Mol Biol (Mosk), 1984 Sep-Oct, 18(5), 1306 - 10 {Photoaffinity modification of Escherichia coli ribosomes near the tRNA-binding centers by tRNAPhe derivatives carrying arylazido groups on guanosine residues}; Babkina GT et al.; Photoreactive derivatives of tRNAPhe (E . coli) bearing arylazido groups scattered statistically over guanosine residues (azido-tRNA) were applied for affinity labelling of E . coli ribosomes in elongation factor-dependent or factor-free model systems mimicking different steps of elongation . It is shown that UV-irradiation of the corresponding complexes of ribosomes with tRNA derivatives results in labelling of both subunits, the 30S one is labelled preferentially . In all experiments only ribosomal proteins were labelled . Comparison of the sets of proteins labelled by tRNA derivatives in different states at P-site allowed us to draw important conclusions concerning the influence of peptidyl moiety and of the occupancy of the A-site with aminoacyl- or peptidyl-tRNA on the arrangement of tRNA located at the P-site . Three of the 30S proteins--S11, S13 S19--are labelled with tRNA derivatives located at P-site in all states. Mol Biol (Mosk), 1984 Sep-Oct, 18(5), 1301 - 5 {Puromycin interacts with the donor (P) site of Escherichia coli ribosomes}; Ivanov IuV et al.; Puromycin inhibits the interaction of peptidyl-tRNA analogs AcPhe-tRNA Phe ox-red, AcPhe-tRNA Phe and FMet-tRNA f Met with the donor (P) site of Escherichia coli ribosomes . It affects both template-free and poly(U)-dependent systems . The inhibition is apparently due to direct competition for the P-site . On isolated 30S ribosomal subunits it was shown that the puromycin binding site is situated far from the peptidyl transferase center . Quantitative measurements of the inhibition revealed that the affinity constant of puromycin for the P-site is not less than its affinity for the A-moiety of the peptidyl transferase center {1.1 divided by 3.8) X 10(3) M-1). J Gen Microbiol, 1984 Sep, 130 ( Pt 9), 2267 - 75 Chemiluminescence induced by phagocytosis of Escherichia coli by polymorphonuclear leucocytes; Fazeli A et al.; Chemiluminescence emitted by phagocytosing human polymorphonuclear leucocytes stimulated by Escherichia coli was measured using a liquid scintillation counter equipped with a multichannel analyser . In the presence of the amplifying agent luminol, light emission can be divided into two channels, one of which ('high energy') appears to correlate directly with phagocytic activity of the PMNL, and the other ('low energy') with the background luminol dioxygenation by the cells . Measuring in the 'high energy' window also eliminates the normal 'out of coincidence' background . The method is applicable to measuring opsonizing capacity of different sera, and responds to PMNL number, age, composition of assay medium and the integrity of the stimulating bacteria . Other bacterial strains produce a similar response, as does the artificial stimulator zymosan . Low temperature and anaerobiosis, which inhibit phagocytic killing, also suppress light emission. J Antibiot (Tokyo), 1984 Sep, 37(9), 1038 - 43 Anticapsin, an active-site directed irreversible inhibitor of glucosamine-6-phosphate synthetase from Escherichia coli; Chmara H et al.; Glucosamine-6-phosphate synthetase from Escherichia coli K-12 is progressively inactivated L-beta-(2,3-epoxycyclohexyl-4-on)alanine (anticapsin) . With increasing concentrations of anticapsin the reaction exhibits rate saturation: the minimum inactivation half-time is 1.15 minutes, with a Kin alpha ct of 2.5 microM . Glutamine and competitive inhibitors protect against inactivation . Fructose-6-phosphate promotes the inactivation rate . It is concluded that anticapsin is an active-site directed glutamine analog in the reaction catalyzed by glucosamine-6-phosphate synthetase. Appl Environ Microbiol, 1984 Sep, 48(3), 550 - 5 Automation of chromogenic substrate Limulus amebocyte lysate assay method for endotoxin by robotic system; Tsuji K et al.; The chromogenic substrate Limulus amebocyte lysate (LAL) assay method for the detection of endotoxin was automated by a Zymate robotic system . The software developed enables the robot to automatically dilute a stock reference endotoxin standard (20,000 endotoxin units per ml) for the construction of a five-point standard curve, make sample dilutions to the proper testing concentration, and perform chromogenic substrate LAL assays in duplicate . The linearity of the standard curve and the endotoxin concentration in each sample are calculated and results are printed automatically . In 48 min the automated system assays three samples and a reference standard in duplicate along with a water blank . Sensitivity of the assay is a function of incubation time . The assay is linear (r greater than 0.99) in the region of 0 to 1.0 endotoxin units per ml or 0 to 0.2 endotoxin units per ml with incubation times of 10 or 16 min, respectively . The method can be made very sensitive, detecting as low as 0.003 endotoxin units per ml with 30 min of incubation . The precision of the assay method, determined by assaying an endotoxin reference solution eight times, is ca . 6% . The LAL reagent designed for gel-clot assay was modified for the chromogenic substrate assay . We describe the optimum conditions for the performance of the chromogenic substrate LAL assay and stability of the LAL reagent. Am J Vet Res, 1984 Sep, 45(9), 1775 - 7 Virulence factors of Escherichia coli isolated from cows with acute mastitis; Sanchez-Carlo V et al.; A total of 184 Escherichia coli isolates recovered from cows with acute mastitis were examined for recognized pathogenic mechanisms and virulence factors commonly found in pathogenic groups of E coli . A modification of the Eng procedure (for detecting complement deficiencies in serum) was used to test for resistance to different animal sera . The Sereny test (for invasiveness), infant mouse test (for heat-stable enterotoxin), and Y-1 adrenal tumor cell assay (for heat-labile enterotoxin) were used . Hemagglutination tests, using rabbit, sheep, and guinea pig RBC, were done with and without added mannose . All of the 184 isolates were serum resistant in all tested sera . None of the isolates was invasive . Only 1 isolate was positive for heat-stable enterotoxin and 2 cultures were positive for heat-labile enterotoxin . Multiple patterns of hemagglutination were observed . The majority of the isolates exhibited both mannose-sensitive and mannose-resistant hemagglutinins with guinea pig and rabbit RBC . A few strains were positive only in mannose-sensitive or mannose-resistant hemagglutination tests . A few strains were negative in all hemagglutination tests . Based on our results, E . coli from cows with acute mastitis lack the virulence factors commonly observed in other E coli groups associated with disease . Serum resistance was the only characteristic that could be related to virulence. Anal Biochem, 1984 Sep, 141(2), 402 - 8 Affinity purification of aminoacyl-tRNA; Louie A et al.; A procedure for separating Escherichia coli aminoacyl-tRNA from unacylated tRNA or components of the aminoacylation reaction, thereby achieving an aminoacyl-tRNA product with a very high specific activity, is described . The method utilizes the specific recognition of aminoacyl-tRNA for E . coli protein synthesis elongation factor Tu which has been immobilized on an affinity matrix . The application of the affinity procedure as a means of purifying a single aminoacyl-tRNA from an unfractionated mixture of tRNAs is also discussed. Anal Biochem, 1984 Sep, 141(2), 351 - 4 An improved bulk purification method for Escherichia coli elongation factor, Ts; Yoder M et al.; A bulk purification procedure has been designed to maximize the yield of Escherichia coli elongation factor, Ts, with a minimum of effort and time . The enzyme purification is achieved by DEAE-Sepharose and elongation factor Tu-affinity chromatographies . The typical yield is 150 mg/kg of E . coli (B) cells. Vet Microbiol, 1984 Sep, 9(5), 477 - 86 Inhibition of adhesion of Escherichia coli K88 antigen by mammary secretions of susceptible and resistant sows; Sellwood R; Anti-adhesive activities of colostrum and milk from genetically susceptible sows, which protected their susceptible offspring in an outbreak of neonatal diarrhoea caused by K88-positive Escherichia coli, were compared with the activities in mammary secretions of resistant dams that did not protect their susceptible progeny . There was significantly more anti-adhesive antibody in the secretions of susceptible sows than in resistant sows, both during the disease period, and 1 year later . Fractionation of colostrum by gel filtration and ion-exchange chromatography led to identification of the anti-adhesive antibodies as including both IgA and IgM. Mutat Res, 1984 Sep-Oct, 132(3-4), 79 - 86 Occurrence and loss of alkali-labile sites in newly synthesized DNA of UV-irradiated Escherichia coli K12; Slezarikova V et al.; In pulse-labelled DNA of ultraviolet-irradiated E . coli, alkali-labile sites were detected . They do not occur in undamaged cells . These sites are produced in wild-type cells as well as in uvrA, uvrB and recA derivatives . Restoration of the synthesis of DNA molecules free of alkali-labile sites requires recA products and involves also uvrA and uvrB products . The chemical nature of alkali-labile sites and their biological function are obscure . They might be stretches of RNA that traverse the lesions, blocking DNA replication and priming recA-dependent DNA replication. J Vet Pharmacol Ther, 1984 Sep, 7(3), 233 - 8 Effects of dipyrone on endotoxin-induced fever, central nervous depression and hypoglycaemia in sheep; Naylor JM et al.; Sixteen sheep were divided into four groups and were given saline, Escherichia coli endotoxin at 35 micrograms/kg0.75, dipyrone at 88 mg/kg0.75, or endotoxin and dipyrone intravenously . Plasma glucose concentrations, rectal temperature and depression of central nervous function were assessed at hourly intervals for 10 h and again at 24 h . Dipyrone had no effect on normal sheep but in sheep given endotoxin, it blocked the febrile response and partially blocked the depression effect upon the central nervous system without affecting the hypoglycaemic response. J Clin Microbiol, 1984 Sep, 20(3), 323 - 9 Human leukocytic pyrogen test for detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli; Dinarello CA et al.; Human growth hormone is biosynthetically produced in recombinant strains of Escherichia coli as methionyl human growth hormone (met-hGH) . When purified from the bacterial culture, met-hGH is biologically active in established assays for growth hormone . Therefore, a phase I trial of met-hGH was carried out in healthy human adults; during the first trial, however, signs, symptoms, and clinical laboratory tests characteristic of an acute-phase response to pyrogenic agents was observed . Prior testing of the met-hGH preparation used in the phase I trial did not reveal evidence of toxicity, and the U.S . Pharmacopeial Convention rabbit pyrogen test, as well as the Limulus amoebocyte lysate (LAL) test, had not detected significant levels of exogenous pyrogens or endotoxin . In addition, standard inhibition studies with added endotoxin showed no inhibition by the LAL test . When this preparation of met-hGH was incubated with human blood mononuclear cells, leukocytic pyrogen (LP) was released into the supernatant medium, suggesting that the preparation contained pyrogenic material . Various lots of met-hGH based on different purification and formulating methods were tested by the human LP assay for contaminating pyrogens . The results of these tests aided in the identification of procedures for met-hGH preparations which did not induce LP in vitro . Thus, subsequent lots of met-hGH which had passed the LP test were used in repeat clinical studies, and no inflammatory or pyrogenic reactions were observed . When the LP test was used, experiments revealed that the original lot of met-hGH was contaminated with endotoxin which had not been detected in the LAL or rabbit pyrogen tests . Lyophilization in glycine-phosphate buffer had resulted in a 10- to 20-fold reduction of endotoxin reactivity in the LAL test and the U.S . Pharmacopeial Convention rabbit pyrogen test . These data provide a probable explanation for the negative result from the LAL and rabbit pyrogen test in the initial lot of met-hGH which induced acute-phase reactions . In addition, these studies demonstrate that the release of LP from human cells is a reliable indicator of the presence of materials that are pyrogenic for humans. Gene, 1984 Sep, 29(3), 293 - 302 A plasmid model to study genetic recombination in yeast; Embretson JE et al.; Chimeric plasmids have been constructed containing two heteroallelic mutant copies of the yeast HIS3 gene as an inverted repetition . Intramolecular exchange events between these two allelic mutant copies are capable of generating a wild-type allele . Plasmids containing two mutant heteroalleles have been transformed into appropriate his3- yeast strains, and the frequency of exchange events generating His+ prototrophs has been measured during mitotic division . After 20 generations of growth under nonselective conditions, between 0.1 and 1% of the transformed yeast cells become His+ prototrophs . This percentage decreases at least ten-fold in a strain with a rad52 mutation . Plasmid molecules having undergone exchange events have been isolated from yeast cells and have been examined after transfer to Escherichia coli . Physical examination shows that less than 10% of the plasmids having undergone genetic exchange have also undergone an internal reciprocal recombination event as evidenced by reorientation of linked restriction sites . The remainder of the plasmids having undergone genetic exchange do not exhibit reciprocal recombination . Characterization of the individual allelic copies within a plasmid having undergone exchange reveals that in 24 of 25 examples only one of the two HIS3 copies has become wild type, and that either copy is equally likely to become wild type . We conclude that the model plasmid we have constructed undergoes intramolecular genetic exchange events and will be useful for studying genetic recombination. Arch Biochem Biophys, 1984 Sep, 233(2), 776 - 84 Reconstitution of the lipid-depleted pyruvate oxidase system of Escherichia coli: the palmitic acid effect; Kiuchi K et al.; Previous work has shown that the coupling of the soluble Escherichia coli pyruvate oxidase to a lipid-depleted membrane terminal electron transport system requires the addition of ubiquinone and a neutral lipid fraction (C . Cunningham and L . P . Hager (1975) J . Biol . Chem . 250, 7139-7146) . The active factor present in the neutral lipid fraction has now been isolated and characterized . NMR, uv, and mass spectroscopic analysis identifies palmitic acid as the active component . A comparison of palmitic acid with other fatty acids of varying chain lengths indicates that most fatty acids having chain lengths in the range C12 to C20 have comparable activity to palmitic acid . Exceptions are stearic and arachidic acid which have greatly reduced activity . Fatty acids of C6 to C10 chain length showed about one third the activity of palmitic acid . Fatty acids having chain lengths of 2 to 5 carbon atoms are essentially inactive . The carboxyl function of the fatty acid is required for activity . Derivatives of fatty acids in which the carboxyl group had been modified to an alcohol, aldehyde, or methyl ester function show greatly diminished activity . Both the cis and trans forms of unsaturated long-chain fatty acids are active . The stimulation of the electron transfer reaction by fatty acids occurs at the ubiquinone level of the electron transport chain . Ubiquinone-30 is rapidly reduced by pyruvate oxidase only in the presence of palmitic acid. Arch Biochem Biophys, 1984 Sep, 233(2), 565 - 72 Tissue specificity of 3'-untranslated sequence of myosin light chain gene: unexpected interspecies homology with repetitive DNA; Saidapet C et al.; Using the 3' noncoding and coding sequences of chick heart myosin light chain mRNA cloned into Escherichia coli as probes, it was observed that, while the coding sequence shared homology with myosin light-chain mRNAs from other sources, the 3' noncoding sequence was specific for chick heart muscle . This property was used to detect chick heart-specific myosin light-chain gene activity in chick blastoderms of very early developmental stages where cells of different muscle origins cannot be distinguished morphologically . However, in spite of the tissue-specific divergence of the 3' noncoding sequence of myosin light-chain gene, which is present in a single copy in the chick genome, a surprising homology with DNA from such a diverse source like Dictyostelium discoideum was noted . The sequence homologous to chick myosin light-chain DNA was apparently present in a high repetition frequency in the Dictyostelium genome. Arch Biochem Biophys, 1984 Sep, 233(2), 475 - 80 Formation of beta,gamma-methylene-7,8-dihydroneopterin 3'-triphosphate from beta,gamma-methyleneguanosine 5'-triphosphate by GTP cyclohydrolase I of Escherichia coli; Ferre J et al.; GTP cyclohydrolase I of Escherichia coli converts {beta,gamma-methylene} GTP to a fluorescent product that is characterized as {beta,gamma-methylene}dihydroneopterin triphosphate . Interaction between the GTP analog and the enzyme gave a Ki of 3.0 microM, which may be compared to the Km of 0.1 microM for GTP . This new analog of dihydroneopterin triphosphate may, in turn, be converted to the same greenish-yellow pteridines (compounds X, X1, and X2) that are obtained from dihydroneopterin triphosphate . Because of its stability to phosphatase action, this analog may be useful for studies in pteridine metabolism. Vet Pathol, 1984 Sep, 21(5), 516 - 20 Ultrastructure of the intestinal mucosa in pigs experimentally inoculated with an edema disease-producing strain of Escherichia coli (0139:K12:H1); Methiyapun S et al.; The intestinal tissues from 11 pigs orally inoculated with Escherichia coli (E . coli, 0139:K12:H1) were examined by transmission electron microscopy . The colonization of E . coli along the small intestinal mucosa was found in seven principals without any major changes in the enterocytes from day 2 to day 7 after inoculation when the experiment was terminated . Lesions of vessels of the intestinal mucosa could be detected as early as two days after inoculation and persisted until the experiment was terminated . Lesions consisted of endothelial swelling and vacuolation, subendothelial fibrin deposition, perivascular edema, microthrombus formation, endothelial proliferation, and necrosis of the tunica media . The possible pathogenesis of the disease is discussed. J Bacteriol, 1984 Sep, 159(3), 986 - 90 Purification and properties of D-mannitol-1-phosphate dehydrogenase and D-glucitol-6-phosphate dehydrogenase from Escherichia coli; Novotny MJ et al.; D-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and D-glucitol-6-phosphate dehydrogenase (EC 1.1.1.140) were purified to apparent homogeneity in good yields from Escherichia coli . The amino acid compositions, N-terminal amino acid sequences, sensitivities to chemical reagents, and catalytic properties of the two enzymes were determined . Both enzymes showed absolute specificities for their substrates . The subunit molecular weights of mannitol-1-phosphate and glucitol-6-phosphate dehydrogenases were 40,000 and 26,000, respectively; the apparent molecular weights of the native proteins, determined by gel filtration, were 40,000 and 117,000, respectively . It is therefore concluded that whereas mannitol-1-phosphate dehydrogenase is a monomer, glucitol-6-phosphate dehydrogenase is probably a tetramer . These two proteins differed in several fundamental respects. J Bacteriol, 1984 Sep, 159(3), 919 - 24 Shrinkage of growing Escherichia coli cells by osmotic challenge; Koch AL; The immediate response of growing Escherichia coli to changing external osmotic pressure was studied with stopped-flow turbidimetric measurements with a narrow-beam spectrophotometer . It is shown theoretically that in such a photometer rod-shaped bacteria have an apparent absorbance which is proportional to the inverse of the surface area . The apparent optical density, corrected for effects of alteration of the index of refraction of the medium, increased continuously as the external osmotic pressure was raised . Because of the short time scale of the measurements, the turbidity increases could result either from shrinkage of the cells or from plasmolysis, or both, but not from growth or metabolic adaptation . With low concentrations of pentose such that the external osmotic pressure was not greater than that inside the cells, plasmolysis would not occur and, consequently, only shrinkage of the previously stretched sacculus remains to account for the observed optical effects . Taking the osmotic pressure of the growing cells as 5 atmospheres (506 kPa), the turbidity changes correspond to the murein fabric having been stretched 20% beyond its unstressed equilibrium area during growth under the conditions used. J Bacteriol, 1984 Sep, 159(3), 1053 - 5 Outer membrane protein NmpC of Escherichia coli: pore-forming properties in black lipid bilayers; Hindahl MS et al.; The purified NmpC outer membrane protein from Escherichia coli, when incorporated into planar lipid bilayers, gave rise to channels with a single-channel conductance of 1.8 nS in 1 M KCl . This suggests that the NmpC protein is a porin. J Bacteriol, 1984 Sep, 159(3), 1034 - 9 Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid; Russel M et al.; To determine whether the fip gene of Escherichia coli, which is required for filamentous phage assembly, is required for cell viability, we replaced the chromosomal copy of the gene with an inactive copy introduced on a plasmid . We found that the fip gene is dispensable . The method we devised, which should be generally useful, was also tested with an inactivated rho gene . As expected, the rho gene is essential. Biochem J, 1984 Sep 1, 222(2), 519 - 34 Nucleotide sequence encoding the flavoprotein and hydrophobic subunits of the succinate dehydrogenase of Escherichia coli; Wood D et al.; The nucleotide sequence of a 3614 base-pair segment of DNA containing the sdhA gene, encoding the flavoprotein subunit of succinate dehydrogenase of Escherichia coli, and two genes sdhC and sdhD, encoding small hydrophobic subunits, has been determined . Together with the iron-sulphur protein gene (sdhB) these genes form an operon (sdhCDAB) situated between the citrate synthase gene (gltA) and the 2-oxoglutarate dehydrogenase complex genes (sucAB): gltA-sdhCDAB-sucAB . Transcription of the gltA and sdhCDAB gene appears to diverge from a single intergenic region that contains two pairs of potential promoter sequences and two putative CRP (cyclic AMP receptor protein)-binding sites . The sdhA structural gene comprises 1761 base-pairs (587 codons, excluding the initiation codon, AUG) and it encodes a polypeptide of Mr 64268 that is strikingly homologous with the flavoprotein subunit of fumarate reductase (frdA gene product) . The FAD-binding region, including the histidine residue at the FAD-attachment site, has been identified by its homology with other flavoproteins and with the flavopeptide of the bovine heart mitochondrial succinate dehydrogenase . Potential active-site cysteine and histidine residues have also been indicated by the comparisons . The sdhC (384 base-pairs) and sdhD (342 base-pairs) structural genes encode two strongly hydrophobic proteins of Mr 14167 and 12792 respectively . These proteins resemble in size and composition, but not sequence, the membrane anchor proteins of fumarate reductase (the frdC and frdD gene products). Proc Natl Acad Sci U S A, 1984 Sep, 81(17), 5389 - 93 Translational regulation of the L11 ribosomal protein operon of Escherichia coli: analysis of the mRNA target site using oligonucleotide-directed mutagenesis; Baughman G et al.; The L11 ribosomal protein operon in Escherichia coli consists of the genes for proteins L11 and L1 and is feedback regulated by the translational repressor L1 . The mRNA target site for this repression is located close to the translation initiation site of the first L11 cistron . Several mutant plasmid molecules carrying altered nucleotide sequences in the L1 target site were constructed by site-directed in vitro mutagenesis using synthetic oligodeoxyribonucleotides . Specifically, we examined the importance of a presumptive double-stranded stem structure that is common among L1 binding sites on rRNA from a variety of organisms and in L11 mRNA . Mutational alterations that disrupt the stem structure were found to abolish translational regulation as analyzed both in vitro and in vivo . Two of the mutations were combined so that the stem structure was restored but with a different primary nucleotide sequence . This double mutant was shown to restore the original phenotype, the ability to be translationally regulated by L1 . These experiments show the importance of the stem structure, but not its primary sequence, for the interaction of L1 with the mRNA and support the concept that mRNA target sites share some structural features with the corresponding ribosomal protein binding sites of rRNA. J Surg Res, 1984 Sep, 37(3), 208 - 25 Naloxone therapy in awake endotoxemic Yucatan minipigs; Fettman MJ et al.; Twelve Yucatan miniature pigs were fitted with jugular, portal, hepatic vein, and carotid artery catheters, and hepatic artery and portal vein flow cuffs to quantitate portosystemic and transhepatic kinetics . Seventy-two hours later they were placed in slings, and following a 3-hr control period were infused with Escherichia coli endotoxin at 15 micrograms/kg/hr for 6 hr . Eight were controls and 4 received a primed (2 mg/kg) continuous infusion (2 mg/kg/hr) of naloxone 1 hr after initiation of endotoxin . In the naloxone group, arterial hypotension developed by 60 min . Arterial pressure increased to 125 mm Hg by 80 min (20 min post naloxone) and maintained this level for 3 hr . {6-3H}Glucose-derived rate of disappearance (Rd) values were increased above their own control period at 60 and 80 min, but were approximately 55% lower than untreated controls . Rate of appearance (Ra) values remained unchanged in the naloxone group resulting in a net glucose deficit . After 1 hr of endotoxin, blood pyruvate increased from 1.0 to 1.8 mg%, reached 3.3 mg% at 80 min, and remained elevated . Blood lactate similarly increased from 11 to 35 mg% by 60 min, increasing further to 82 mg% 20 min after naloxone infusion, in concert with signs of severe distress . Portal and hepatic total O2 decreased by 60 min and 100 min, respectively, reflecting depressed hepatic O2, input . Lethality was 75% in naloxone-treated pigs by the end of the experiment, vs 0% in untreated animals . Naloxone improves some hemodynamic parameters . However, in these conscious postsurgical patients, naloxone blocked some beneficial effects of endogenous opiates resulting in severe metabolic derangements and increased mortality. J Surg Res, 1984 Sep, 37(3), 197 - 201 The clearance capacity of the canine liver for a portal vein endotoxin infusion; Caruana JA et al.; The capacity of the livers of anesthetized dogs to clear a portal vein infusion of Escherichia coli 026 endotoxin was evaluated . Appearance of the endotoxin in arterial blood was quantitated by immunoradiometric assay . Various hemodynamic and metabolic parameters were monitored throughout the infusion to corroborate the development of systemic endotoxemia . Significant amounts of E . coli 026 endotoxin were detected in arterial blood after infusion of 240 micrograms endotoxin . As expected, systemic endotoxemia was associated with decreased cardiac index, mean arterial blood pressure, heart rate, and splanchnic (portal vein) blood flow . Changes in plasma levels of glucose, insulin, and glucagon and in the pancreatic outputs of insulin and glucagon did not occur before the development of severe hypotension and the termination of the study . It was concluded that the liver clearance capacity for endotoxin in the dog is 0.72 microgram/gm liver/hour and that severe hemodynamic alterations develop in this animal model before changes in carbohydrate balance. Arch Biochem Biophys, 1984 Sep, 233(2), 796 - 9 Inhibition of ATPase activity of the recA protein by ATP ribose-modified analogs; Karasaki Y et al.; The single-stranded, DNA-dependent ATPase activity of purified recA protein was found to be inhibited competitively by ribose-modified analogs of ATP, 3'-O-anthraniloyl-ATP (Ant-ATP), and 3'-O-(N-methylanthraniloyl)-ATP (Mant-ATP) . The Ki values for Ant-ATP and Mant-ATP were around 7 and 3 microM at pH 7.5, respectively . The inhibitions by these analogs were much stronger than that by ADP, which is also a competitive inhibitor for the ATPase activity of the recA protein . The Ki value for ADP is 76 microM . Ant-ATP and Mant-ATP reduced the Hill coefficient for ATP hydrolysis and thus contributed to the cooperative effect of ATP. Proc Natl Acad Sci U S A, 1984 Sep, 81(17), 5394 - 8 cDNA clone for the alpha-chain of human beta-hexosaminidase: deficiency of alpha-chain mRNA in Ashkenazi Tay-Sachs fibroblasts; Myerowitz R et al.; We have isolated a cDNA clone containing sequences complementary to mRNA encoding the alpha-chain of the lysosomal enzyme beta-hexosaminidase . RNA from a human lung fibroblast strain, IMR90, was enriched for beta-hexosaminidase messenger by polysome immunoselection with antiserum against beta-hexosaminidase A . This preparation was used to construct cDNA recombinant plasmids by the Okayama-Berg vector primer procedure . After transformation of Escherichia coli, 385 ampicillin-resistant colonies were obtained, 44 of which contained inserts in the plasmid DNA . Differential hybridization, with cDNA probes prepared from polysomal RNA enriched or depleted for beta-hexosaminidase messenger, was used to screen the recombinant plasmids for sequences encoding beta-hexosaminidase . One clone, p beta H alpha-1, containing a cDNA insert of approximately equal to 240 base pairs, was identified in this manner . The plasmid hybrid-selected a messenger from placental RNA that programed a translation system to synthesize the alpha-chain of beta-hexosaminidase . p beta H alpha-1 hybridized to an mRNA of approximately equal to 1.9 kilobases in preparations enriched separately in messenger for the alpha-chain or for both alpha- and beta-chains (by polysome immunoselection with antiserum against isolated alpha-chain or against beta-hexosaminidase A, respectively) . It did not hybridize to an RNA preparation enriched for messenger of beta-chain by immunoselection with antiserum against beta-hexosaminidase B . The 1.9-kilobase mRNA was observed in poly(A)+ RNA preparations from control fibroblasts and from fibroblasts of a Tay-Sachs patient that synthesize an altered alpha-chain; however, it was not seen in similar preparations from fibroblasts of four Ashkenazi Tay-Sachs patients. Mutat Res, 1984 Sep, 128(2), 127 - 35 Mutagenicity of neocarzinostatin in Neurospora crassa; DeGraff WG; Neocarzinostatin (NCS) is an acidic, single-chain polypeptide of 109 amino acids that has shown some antitumor activity in clinical trials . NCS is mutagenic in recA+ strains of Escherichia coli, but not in recA strains; on the other hand, a defect in the nucleotide-excision-repair pathway has no effect on the mutagenicity of NCS in E . coli . Similar results are seen in mammalian cells . Excision-repair-deficient xeroderma pigmentosum (XP) cells repair NCS-induced DNA damage at the same rate as repair-proficient XP heterozygotes, and X-ray-sensitive ataxia telangiectasia fibroblasts are also sensitive to NCS . I have investigated the mutagenicity of NCS in the ad-3 forward-mutation test in nucleotide excision-repair-sufficient and -deficient heterokaryons of Neurospora crassa . Resting conidia from a repair-sufficient strain, H-12, and a nucleotide-excision-repair-deficient strain (uvs-2) H-59, were exposed to NCS . These conidia were assayed for survival and ad-3 forward mutation . The results show that H-59 is more sensitive to the killing and mutagenic activities of NCS than is H-12 . These data indicate, in contrast to E . coli and mammalian cells, that the nucleotide-excision-repair pathway of N . crassa does repair NCS-induced lesions . In other experiments, ad-3 mutants induced by NCS in H-59 were characterized to determine the spectrum of NCS-induced mutation . The results show that NCS induces both intracistronic mutations and multilocus deletions in H-59. Ukr Biokhim Zh, 1984 Sep-Oct, 56(5), 503 - 14 {Isolation of reverse transcriptase from the avian myeloblastosis virus in preparative quantities}; Staverskaia OV et al.; Inverse transcriptase of bird myeloblastosis virus is a unique instrument for artificial synthesis of structural genes of viruses, plants, animals . Methods for the virus production in preparative amounts are developed due to selection of the corresponding line of chickens, conditions of their maintenance, diet infection methods and myeloblastosis diagnostics . Main demands to the inverse transcriptase preparations (their high activity, absence of nuclease impurities, high concentration of the enzyme preparation solutions and their stability in storage) are ensured by zonal centrifugation purification of the virus in a sucrose density gradient, described methods of inverse transcriptase isolation and purification as well as conditions of its storage. Mol Biol (Mosk), 1984 Sep-Oct, 18(5), 1419 - 23 {Selective alkylation of G24 residue in tRNAPhe}; Gimautdinova OI et al.; Alkylation of E . coli tRNAPhe with 4-(N-2-chloroethyl-N-methylamino) benzyl-5'-phosphamide of oligonucleotide d(pAACCA) was studied . G24 residue located near the sequence C17GGDA21 partially complementary to the oligonucleotide moiety of the reagent was shown to be alkylated . Oligonucleotide d(pAACCA) inhibited the alkylation . Association constant of oligonucleotide derivative with tRNAPhe (10(3) M-1) was evaluated from the dependence of the extent of tRNA modification on the concentration of the reagent . The reported method for selective alkylation of tRNA may be used for preparing photoaffinity derivatives of tRNA bearing an arylazidogroups in desired position. Mol Biol (Mosk), 1984 Sep-Oct, 18(5), 1336 - 41 {Aminoacyl-tRNA-synthetases and their high molecular weight complexes in the regenerating rat liver}; Iaremchuk AD et al.; Glutamyl- and lysyl-tRNA synthetase activities in total preparations and high-molecular complexes from rat liver increase 21 h after partial hepatectomy . Glutamyl-tRNA synthetase has been highly purified from normal and regenerating liver . Km, Vmax values and molecular activity were practically the same for both enzyme preparations . Total methyltransferase activity and that as a part of high-molecular complexes increase 1.5 fold 21 h after the operation but the changes of individual methyltransferase activities differ . The level of tRNA methylation in vivo is also higher during regeneration . Proteinkinases of high-molecular complexes from regenerating liver are sensitive to cyclic AMP and GMP in contrast to control. Mol Biol (Mosk), 1984 Sep-Oct, 18(5), 1287 - 96 {Affinity modification of Escherichia coli ribosomes near the acceptor tRNA-binding site}; Babkina GT et al.; It was shown that Phe-tRNA Phe derivatives bearing arylazidogroups scattered statistically on N7 guanosine residues retain the ability to EF-Tu-dependent binding to E . coli ribosomes . UV-irradiation of the corresponding complex with the derivative of Phe-tRNA Phe located at A-site results in a specific modification of both ribosomal subunits to an approximately equal extent . It was found that proteins S9, S15, S16, S17, S18, S19 and L8/L9, L13, L15, L27 are labelled at A-site. Mol Biol (Mosk), 1984 Sep-Oct, 18(5), 1194 - 207 {tRNA-binding centers of Escherichia coli ribosomes and their structural organization}; Karpova GG; Problems concerning the interaction of tRNA with Escherichia coli ribosomes in different functional states were studied . These problems deal first of all with the number of tRNA-binding sites on ribosome, the conservation of the codon-anticodon interaction at the P-site and with regions of tRNA interacting with ribosome . The problems concerning structural organization of tRNA-binding centers are discussed in more detail. J Gen Microbiol, 1984 Sep, 130 ( Pt 9), 2247 - 51 Changes in ATP concentration in Escherichia coli during induction of the SOS system by mitomycin C and bleomycin; Guerrero R et al.; Treatment of Escherichia coli with bleomycin induced a dramatic increase in ATP concentration in the first 30 min . Afterwards, in RecA+ strains, ATP dropped quickly to values similar to those of untreated cells . Mutants of E . coli defective in either RecA protein or RecA protease activity did not show this decrease, indicating that it was due to the action of RecA protease . The increase in ATP in the first 30 min was dependent on RecBC exonuclease activity and must have been due to substrate level phosphorylation, since an uncoupler such as dinitrophenol did not affect it . Nevertheless, mitomycin C did not induce any change in ATP pools of RecA+ strains, at least during 120 min following treatment . The implications of these findings are discussed in relation to the possible pathways of activation of RecA protease. J Hosp Infect, 1984 Sep, 5(3), 283 - 8 An outbreak of gastroenteritis due to Escherichia coli 0142 H6 in a neonatal department; Gerards LJ et al.; An outbreak of gastroenteritis due to Escherichia coli 0142 H6 in a neonatal ward is described . The epidemic affected 16 of 24 infants (infection-rate 66 per cent), of whom one died due to necrotizing enterocolitis . Administration of antibiotics was of limited value in treatment or in eradicating E . coli 0142 H6 from the stools . Termination of the epidemic was only accomplished by isolating the patients, accompanied by strict hygienic measures, including the use of disposable gloves . Gastroenteritis due to this organism occurred only in prematurely born infants during the first 2 weeks of life. Gene, 1984 Sep, 29(3), 263 - 9 Expression of canine parvovirus-beta-galactosidase fusion proteins in Escherichia coli; Smith S et al.; Cloned DNA fragments encoding portions of canine parvovirus (CPV) structural proteins were inserted into plasmid expression vectors . These plasmids expressed CPV-beta-galactosidase fusion proteins under the transcriptional control of the Escherichia coli lac promoter-operator . The fusion proteins were purified and used to immunize rabbits . Rabbit antibodies raised against these fusion proteins were shown to immunoprecipitate authentic CPV structural proteins from infected cell extracts . This demonstrated that the CPV-beta-galactosidase fusion proteins expressed in bacteria elicit antibodies which can recognize determinants of authentic CPV proteins . However, none of the antibodies neutralizes CPV virus particles. J Bacteriol, 1984 Sep, 159(3), 877 - 82 Serum-resistant mutants of Escherichia coli O111 contain increased lipopolysaccharide, lack an O antigen-containing capsule, and cover more of their lipid A core with O antigen; Goldman RC et al.; Escherichia coli strains of group O111 were characterized with respect to sensitivity to complement killing, amount of lipopolysaccharide and O antigen-containing capsule, and distribution of O antigen . All wild-type E . coli O111 strains were resistant to complement killing in the absence of specific antibody . Presensitization of strains with antibody to whole cells (OK antibody), followed by incubation in 50% pooled normal human serum as a source of complement, subdivided wild-type strains into three types: completely resistant, partially resistant, and sensitive . Completely and partially resistant mutants were isolated by cycles of serum killing, starting with one sensitive strain . Completely resistant mutants had no O antigen-containing capsule, but had 50% more lipopolysaccharide than did the parent, and this lipopolysaccharide had 30% fewer lipid A core molecules devoid of O antigen . Partially resistant mutants still had O antigen-containing capsule, but contained 40% more lipopolysaccharide than did the parent; the extent of coverage of lipid A core with O antigen remained unchanged . No correlations were found between outer membrane protein composition and the degree of serum resistance . Since the terminal membrane attack complex (C5b-9) must stably insert into a hydrophobic membrane site to effect killing, we conclude that both increased lipid A core and increased coverage of lipid A core with O antigen preclude access of C5b-9 to lethal sites on the cell surface. Proc Natl Acad Sci U S A, 1984 Sep, 81(17), 5504 - 8 Receptors for human alpha and beta interferon but not for gamma interferon are specified by human chromosome 21; Raziuddin A et al.; We examined the proposed role of human chromosome 21 in determining the cellular sensitivity to human alpha, beta, and gamma interferons (HuIFN-alpha, -beta, and -gamma) and the expression of the receptors for the HuIFNs with the use of mouse-human hybrid cells containing human chromosome 21 . Hybrid cells (WA17) containing three copies of human chromosome 21 showed specific displaceable binding of 125I-labeled HuIFN-alpha 2 (125I-HuIFN-alpha 2), which was not observed with mouse parent (A9) cells . Crosslinking of 125I-HuIFN-alpha 2 bound to WA17 cells with disuccinimidyl suberate yielded a complex of Mr approximately equal to 150,000 similar to the 125I-HuIFN-alpha 2-receptor complex obtained with human cells as described earlier . Such a complex was not obtained with mouse parent (A9) cells or with hybrid cells containing certain other human chromosomes but not chromosome 21 . Mice inoculated with mouse-human hybrid cells containing human chromosome 21 produce antibodies that block the antiviral action of HuIFN-alpha and -beta on human cells . Such antibodies could immunoprecipitate the 125I-HuIFN-alpha 2-receptor complex obtained from human cells but not free 125I-HuIFN-alpha 2, indicating that these antibodies were directed against the receptor . WA17 hybrid cells were highly sensitive to the antiviral action of HuIFN-alpha 2, -alpha (Le) and -beta but were completely insensitive to HuIFN-gamma . Furthermore, 125I-HuIFN-gamma showed specific binding to human WISH cells but not to WA17 hybrid cells or A9 mouse cells . The results indicate that the receptors for HuIFN-alpha and -beta but not for HuIFN-gamma are specified by human chromosome 21 . Hybrid cells containing one, two, or three copies of human chromosome 21 were found to be increasingly sensitive to HuIFN-alpha 2, indicating that a chromosome 21-specified component (possibly the HuIFN-alpha receptor) may be a limiting factor in the cellular sensitivity to HuIFN-alpha. J Virol, 1984 Sep, 51(3), 754 - 9 Cloning of the bluetongue virus L3 gene; Purdy M et al.; The genes of the bluetongue virus (BTV) serotype 17 have been cloned into pBR322 by tailing both strands of the double-stranded RNA with polyadenylic acid, transcribing them with reverse transcriptase with an oligodeoxythymidylic acid primer, hybridizing the cDNA products, and completing them into duplex structures with the Klenow fragment of Escherichia coli DNA polymerase . After cloning the double-stranded cDNA molecules into pBR322, the complete sequence of the cloned L3 gene was determined . The clone is 2,772 nucleotides long (1.78 X 10(6) daltons), excluding the 3' polyadenylic acid sequence, and has an open reading frame which codes for a protein of some 901 amino acids (103,412 daltons) . This clone can hybridize L3 RNA segments of three other U.S . BTV serotypes, BTV-10, -11, and -13 in addition to -17 but not the equivalent RNA segment of epizootic hemorrhagic disease virus of deer, an orbivirus related to BTV. Carcinogenesis, 1984 Sep, 5(9), 1165 - 71 Assessment of mutagenic efficiency of two carcinogen-modified nucleosides, 1,N6-ethenodeoxyadenosine and O4-methyldeoxythymidine, using polymerases of varying fidelity; Singer B et al.; Terminal deoxynucleotidyl transferase (TdT) was used to prepare copolymers of dA and 1,N6-ethenodeoxyadenosine (epsilon dA) . When used as templates for Escherichia coli DNA polymerase I (Pol I) and compared with poly (dA), normal dTTP incorporation was not significantly affected by the presence of 7% epsilon dA . dGTP misincorporation was only slightly increased and occurred about once for every 500 epsilon dA residues . The error-prone polymerase from avian myeloblastosis virus (AMV reverse transcriptase) increased this error rate 5- to 20-fold to a maximum of 1 dG/25 epsilon dA . No dCTP misincorporation was detected with either polymerase . In transcription with E . coli DNA-dependent RNA polymerase, no errors were revealed by nearest neighbor analysis . Poly (dA) treated with chloroacetaldehyde under conditions producing the same proportion of epsilon dA (without the hydrated form) as the synthesized template behaved in the same manner with a similar low level of misincorporation of dG . Such treatment of alternating poly d(A-T) caused structural changes indicative of crosslinks but did not alter its template properties . Increasing the amount of epsilon dA in either synthesized or modified polymers greatly decreased the template activity without increasing the error rate . It is suggested that epsilon dA generally does not prevent dT incorporation but behaves as a bulky lesion which is bypassed . In contrast to the low mutagenic efficiency of epsilon dA, O4-methyldeoxythymidine (m4dT), in copolymers with dA, directed the misincorporation of 1 dG/12 m4dT with Pol I and 1 dG/3 m4dT with reverse transcriptase . Nearest neighbor analysis of transcripts showed the incorporation of 1 dG/12 m4dT . These data are in agreement with the previous reported mutagenicity of m4dT in alternating poly d(A-T, m4T). Proc Natl Acad Sci U S A, 1984 Sep, 81(18), 5704 - 8 Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules; Gibbs JB et al.; The 21-kilodalton protein (p21) encoded by normal cellular Harvey-ras has been expressed in Escherichia coli as a fusion protein by using the pUC8 vector and has been purified to greater than 95% homogeneity by ion-exchange chromatography and gel filtration . The purified protein molecules possess intrinsic GTPase activity on the basis of the following criteria: (i) elution of the GTPase activity with p21 GDP-binding activity in two different chromatography systems, (ii) parallel thermal inactivation of GTPase activity and p21 GTP-binding activity, and (iii) immunoprecipitation of the GTPase activity with monoclonal antibodies to p21 . At 37 degrees C, the rate of GTP hydrolysis by the purified normal p21 assayed in solution was 5.3-6.6 mmol/min per mol of p21 . The rate of GTP hydrolysis by a form of p21 {Val12} encoded by a human oncogene was significantly lower (1.4-1.9 mmol/min per mol of p21) . The presence of a threonine phosphate acceptor site at residue 59 also decreased p21 GTPase activity . For regulatory proteins that use GTP as part of their biochemical mechanism, the hydrolysis of GTP to GDP reverses the biological activity of the respective proteins . The observation that oncogenic forms of p21 lose GTPase activity suggests that GTP hydrolysis may be a biochemical event that inactivates the growth-promoting effects of a p21 X GTP complex. J Bacteriol, 1984 Sep, 159(3), 1077 - 9 Dependence of secretion and assembly of type 1 fimbrial subunits of Escherichia coli on normal protein export; Dodd DC et al.; The export of fimbrial subunits was found to be diminished at the restrictive temperature in a strain bearing a secA(Ts) mutation . Likewise, export was inhibited in a strain harboring a malE-lacZ protein fusion upon induction of hybrid protein synthesis . Both conditions resulted in the accumulation of a precursor protein ca . 2,000 daltons larger than the mature fimbrial subunit. Infect Immun, 1984 Sep, 45(3), 737 - 40 Capsule reduces adherence of enterotoxigenic Escherichia coli to isolated intestinal epithelial cells of pigs; Runnels PL et al.; Previous reports have demonstrated that heat-stable (A-type) capsule on piliated enterotoxigenic Escherichia coli enhances colonization of enterotoxigenic E . coli in the small intestine and enhances virulence of enterotoxigenic E . coli . In this report, four encapsulated enterotoxigenic E . coli strains and one encapsulated nonenterotoxigenic strain of E . coli and their nonencapsulated mutants were tested for adhesion to isolated intestinal epithelial cells or brush borders from neonatal pigs . The enterotoxigenic E . coli also expressed the K99 pilus antigen . The nonencapsulated mutants of the four enterotoxigenic E . coli adhered in higher numbers than did the encapsulated parental strains . Both the encapsulated and nonencapsulated forms of enterotoxigenic E . coli 431 grown at 18 degrees C (K99 production suppressed) adhered poorly to the isolated cells . The nonenterotoxigenic E . coli 1793 which does not express K99 antigen also adhered poorly in both encapsulated and nonencapsulated forms . Fab fragments of anticapsular immunoglobulin G failed to block the effect of capsule on adherence of strain 431 . The results indicated that K99 was the principal mediator of in vitro adhesion of the enterotoxigenic E . coli strains and that capsule impedes the in vitro adhesion . They also suggested that the capsular enhancement of colonization by such strains in vivo probably is by some mechanism other than enhanced adhesion to epithelium. Isr J Med Sci, 1984 Sep, 20(9), 793 - 6 Cloning of L-2 DNA in Escherichia coli pOL4 plasmid; Honigman A et al.; A physical map of L-2 DNA was constructed using restriction endonucleases . Based on this map the five HincII-generated L-2 DNA fragments (A-E) were cloned into the SmaI site of Escherichia coli vector plasmid pOL4, that was designed to analyze promoters and transcriptional terminators . The insertion of the HincII-generated L-2 DNA fragments into this plasmid clearly demonstrated that a fragment (fragment E) with a size of 1.1 kbp carried a sequence that initiated transcription in E . coli. Plasmid, 1984 Sep, 12(2), 103 - 10 Plasmid vectors based on Tn10 DNA: gene expression regulated by tetracycline; de la Torre JC et al.; The regulatory region of the tetracycline resistance determinant from transposon Tn10 has been used to construct plasmid vectors for gene expression regulated by tetracycline . Plasmids pRS tetBam-8 and pRS tetBam-16 include the tet regulatory region, the segment coding for the first four amino acids of the tetracycline resistance protein (tetA protein), and a linker region with SalI, HpaII, and BamHI restriction sites for gene fusions . Plasmid pTB-1, a derivative of pRS tetBam-8 and of the beta-galactosidase gene-containing plasmid pMC1403, constitutively expresses a tetA fragment-beta-galactosidase fusion protein . If a multicopy runaway replication plasmid, pMOBglII-16 that includes a 2.7-kb BglII DNA fragment from Tnl10 that provides tetR protein is present along with pTB-1, the expression of beta-galactosidase is reduced eightfold . Tetracycline acts as an inducer of the system and restores the level of beta-galactosidase activity measured in transformants containing pTB-1 alone . Plasmid mutants unable to produce active tetR protein are ineffective in reducing expression . Escherichia coli carrying plasmids that express both tetA protein and tetR protein show an increase in the tetracycline resistance level after incubation with the drug . The observations are consistent with the previously proposed mechanism of regulation of tetracycline resistance in Tn10. Mol Biol (Mosk), 1984 Sep-Oct, 18(5), 1352 - 62 {Organization of genes coding for 5S rRNA in the loach Misgurnus fossilis L.}; Shostak NG et al.; The organization of 5S rRNA genes in the loach Misgurnus fossilis L . was studied . 5S rDNA was cloned in Escherichia coli using the HindIII-fragments of loach genome DNA fused into the plasmid pBR322 restricted at the same site . The recombinant clones were tested by colony hybridization . The presence of the 5S rDNA structural sequences in cloned fragments was determined by the Southern procedure of hybridization with 5S {32P}rRNA of the loach and transcription in the oocyte test system . It was found that the size of 5S rDNA repetitive units corresponds to 240-250 bp and 450-460 bp . By the CsCl centrifugation and restriction analysis it was shown that the 5S genes in the loach genome are arranged in clusters (5-30 repeats per cluster), the smaller repeat was found to contain one coding sequence while the larger repeat contains two coding sequences of 5S rDNA. Cell Biol Int Rep, 1984 Sep, 8(9), 773 - 86 Inhibition of postreplication recombinational repair by DNA-gyrase antagonists in Escherichia coli; Priel E; In the present study we investigated the possible involvement of DNA-Gyrase in postreplication repair in E . Coli . It was observed that nalidixic acid and oxolinic acid (which are known-antagonists of DNA-Gyrase) inhibited recombinational repair . These results strongly suggest that the nicking closing activity of DNA-Gyrase is essential for efficient recombinational repair. Mol Cell Biol, 1984 Sep, 4(9), 1730 - 7 Amphotropic retrovirus vector system for human cell gene transfer; Sorge J et al.; Retroviral vectors have been constructed for gene transfer in mammalian and avian cells, however most retroviral vector systems are complicated by the spread of a replication-competent helper virus . This problem has been circumvented by segregating the viral genome into cis- and trans-acting components . By establishing helper cell lines that produce the trans-acting viral gene products, one can propagate the cis-acting component in them and harvest defective viral particles that contain only the cis-acting component . The cis-acting component can provide a useful vehicle for the highly efficient transfer of genes into target cells . The defective vector systems described to date, however, are restricted in host range to murine, avian, rat, and dog cells . We describe a helper-free vector system based entirely on an amphotropic murine virus with a wide mammalian host range, including the ability to carry out efficient gene transfer into human cells . We also describe a double mutation constructed in the trans-acting genome which reduces the frequency of replication-competent recombinant viruses to undetectable levels. Gene, 1984 Sep, 29(3), 255 - 61 Recombination following transformation of Escherichia coli by heteroduplex plasmid DNA molecules; Chang S et al.; Circular heteroduplex DNA molecules introduced into Escherichia coli-competent cells are converted to new recombinant plasmids as a result of enzymatic actions in vivo . A pair of plasmids with partial sequence homology were each linearized at a different position with restriction enzymes, and the termini were made flush with the single-strand-specific S1 nuclease . Duplex molecules were then formed by melting and annealing these plasmid DNAs together . In contrast to linear homoduplex molecules, heteroduplexes circularize and therefore transform E . coli efficiently . Unique DNA sequences on each of the parental strands in the transforming heteroduplexes can be selectively incorporated or deleted as a result of in vivo enzymatic activities in transformed cells . This method permits the generation of new recombinant sequences in vivo without relying solely on the presence of convenient restriction sites for manipulation of DNA fragments in vitro. EMBO J, 1984 Sep, 3(9), 2159 - 64 Formation of covalently closed heteroduplex DNA by the combined action of gyrase and RecA protein; Cassuto E; The concerted action of DNA gyrase and RecA protein of Escherichia coli on intact and gapped homologous or partially homologous plasmid DNA molecules leads to the formation of covalently closed DNA containing one strand of each parental molecule . Large regions of non-homology can be incorporated into the closed circular duplex . Both proteins are essential for the reaction to take place, and type I topoisomerase cannot substitute for DNA gyrase. Arch Biochem Biophys, 1984 Sep, 233(2), 611 - 6 Reactions of the sulfhydryl groups of alanyl-tRNA Synthetase; Chen ZQ et al.; The six sulfhydryl groups in each subunit of the alanyl-tRNA synthetase of Escherichia coli react with sulfhydryl reagents with at least four different rates . One reacts very rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and a second reacts somewhat less rapidly with this reagent . These two groups are required for transfer activity, which is lost in proportion to the extent of derivatization . Two other groups react more slowly, with a consequent loss of exchange activity . The remaining two sulfhydryl groups do not react with DTNB until the protein is denatured . The inactivations are reversed by dithiothreitol . Two sulfhydryl groups react with N-ethylmaleimide (NEM) and with a spin-label derivative of NEM . These reactions resemble the modification of two sulfhydryl groups with DTNB, in that they also inactivate the transfer reaction but not the ATP:PPi exchange . The two spin labels are incorporated at similar rates but are in very different environments, one highly exposed and one highly immobilized . These groups do not interact with Mn2+, which is bound to the enzyme in the absence of ATP. Virology, 1984 Sep, 137(2), 439 - 44 Effects of orientation and position on the activity of a herpes simplex virus immediate early gene far-upstream region; Preston CM et al.; It has previously been shown that a far-upstream region of the herpes simplex virus immediate early (IE) gene 3 increases the expression of a linked thymidine kinase (TK) gene and also contains sequences which respond to stimulation of transcription by a virion component . The effects of altering the orientation and distance of the far-upstream region with respect to the normal IE gene 3 promoter are described . Reversal of the orientation whilst retaining the normal position of the far-upstream region did not affect its activity, but placing it downstream from the TK gene abolished any detectable effect . When the far-upstream region was separated from the promoter by insertion of Escherichia coli DNA fragments (approximately 1000 base pairs), its activity was reduced but not prevented . A similar effect was observed for unstimulated expression of TK and stimulated expression in the presence of the virion component . The IE gene 3 far-upstream region therefore resembles enhancer sequences in some respects but also shows significant differences. Proc Natl Acad Sci U S A, 1984 Sep, 81(18), 5836 - 40 Cloning of terminal transferase cDNA by antibody screening; Landau NR et al.; A cDNA library was prepared from a terminal deoxynucleotidyltransferase-containing thymoma in the lambda phage vector lambda gt11 . By screening plaques with anti-terminal transferase antibody, positive clones were identified of which some had beta-galactosidase-cDNA fusion proteins identifiable after electrophoretic fractionation by immunoblotting with anti-terminal transferase antibody . The predominant class of cross-hybridizing clones was determined to represent cDNA for terminal transferase by showing that one representative clone hybridized to a 2200-nucleotide mRNA in close-matched enzyme-positive but not to enzyme-negative cells and that the cDNA selected a mRNA that translated to give a protein of the size and antigenic characteristics of terminal transferase . Only a small amount of genomic DNA hybridized to the longest available clone, indicating that the sequence is virtually unique in the mouse genome. Proc Natl Acad Sci U S A, 1984 Sep, 81(18), 5821 - 5 Yeast gene CDC8 encodes thymidylate kinase and is complemented by herpes thymidine kinase gene TK; Sclafani RA et al.; The herpes simplex virus type 1 thymidine kinase gene TK complements the defect in five temperature-sensitive mutants and in vitro constructed insertion and deletion mutants of the CDC8 gene of Saccharomyces cerevisiae . The herpes thymidine kinase enzyme acts as both a thymidine kinase and a thymidylate kinase (dTMP kinase) . The latter activity is responsible for the cdc8 complementation since all thermosensitive cdc8 mutants are deficient in dTMP kinase activity at all temperatures . However, an intragenic revertant, cdc8-320, which was selected by demanding mitotic growth at the restrictive temperature, exhibits thermolabile dTMP kinase activity . We conclude that CDC8 is the structural gene for dTMP kinase, which catalyzes an essential step in DNA precursor biosynthesis . Previously, it has been shown that the DNA replication defect of cdc8 mutants could not be bypassed by the addition of deoxyribonucleoside triphosphates to permeabilized cells . This apparent discrepancy can be explained by hypothesizing a multiprotein yeast DNA replication complex containing the CDC8 protein. Proc Natl Acad Sci U S A, 1984 Sep, 81(18), 5792 - 6 Processing of complex heteroduplexes in Escherichia coli and Cos-1 monkey cells; Abastado JP et al.; Upon transformation into Escherichia coli or Cos-1 monkey cells, heteroduplex DNA made of two sequences containing many nucleotide mismatches yields a wide array of different molecules, some with a patchwork structure . Thus, complex heteroduplexes can be processed to generate many genetic variants. J Pediatr Gastroenterol Nutr, 1984 Sep, 3(4), 593 - 601 Effects of loperamide on intestinal ion transport; Guandalini S et al.; The synthetic opiate loperamide (LPMD) is an antidiarrheal compound which affects both intestinal motility and the transport of water and electrolytes . We have investigated its effects on ion transport in the stripped ileal mucosa of rabbits, mounted in Ussing chambers in vitro . Addition of LPMD to serosal bathing medium resulted in a dose-dependent decrement of short-circuit current (Isc) . LPMD (50 microM) significantly increased net Cl absorption (JClnet) and JRnet (assumed to represent HCO3 secretion) . LPMD also inhibited the Isc increments evoked either by agents that increase 3',5'-cyclic adenosine monophosphate (cAMP) content (theophylline and prostaglandin E2) or by the Ca2+ ionophore A23187; the opiate, however, did not prevent the Isc change induced by 3',5'-cyclic guanosine monophosphate (cGMP)-related agents such as 8-Br-cGMP and Escherichia coli heat-stable enterotoxin . LPMD did not alter basal or secretagogue-stimulated tissue cAMP . In contrast, LPMD caused a small increase in cGMP content of stripped ileal mucosa, but not in that of isolated enterocytes . The role of Ca2+ in LPMD action is suggested by the significantly different effect of the drug on JClnet in the presence and in the absence of Ca2+ in the serosal solution. J Bacteriol, 1984 Sep, 159(3), 991 - 9 Gene sequence and predicted amino acid sequence of the motA protein, a membrane-associated protein required for flagellar rotation in Escherichia coli; Dean GE et al.; The motA and motB gene products of Escherichia coli are integral membrane proteins necessary for flagellar rotation . We determined the DNA sequence of the region containing the motA gene and its promoter . Within this sequence, there is an open reading frame of 885 nucleotides, which with high probability (98% confidence level) meets criteria for a coding sequence . The 295-residue amino acid translation product had a molecular weight of 31,974, in good agreement with the value determined experimentally by gel electrophoresis . The amino acid sequence, which was quite hydrophobic, was subjected to a theoretical analysis designed to predict membrane-spanning alpha-helical segments of integral membrane proteins; four such hydrophobic helices were predicted by this treatment . Additional amphipathic helices may also be present . A remarkable feature of the sequence is the existence of two segments of high uncompensated charge density, one positive and the other negative . Possible organization of the protein in the membrane is discussed . Asymmetry in the amino acid composition of translated DNA sequences was used to distinguish between two possible initiation codons . The use of this method as a criterion for authentication of coding regions is described briefly in an Appendix. J Bacteriol, 1984 Sep, 159(3), 979 - 85 Regulation of the phosphate regulon in Escherichia coli K-12: regulation of the negative regulatory gene phoU and identification of the gene product; Nakata A et al.; The phoU gene is one of the negative regulatory genes of the pho regulon of Escherichia coli . The DNA fragment carrying phoU has been cloned on pBR322 (Amemura et al., J . Bacteriol . 152:692-701, 1982) . Further subcloning, Tn1000 insertion inactivation, and complementation tests localized the phoU gene within a 1.1-kilobase region on the cloned DNA fragment . The gene product of phoU was identified by the maxicell method as a protein with an approximate molecular weight of 27,000 . A hybrid plasmid that contains a phoU'-lac'Z fused gene was constructed in vitro . This plasmid enabled us to study phoU gene expression by measuring the beta-galactosidase level in the cells . The plasmid was introduced into various regulatory mutants related to the pho regulon, and phoU gene expression in these strains was studied under limited and excess phosphate conditions . It was found that phoU is expressed at a higher level when the cells are cultured under the excess phosphate condition . The higher phoU expression was observed in a phoB mutant and a phoR-phoM double mutant . The implications of these findings for the regulation of pho genes are discussed. J Bacteriol, 1984 Sep, 159(3), 1068 - 71 Isolation and expression of the Bradyrhizobium japonicum adenylate cyclase gene (cya) in Escherichia coli; Guerinot ML et al.; A 5.0-kilobase-pair HindIII fragment of Bradyrhizobium japonicum DNA containing the cya gene which encodes adenylate cyclase was isolated as an insert in pBR322, using marker rescue of the maltose-negative phenotype of an Escherichia coli cya mutant for identification . The isolated B . japonicum DNA fragment was capable of reversing the pleiotropic phenotype of cya mutations when inserted in either orientation in the HindIII site of pBR322 . The complemented E . coli strains produced high levels of cyclic AMP . No sequence homology between the B . japonicum cya gene and that of E . coli was detected by hybridization analysis. J Bacteriol, 1984 Sep, 159(3), 1060 - 2 Mapping of trxB, a mutation responsible for reduced thioredoxin reductase activity; Haller BL et al.; The mutation (trxB) responsible for reduced thioredoxin reductase activity has been mapped on the Escherichia coli K-12 chromosome clockwise from aroA between 20 and 21 min . The gene order in this region of the E . coli chromosome was found to probably be serC-aroA-trxB . The location of gshA, the structural gene for gamma-glutamylcystein synthetase, relative to srl and recA also was determined. J Bacteriol, 1984 Sep, 159(3), 1027 - 33 Multiple copies of hemolysin genes and associated sequences in the chromosomes of uropathogenic Escherichia coli strains; Knapp S et al.; The O6 serogroup Escherichia coli strain 536 carries two hemolysin (hly) determinants integrated into the chromosome . The two hly determinants are not completely identical, either functionally or structurally, as demonstrated by spontaneous deletion mutants carrying only one of them and by cloning each of the two determinants separately into cosmid vectors . Each hly determinant is independently deleted at a frequency of 10(-4), leading to variants which exhibit similar levels of internal hemolysin but different amounts of secreted hemolysin . The two hly determinants were also identified in the O4 E . coli strain 519 . The three E . coli strains 251, 764, and 768, which belong to the serogroup O18, and the O4 strain 367 harbor a single chromosomal hly determinant, as demonstrated by hybridization with hly-gene-specific probes . However, a hybridization probe derived from a sequence adjacent to the hlyC-proximal end of the plasmid pHly 152-encoded hly determinant hybridizes with several additional chromosomal bands in hemolytic O18 and O6 E . coli strains and even in E . coli K-12 . The size of the probe causing the multiple hybridization suggests a 1,500- to 1,800-base pair sequence directly flanking hlyC . Spontaneous hemolysin-negative mutants were isolated from strains 764 and 768, which had lost the entire hly determinant but retained all copies of the hlyC-associated sequence.2+. Genetics, 1984 Sep, 108(1), 39 - 52 The effect of nonhomologous DNA sequences on interplasmidic recombination; Laban A et al.; The effect of nonhomologous DNA sequences at one or both sides of short genetic intervals on recombination within that interval was investigated, using an interplasmidic recombination system in Escherichia coli K-12 . The recombining plasmids were derivatives of pBR322 and pACYC184, which share a 1330-nucleotide sequence that includes the tet gene . The genetic interval was defined by the HindIII and BamHI or BamHI and the SalI restriction endonuclease sites of this gene . The substantial differences between recombination frequencies measured within intervals bracketed or bounded on one side by major nonhomologies suggests that, in this system, strand exchange is polar and is blocked by major nonhomologies . This conclusion is substantiated by results of three-factor crosses and by structural analysis of recombination products . Results of two-factor crosses in recA genetic background and structural analysis of recombination products suggest that strand exchange occurs in the absence of a functional recA gene . "Opening" a bracketed BamHI-SalI genetic interval of the tet gene, at the SalI site, by substituting a major insertion with a short deletion, results in an increase in recombination frequency, within this genetic interval, which is greater than expected on the basis of the ratio of the length of homology on the two sides of the SalI site . This observation suggests that a genetic element that may affect rate of recombination initiation, polarity of strand exchange, template specificity in mismatch repair or more than one of these events may be present on the outer side of the SalI site of the tet gene. Somat Cell Mol Genet, 1984 Sep, 10(5), 495 - 502 High-efficiency polyethylene glycol-mediated transformation of mammalian cells; Klebe RJ et al.; A new, high-efficiency method for transformation of mammalian cells with nucleic acids is described which yields 10(5)-10(6) plaques/micrograms poliovirus infectious RNA (iRNA) . The optimized procedure consists of two steps: (1) exposure of cells to iRNA in a high ionic-strength buffer followed by (2) a brief exposure to a 35% polyethylene glycol (PEG) solution . Optimized conditions for each variable in the procedure are described . Under optimized conditions for PEG-mediated transformation with RNA, large numbers of transformants are recovered with plasmid DNA as well . The procedure presented is similar to other high-efficiency PEG-mediated methods previously described for the genetic transformation of both nonprotoplasted Escherichia coli and yeast. Proc Natl Acad Sci U S A, 1984 Sep, 81(17), 5528 - 31 Demonstration of the production of frameshift and base-substitution mutations by quasipalindromic DNA sequences; de Boer JG et al.; The in vivo production of frameshift and base-substitution mutations predicted as a consequence of the metabolic processing of misaligned quasipalindromic DNA sequences has been confirmed . Spontaneous frameshift mutations of the T4 rII gene that had been genetically mapped to quasipalindromic DNA sequences were sequenced . Some of the mutant sequences are exactly those predicted by a mutational model based on misaligned quasipalindromes . Furthermore, these sequences are distinct from those predicted by the classical frameshift model based on misaligned repeated sequences . The rII frameshift mutant sequences reported here result from the deletion of a specific base or bases that would remain looped out should the quasipalindromes assume a hairpin secondary structure . One hairpin predicted not only the deletion of two bases (a frameshift) but the concomitant production of nearby but noncontiguous base substitutions . The substitution of as many as three bases as well as the frameshift were predicted to arise as a consequence of a single mutational event in the palindrome . Two independent examples of the predicted deletion frameshift were found among the small sample of sequenced spontaneous frameshifts examined and both were associated with the predicted transversion and transition base substitutions. Proc Natl Acad Sci U S A, 1984 Sep, 81(17), 5403 - 7 Role of mRNA translational efficiency in bovine growth hormone expression in Escherichia coli; Schoner BE et al.; The conditions necessary for high-level expression of methionyl bovine growth hormone (Met-bGH) in Escherichia coli were investigated . Plasmids were constructed that contain a thermoinducible runaway replicon and either the E . coli tryptophan or lipoprotein promoter and ribosome binding sites, which served as transcriptional and translational initiation sites for the expression of the bGH gene . The expression of Met-bGH was low with either system . However, expression levels of up to 30% of total cell protein were obtained after the introduction of additional codons 3' to the initiating AUG codon, thus altering the NH2-terminal amino acid sequence of bGH . To obtain high-level expression of Met-bGH a two-cistron system was constructed in which the codons that enhanced the expression of bGH were incorporated into the first cistron, and the coding region for Met-bGH was incorporated into the second cistron . This approach may be generally applicable to achieving high-level expression of a gene that contains NH2-terminal sequences that do not allow for its efficient expression . Analyses of the stabilities of the bGH derivatives and their transcripts in vivo suggested that the variations in the level of expression were due to variations in the efficiency of mRNA translation. Proc Natl Acad Sci U S A, 1984 Sep, 81(17), 5369 - 73 Expression of a biologically active fragment of human IgE epsilon chain in Escherichia coli; Liu FT et al.; cDNA corresponding to human IgE heavy (epsilon) chain mRNA was cloned from human IgE-secreting myeloma U266 cells . Partial nucleotide sequence analysis demonstrated that the cloned cDNA contained the coding region for about two-thirds of the CH2 and all of the CH3 and CH4 domains as well as the 3'-untranslated region . This epsilon cDNA was inserted into expression vector pUC7 and expression of an epsilon-chain fragment in Escherichia coli was demonstrated by protein blot analysis using 125I-labeled goat anti-human IgE as probe . The expression product was purified on a column of goat anti-human IgE-conjugated Sepharose 4B and the polypeptide was found to retain binding activity to human basophils. Cell, 1984 Sep, 38(2), 463 - 9 Mutants of the gamma delta resolvase: a genetic analysis of the recombination function; Newman BJ et al.; The resolvase protein encoded by the gamma delta transposon has two functions . It catalyzes a site-specific recombination, and it negatively regulates the expression of two transposon genes . Both functions involve the action of resolvase at the res site . To define regions of resolvase that are involved specifically in the recombination reaction, we have isolated and characterized mutants that are defective in cointegrate resolution but retain the ability to bind to res (as measured by regulatory activity) . Nine independent mutants were found to contain six different amino acid substitutions among just four distinct residues . The altered residues all lie within the 140 amino acid amino-terminal domain of resolvase and fall within two clusters of amino acids that are highly conserved in other related recombinases . The regulatory properties of the mutants suggest that one of these clusters may be involved in the interaction of the catalytic domain with the crossover site. Clin Chim Acta, 1984 Aug 31, 141(2-3), 169 - 77 Distribution of immunoreactive carbonic anhydrase III in various human tissues determined by a sensitive enzyme immunoassay method; Kato K et al.; A sensitive sandwich enzyme immunoassay method for measurement of carbonic anhydrase III (CA-III) was established by use of purified antibodies to CA-III . The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from E . coli . The assay was highly sensitive and pg levels of CA-III were measureable . Coefficients of variation in within-run and between-run precision studies for serum CA-III were less than 10% . Serum CA-III levels in healthy subjects of various ages ranged from 0.8 to 24 ng/ml . Concentrations of immunoreactive CA-III in the extracts of various human tissues were also determined . Tissues composed of striated muscle contained more than 10 micrograms/mg protein of CA-III, whereas other tissues, including heart muscle, contained less than 0.5 microgram/mg protein . These results were consistent with other data showing that serum CA-III levels were raised in patients with progressive muscular dystrophy but not in those with acute myocardial infarction. Biochem Biophys Res Commun, 1984 Aug 30, 123(1), 8 - 15 Synthesis of the disaccharide 6-O-beta-D-galactopyranosyl-2-acetamido-2-deoxy-D-galactose using immobilized beta-galactosidase; Hedbys L et al.; The disaccharide 6-O-beta-D-galactopyranosyl-2-acetamido-2-deoxy-D-galactose has been synthesized by transfer of the beta-D-galactopyranosyl residue from lactose to 2-acetamido-2-deoxy-D-galactose utilizing the transferase activity of beta-galactosidase from E . coli . To make the enzyme reusable, it was applied in an immobilized form covalently bound to Sepharose CL-4B . The yield of the disaccharide was about 20%, calculated on the amount of acetamido-deoxy-D-galactose added . The disaccharide could also be obtained by reversal of the hydrolytic activity of the enzyme, using D-galactose and 2-acetamido-2-deoxy-D-galactose as substrate . The yield in this reaction, however, was only 2-3% under the conditions applied. Biochem Biophys Res Commun, 1984 Aug 30, 123(1), 324 - 30 Comparison of repair of methylated pyrimidines in poly(dT) by extracts from rat liver and Escherichia coli; Dolan ME et al.; Partially purified preparations of O6-alkylguanine-DNA alkyltransferase from rat liver and E . coli were tested for their ability to repair O4-methylthymine in a methylated poly(dT) X poly(dA) substrate . The bacterial preparation readily carried out this reaction, but no loss of O4-methylthymine was obtained with the rat liver protein . These results indicate a significant difference in specificity between the mammalian and bacterial proteins which could have important consequences for carcinogenesis and mutagenesis by alkylating agents in mammalian cells. Biochem Biophys Res Commun, 1984 Aug 30, 123(1), 254 - 61 Monoclonal antibodies to mitochondrial F1-ATPase and oligomycin sensitivity conferring protein (OSCP) . Tools for recognition of well conserved and essential antigenic sites; Archinard P et al.; The preparation of anti-OSCP monoclonal antibodies is described for the first time . One of these antibodies prevents the activating effect of OSCP in reconstitution experiments . These antibodies and antibodies previously obtained against the alpha- and beta-subunits of pig heart mitochondrial F1-ATPase have been used to look for well conserved epitopes in various species . One anti-beta antibody can recognize all species tested while the anti-OSCP antibodies only recognize the pig or beef enzyme . The above anti-beta antibody inhibits ATP synthesis without modifying the rate of ATP hydrolysis . This antibody also prevents the ADP-induced hysteretic inhibition of F1-ATPase. Biochem Biophys Res Commun, 1984 Aug 30, 123(1), 1 - 7 Human apolipoprotein A-II: nucleotide sequence of a cloned cDNA, and localization of its structural gene on human chromosome 1; Moore MN et al.; Using the technique of oligonucleotide hybridization, we have isolated two dscDNA clones to human apolipoprotein A-II . One of the clones (pAII-1) has been completely sequenced . It has 433 nucleotides which includes a poly A tail of 10 adenylic acid residues, all the coding and 3'-non-translated regions of the mRNA and part of the 5'-non-translated region . The amino acid sequence derived from the cDNA clone includes 100 amino acids (including the 23 amino acid prepropeptide) which is very similar to the sequence reported by Brewer et al . (Proc . Natl . Acad . Sci . USA 69, 1304-1308) . {32P}-labeled pAII-1 was used as a probe in chromosome mapping studies to detect the human apoAII structural gene sequence in human-Chinese hamster cell hybrids . Southern blot analysis of 10 hybrids localized the gene to human chromosome 1. Biochemistry, 1984 Aug 28, 23(18), 4193 - 9 Amino acid sequence of the regulatory subunit of bovine type I adenosine cyclic 3',5'-phosphate dependent protein kinase; Titani K et al.; The complete amino acid sequence of the regulatory subunit of type I cAMP-dependent protein kinase from bovine skeletal muscle is presented . The S-carboxymethylated protein was cleaved with cyanogen bromide to provide a complete set of nonoverlapping fragments . These fragments were overlapped and aligned by using peptides generated by proteolytic cleavage . The protein contains 379 amino acid residues corresponding to a molecular weight of 42 804 . As in the type II regulatory subunit of cAMP-dependent protein kinase, a pattern of internal gene duplication is observed, which is consistent with two cAMP-binding domains . The two types of regulatory subunit from type I and type II kinase display similarities in domain substructure and in amino acid sequence, which provide a molecular basis for new insight into their regulatory roles . Detailed analyses of the homology of the regulatory subunits of type I and type II cAMP-dependent protein kinase and of similar relationships to cGMP-dependent protein kinase and Escherichia coli catabolite gene activator protein are presented in accompanying reports from this laboratory {Takio, K., Smith, S . B., Krebs, E . G., Walsh, K., & Titani, K . (1984) Biochemistry (second paper of three in this issue); Takio, K., Wade, R . D., Smith, S . B., Krebs, E . G., Walsh, K . A., & Titani, K . (1984) Biochemistry (third paper of three in this issue)}. Biochemistry, 1984 Aug 28, 23(18), 4200 - 6 Amino acid sequence of the regulatory subunit of bovine type II adenosine cyclic 3',5'-phosphate dependent protein kinase; Takio K et al.; Evidence is presented that establishes the amino acid sequence of the regulatory subunit of type II cAMP-dependent protein kinase from bovine cardiac muscle . Complementary sets of overlapping peptides were generated primarily by tryptic digestion and by chemical cleavage at methionyl residues . The analysis was augmented by chemical cleavage at a single tryptophanyl residue and at three of the four aspartyl-proline bonds . Several large fragments generated by limited proteolysis contributed to the proof of structure . The subunit is a single chain of 400 residues corresponding to a molecular weight of 45 004 . An amino-terminal segment of about 100 residues is believed to include the region responsible for oligomeric association . The remainder of the molecule consists of two tandem homologous domains, each of which is thought to bind a single molecule of cAMP . Comparison of the three domains with corresponding regions of the type I isozyme, of the Escherichia coli catabolite gene activator protein, and of cGMP-dependent protein kinase indicates extensive regions of homology and as much as 50% identity with the sequence of an internal segment of the type I isozyme. Biochemistry, 1984 Aug 28, 23(18), 4140 - 3 Interconversion of high and low adenosinetriphosphatase activity forms of Escherichia coli F1 by the detergent lauryldimethylamine oxide; Lotscher HR et al.; The amphipathic detergent lauryldimethylamine oxide (LDAO) stimulated ATP hydrolytic activity of Escherichia coli membranes and isolated ECF1 and ECF1-F0 ATPase complexes in a concentration-dependent manner . The enzyme was maximally activated 3-fold in membranes and 5-6-fold for isolated ECF1 or the ECF1-F0 complex . The maximal specific activity of activated ECF1 was 140-160 mumol of ATP hydrolyzed min-1 mg-1 . The activation by LDAO was reversible . LDAO specifically released subunit delta from ECF1, generating a four subunit enzyme (alpha, beta, gamma, and epsilon subunits) . The removal of subunit delta was not responsible for the stimulation of ATPase activity as evidenced by the full activation of the four subunit enzyme by LDAO . Treatment of ECF1 with 1-ethyl-3-{3-(dimethylamino)propyl}carbodiimide generated a beta-epsilon cross-link in high yield {Lotscher, H.R., DeJong, C., & Capaldi, R . A . (1984) Biochemistry (accompanying paper in this issue)} . The formation of this cross-link was greatly reduced in the presence of LDAO, indicating that the detergent perturbated the interaction between epsilon and beta subunits although epsilon was not removed from the ECF1 complex . The results suggest that the interconversion of ECF1 from a low to a high ATPase activity form by LDAO is in major part due to a release of the inhibitory action of subunit epsilon on subunit beta. Biochemistry, 1984 Aug 28, 23(18), 4134 - 40 Inhibition of the adenosinetriphosphatase activity of Escherichia coli F1 by the water-soluble carbodiimide 1-ethyl-3-{3-(dimethylamino)propyl}carbodiimide is due to modification of several carboxyls in the beta subunit; Lotscher HR et al.; Reaction of the ATPase of Escherichia coli (ECF1) with 1-ethyl-3-{3-(dimethylamino)propyl}carbodiimide (EDC) resulted in a time- and concentration-dependent inhibition of ATPase activity . The inactivation was greatly reduced by Mg2+ ions . Close to 13 mol of EDC per mol of ECF1 was incorporated into the enzyme at 95% inhibition of ATPase activity . Two-thirds of the label was found to be associated with subunit beta with a stoichiometry of about 3 mol of EDC per mol of beta . Cleavage of EDC-modified subunit beta with cyanogen bromide and fractionation of the peptides by high-pressure liquid chromatography revealed a short segment of 33 amino acids (CB8, residues 162-194) containing 3 mol of EDC per mol of peptide . In tryptic peptide maps, two EDC-labeled fragments could be identified (T18, residues 166-183, and T20, residues 186-202) . The analyses were complicated by significant internal cross-linking within the beta subunit induced by EDC . The results show that EDC modifies multiple sites in a short segment of subunit beta which includes the glutamic acids modified by dicyclohexylcarbodiimide in F1 from both E . coli and PS3 . In addition to covalent modification, EDC also promoted the formation of intersubunit cross-links . The predominant cross-linked product was identified as a beta-epsilon complex by antibody binding experiments. Biochemistry, 1984 Aug 28, 23(18), 4128 - 34 Modification of the F0 portion of the H+-translocating adenosinetriphosphatase complex of Escherichia coli by the water-soluble carbodiimide 1-ethyl-3-{3-(dimethylamino)propyl}carbodiimide and effect on the proton channeling function; Lotscher HR et al.; 1-Ethyl-3-{3-(dimethylamino)propyl}carbodiimide (EDC), a water-soluble carbodiimide, inhibited ECF1-F0 ATPase activity and proton translocation through F0 when reacted with Escherichia coli membrane vesicles . The site of modification was found to be in subunit c of the F0 portion of the enzyme but did not involve Asp-61, the site labeled by the hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) . EDC was not covalently incorporated into subunit c in contrast to DCCD . Instead, EDC promoted a cross-link between the C-terminal carboxyl group (Ala-79) and a near-neighbor phosphatidylethanolamine as evidenced by fragmentation of subunit c with cyanogen bromide followed by high-pressure liquid chromatography and thin-layer chromatography. Biochemistry, 1984 Aug 28, 23(18), 4207 - 18 Guanosine cyclic 3',5'-phosphate dependent protein kinase, a chimeric protein homologous with two separate protein families; Takio K et al.; The amino acid sequence of bovine lung cGMP-dependent protein kinase has been determined by degradation and alignment of two primary overlapping sets of peptides generated by cleavage at methionyl or arginyl residues . The protein contains 670 residues in a single N alpha-acetylated chain corresponding to a molecular weight of 76 331 . The function of the molecule is considered in six segments of sequence which may correspond to four folding domains . From the amino terminus, the first segment is related to the dimerizing property of the protein . The second and third segments appear to have evolved from an ancestral tandem internal gene duplication, generating twin cGMP-binding domains which are homologous to twin domains in the regulatory subunits of cAMP-dependent protein kinase and to the cAMP-binding domain of the catabolite gene activator of Escherichia coli . The fourth and fifth segments may comprise one domain which is homologous to the catalytic subunits of cAMP-dependent protein kinase, of calcium-dependent phosphorylase b kinase, and of certain oncogenic viral protein tyrosine kinases . The regulatory, amino-terminal half of cGMP-dependent protein kinase appears to be related to a family of smaller proteins that bind cAMP for diverse purposes, whereas the catalytic, carboxyl-terminal half is related to a family of protein kinases of varying specificity and varying sensitivity to regulators . These data suggest that ancestral gene splicing events may have been involved in the fusion of two families of proteins to generate the allosteric character of this chimeric enzyme. J Biol Chem, 1984 Aug 25, 259(16), 10247 - 51 Gap-filling DNA synthesis by HeLa DNA polymerase alpha in an in vitro base excision DNA repair scheme; Mosbaugh DW et al.; The ability of HeLa DNA polymerase alpha to utilize gapped PM2 DNAs for synthesis in a model base excision DNA repair scheme was examined . Partially depurinated PM2 DNA was incised on the 5' side of apurinic sites with HeLa apurinic/apyrimidinic endonuclease II, then the baseless sugar was removed and gaps of defined mean lengths were introduced at these sites by exonucleolytic digestion with HeLa DNase V . Gaps smaller than approximately 15 nucleotides did not serve as efficient primer-templates for DNA polymerase alpha . Gaps with mean lengths of 20-63 nucleotides did support limited DNA synthesis, but such synthesis terminated after the gap was reduced to roughly 15 nucleotides . These products were not substrates for Escherichia coli DNA ligase . In contrast, HeLa DNA polymerase beta utilize as primer-templates all of the gapped DNA substrates tested though it acted more efficiently with the smaller gaps . Moreover, the beta-polymerase was capable of filling these gaps to completion . In the case of the gaps that remained after partial closure by DNA polymerase alpha, DNA polymerase beta incorporated roughly 15 nucleotides and formed a product which was a substrate for DNA ligase . These results suggest that in vivo DNA repair pathways that involve a gap-filling DNA synthesis reaction might utilize DNA polymerase alpha only for larger gaps. J Biol Chem, 1984 Aug 25, 259(16), 9971 - 4 Proximity of 5.8 S RNA-binding proteins and A-site proteins in yeast ribosomes inferred from cross-linking; Lee JC et al.; In order to probe the spatial arrangement of proteins within the 5.8 S rRNA domain of the intact ribosomal subunit of Saccharomyces cerevisiae, 60 S ribosomal subunits were treated with 2-iminothiolane . Proteins were extracted from the cross-linked ribosomes, fractionated by Sephadex G-150 column chromatography, and analyzed by diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis . Constituent proteins of cross-linked pairs were identified by two-dimensional polyacrylamide gel electrophoresis . Nine cross-links involving five of the 11 5.8 S rRNA-binding proteins were analyzed . A model showing the network of cross-links is presented . Several of the 5.8 S rRNA-binding proteins are in sufficiently close proximity of the ribosomal A-site proteins to be cross-linked. J Biol Chem, 1984 Aug 25, 259(16), 10493 - 8 High resolution localization of the tRNA anticodon interaction site on the Escherichia coli 30 S ribosomal subunit; Gornicki P et al.; A body of previous work has shown that when Escherichia coli tRNAVal1 is placed in the P site of E . coli ribosomes and irradiated, the 5'-anticodon base of this tRNA, 5-carboxymethoxyuridine, is cross-linked to C-1400 of the 16 S rRNA . By tagging the carboxyl group of the cross-linked tRNA residue with a 2,4-dinitrophenyl (DNP) group attached via a 9 A spacer, it has been possible to directly visualize this cross-linking site by immunoelectron microscopy . The DNP group was attached by addition of ethylenediamine to the carboxyl group, followed by condensation of the newly formed free amino group with the N-hydroxysuccinimide ester of N-2,4-dinitrophenyl-gamma-aminobutyric acid . When reacted with anti-DNP antibody, this modification brings the surface of the antibody to within 9 A of the pyrimidine ring which was cross-linked . Neither codon-dependent binding nor cross-linking were materially affected by the tRNA modification . The tRNA-ribosome adduct formed a stable complex with anti-DNP antibody only when 50-30 S subunit association was prevented . Electron microscopic examination of the immune complexes showed that greater than 95% of those detected had the antibody localized deep in the cleft which separates the head and neck of the 30 S from the large protrusion . Since this is the site of cross-linking of the anticodon of tRNA, we conclude that this region on the 30 S subunit corresponds to the decoding site. J Biol Chem, 1984 Aug 25, 259(16), 10469 - 74 Amplification and purification of plasmid-encoded thioredoxin from Escherichia coli K12; Lunn CA et al.; The thioredoxin gene (trxA) from Escherichia coli K12 has been cloned on a 3-kilobase pair PvuII fragment in a derivative of pBR325 (pBHK8) . Thioredoxin protein production was amplified 150-200-fold in a strain containing pBHK8 (SK3981), with the greatest increase/cell observed after cultures reached stationary phase . A simple purification procedure, involving DEAE and AcA-54 column chromatography, yielded homogeneous protein with approximately 70% yield . The high amplification of thioredoxin in these cells (i.e . 10(6) copies/cell representing 40% of total cell protein) approaches the maximum yields seen in genetically constructed cloning vehicles (Bernard, H.U., and Helinski, D.R . (1980) in Genetic Engineering (Setlow, J . K., and Hollaender, A., eds) Vol . 2, pp . 133-167, Plenum Press, New York) . This tremendous overproduction of thioredoxin protein is attributed to the high plasmid copy number observed in SK3981 (1700/cell) . These results suggest a role for thioredoxin in plasmid DNA replication. J Biol Chem, 1984 Aug 25, 259(16), 10150 - 8 The phospholipid requirement for activity of the lactose carrier of Escherichia coli; Chen CC et al.; The transport activity of the lactose carrier of Escherichia coli has been reconstituted in proteoliposomes composed of different phospholipids . The maximal activity was observed with the natural E . coli lipid as well as mixtures containing phosphatidylethanolamine or phosphatidylserine . Phosphatidylcholine or mixtures of phosphatidylcholine with phosphatidylglycerol, phosphatidic acid, or cardiolipin showed low activity . The lactose carrier reconstituted with amino phospholipids of increasing degrees of methylation (dioleoylphosphatidylethanolamine, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine, and dioleoylphosphatidylcholine) revealed a progressive decrease in both counterflow and proton motive force-driven lactose uptake activities . Trinitrophenylation of phosphatidylethanolamine in the E . coli proteoliposomes resulted in a marked reduction in lactose carrier activity . Partial restitution of transport activity was obtained by detergent extraction of the carrier from these inactive proteoliposomes and reconstitution of the carrier into proteoliposomes containing normal E . coli lipid . These results suggest that the amino group of the amino phospholipids (e.g . phosphatidylethanolamine and phosphatidylserine) is required for the full function of the lactose carrier from E . coli. J Biol Chem, 1984 Aug 25, 259(16), 10012 - 9 Malyl-CoA formation in the NAD-, CoASH-, and alpha-ketoglutarate dehydrogenase-dependent oxidation of 2-keto-4-hydroxyglutarate . Possible coupled role of this reaction with 2-keto-4-hydroxyglutarate aldolase activity in a pyruvate-catalyzed cyclic oxidation of glyoxylate; Gupta SC et al.; The alpha-ketoglutarate dehydrogenase complex of either pig heart or Escherichia coli catalyzes a NAD- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate which is stereoselective toward the L-isomer of this hydroxyketo acid . L-Malyl-CoA is the product of the reaction; the evidence includes observing (a) a steady increase in absorbance at 230 nm during the oxidation of 2-keto-4-hydroxyglutarate, (b) a positive response of oxidation reaction mixtures to neutral hydroxylamine, (c) loss of the two foregoing results concomitant with release of thiol-reacting material and the formation of free malate when reaction mixtures are heated, (d) formation of a hydroxamate which has chromatographic mobilities identical to that of chemically synthesized malate hydroxamate, (e) enzymatic formation of a radioactive product from 14C-labeled 2-keto-4-hydroxyglutarate which co-migrates with chemically synthesized malyl-CoA, and (f) hydrolysis of the product by citrate synthase, an enzyme absolutely specific for citryl-CoA and L-malyl-CoA . A 1:1:1 stoichiometric relationship exists between the amount of 2-keto-4-hydroxyglutarate oxidized, NAD reduced, and malate (or malyl-CoA) formed . Results from studies in which either 14C-labeled 2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate is incubated with mixtures of purified enzymes or extracts of E . coli support the suggestion that the aldolase which preferentially catalyzes formation of L-2-keto-4-hydroxyglutarate from pyruvate plus glyoxylate in E . coli is coupled with the oxidative decarboxylation of this substrate, as reported here, and other enzymes in a multistep pyruvate-catalyzed cyclic oxidation of glyoxylate. J Biol Chem, 1984 Aug 25, 259(16), 10048 - 52 The activation of protein kinase C by biologically active lipid moieties of lipopolysaccharide; Wightman PD et al.; The monosaccharide lipid A precursor, N2,O3-diacylglucosamine 1-phosphate (Escherichia coli lipid X), has been shown previously to be a potent B-lymphocyte mitogen . We now report that lipid X interacts with macrophages, stimulating turnover of phosphatidylinositol, deacylation of phospholipids, and release of arachidonic acid . In addition, the monosaccharide lipid X, the incomplete lipid A disaccharides found in KDO-deficient mutants, and crude free lipid A by itself activate protein kinase C isolated from RAW 264.7 macrophages . This activation is augmented by diglyceride, a product of phosphatidylinositol turnover . Like the lipid X-induced mitogenesis of B-lymphocytes, lipid X activation of macrophages and the cell-free activation of protein kinase by lipid X require the presence of the O-linked hydroxymyristoyl residue at position 3 . We suggest, therefore, that some of the biological effects of lipid A may be mediated by its interaction with protein kinase C. J Mol Biol, 1984 Aug 25, 177(4), 627 - 44 Identification of transfer RNA suppressors in Escherichia coli . IV . Amber suppressor Su+6 a double mutant of a new species of leucine tRNA; Yoshimura M et al.; An Escherichia coli DNA fragment containing an Su+6 amber suppressor gene (supP) was cloned into a lambda gt lambda Ch vector by the shotgun method, selecting a Su+6 transducing phage lambda pSu+6 . Through prophage integration followed by induction occurring at the transducing region of the lambda pSu+6 in Su- E . coli, a counterpart transducing phage carrying the wild-type allele (Su degrees 6) was isolated (lambda pSu degrees 6) . The fingerprint of a tRNA encoded by lambda pSu degrees 6 was identical to that of an unidentified tRNAE previously reported (Ikemura & Ozeki, 1977) . The cloverleaf structure of this tRNA was determined by combining the results of tRNA analysis and DNA sequencing of the gene . Judging from the anticodon of 5'-CAA-3', Su degrees 6 tRNA was identified as a new type of leucine isoacceptor in E . coli . Unlike other suppressors analyzed, Su+6 tRNA differed by two nucleotides from Su degrees 6 tRNA; one at the anticodon (CAA to CUA) and the other at the junction of D- and anticodon-stem (A27 to G27) . DNA sequence analysis revealed that a single stretch of tRNA is flanked by the putative sequences of promoter and terminator . Thus a single copy of the Su degrees 6 tRNA gene constitutes a solitary tRNA transcription unit . Southern blotting showed only one copy of Su degrees 6 tRNA gene per haploid genome of E . coli . Since this single gene can mutate to the Su+6 suppressor, the Su degrees 6 leucine tRNA may be accounted as a dispensable species among the leucine isoacceptor tRNAs . Two possible open reading frames are found immediately following the Su degrees 6 tRNA gene. J Mol Biol, 1984 Aug 25, 177(4), 609 - 25 Identification of transfer RNA suppressors in Escherichia coli . III . Ochre suppressors of lysine tRNA; Yoshimura M et al.; Transducing phages of lambda carrying suppressors, lysT (Su+ beta), supG and and supL, were isolated in vivo . Upon infection with each of these phages, the production of tRNALys and tRNAVal1 was markedly enhanced . Fingerprint analysis of these tRNAs revealed that they consisted of normal tRNALys, mutant tRNALys and tRNAVal1 in equimolar ratios . The mutant tRNALys carried a single-base alteration at the anticodon, from 5'-UUU-3' to 5'-UUA-3', which makes it an ochre suppressor . DNA sequence analysis of the entire transducing fragment (730 base-pairs) of lambda pSu+ beta revealed that three tRNA genes are tightly clustered within a transcription unit in the following order; i.e . promoter-(48 base-pairs)-wild-type tRNALys-(132 base-pairs)-tRNAVal1-(2 base-pairs)-Su+ beta tRNALys- . In wild-type bacteria there are two identical tRNALys genes in one operon . Although we have shown that in Su+ beta it is the distal tRNALys that has been mutated to the ochre suppressor by a single base change at the anticodon (U36 to A36), we have not determined which of the two genes bears the supG or the supL mutation . The sequences following both tRNALys genes are highly homologous: both are about 100 base-pairs long and both terminate with an 18 base-pair sequence homologous to the last 18 bases of each tRNA . The sequences of tRNALys and tRNAVal1 are also very similar . Thus, including the 3'-portions of these tRNA genes, the 18 base-pair sequence is more or less periodically repeated five times in the DNA sequence. J Mol Biol, 1984 Aug 25, 177(4), 645 - 61 Role of the IS50 R proteins in the promotion and control of Tn5 transposition; Johnson RC et al.; IS50R, the inverted repeat sequence of Tn5 which is responsible for supplying functions that promote and control Tn5 transposition, encodes two polypeptides that differ at their N terminus . Frameshift, in-frame deletion, nonsense, and missense mutations within the N terminus of protein 1 (which is not present in protein 2) were isolated and characterized . The properties of these mutations demonstrate that protein 1 is absolutely required for Tn5 transposition . None of these mutations affected the inhibitory activity of IS50, confirming that protein 2 is sufficient to mediate inhibition of Tn5 transposition . The effects on transposition of increasing the amount of protein 2 (the inhibitor) relative to protein 1 (the transposase) were also analyzed . Relatively large amounts of protein 2 were required to see a significant decrease in the transposition frequency of an element . In addition, varying the co-ordinate synthesis of the IS50 R proteins over a 30-fold range had little effect on the transposition frequency . These studies suggest that neither the wild-type synthesis rate of protein 2 relative to protein 1 nor the amount of synthesis of both IS50 R proteins is the only factor responsible for controlling the transposition frequency of a wild-type Tn5 element in Escherichia coli. J Biol Chem, 1984 Aug 25, 259(16), 10606 - 13 Sequences of the malE gene and of its product, the maltose-binding protein of Escherichia coli K12; Duplay P et al.; The sequences of the malE gene and of its mature product, the maltose-binding protein, have been determined and are in good agreement . The malE gene encodes the pre-protein (396 amino acid residues) which yields, upon cleavage of the NH2-terminal extension (26 amino acid residues), the mature maltose-binding protein (370 amino acid residues) . The malE mRNA could form stable stem and loop structures, some of which may account for translational pauses observed by Randall et al . (Randall, L., Josefsson, L . G . & Hardy, S . J . S . (1980) Eur . J . Biochem . 107, 375-379) . The sequence change due to an in-frame nonpolar deletion of 765 nucleotides in malE is also presented as well as homologies between the maltose-binding protein and other sugar-binding proteins. J Biol Chem, 1984 Aug 25, 259(16), 10386 - 92 Escherichia coli DNA polymerase I . Construction of a polA plasmid for amplification and an improved purification scheme; Minkley EG Jr et al.; Previous attempts to clone the Escherichia coli polA+ gene onto a high copy number plasmid were unsuccessful . The apparent lethality of unregulated overproduction of DNA polymerase I can be eliminated by cutting at a BglII site 100 nucleotides upstream from the ATG start codon of the polA gene . This permitted the construction of plasmid pMP5 which contains both the coding sequence for DNA polymerase I and the lambda pL promoter for conditional control of polA gene expression . BglII cutting only damages but does not eliminate the polA promoter activity; the BglII site thus lies within the polA promoter region . Leakiness of the damaged polA promoter results in overproduction of DNA polymerase I even under conditions where pL is fully repressed . This overproduction is inhibitory of cell growth, as reflected in both growth rate and in the frequency of appearance of mutant plasmids which are nonproducers of DNA polymerase I . Transformation of plasmid pMP5 into E . coli N4830 yields strain ATL100 which under inducing conditions provides 138-fold amplification of DNA polymerase I . Optimization of growth and expression conditions are presented together with an optimized rapid polymerase purification scheme . In addition to providing a convenient source for preparation of DNA polymerase I, this work serves as the basis for a future detailed molecular genetic analysis of the polA gene product. J Biol Chem, 1984 Aug 25, 259(16), 10076 - 9 Replacement of serine 373 by phenylalanine in the alpha subunit of Escherichia coli F1-ATPase results in loss of steady-state catalysis by the enzyme; Noumi T et al.; The mutant allele (uncA401) of the gene for the alpha subunit of Escherichia coli F1-ATPase was cloned from the total DNA of the mutant AN120 on a hybrid plasmid pAN120 . Determination of the DNA sequence of the alpha subunit gene from pAN120 revealed a single base change of cytosine at nucleotide residue 1118 to thymine and indicated that serine 373 was replaced by phenylalanine . It has been reported that the mutant F1 is defective in a step of steady-state catalysis, whereas its single turnover process is normal (Kanazawa, H., Noumi, T., Matsuoka, I., Hirata T., and Futai, M . (1984) Arch . Biochem . Biophys . 228, 258-269) . Thus, we concluded that serine 373 in the alpha subunit is essential for steady-state catalysis by F1-ATPase. J Biol Chem, 1984 Aug 25, 259(16), 10071 - 5 A phenylalanine for serine substitution in the beta subunit of Escherichia coli F1-ATPase affects dependence of its activity on divalent cations; Noumi T et al.; A mutant (KF11) of Escherichia coli H+-translocating ATPase (F1-F0) has a single point mutation in the beta subunit of F1 that has lost 90% of its Mg2+-dependent ATPase activity (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., and Futai, M . (1980) J . Biochem . (Tokyo) 88, 695-703) . The mutation was mapped at about the 500th nucleotide residue from the 5' end of the beta subunit gene by a genetic recombination test on the physical map of the cistron coding for the beta subunit . The mutant allele of KF11 (uncD11) was cloned on a hybrid plasmid (pKF11) via DNA isolated from a lambda uncD11 transducing phage . Restriction fragments of pKF11 containing the estimated mutation site were subjected to polyacrylamide gel electrophoresis under conditions where strands were separated into single strands . The two strands of a DNA segment, which was shown to carry an altered base, showed anomalous migration compared with those from the wild-type fragment . The results confirmed the result of mapping of the altered site by genetic tests . On the basis of these results, the nucleotide sequence of the mutated gene was determined, and a single base change of the 524th cytosine to thymine resulting in a phenylalanine for serine substitution at residue 174 of the beta subunit was found . This result, together with results on the altered properties of F1 from KF11 reported previously, indicates that residue 174 is essential for the Mg2+-dependent ATPase activity of F1 but not for the Ca2+-dependent ATPase activity. Nucleic Acids Res, 1984 Aug 24, 12(16), 6359 - 67 Enzymatic excision from gamma-irradiated polydeoxyribonucleotides of adenine residues whose imidazole rings have been ruptured; Breimer LH; The main forms of base damage in polydeoxyadenylic acid gamma-irradiated under hypoxic conditions are due to saturation and fragmentation of the adenine imidazole ring . An irradiated polymer was annealed with an equimolar amount of poly (dT) to generate a double-stranded polydeoxyribonucleotide containing scattered damaged base residues . On incubation of the latter with partially purified cell extracts of E.coli, imidazole ring-opened adenine, i.e . 4,6-diamino-5-formamidopyrimidine, was released in free form by a DNA glycosylase activity . The enzyme has been purified 4,500-fold, has Mr = 29,000, and appears to be identical with the previously described DNA repair enzyme formamidopyrimidine-DNA glycosylase. Nucleic Acids Res, 1984 Aug 24, 12(16), 6547 - 58 Junctions of the large single copy region and the inverted repeats in Spinacia oleracea and Nicotiana debneyi chloroplast DNA: sequence of the genes for tRNAHis and the ribosomal proteins S19 and L2; Zurawski G et al.; This work describes the organization, at the nucleotide sequence level, of genes flanking the junctions of the large single copy regions and the inverted repeats of Spinacia oleracea (spinach) and Nicotiana debneyi chloroplast DNAs . In both genomes, trnH1, the gene for tRNA-His(GUG) is lo