site lysyl residue.

J Biol Chem, 1983 May 10, 258(9), 5340 - 3
RNase III is positively regulated by T7 protein kinase; Mayer JE et al.; RNase III activity of Escherichia coli is stimulated 4-fold after infection with bacterial virus T7 . The mechanism of stimulation is based on phosphate transfer to RNase III by the T7 coded protein kinase . In vitro synthesized protein kinase also stimulates RNase III . Serine is the phosphate acceptor.

Biochemistry, 1983 May 10, 22(10), 2378 - 84
Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro; Kunkel TA et al.; Removal of purine bases from phi X174 single-stranded DNA leads to increased reversion frequency of amber mutations when this DNA is copied in vitro with purified DNA polymerases . This depurination-induced mutagenesis is observed at three different genetic loci and with several different purified enzymes, including Escherichia coli DNA polymerases I and III, avian myeloblastosis virus DNA polymerase, and eukaryotic DNA polymerases alpha, beta, and gamma . The extent of mutagenesis correlates with the estimated frequency of bypass of the lesion and is greatest with inherently inaccurate DNA polymerases which lack proofreading capacity . With E . coli DNA polymerase I, conditions which diminish proofreading result in a 3-5-fold increase in depurination-induced mutagenesis, suggesting a role for proofreading in determining the frequency of bypass of apurinic sites . The addition of E . coli single-stranded DNA-binding protein to polymerase I catalyzed reactions with depurinated DNA had no effect on the extent of mutagenesis . Analysis of wild-type revertants produced during in vitro DNA synthesis by polymerase I or avian myeloblastosis virus DNA polymerase on depurinated phi X174 amber 3 DNA indicates a preference for insertion of dAMP opposite the putative apurinic site at position 587 . These results are discussed in relation both to the mutagenic potential of apurinic sites in higher organisms and to studies on error-prone DNA synthesis.

Biochemistry, 1983 May 10, 22(10), 2339 - 46
Identification of tyrosine residues that are susceptible to lactoperoxidase-catalyzed iodination on the surface of Escherichia coli 30S ribosomal subunit; Wower J et al.; The detailed surface topography of the Escherichia coli 30S ribosomal subunit has been investigated, with iodination catalyzed by immobilized lactoperoxidase as the surface probe . Under mild conditions, only proteins S3, S7, S9, S18, and S21 were iodinated to a significant and reproducible extent . These proteins were isolated from the iodinated subunits, and in each case, the individual tyrosine residues that had reacted were identified by standard protein sequencing techniques . The targets of iodination that could be positively established were as follows: in protein S3 (232 amino acids), the tyrosines at positions 167 and 192; in S7 (153 amino acids), tyrosines 84 and 152; in S9 (128 amino acids), tyrosine 89; in S18 (74 amino acids), tyrosine 3 (tentative); in S21 (70 amino acids), tyrosines 37 and 70 . The results represent part of a broader program to investigate ribosomal topography at the amino acid-nucleotide level.

J Biol Chem, 1983 May 10, 258(9), 5819 - 27
Cytochrome b from Escherichia coli nitrate reductase . Its properties and association with the enzyme complex; Chaudhry GR et al.; Membrane-bound nitrate reductase purified from Escherichia coli was resolved into two separate forms . The majority of the enzyme complex had a subunit composition of 2A:2B:4C, exhibited cytochrome b spectra, and was found to be stable after purification . A second form of nitrate reductase activity was a modified complex with a subunit composition of 2A:2B and lacked cytochrome . The subunit B from this complex was altered in its mobility on sodium dodecyl sulfate-polyacrylamide gels . The cytochrome-containing enzyme had 28 +/- 2 atoms of iron and 1.35 atoms of molybdenum whereas iron and molybdenum in cytochromeless enzyme were 24 +/- 2 atoms and 1.18 atoms/molecule, respectively . Besides cytochrome-containing nitrate reductase, two other cytochrome b-containing fractions were also resolved . These were cytochrome b associated with formate dehydrogenase and a novel cytochrome b with reduced absorption maxima at 430, 529.5, and 560 nm . Nitrate reductase cytochrome b (subunit C) was isolated from subunits A and B as a partially denatured form and its renaturation was accomplished by dialyzing against hemin . The renatured cytochrome yielded absorption spectra similar to the holoenzyme . The pure cytochrome aggregated upon heating, even in the presence of sodium dodecyl sulfate . It had a high isoelectric point (pH greater than 9.5) and had 45% hydrophobic amino acids.

J Biol Chem, 1983 May 10, 258(9), 5467 - 76
Purification and characterization of an RNA processing enzyme from Caulobacter crescentus; Bellofatto V et al.; An RNA processing enzyme has been isolated from Caulobacter crescentus which is specific for double-stranded RNA, has an absolute requirement for monovalent cations, and can be eluted from a poly I:C agarose affinity column in pure form . This enzyme, like RNase III isolated from Escherichia coli, processes precursor ribosomal RNAs and polycistronic phage mRNAs and has a monomeric Mr of approximately 20,000 . The two enzymes differ, however, in the recognition of specific cleavage sites and yield different digestion products when either coliphage T7 or C . crescentus phage phi Cdl early mRNA is used as substrate . Two lines of evidence are presented which show that an RNase III activity functions as a processing enzyme in C . crescentus . (a) In an in vitro reaction, C . crescentus phage phi Cdl major early mRNA synthesized in vitro by host RNA polymerase was processed by RNase III to yield RNA species which co-migrated with phage RNA synthesized in vivo in phi Cdl-infected cells, and (b) an in vitro transcript of a C . crescentus DNA clone containing the entire 16 S gene and part of the 23 S gene was processed by C . crescentus RNase III to yield an RNA product which co-migrated with 16 S RNA . The RNase III activity isolated from C . crescentus cell extracts has potential use in the analysis of specific RNA species because it was found to be more stringent in the recognition of cleavage sites than the E . coli enzyme.

J Biol Chem, 1983 May 10, 258(9), 5428 - 32
Periplasmic glycerophosphodiester phosphodiesterase of Escherichia coli, a new enzyme of the glp regulon; Larson TJ et al.; The promoter-proximal gene (glpT) of the glpT-glpQ operon of Escherichia coli encodes a membrane permease responsible for active transport of sn-glycerol 3-phosphate . Promoter-distal glpQ encodes a periplasmic protein which is not required for active transport of sn-glycerol 3-phosphate (Larson, T.J., Schumacher, G., and Boos, W . (1982) J . Bacteriol . 152, 1008-1021) . This periplasmic protein has now been identified as a phosphodiesterase which hydrolyzes glycerophosphodiesters into sn-glycerol 3-phosphate plus alcohol . The enzyme exhibited broad substrate specificity with respect to the alcohol moiety; sn-glycerol 3-phosphate was released from glycerophosphoethanolamine, glycerophosphocholine, glycerophosphoglycerol, and bis(glycerophospho)glycerol . The enzyme was specific for glycerophosphodiesters; bis(p-nitrophenyl)phosphate, a substrate for other phosphodiesterases, was not hydrolyzed . In a coupled spectrophotometric assay utilizing sn-glycerol 3-phosphate dehydrogenase and NAD, apparent activity was optimal at pH 9 and was stimulated by Ca2+ . The substrates of the phosphodiesterase had no affinity for the glpT-encoded active transport system . Thus, the glpQ gene product expands the catabolic capability of the glp regulon to include a variety of glycerophosphodiesters.

FEBS Lett, 1983 May 8, 155(2), 197 - 200
NADH oxidation in phospholipid-enriched cytoplasmic membrane vesicles from Escherichia coli; Demant EJ et al.; NADH oxidation in Escherichia coli cytoplasmic membrane vesicles enriched in anionic phospholipids by de novo synthesis of lipid in the vesicles from acyl-CoA esters and sn-glycerol 3-phosphate has been studied . NADH-oxidase but not NADH-dehydrogenase activity was found to decrease during synthesis and accumulation of phospholipid in the vesicles . Density gradient fractionation showed that NADH-oxidase activity was reduced to approximately 30% in vesicles with a 3-6 fold increase in anionic phospholipid, whereas vesicles with a greater than 10-fold increase in phospholipid had virtually no NADH oxidase activity.

J Theor Biol, 1983 May 7, 102(1), 101 - 20
Cell division in Escherichia coli after changes in the velocity of DNA replication; Bremer H et al.; A method of computer analysis was developed to evaluate the kinetic changes in the rate of cell division in non-synchronous cultures of E . coli resulting from changes in the velocity or initiation of chromosome replication . This method takes into account that the cell division pathway in E . coli includes a reaction of indeterminate length described by a probability function that applies to the cell population . The analysis yields a hypothetical cell number kinetics as it would be observed if the stochastic element in the division pathway were absent . Since this derived cell number curve responds to experimentally induced perturbations of replication at defined times whereas the actual cell number curve reflects these perturbations only in a blurred fashion, replication and division events can be precisely correlated with this method . The method was applied to the evaluation of thymine starvation experiments with two Thy- derivatives of E . coli B/r; one of the strains has a mutationally altered (60% increased) cell mass at initiation of chromosome replication . In both strains, the stochastic phase of the cell cycle had the same half-life value of 10 min and began 18 min after each termination of replication . This suggests that the time of cell division is linked to replication, not to cell mass or length . This interpretation is supported by results of experiments in which the rate of cell growth was altered at the time of thymine starvation.

Biochim Biophys Acta, 1983 May 5, 730(2), 379 - 86
The effects of weak acids on potassium uptake by Escherichia coli K-12 inhibition by low cytoplasmic pH; Bakker EP et al.; The activity of the Escherichia coli K+ transport system TrkA was measured as a function of the cytoplasmic pH of the cell . For this purpose, pHin was decreased by the addition of the weak acids acetic acid, benzoic acid or salicylic acid to K+-depleted cells . Under these conditions, the initial rate of K+ uptake decreased strongly with pHin, and was almost independent of the acid used . This inhibition was due to a strong decrease in the Vmax for K+ uptake, which indicates that low cytoplasmic pH inactivates the TrkA K+ uptake system . The relevance of this inhibition for growth and metabolism at low pHin is discussed.

J Mol Biol, 1983 May 5, 166(1), 1 - 19
Sequence diversity among related genes for recognition of specific targets in DNA molecules; Gough JA et al.; Escherichia coli strains K12 and B, and a new strain designated D, each encode a characteristic restriction and modification enzyme . These enzymes (EcoK, EcoB and presumably EcoD) comprise three subunits of which one, that encoded by the so-called specificity gene (hsdS), is responsible for recognition of the DNA sequence specific to that system . The other two subunits, encoded by hsdR and hsdM, are interchangeable between systems, and the available molecular evidence suggests that the hsdR and hsdM genes are highly conserved . The DNA sequence of a segment of the hsd region that includes the hsdS gene has been determined for each of the three strains . The hsdS gene varies in length from 1335 to 1425 base-pairs and the only regions showing obvious homology, one of about 100 base-pairs and a second of about 250 base-pairs, are highly conserved . The remainder of each hsd S gene shares little, or no, homology with either of the other related specificity genes . Thus, the specificity subunits, though components of a family of closely related enzymes with very similar functions, have remarkably dissimilar primary structure.

Biochim Biophys Acta, 1983 May 4, 757(1), 92 - 100
Action of ions and pH on the binding of virginiamycin S to ribosomes; Di Giambattista M et al.; Virginiamycin S (VS) binds to the 50 S ribosomal subunits (KaVS = 2.5 X 10(6) M-1); the affinity of ribosomes for VS undergoes a 6-fold increase (KaVS = 15 X 10(6) M-1) in the presence of virginiamycin M (VM) . In the present work, the action of inorganic ions and pH on the binding reaction of VS to ribosomes was analyzed by a spectrofluorimetric technique, in the presence and in the absence of VM . Preliminary to this study, the interaction of ions with free VS was also explored . In aqueous solution and in the absence of VM, chelation by VS of K+, NH4+, Na+, Mg2+ and Ca2+ was observed . The binding of Mg2+ was strongly influenced by monovalent ions and pH between 7 and 8: the association constants for the VS:Mg2+ complex were 12.5 M-1 (+NH4+) and 45 M-1 (-NH4+) . Binding of VS to ribosomes occurred as well in the presence of NH4+ as with K+, but was suppressed upon replacement of these ions by Na+ . The association constant of the VS binding reaction to ribosomes was strongly enhanced by VM in the presence of either NH4+ or K+ but, while a plateau value was observed over the entire range of NH4+ concentrations, 100-fold higher values were obtained at low (25 mM) K+ . This VM-induced increase of ribosome affinity for VS (KaVS) was dependent also on Mg2+, which displayed a higher synergistic action with K+ than with NH4+ (the highest KaVS values were recorded for 5 mM Mg2+ and 100 mM K+) . In this reaction, Mg2+ could be replaced by Ca2+, whereas spermidine was ineffective . Also, the VM-promoted enhancement of the KaVS value increased with the pH . In conclusion, the two ribosomal functions analyzed in the present work, VS-binding to ribosome and VM-promoted enhancement of ribosome affinity for VS, rely on the concentration of monovalent and bivalent ions and on the pH . Both functions require either NH4+ or K+, and either Mg2+ or Ca2+ (the elements of each couple are not truly equivalent), from which ribosome conformation depends.

FEBS Lett, 1983 May 2, 155(1), 167 - 72
Structural study of translating 70 S ribosomes from Escherichia coli . I . Electron microscopy; Vasiliev VD et al.; Translating 70 S ribosomes of Escherichia coli either in the pre-translocation or in the post-translocation state have been prepared by using the cell-free translation system in poly(U)-S-S-Sepharose columns {Methods Enzymol . (1979) 59, 382-398} . Electron microscopy study of the preparations has demonstrated that: (1) the mutual orientation of the ribosomal subunits in the translating ribosomes is the same as proposed by Lake for routine 30 S.50 S couples {J . Mol . Biol . (1976) 105, 111-130}; (2) the L7/L12 stalk of the 50 S subunit sticks out from the 70 S particle and does not join the 30 S subunit; (3) pre-translocation and post-translocation state ribosomes do not differ in mutual orientation of the subunits and in the position of the L7/L12 stalk, within the limits of electron microscopy resolution.

Eur J Biochem, 1983 May 2, 132(2), 411 - 5
Gamma-ray induction of deoxyribonucleic acid synthesis in temperature-sensitive DNA initiation mutants of Escherichia coli; Kohiyama M et al.; DNA synthesis was followed after gamma-ray irradiation of several different temperature-sensitive mutants with defects in the initiation process . The results indicate that only dnaA and dnaI mutants show induction of supplementary DNA synthesis after gamma-ray irradiation . The induction of DNA synthesis by gamma-ray irradiation was also shown to be recA+ dependent in the dna5 mutant.

Eur J Biochem, 1983 May 2, 132(2), 321 - 7
NMR study of the interaction between the lac repressor and the lac operator; Buck F et al.; Binding of the lac repressor headpiece, the N-terminal region of the lac repressor, to the lac operator of Escherichia coli was studied by 1H-NMR spectroscopy . Two DNA fragments, of 51 base pairs and 62 base pairs, containing the lac operator region, were investigated . The signals of their hydrogen-bonded imino protons were well resolved in the 500-MHz NMR spectra . The spectra of the free lac operator DNA are similar to those obtained from ring-current-shift calculations for a B-DNA structure . Complex formation with the headpiece led to small but nevertheless characteristic changes in the spectra . The fact that very few imino resonances shifted upon addition of headpiece, as well as the variety in direction and size of these chemical shifts, indicate the formation of a specific complex between the lac repressor and the lac operator . The observed changes in the resonance positions exclude the intercalation of tyrosine residues of the headpiece between adjacent base pairs of the lac operator as well as the formation of a cruciform structure . They rather reflect a small conformational transition in the DNA itself, caused for example by an alteration in the tilt of a few base pairs or a shift of the keto-enol tautomeric equilibrium of the bases towards the enolic form.

Eur J Biochem, 1983 May 2, 132(2), 389 - 94
Unbalanced ribosomal protein synthesis in a strain of Escherichia coli containing a cloned, truncated 16-S ribosomal RNA gene; Barnsley PG et al.; An Escherichia coli K12 strain, carrying the promotor and proximal portion of the 16-S rRNA gene from rrnB cloned in the high-copy-number plasmid psF2124, has been examined for abnormalities in ribosome biogenesis . Both ribosomal RNA accumulation and ribosome content are depressed in this strain as compared to the control strain carrying the plasmid vector alone . The rate of total protein synthesis, however, appears to be normal . In contrast, the rate of ribosomal protein synthesis, relative to total protein synthesis, is elevated . The rates of synthesis of individual ribosomal proteins were determined and found to vary greatly, ranging from severe under-synthesis (displayed especially by proteins L7/L12) to massive over-synthesis (displayed particularly in the case of protein S7) . Analysis of the rates of synthesis of other proteins coded for by the S12 operon revealed that protein S12 was moderately over-produced, but elongation factors EF-G and EF-Tu appear to be synthesized at the same rate as EF-Ts, all three being moderately under-synthesized relative to total soluble proteins.

Arch Biochem Biophys, 1983 May, 223(1), 140 - 8
Cloning of total mRNA populations from adult and embryonic mice; Tocci MJ et al.; Total clone banks of cDNAs synthesized from poly(A)-RNA obtained from three stages of the developing mouse were constructed . The stages chosen were 13-day-old embryo, neonatal, and fully grown adult . To have as complete a bank as possible, large numbers of individual clones were generated approximately 400,000 for the 13th day embryo and neonatal mouse and approximately 610,000 for the adult bank . In each case the clone bank was constructed by inserting double stranded cDNA into the PstI site of pBR322 by the "G-C tailing" method . Sequences cloned in this way could be separated from the plasmid host DNA by treatment of the resultant total chimeric plasmid population with PstI . Aliquots of the cloned cDNA material were labeled with 32P by "nick translation" using Escherichia coli DNA polymerase I for the preparation of hybridization probes . Back-hybridization of these probes to the total clone banks allowed the determination of the sequence diversity among the above three very different developmental stages . The use of such clone banks should allow the identification of developmental stage specific mRNAs.

Am J Hematol, 1983 May, 14(3), 271 - 8
Migration of human leukocytes from soft agarose droplet: a simplified method for studying chemotaxis and spontaneous migration; Barak Y et al.; Studies of in vitro chemotaxis and spontaneous migration of human leukocytes using the accepted method with the Boyden-chamber-filter are troublesome, because of the need for specially constructed vessels as well as the difficulties caused by the use of membrane filters . We describe a new and simplified method for measuring human leucocyte chemotaxis, which is a modification of the recently described underagarose migration method and which is based upon spontaneous migration of cells from a soft agarose droplet and in response to a chemotactic gradient . We examined suspensions of leukocytes, purified granulocytes, and mononuclear cells from 10 healthy normal adults and from 10 samples of cord blood using E Coli O111B4 endotoxin-activated human serum as attractant . Our results showed that the mean chemotactic indices (C.I.-chemotaxis/migration) for purified granulocytes and for mononuclear cells from normal individuals were 3.0 +/- 1.2 and 2.7 +/- 1.5, respectively . Chemotaxis was significantly reduced when unwashed leukocytes were studied, indicating a detrimental effect of autologous plasma on leukocytic response to a chemotactic stimulus in this system . Cord blood cells showed normal spontaneous migration, but significantly decreased chemotaxis . This preliminary report shows that the technique is simple, rapid, and reproducible, and can detect abnormalities of chemotaxis in both granulocytes and mononuclear cells.

J Reticuloendothel Soc, 1983 May, 33(5), 353 - 67
Interaction of latex-insolubilized endotoxins with murine macrophages: phagocytic responses of endotoxin-responsive (C3HeB/FeJ) and -unresponsive (C3H/HeJ) macrophages in vitro; Lubinsky-Mink S et al.; Insolubilized lipopolysaccharides (LPS) were prepared by covalently coupling LPS from polysaccharide-deficient S . minnesota R595 and polysaccharide-rich E . coli 055:B5 to carboxylated latex particles . The stability of these LPS-latex complexes was determined using several assays to detect soluble LPS following incubation at ambient and elevated temperatures . Resident and thioglycollate-elicited macrophages from both LPS-responder C3HeB/FeJ and LPS nonresponder C3H/HeJ mice were examined for their capacity to phagocytose the LPS particles following in vitro culture for various time periods . Uptake was demonstrated by an increase in the number of particles within the macrophages with increasing time of incubation . Rough polysaccharide-deficient LPS-latex particles were found to be more readily phagocytosed than control particles, whereas smooth polysaccharide-rich LPS particles were phagocytosed less readily than the controls . Qualitatively similar results were found in the relative rate of uptake of particles by the macrophages from the endotoxin-responsive and -unresponsive mouse strains used in this study.

Infect Immun, 1983 May, 40(2), 613 - 21
Down regulation of hepatic glucocorticoid receptors after endotoxin treatment; Stith RD et al.; The mechanism by which endotoxin administration results in hypoglycemia was evaluated by characterizing {3H}dexamethasone binding and phosphoenolpyruvate carboxykinase activity in hepatic cytosol preparations from treated and control mice . Starved mice were given Escherichia coli O111:B4 endotoxin or saline intraperitoneally on day 3 after bilateral adrenalectomy . {3H}dexamethasone binding was measured by the charcoal method after the incubation of cytosol preparations with {3H}dexamethasone in the presence or absence of unlabeled dexamethasone . Changes in {3H}dexamethasone binding were found to be time and dose dependent in treated mice . When mice given different doses of endotoxin reached the same stage of morbidity, as indicated by the average time of death, significantly lower glucocorticoid binding was measured . Scatchard analysis of binding isotherms defined a single class of binding sites . Association and dissociation rate constants and the equilibrium dissociation constant (Kd) were not altered, but the maximum number of binding sites was depressed by endotoxin . The rank order of potency of competitors for {3H}dexamethasone binding, dexamethasone greater than hydrocortisone = corticosterone greater than deoxycorticosterone greater than progesterone greater than testosterone = estradiol, was consistent with a glucocorticoid receptor, although the competition was not altered by endotoxin . Endotoxin treatment prevented the glucocorticoid-induced increase in hepatic phosphoenolpyruvate carboxykinase activity . We conclude that the hypoglycemia of endotoxin poisoning is effected, in part, by the inhibition of the glucocorticoid-mediated induction of phosphoenolpyruvate carboxykinase via the down regulation of hepatic glucocorticoid receptors.

Scand J Immunol, 1983 May, 17(5), 397 - 401
Chronic lymphocytic leukaemia cells activated in vitro reveal cellular changes that characterize B-prolymphocytic leukaemia and immunocytoma; Robert KH et al.; Leukaemic blood lymphocytes from a patient in the terminal stage of chronic lymphocytic leukaemia (CLL) according to the Kiel classification were stimulated by lipopolysaccharide from Escherichia coli . During progression to blast transformation, stimulated cells lost receptors for mouse erythrocytes (MRBC), accumulated intracellular Ig (cIg), and expressed more surface membrane immunoglobulin (SmIg) . In addition, transforming cells expressed receptors for the monoclonal antibody FMC7, known to distinguish between CLL cells and prolymphocytic leukaemia (B-PLL) cells . Thus, activation of the patient's CLL cells induced changes in the cellular phenotype so as to resemble B-PLL . Some clonal cells progressed further to a secretory stage of differentiation . Thus, in vitro stimulation induced cellular changes reminiscent of not only B-PLL but also immunocytoma . Further studies of this kind may elucidate the relationship between these lymphocytic malignancies.

Proc Natl Acad Sci U S A, 1983 May, 80(10), 3015 - 9
High mutation frequency in DNA transfected into mammalian cells; Calos MP et al.; The lacI gene of Escherichia coli was used to score mutation in mammalian cells of simian virus 40-based recombinant DNA vectors that provide for replication and selection in both bacterial and mammalian cells . Plasmid DNA was introduced into COS7 simian cells by DEAE-dextran transfection, allowed to replicate in the mammalian cells, and then returned to E . coli for analysis . Mutants in lacI were observed at frequencies of one to several percent, compared with a spontaneous mutation rate in E . coli of less than 10(-5) . The lesions include a large number of base substitutions, in addition to deletions, duplications, and more complex rearrangements, including insertion into the plasmid of sequences originating in the host genome . We discuss possible sources of the high mutation frequency and its implications for experiments involving DNA transfer.

Proc Natl Acad Sci U S A, 1983 May, 80(9), 2544 - 8
Mapping of single-stranded regions in duplex DNA at the sequence level: single-strand-specific cytosine methylation in RNA polymerase-promoter complexes; Kirkegaard K et al.; A method based on the differential rate of cytosine methylation in single- and double-stranded nucleic acids by dimethyl sulfate {Peattie, D.A . & Gilbert, W . (1980) Proc . Natl . Acad . Sci . USA 77, 4679-4682} has been developed for probing unpaired cytosines in DNA and DNA-protein complexes at the sequence level . Application of the method to the complexes between Escherichia coli RNA polymerase (EC 2.7.7.6) and three related promoters, lac UV5, trp, and a hybrid promoter tac resulting from the fusion of the two, reveals distinct differences in the way RNA polymerase unpairs DNA in these promoters . No single-stranded region is detectable in the complex with the trp promoter . For the lac UV5 promoter, the cytosines at positions -6, -4, -2, and -1 are in an unpaired region . The same cytosines in the tac promoter, which is homologous in sequence to lac UV5 in this region, are also found to be single stranded . For the pair of promoters lac UV5 and tac, the cytosine methylation reaction has also been used to demonstrate the steep temperature dependence of opening of base pairs by RNA polymerase . One striking feature is that the midpoint of this transition for the tac promoter is 3 degrees C lower than the corresponding value for lac UV5, even though the sequence of the unpaired region in the two promoters is identical.

Proc Natl Acad Sci U S A, 1983 May, 80(9), 2695 - 8
Base substitution mutations induced by metabolically activated aflatoxin B1; Foster PL et al.; We have determined the base substitutions generated by metabolically activated aflatoxin B1 in the lacI gene of a uvrB- strain of Escherichia coli . By monitoring over 70 different nonsense mutation sites, we show that activated aflatoxin B1 specifically induced GxC leads to TxA transversions . One possible pathway leading to this base change involves depurination at guanine residues . We consider this mechanism of mutagenesis in the light of our other findings that the carcinogens benzo{a}pyrene diol epoxide and N-acetoxyacetylaminofluorene also specifically induce GxC leads to TxA transversions.

What Is Salmonella?, What Is Yeast?, What Is Biotechnology?, What Is Functional Genomics?, What Is Activated Sludge?, c, Microbiology, e, Microbes, s, Bacteria, a, Bacterium, r, Microorganisms, o, Escherichia coli, a, Escherichia coli, i, Minimal inhibiting concentration, a, Sepsis, e, Bacteriophages, r, Phage, o, Antimicrobial, c, Escherichia coli, s, Escherichia coli, c, Lactococci, i, Antibiotics, o, Yeasts, o, Bacillus, e, Escherichia coli, i, Yeasts, r, Microorganisms, n, Cryptococci, s, Pseudomonas aeruginosa, e, Enterobacters, r, Beta lactamase, s, Antibiotics

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005