Nucleic Acids Res, 1980 Dec 20, 8(24), 6175 - 88 A cDNA clone from Zea mays endosperm sucrose synthetase mRNA; Geiser M et al.; A cDNA clone for maize endosperm sucrose synthetase of 62o nucleotide pairs length was obtained by cloning double stranded DNA obtained from the total maize endosperm poly(A) RNA in pBR322, and identifying the appropriate clone by hybrid-promoter translation . In Southern blotting to genomic BamHI-digested DNA, a single band only of approximately 20 Kb lights up, indicating that the sucrose synthetase gene is unique, or that closely linked copies are located on this DNA fragment. Nucleic Acids Res, 1980 Dec 20, 8(24), 6143 - 62 On the influence of thymidine analogues on the activity of phage fd promoters in vitro; Hofer B et al.; RF I DNA of phage fd containing 5-bromo-deoxyuridine (br5Ud) or deoxyuridine (Ud) instead of deoxythymidine (Td) inthe codogenic strand was synthesized in vitro . The modified genomes could be cleaved by restriction endonuclease Hpa II . Although the recognition site of Hpa II is CCGG, the cleavage rate was significantly reduced with Ud-containing DNA . Both base substitutions altered the mobilities of several DNA fragments under the conditions of polyacrylamide gel electrophoresis . The fragments containing binding sites for RNA polymerase were assayed for the rates of stable complex formation . The substitution of Td for both, Ud and br5Ud, strongly influenced this parameter . Thus the methyl group of Td has to be regarded as one of the sites in DNA which determine the rate of stable RNA polymerase binding and thereby possibly mediate promoter activity in vitro (24,25,26) . In most cases the rate of complex formation was decreased by Ud, but increased by br5Ud. Experientia, 1980 Dec 15, 36(12), 1416 - 7 Estimation of nonspecific lectin-mediated staining of glutaraldehyde-fixed cells; Gilboa-Garber N et al.; Lectin-mediated stainings are widely used for the visualization of carbohydrate-carrying cellular components using the electron microscope . The use of glutaraldehyde-fixed cells for these stainings introduces the possibility of low nonspecific lectin-trapping by the glutaraldehyde which coats the cells . This trapping was estimated by means of peroxidase-binding to human leukocytes . Tetrahymena pyriformis and Escherichia coli cells and was shown to be prevented by rinsing the glutaraldehyde-fixed cells in an amino solution before exposure to the lectin. Biochim Biophys Acta, 1980 Dec 15, 633(3), 457 - 64 Effects of macrocyclic polyamines and polymethylenediamines on reactions influenced by polyamines; Igarashi K et al.; The effects of macrocyclic polyamines and polymethylenediamines on various reactions influenced by polyamines have been studied . Among the amines tested, 2,3,4,3- and 3,3,3,4-cyclic polyamines, NH2(CH2)6NH2 and NH2(CH2)8NH2 had some ability to stimulate polyphenylalanine synthesis, globin synthesis and rat liver isoleucyl-tRNA formation . The degree of stimulation was at most 40% of that obtained by polyamines . In the degradation of poly(C) by bovine pancreatic RNAase A, all tested amines stimulated the degradation . In the NADPH-dependent lipid peroxidation of rat liver microsomes, the degree of inhibition by 2,3,2,3- or 2,3,3,3-cyclic polyamine was greater than that by spermine . The hydrolysis of ATP by an oligomycin-sensitive ATPase was inhibited by 2,3,4,3- and 3,3,3,4-cyclic polyamines, NH2(CH2)10HN2 and spermine at somewhat comparable levels . None of the macrocyclic polyamines or polymethylenediamines stimulated the growth of a polyamine-requiring mutant of Escherichia coli . Possible explanations for the differences in the effects of amines on the various reactions are discussed. Vet Rec, 1980 Dec 13, 107(24), 549 - 51 Comparison between milk deprivation and oral rehydration with a glucose-glycine-electrolyte formulation in diarrhoeic and transported calves; Bywater RJ; Treatment of diarrhoeic calves by oral administration of a glucose-glycine-electrolyte solution (GGES) was compared with milk deprivation (water given instead) and with no treatment (milk-fed controls) . The diarrhoea followed challenge with enteropathogenic Escherichia coli . The GGES group showed a significantly lower mortality (5 per cent) in comparison with the controls (37 per cent) . The milk-deprived group had a slightly lower mortality (30 per cent) in comparison with the controls . The milk deprived group showed a significantly prolonged duration of diarrhoea in survivors compared with the control group, while the GGES group did not differ significantly from the controls . Calves arriving on farms were allocated randomly to either GGES or to an alternative control treatment (usually partial milk deprivation) . The incidence of diarrhoea during the subsequent two weeks was significantly less in the GGES group . Clotting of milk by rennet was impaired by addition with either water or an alkaline electrolyte solution but was enhanced by dilution with GGES. Nucleic Acids Res, 1980 Dec 11, 8(23), 5895 - 912 DNA strand specificity in promoter recognition by RNA polymerase; Park CS et al.; DNA strand and enzyme subunit specificities involved in the interaction between E . coli RNA polymerase and T7 DNA were studied by photo-crosslinking techniques . In non-specific enzyme-DNA complexes, subunits, sigma, beta, and beta' were crosslinked to both strands of the DNA . Under conditions leading to specific enzyme-promoter complexes, however, only sigma and beta subunits were crosslinked . The sigma subunit was crosslinked preferentially to the non-sense strand at promoter sites . No such strand specificity was observed for the beta subunit . These results provide insight into the molecular mechanism of promoter recognition and indicate that the interaction between RNA polymerase and DNA template is different at promoters and at non-specific sites. Nucleic Acids Res, 1980 Dec 11, 8(23), 5859 - 73 Two classes of Mu lig mutants: the thermosensitives for integration and replication and the hyperproducers for ligase; Paolozzi L et al.; We have previously shown that Mu restores an active DNA replication at non-permissive temperature in E . coli K12 ligts7 strains . In this paper we describe two new types of phage mutants for the Mu lig function . The Mu ligts mutants are conditional lethals defective for both integration and replication of DNA, unable to 'complement' the bacterial lig mutation; the map between B and lys . The mutants of the other type, on the other hand, are able to restore to a maximum level the activity impaired by the ligts7 mutation in the host . We suggest the hypothesis that the product of Mu lig gene could be part of a complex as a topoisomerase. Nucleic Acids Res, 1980 Dec 11, 8(23), 5649 - 67 Alcohol dehydrogenase in Drosophila: isolation and characterization of messenger RNA and cDNA clone; Benyajati C et al.; The mRNA for alcohol dehydrogenase (ADH) in D . melanogaster has been identified by translation in a cell-free system . The in vitro synthesized polypeptide, specifically precipitated by anti-ADH antibody, has identical subunit molecular weight (25,000 daltons) and tryptic peptide profile to the in vivo synthesized ADH . The poly A containing ADH-mRNA has been purified by specific precipitation of ADH-polysomes using anti-ADH antibody and S . aureus . Transformation of E . coli with the dA-tailed ADH-mRNA-complementary DNA hybrid annealed to the dT-tailed pBR322 yielded one plasmid which has been identified as the ADH-cDNA clone . The identification involved hybridization selection of ADH-mRNA and in vitro translation, in situ hybridization to the Adh locus on salivary gland polytene chromosomes and DNA sequencing . This ADH-cDNA plasmid contains 349 bases of the C-terminal protein coding and 180 bases of the 3' untranslated region. Nucleic Acids Res, 1980 Dec 11, 8(23), 5825 - 33 Deletions and DNA rearrangements within the transposable DNA element IS2 . A model for the creation of palindromic DNA by DNA repair synthesis; Besemer J et al.; Three derivatives of mutant ga10P-308::IS2-I of Escherichia coli were characterized by DNA sequence analysis . Deletions and DNA sequence rearrangements were observed which apparently were initiated at short A-T rich inverted repeats within IS2 . Two of the mutants carried newly synthesized DNA sequences which were inverted copies of already existing IS2 sequences . Thus long stretches with twofold symmetry were formed . It is discussed whether these inverted repeats were formed by DNA repair synthesis which was initiated at the A-T rich palindromes of IS2. Nucleic Acids Res, 1980 Dec 11, 8(23), 5767 - 77 The translocation inhibitor tuberactinomycin binds to nucleic acids and blocks the in vitro assembly of 50S subunits; Yamada T et al.; Binding studies were performed with a {14C}-labelled derivative of viomycin, tuberactinomycin 0 (TUM O) . TUM O bound to 30S and 50S subunits . The binding component was the RNA, since ribosomal proteins did not bind the drug . Other RNAs such as tRNA, phage RNA (MS2), and homopolynucleotides also bound the drug . Striking differences in the binding capacity of the various homopolynucleotides were found . Poly(U) bound strongly, poly(G) and poly(C) bound intermediately, whereas poly(A) showed a very low binding . DNA also bound TUM O, although with native DNA the binding was only weak . Finally the effects of viomycin on the assembly in vitro of the 50S subunit from E . coli were tested . A very strong inhibition was found: when the reconstitution was performed at 0.5 x 10(-6) M viomycin the particles formed sedimented at about 50S, but showed a residual activity of less than 10% . The inhibitory power of viomycin with respect to the in vitro assembly is more pronounced than that found in in vitro systems for protein synthesis. Nucleic Acids Res, 1980 Dec 11, 8(23), 5669 - 83 Synthesis, restriction analysis, and molecular cloning of near full length DNA complementary to bovine parathyroid hormone mRNA; Gordon DF et al.; DNA complementary (cDNA) to a partially purified preparation of bovine parathyroid hormone mRNA was synthesized using avian myeloblastosis viral reverse transcriptase . The PTH cDNA contained about 750 bases and was greater than 95% sensitive to digestion by S1 nuclease . Analysis of the mRNA preparation by excess RNA hybridization to the PTH cDNA revealed one rapidly hybridizing component consisting of 50% of the PTH cDNA . Sequential incubation of the PTH mRNA with reverse transcriptase and E . coli DNA polymerase I produced near full length double-stranded PTH cDNA . Of the 22 restriction endonucleases tested, double-stranded PTH cDNA could be cleaved with Alu I, Mbo II, Sau 3A, Sst I, and Taq I . The restriction fragments corresponding to the 5' terminus of the sense strand were identified for the last three enzymes by comparing the size of fragments obtained from PTH cDNA before and after cleavage of the hairpin loop connecting the two strands by S1 nuclease . The restriction map of the cDNA was used to detect clones of bacteria containing recombinant plasmids with near full length PTH cDNA inserts. Nucleic Acids Res, 1980 Dec 11, 8(23), 5541 - 9 A subcloning strategy for DNA sequence analysis; Frischauf AM et al.; We describe here a new strategy of fragment preparation for sequencing procedures using endlabelled DNA fragments as substrates (2,3) which is directly applicable to DNA fragments cloned into the Pst I site of pBR322, or in modified form, to inserts into the BamH I or Sal I site of the same plasmid . Ordered sets of subclones of predetermined overlap are are generated . These can be sequenced directly without further strand- or fragment separation steps. Nucleic Acids Res, 1980 Dec 11, 8(23), 5813 - 24 Dinucleotide codon-anticodon interaction as a minimum requirement for ribosomal aa-tRNA binding: stabilisation by viomycin of aa-tRNA in the A site; Luhrmann R; The requirements for the decoding process at the ribosomal A site have been investigated in the presence of viomycin . For these studies natural mRNA was replaced either by the synthetic oligonucleotide A-U-G(-U)n, with 0 less than or equal to n less than or equal to 4, or by a physical mixture of the oligonucleotides A-U-G and various oligo(U) sequences . Thus the effect of the "removal" of selected covalent bonds from the sequence A-U-G(U)n could be studied . When the ribosomal P site contains tRNAMetf, then normally the full hexanucleotide "messenger" A-U-G-U-U-U is needed for the EF-Tu-mediated binding of Phe-tRNA into the A site . However in presence of viomycin the pentanucleotide A-U-G-U-U suffices for this . It is also possible in the presence of viomycin to replace A-U-G-U and U-U . In all the above systems the binding of Phe-tRNA required the presence of EF-Tu and GTP . The results suggest that viomycin reinforces interactions between aa-tRNA and the A site after the codon-anticodon recognition step. Nucleic Acids Res, 1980 Dec 11, 8(23), 5567 - 77 Characterization of the Escherichia coli 23S Ribosomal RNA region associated with ribosomal protein L1 . Evidence for homologies with the 5'-end region of the L11 operon; Branlant C et al.; The results previously obtained upon studying the L1-23S RNA complex by the fingerprint technique have been reexamined in the light of new data on 23S RNA primary structure . The 23S RNA region that remains associated with the L1 ribosomal protein after RNase digestion of the synthetic complex lies between nucleotides 2067 and 2235 from the 5'-end of the molecule . This region contains a m7G near to the 5'-end and possesses a high degree of mutability in E . coli . Three different sequences were observed in E . coli MRE 600 . All three sequences differ in two positions relative to the corresponding sequence in rrnB cistron from E . coli K12 . Striking homology is observed between the 23S RNA region associated with protein L1 and the 5'-part of L11 operon . This observation supports the model of feedback regulation of r-proteins synthesis proposed by Yates et al . (PNAS, 77, 1837) and strongly suggests that the region of 23S RNA located between positions 2155 and 2202 is essential for the binding of protein L1. J Biol Chem, 1980 Dec 10, 255(23), 11435 - 41 Biosynthetic control of the natural abundance of carbon 13 at specific positions within fatty acids in Escherichia coli . Evidence regarding the coupling of fatty acid and phospholipid synthesis; Monson KD et al.; Stable carbon isotope ratios (13C/12C) at natural abundance levels have been determined for individual carbon atoms in each of the major phospholipid fatty acids of Escherichia coli grown on glucose as the sole carbon source . Two models were constructed for the isotope effects and carbon flow pathways which must be responsible for the observed isotopic fractionations . Both models incorporate a branch in the carbon flow at which fatty acyl-acyl carrier protein (acyl-ACP) is utilized either for complex lipid synthesis or for elongation by fatty acid synthetase . Depletion of carbon 13 in the carboxyl groups of myristic and palmitoleic acids (relative to carbonyl groups in precursor acyl-ACP's) was observed to occur at this branching site . Only one of the models was consistent both with this observation and with the observation (Silbert, D . F., Ruch, F., and Vagelos, P . R . (1968) J . Bacteriol . 95, 1658-1665) that exogenous fatty acids are incorporated into phospholipids but are not elongated . The successful model has free fatty acid as the intermediate product coupling fatty acid biosynthesis to phospholipid synthesis . Essential to this pathway are those reactions catalyzed by thioesterases I and II as well as acyl-ACP synthetase, enzymes whose roles have previously been unknown in vivo. J Biol Chem, 1980 Dec 10, 255(23), 11088 - 90 Single turnover kinetic studies of guanosine triphosphate hydrolysis and peptide formation in the elongation factor Tu-dependent binding of aminoacyl-tRNA to Escherichia coli ribosomes; Thompson RC et al.; The rates of GTP hydrolysis and peptide formation during the reaction of Phe-tRNA . elongation factor Tu . GTP complex with acetyl-Phe-tRNA polyuridylate-programmed ribosomes have been measured . The GTPase reaction is second-order up to reactant concentrations of 0.2 microM and has a rate constant of 5 X 10(6) M-1 s-1 at 5 degrees C and 5 mM Mg2+, pH 7.2 . The formation of peptide shows a lag phase and has a rate constant of 0.4 S-1 under these conditions . The results of a series of experiments between 5 degrees C and 25 degrees C show that GTP hydrolysis and peptide formation have Arrhenius activation energies of 13.1 and 15.3 kcal mol-1, respectively . The results indicate that these reactions proceed in vitro at rates comparable to those observed for protein biosynthesis in vivo, and that peptide bond formation occurs after GTP hydrolysis. J Biol Chem, 1980 Dec 10, 255(23), 11075 - 7 Three origins of replication are active in vivo in the R plasmid RSF1040; Crosa JH; Replicating DNA molecules of RSF1040, a deletion derivative of the conjugative R plasmid R6K, are cleaved at a single site by the Eco RI restriction endonuclease . Microscopic analysis of Eco RI-cleaved RSF1040 replicative intermediates synthesized in vivo indicates that initiation of replication occurs at three unique sites, ori alpha, ori beta, and ori gamma . The relative frequencies of initiations at these three origins are different from those found in vitro. Biochemistry, 1980 Dec 9, 19(25), 5874 - 83 Reversible phosphorylation of a nucleosome binding protein that stimulates transcription of nucleosome deoxyribonucleic acid; Saffer JD et al.; A nonhistone chromosomal protein (D-55) of Mr 55000 has been isolated in homogeneous form from calf thymus by using the standard salt-extraction procedures for the isolation of nonhistone chromosomal proteins followed by hydroxyapatite chromatography . D-55 is further characterized as coming from the group of nonhistone chromosomal proteins easily phosphorylated by an endogenous nuclear protein kinase . The kinase incorporates 1 mol of phosphate per mol of protein from {gamma-32P}ATP . The unphosphorylated form of D-55 binds to DNA, histones, and nucleosomes . Phosphorylation of D-55 does not significantly alter the binding of D-55 to DNA but greatly enhances its binding to histones and nucleosomes . Binding of D-55 to reconstituted nucleosomes enhances transcription of the nucleosome DNA by E . coli RNA polymerase by approximately 100-fold, to a level approximately 4 times that observed with naked calf thymus DNA as template . Phosphorylation of D-55 abolishes this enhancement . Binding of D-55 produces no apparent alteration in nucleosome structure as assayed by nuclease digestion patterns . In contrast, phospho-D55 alters nucleosome structure significantly. Biochemistry, 1980 Dec 9, 19(25), 5857 - 64 Mechanism of translocation: effect of cognate transfer ribonucleic acids on the binding of AUGUn to 70S ribosomes; Holschuh K et al.; We try to mimic the unidirectional sliding-type movement of the PP-tRNA . mRNA complex with respect to the ribosome by looking at the effect of different combinations of cognate tRNAs on the stability of the 70S-AUGUn complex . The association constant for the binary complex 70S-AUGU3 was determined as 6.8 x 10(5) M-1 . Addition of tRNAfMet resulted in a 67-fold increase in the association constant, which with both cognate tRNAs is revised to Kassoc = 2.2 x 10(8) M-1 . Increasing the chain length of the oligonucleotide from AUGU3 to AUGU13 did not further raise the association constant . The data indicate that the stability of the 70S ribosome . mRNA interaction is governed by the presence of the cognate tRNAs and is topographically restricted to the decoding domains . Since a peptidyl group in the tRNA increases the affinity of AUGU3 for the ribosome by up to 15-fold, we conclude that the affinity of the peptidyl transfer center for the peptidyl moiety pulls the PP-tRNA . mRNA complex from the A (aminoacyl-tRNA) site to the P (peptidyl-tRNA) site . EF-G . GTP or EF-G . GMPPCP 5'-(beta, gamma-methylene)triphosphate} displace tRNAfMet from the quaternary complex 70S . AUGUn . tRNAfMet . tRNAPhe (n = 3 and 6) at Mg2+ less than 25 mM . From the amount of EF-G . GTP bound to a 70S ribosome, it follows that the elongation factor replaces the deacylated tRNA in a stoichiometric way . These data indicate that the EF-G . GTP-dependent release of the deacylated tRNA from the P site, followed by removal of EF-G . GDP from the 50S subunit, is sufficient to trigger the translocation of the mRNA . PP-tRNA complex. Biochemistry, 1980 Dec 9, 19(25), 5768 - 73 Binding of regulatory nucleotides to aspartate transcarbamylase: nuclear magnetic resonance studies of selectively enriched carbon-13 regulatory subunit; Moore AC et al.; Specifically enriched {gamma-13C}phenylalanine, -tyrosine, and -histidine have been biosynthetically incorporated into aspartate transcarbamylase from Escherichia coli . These nonperturbing NMR probes have been used to characterize the interaction of the regulatory sites on the enzyme with nucleotide effectors . The C gamma carbons of the three tyrosines and four histidines per regulatory chain give narrow, well-resolved resonances, and the signals from the five phenylalanines per chain are partially resolved in the presence of bound inhibitor . Spectral changes in regulatory subunit were monitored as a function of concentration of the inhibitor, CTP, and the activator, ATP . Three histidine residues responded to ATP and CTP in an identical manner while two phenylalanine residues were sensitive to CTP but not ATP binding . The tyrosine resonances were not perturbed by effectors . The chemical shift response of the single observable histidine resonance to bound nucleotides in the reconstituted enzyme was identical with that observed for isolated regulatory subunit . This histidine spectrum was undisturbed by the T to R conformational transition of the enzyme . The results suggest that the regulatory subunit experiences minimal rearrangement of tertiary structure on binding effectors and that at least one phenylalanine and one histidine residue are present in the region of the CTP binding site. Biochemistry, 1980 Dec 9, 19(25), 5864 - 9 Productive and abortive initiation of transcription in vitro at the lac UV5 promoter; Gralla JD et al.; The rates of productive and abortive initiation of transcription in vitro at the lac UV5 promoter have been determined and compared to values determined for phage lambda and T7 promoters . The rate constants for productive initiation of lac transcript are consistently lower over a range of low to moderate concentration of initiating nucleoside triphosphate (ATP) . Abortive initiation of lac dinucleoside tetraphosphate is also slower at low to moderate concentrations of ATP . These data demonstrate the existence of significant differences in initiation rate among promoters . We suggest that these differences may be a consequence of the initial mRNA sequences and extents of RNA polymerase cycling during initiation of promoter-specific transcription. Biochemistry, 1980 Dec 9, 19(25), 5939 - 46 On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta; Fry M et al.; Accuracy of poly{d(A-T)} synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types . A new procedure was developed for the isolation of copied poly{d(A-T)} from chromatin DNA . This method involved in vitro copying of poly{d(A-T)} by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation . The fragmented natural DNA is then separated from the high molecular weight poly{d(A-T)} by gel filtration . The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly{d(A-T)} synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA . Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin . Synthesis of poly{d(A-T)} by chromatin is catalyzed mainly by DNA polymerase-beta . By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized. Biochemistry, 1980 Dec 9, 19(25), 5869 - 73 Single-stranded poly(deoxyguanylic acid) associates into double- and triple-stranded structures; Dugaiczyk A et al.; Circular plasmid deoxyribonucleic acid (DNA), pBR322, was digested with the restriction endonuclease PstI to give full-length double-stranded DNA molecules, terminated by two self-complementary single-stranded sequences: (formula: see text) . The protruding 3' termini were extended with dG by using calf thymus terminal deoxynucleotidyl transferase and dGTP, to form single-stranded tails of oligo(dG) . At a length of about dG15, such tails become resistant to single strand specific endonuclease S1, and also cease to function as substrate (initiator) for the terminal deoxynucleotidyl transferase . This altered reactivity arises from association of the oligo(dG) tails into double- and triple-stranded structures, resulting in linear, circular, and branched polymers of the monomeric linear plasmid DNA . All these polymeric structures of the plasmid DNA are stable at room temperature, can be observed in the electron microscope, and can be separated from each other by agarose gel electrophoresis . At 60 degrees C or in 50% formamide, most of the oligo(dG) self-association can be reversed (melted), and the plasmid DNA is again found as the original linear monomer. Biochemistry, 1980 Dec 9, 19(25), 5774 - 81 Subunit interaction during catalysis: ammonium ion modulation of catalytic steps in the Escherichia coli glutamine synthetase reaction; Bild GS et al.; A new approach for assessing if catalytic cooperativity may occur between subunits has been applied to Escherichia coli glutamine synthetase . The extent of oxygen exchange between bound {18O}glutamate and phosphate per molecule of glutamine formed was evaluated at various NH4+ concentrations . This allows calculation of the minimum number of reaction reversals in which bound glutamine is converted to bound glutamate prior to release of glutamine . At 1000 microM NH4+ no detectable reversals occurred, and only one glutamate oxygen appeared in product phosphate as required by the reaction mechanism . However, at 10 microM NH4+ over 15 reversals of bound glutamine formation occurred . Controls showed that under the experimental conditions free glutamine does not become significantly involved in exchange and, therefore, the reversal of the oxygen exchange steps appears to be limited to bound glutamine . In contrast to the effect seen with NH4+, adenosine 5'-triphosphate concentration appears to modulate the exchange of oxygen between glutamate and phosphate only slightly . These findings are interpreted as showing that NH4+ either promotes the dissociation of one of the reaction products or decreases the participation of bound products in the exchange . The NH4+ modulation of the oxygen exchange is consistent with binding of NH4+ at one catalytic site promoting catalytic events at an alternate catalytic site but does not eliminate all other explanations. C R Seances Acad Sci D, 1980 Dec 8, 291(12), 937 - 40 {Secondary and topographic structure of ribosomal RNA 16S of Escherichia coli}; Stiegler P et al.; We present a model for the secondary structure of 16S ribosomal RNA from E . coli . This model has been deduced by restricting the total number of theoretical base pairings using the following criteria: (1) susceptibility of residues towards enzymatic probes that are specific for either paired or single stranded regions; (2) reactivity of certain residues to chemical modification; (3) evidence for medium and long range interactions; (4) comparative analysis of ribosomal RNA sequences from other organisms. C R Seances Acad Sci D, 1980 Dec 8, 291(12), 933 - 6 {Nucleotide sequence of the gene for the mitochondrial 15S ribosomal RNA of yeast}; Sor F et al.; We have determined the nucleotide sequence of a DNA segment carrying the entire 15S ribosomal RNA gene of yeast mitochondrial genome . Many stretches of sequence are present which are homologous to the E . coli 16S ribosomal RNA gene . The gene sequence can be folded into a secondary structure according to the {1} model on bacterial ribosomal RNAs . The structure reveals a striking similarity between the two RNAs despite the large difference in their base compositions . In the middle of the gene, we found a guanine-cytosine rich sequence that is also present in several other regions of the mitochondrial genome. Biochim Biophys Acta, 1980 Dec 5, 620(3), 356 - 63 Acyl phosphatidylglycerol of Escherichia coli; Kobayashi T et al.; Acyl phosphatidylglycerol, isolated from Escherichia coli, has been identified as 3-sn-phosphatidyl-1'-(3'-acyl)-sn-glycerol . The fatty acids of the diacylglycerol moiety of acyl phosphatidylglycerol resemble those of phosphatidylglycerol in composition . However, the monoacylglycerol moiety of this lipid contains more unsaturated fatty acids than the diacylglycerol part of this lipid or other phospholipids in E . coli . Furthermore, the fatty acids present in the monoacylglycerol moiety, were found to contain major amounts of an unsaturated acid identified as 7-tetradecenoic acid by combined gas-liquid chromatography-mass spectrometry . This acid was present only in low concentrations in most phospholipids of E . coli. Biochim Biophys Acta, 1980 Dec 4, 616(2), 319 - 28 Studies on aspartase . VI . Trypsin-mediated activation releasing carboxy-terminal peptides; Yumoto N et al.; Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) of Escherichia coli, already of full activity, is 3-5-fold activated by a limited proteolysis with trypsin (Mizuta, K . and Tokushige, M . (1976) Biochim . Biophys . Acta 452, 253-261) . Structural bases for the activation were investigated . The NH2-termini of the native enzyme and of the trypsin-activated enzyme were found to be equally serine, as analyzed by the dansylation method . However, the COOH-terminal glutamate of the native enzyme was altered to arginine upon activation, as revealed by treatments with carboxypeptidases Y, A and B . The released peptides were obtained by molecular sieve membrane filtration following trypsin activation of the enzyme . The peptides were separated by high voltage paper electrophoresis, and the amino acid composition and the terminal residues were determined . The results showed that one or a few related peptides consisting of 7-17 residues were released from the COOH-terminal upon activation . The circular dichroism spectrum of the enzyme suggested that the helical content of the activated enzyme was about 5% less than that of the native enzyme, an indication that the trypsin-activated enzyme has a somewhat looser conformation than the native enzyme . Determination of the fluorescence decay time of the enzyme protein indicated that the tryptophan residue became more exposed to outer environment than that of the native enzyme upon trypsin-activation. Biochim Biophys Acta, 1980 Dec 2, 603(1), 13 - 26 Studies on the damage to Escherichia coli cell membrane caused by different rates of freeze-thawing; Souzu H; Freeze-thawing of Escherichia coli cells caused a release of cell membrane components such as protein, phospholipids and lipopolysaccharides . A greater amount of release and a lesser extent of cell survival were seen in slow freeze-thawing than in rapid freeze-thawing . Several dehydrogenases in the cells were also freed . The mode of release was also dependent on the rate of freeze-thawing . The materials released by slow freeze-thawing were found to be mostly composed of outer membrane components, whereas the materials released by rapid freeze-thawing contained cytoplasmic as well as outer membrane components . The chemical composition of these fragments differed significantly from that of the original membranes . The relative content of cytoplasmic membrane-bound enzymes in these fragments also differed from that of the cytoplasmic membrane . The fragmentation was assumed to have resulted mainly from the crystallization of external water . In slow fraeeze-thawing, it was considered that the phase separation of the membrane phospholipid bilayer increased the possibility of outer membrane fragmentation . Rapid freeze-thawing caused cytoplasmic membrane damage to the cells as well as to the outer membrane . In rapid freeze-thawing, the effect of phase separation appeared to be small because of rapid passage through the transition temperatures . The presence of 10% glycerol completely inhibited the release of cellular materials and enzymes . Cell survival was maintained at a high level in the glycerol-treated samples whether freeze-thawed slowly or rapidly. Immunopharmacology, 1980 Dec, 2(4), 285 - 91 Endotoxin depression of hepatic mixed function oxidase system in C3H/HeJ and C3H/HeN mice; Williams JF et al.; The effect of endotoxin to depress the hepatic drug-metabolizing enzyme activity has been studied in the C3H/HeJ and C3H/HeN strains of mice . The C3H/HeJ mouse strain is generally considered to be unresponsive to the biological effects of endotoxin . However, injection of these mice with 0.5 mg/kg body weight of E.coli endotoxin (Westphal extracted) produced a decrease in the rate of N-demethylation of ethylmorphine and in the levels of cytochrome P-450 and cytochrome b5 comparable to that observed in the endotoxin-sensitive C3H/HeJ mouse strain . Although the mechanism of endotoxin action to decrease hepatic microsomal drug-metabolizing activity is presently unknown, the results suggest that: 1) the C3H/HeJ mouse stain is responsive to this endotoxin effect, and 2)that cellular constituents other than B-cells or macrophages are probably involved in eliciting the response, since these cells of the C3H/HeJ mouse are unresponsive to endotoxin. Cell, 1980 Dec, 22(3), 875 - 86 A family of inverted repeat sequences and specific single-strand gaps at the termini of the Physarum rDNA palindrome; Johnson EM; Ribosomal genes of Physarum polycephalum are located on multiple copies of an extrachromosomal, 61 kb rDNA palindrome . Each terminus of the palindrome consists of a nontranscribed spacer, averaging 5.4 kb in length, which includes a region of multiple inverted repeat sequences . Electron microscopy of denatured and reannealed termini reveals foldback segments that are multiples of a unit length of 50 bp, a length confirmed by sizing of restriction fragments from labeled termini . The total length of this inverted repeat region averages 600 bp per terminus, and the region is centered approximately 4 kb from the 3' end of the 26S gene . Restriction fragments containing the termini are heterogeneous, varying in length by +/- 400 bp . This heterogeneity is due to variability in both the number of 100 bp inverted repeats and the length of additional terminal spacer sequences . The inverted repeat region contains selectively located single-strand discontinuities as revealed by brief incubation with deoxynucleoside alpha-32P-triphosphates and E . coli DNA polymerase I (nick translation) . Labelling at the discontinuities begins specifically with the sequence CCCTA . Discontinuities are nonligatable and are most likely gaps one nucleotide long . Each terminus contains 3 to 5 such gaps spaced approximately 200 bp apart . As measured by hybridization with selectively labeled foldback DNA, sequences homologous to the inverted repeats are widely distributed throughout chromosomal DNA. Br J Cancer, 1980 Dec, 42(6), 900 - 7 Hyperphagocytosis and the effect of lipopolysaccharide injection in tumour-bearing mice; Bradfield JW et al.; (AxT6)F1 hybrid mice received s.c . transplants from (AxT6)F1 mammary carcinomas . At 1, 2 or 4 weeks after tumour transplantation, the mice were bled to obtain plasma and then challenged with 25 micron E . coli lipopolysaccharide (LPS) endotoxin i.v . The mice were killed 24 hr later, further plasma was obtained and their liver ratios and spleen ratios were determined . A similar procedure was carried out on non-tumour-bearing mice . Progressive tumour growth was associated with an increase in the liver ratio . In parallel, mice with 4-week tumour transplant showed increased uptake of colloidal carbon particles and 51Cr-labelled sheep red blood cells in the liver . The plasma amino aspartate transaminase (AST) and the ornithine carbamoyl transferase (OCT) showed a constant rise in all groups of mice after LPS injection . However, at 24 hr after LPS injection, the AST level showed the greatest rise in mice with 4-week tumour transplants . By contrast, OCT, which is liberated only from hepatocytes, showed the greatest rise in non-tumour-bearing mice. Arch Toxicol, 1980 Dec, 46(3-4), 277 - 85 Comparison of alkylation rates and mutagenicity of directly acting industrial and laboratory chemicals: epoxides, glycidyl ethers, methylating and ethylating agents, halogenated hydrocarbons, hydrazine derivatives, aldehydes, thiuram and dithiocarbamate derivatives; Hemminki K et al.; Groups of industrial and laboratory chemicals were tested for their alkylation activity using 4-(p-nitrobenzyl)-pyridine and deoxyguanosine as nucleophiles . The alkylation activity was compared with mutagenicity of the chemicals to E . coli WP2 uvrA without metabolic activation . All the epoxide-containing compounds including simple epoxides and glycidyl ethers elicited alkylation activity and mutagenicity . Furthermore there was a reasonable correlation between the rate of alkylation and the mutagenic potency . All the methylating and ethylating compounds tested were active but no correlation was observed between the rate of alkylation and the mutagenic potency, apparently due to the different types of alkylation products formed . The other compounds tested including halogenated hydrocarbons, hydrazine derivatives, aldehydes, thiuram and dithiocarbamate derivatives elicited a slow or no alkylation activity while many of the compounds were mutagenic . There was no evidence among the chemicals tested of an alkylating non-mutagen . Thus evidence of alkylation activity appears to indicate mutagenic risk. J Clin Microbiol, 1980 Dec, 12(6), 768 - 71 Value of passive immune hemolysis for detection of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli; Tsukamoto T et al.; The method of passive immune hemolysis of Evans and Evans (Infect . Immun . 16:604-609, 1977) for detection of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli was modified . A total of 373 strains of E . coli were tested by this method using materials obtained by treating the cells with polymyxin B and rabbit antiserum against cholera enterotoxin, purified by affinity gel column coupled with purified cholera enterotoxin, in N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (pH 6.7) . The results correlated very well with those obtained in an assay with Chinese hamster ovary cells . It is concluded that passive immune hemolysis is useful as a routine clinical method for identifying E . coli strains that produce heat-labile enterotoxin. J Clin Microbiol, 1980 Dec, 12(6), 738 - 43 Detection of enterotoxigenic Escherichia coli colonization factor antigen I in stool specimens by an enzyme-linked immunosorbent assay; Evans DG et al.; An enzyme-linked immunosorbent assay (ELISA) was employed to detect and quantitate the fimbrial colonization factor antigen (CFA/I) of enterotoxigenic Escherichia coli in stool specimens obtained from adult cases of diarrhea in which CFA/I-positive E . coli was the known causative agent . The inhibition method, or blocking technique, was used . In this method, a standardized dilution of human anti-CFA/I serum was preincubated with dilutions of stool extract before transfer to CFA/I-coated microtiter plate wells, and then ELISA was performed with alkaline phosphatase-conjugated anti-human immunoglobulin . CFA/I purified from E . coli strain H-10407 (O78:H11) was used . Acute-phase diarrheal stool specimens were found to contain approximately 3.0 mg of antigen (mean value) per g stool, whereas control (CFA/I-negative) specimens contained insignificant amounts (less than 0.03 mg/g) of antigen . Also, CFA/I was detected in culture fluids of CFA/I positive enterotoxigenic E . coli belonging to a variety of serotypes and was undetectable in similar preparations from P-strains (spontaneous CFA/I-negative derivatives) of the same test cultures . Equivalent results were obtained in ELISA tests by using bacterial cells taken from isolated colonies grown on CFA agar . These results indicate that the ELISA technique will be useful for the diagnosis of diarrhea caused by CFA/I-positive enterotoxigenic E . coli. Q Rev Biol, 1980 Dec, 55(4), 335 - 52 Folding up a transfer RNA molecule is not simple; Guthrie C; For much of its history, molecular biology has concerned itself with the implications and consequences of the "Central Dogma" (Crick, 1958): DNA leads to RNA leads to protein . This "pathway" is of course atypical in that it describes, not chemical interconversions, but rather the flow of information from gene to product . It has been only recently appreciated, however, that in those cases where the final gene product is an RNA molecule, this pathway is not simply truncated . To the contrary, in virtually every instance for which sufficient data are now available, it is found that the RNA transcript is subject to a series of reactions in which the primary gene product undergoes nucleotide additions, deletions, and modifications . The set of biosynthetic reactions that intervene between transcription of the gene and production of the mature functional product is collectively referred to as RNA processing . It now appears that the existence of these complex biosynthetic events cannot always be adequately explained by the necessity to overcome otherwise insurmountable topological or energetic constraints: the final product can, at least in some cases, "self-assemble." The genesis of transfer RNA by this indirect route appears to insure the delivery of a functional product, at the right rate, at the right time . We suggest, moreover, that this process is primarily determined by recognition of some of the same structural elements in the precursor that are required for the function of the mature tRNA molecule. J Gen Microbiol, 1980 Dec, 121(2), 387 - 400 The association of the O18, K1 and H7 antigens and the Co1V plasmid of a strain of Escherichia coli with its virulence and immunogenicity; Smith HW et al.; From an O18ac:K1:H7 ColV+ strain of Escherichia coli (designated MW) that had caused meningitis in a human baby, mutant forms were isolated that lacked different combinations of its O, K and H antigens and its ColV plasmid . These characters were also transmitted by conjugation to E . coli K12 and the virulence, immunogenicity and other properties of the different forms of both strains were studied . All the forms of the MW strain that lacked either the O18 or K1 antigens or the ColV plasmid, but not the H7 antigen, were much less virulent for chickens and mice than the parent form of MW . Another form derived by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment of the parent strain and that possessed all these four characters was also much less virulent . Some of the forms of the K12 strain to which the characters had been transferred were slightly more virulent than the K12 parent, but the virulence of all of them, including one possessing the O18 and K1 antigens and the ColV plasmid, did not approach that of the MW parent . Pathogenesis studies in chickens and colostrum-deprived calves revealed that the loss of virulence exhibited by the forms of the MW strain that lacked O18, K1 and ColV and by the NTG-derived form was associated with decreased ability to invade the body . This was also the reason for the ow virulence of the forms of the K12 strain that had acquired these characters . Possession of both the O18 and K1 antigens was largely responsible for the ability of the different forms of the MW strain to survive in fresh chicken serum; organisms of K12 that possessed the K1 antigen survived as long as those of the parent form of the MW strain . A substantial degree of immunity against lethal infection with the parent form of the MW strain was produced in chickens and mice by all the forms of the MW and K12 strains that possessed the O18 antigen . The K1 and H7 antigens and the ColV plasmid produced no detectable immunity. J Gen Microbiol, 1980 Dec, 121(2), 373 - 80 Major outer membrane proteins of Escherichia coli strains of human origin; Overbeeke N et al.; The major outer membrane protein patterns of 45 Escherichia coli strains of human origin were compared with that of E . coli K12 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis . Preparations of the former strains contained between two and five major bands in the molecular weight range between 30 000 and 42 000 . The patterns were very heterogeneous with respect to the numbers and electrophoresis mobilities of the major outer membrane protein bands . In all cases the fastest moving band was characterized as a protein similar to the ompA protein of strain K12 as it was partially degraded by trypsin and reacted specifically with antiserum against the purified omp A protein in a gel immuno-radioassay . All the other major outer membrane proteins are related to the ompC and ompF proteins (the porins) of strain K12 as they were peptidoglycan-associated and reacted with antisera against the purified ompC and/or ompF proteins. Genetics, 1980 Dec, 96(4), 779 - 99 Genetic factors affecting recovery of nonpoint mutations in the region of a gene coding for ornithine transcarbamylase: involvement of both the F factor in its chromosomal state and the recA gene; Jessop AP et al.; Mutants of E . coli K12 that overproduce ornithine transcarbamylase can be identified in Car- strains because they permit utilization of citrulline as a carbamyl phosphate source, due to reversal of the normal OTCase reaction; they are called Cut mutants (citrulline utilizers) . Hfr strains that carry the F factor adjacent to argF (one of two duplicate genes that code for ornithine transcarbamylase in E . coli K12) yield more Cut mutants than do F+ or F- strains, or Hfr strains in which the F factor is not adjacent to argF . When Hfr strains in which the F factor is integrated adjacent to argF are made recA, they yield few Cut mutants . Many of the Cut mutants recovered from one of the Hfr strains used in the investigation (Hfr P4X) are unstable; the properties of these unstable mutations suggest that they carry aberrations in the region of the argF gene . Thus, the increased yields of Cut mutants probably result from aberrations that occur when the F factor is integrated adjacent to argF . The nature of these aberrations is not yet known . The unstable Cut mutants are to a large extent stabilized by recA; such stabilization is one of the properties of duplications . Other data indicate that the aberrations may be more complex than simple gene duplications; in particular properties of segregants and some recombinants derived from unstable Cut mutants are most easily interpreted by assuming that segregation from, and possibly formation of, the unstable mutants occurs in several stages. Res Commun Chem Pathol Pharmacol, 1980 Dec, 30(3), 419 - 30 Chloroquine pharmacokinetics in tissues of pyrogen treated rats and implications for chloroquine related pruritus; Osifo NG; The concentrations of chloroquine in the tissues and plasma of control and pyrogen treated Long Island rats were serially determined over 16 days . Significant alterations of pharmacokinetic parameters, a delayed completion of distribution with a biphasic pattern of uptake of chloroquine into peripheral organs (skin and skeletal musculature) and increased tissue uptake in visceral organs (heart, liver and kidneys) of pyrogen treated rats were found . It is suggested that the known hemodynamic changes in the febrile state produced these unusual pharmacokinetic changes in the peripheral tissues and may contribute significantly to the occurrence of pruritus and increased acute toxicity of chloroquine during a febrile illness . The uptake patterns of chloroquine into pyrogen treated rat skin and muscle strongly suggest the involvement of a blood flow-dependent process in the movement of the drug into the tissues. Gene, 1980 Dec, 12(3-4), 243 - 8 Isolation of plasmids carrying either the uvrC or uvrC uvrA and ssb genes of Escherichia coli K-12; Yoakum GH et al.; A 3.4 kb PstI fragment containing the uvrC gene of Escherichia coli K-12 has been cloned into pBR322 . Plasmids carrying this PstI fragment, in either orientation (pGY3233, pGY4211) relative to the cloning vehicle, complement uvrC mutants . A second plasmid (pGY3243) with a 21 kb HindIII fragment is shown to complement mutations in uvrA and ssb (single-strand binding protein) . A composite plasmid (pGY4610) containing pBR322 and PstI fragments derived from pGY3233 (3.4 kb) and pGY3243 (11.05 kb) complements the uvrC, uvrA and ssb mutations. Gene, 1980 Dec, 12(3-4), 223 - 34 Cloning and expression of Trypanosoma brucei kinetoplast DNA in Escherichia coli; Brunel F et al.; The kinetoplast DNA of Trypanosoma brucei is made of two components: mini-circles (1 kb, 90% of total kDNA) and maxi-circles (20 kb, 10%) of total kDNA) . These are interlocked to form a network of about 10 000 kb . In order to analyse the components of such a network structure, we have cloned individual mini-circle molecules and two of the three EcoRI maxi-circle fragments in E . coli . Cloned mini-circles are somewhat heterogeneous in size and their restriction patterns are completely different . Despite this heterogeneity all are found to contain a homologous region(s) defined by DNA/DNA hybridization . The maxi-circles probably correspond to the mitochondrial DNA of other organisms and, in contrast to mini-circles, do not show sequence heterogeneity . One of the two cloned maxi-circle EcoRI fragments is able to direct the synthesis of two polypeptides of 10 300 and 13 500 daltons in E . coli mini-cells . Detailed analysis of this phenomenon shows that both structural genes and promoter(s) are located within the cloned maxi-circle fragment. Gene, 1980 Dec, 12(3-4), 179 - 90 The araC gene of Escherichia coli: transcriptional and translational start-points and complete nucleotide sequence; Wallace RG et al.; The nucleotide sequence of the Escherichia coli araC gene and flanking regions have been determined from a series of overlapping fragments using the technique of base-specific chemical cleavage of Maxam and Gilbert (1980) . The nucleotide sequence of araC gene was confirmed by the partial amino acid sequences of araC protein and its methionine peptides . The primary structure of araC polypeptide consists of 291 amino acid residues, giving it a chemical molecular weight of 33 314 daltons . The transcriptional start-point has been deduced from the sequence of araC mRNA synthesized in vitro and in vivo, and it is located 148 bp away from the transcriptional start-point of the araBAD operon . The translational start-point for the araC gene was deduced from the N-terminal amino acid sequence of the protein, and is located 165 bp from the 5'-end of araC mRNA . There is, therefore, a leader sequence of 164 bp preceding the araC gene. Can J Biochem, 1980 Dec, 58(12), 1381 - 6 The uptake and acylation of exogenous lysophosphatidylethanolamine by Escherichia coli cells; Hellion P et al.; Escherichia coli envelopes were fractionated to yield inner and outer membrane fractions . Both these fractions were found to convert {14C}lysophosphatidylethanolamine to its diacyl analogue . Intact Escherichia coli cells were capable of absorbing exogenous labelled lysophosphatidylethanolamine and converting it to phosphatidylethanolamine . When the 14C- and 32P-labelled lyso analogue was used, both the absorption process and the conversion to diacyl analogue proceeded without a significant change in isotope ratio either in the presence or in the absence of added inorganic phosphate . The absorption process was not markedly stimulated by Ca2+ in the medium; it proceeded to an amount representing 25--30% of the endogenous membrane lipid and was accompanied by some degradation to water-soluble products which accumulated in the cell mainly, but also in the incubation medium . The absorbed lipid was recovered in both the inner and outer membrane fractions of the cell envelope . The results indicate that Escherichia coli inner and outer membranes are capable of absorbing exogenous lysophosphoglyceride and converting it into structurally useful diacyl analogue. Biokhimiia, 1980 Dec, 45(12), 2189 - 97 {Heparin complexes with DNA: formation, properties and methylation in vitro}; Liapina LA et al.; Heparin complexes with DNA were obtained in vitro, the components being taken in weight ratios of 1 : 6 or 1 : 12, respectively . The complexes obtained exhibited the anticoagulating activity and antipolymerizing properties in the presence of a fibrin-monomer and an ability to dissolve nonstabilized fibrin . An intravenous injection of these complexes to rats increased the total and non-enzymatic fibrinolytic activity of blood plasma and its anticoagulating properties, while an intravenous injection of DNA caused only a short-term hypercoagulating effect . The DNA modified by heparin altered its acceptor abilities in an in vitro methylation reaction in the presence of DNA-methylases from animal and bacterial cells . Incorporation of methyl groups into DNA showed a decrease, apparently due to heparin blocking of some methylable sequences . An addition of heparin to a system containing purified DNA, DNA-methylase and S-adenosylmethionine was accompanied by a concentration-dependent inhibition of the methyl group incorporation into DNA. Can J Microbiol, 1980 Dec, 26(12), 1514 - 8 Inhibition of in vitro peptidoglycan biosynthesis in Escherichia coli by guanosine 5'-diphosphate 3'-diphosphate; Ishiguro EE et al.; Physiological concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the synthesis of lipid intermediates and peptidoglycan catalyzed by a particulate enzyme preparation from Escherichia coli . The inhibition of these reactions was dependent on the concentrations of ppGpp and MgCl2 in the assay . The degree of inhibition of lipid intermediate synthesis decreased as the molar ratio of MgCl2 to ppGpp was increased, and no inhibition was observed above a MgCl2 to ppGpp ratio of 2.5 . The synthesis of peptidoglycan was more sensitive to inhibition by ppGpp, and significant inhibition occurred under conditions where lipid intermediate synthesis was unaffected (i.e., at MgCl2 to ppGpp ratios of 2.5 or more) . A variety of other nucleotides did not inhibit the synthesis of lipid intermediates and peptidoglycan. Antimicrob Agents Chemother, 1980 Dec, 18(6), 887 - 92 Inhibition of peptidoglycan synthesis in ether-permeabilized Escherichia coli cells by structural analogs of D-alanyl-D-alanine; Pelzer H et al.; Several analogs of D-alanyl-D-alanine have been proved to be competitive inhibitors of murein (cross-linked peptidoglycan) synthesis in either-permeabilized cells of Escherichia coli . Some analogs, distinguished from D-alanyl-D-alanine only by minor structural deviations, were incorporated into a murein-like sodium dodecyl sulfate-insoluble material in place of the natural substrate . These analogs therefore could be designated as competitive substrates of the cross-linked end product of murein synthesis . In contrast, others, even those containing bulky residues at the methyl group of the amino-terminal D-alanine, exhibited inhibition of murein synthesis . The last-mentioned group of analogs also inhibited a blank value which seems to be a characteristic feature of this system without added D-alanyl-D-alanine . From this data, a steady-state concentration of D-alanyl-D-alanine or D-alanine or both in growing cells of E . coli could be calculated as approximately 0.4 x 10(-3) M. Infect Immun, 1980 Dec, 30(3), 894 - 8 Translocation of Escherichia coli from the gastrointestinal tract to the mesenteric lymph nodes in gnotobiotic mice receiving Escherichia coli vaccines before colonization; Berg RD et al.; Germfree mice were immunized orally or intraperitoneally for 6 weeks with heat-killed vaccines of indigenous Escherichia coli or nonindigenous E . coli O 127: B8 before colonization with these strains . The mice exhibited increases in specific serum antibodies and intestinal immunoglobulin A reacting with the E coli antigens . Prior immunization did not reduce the gastrointestinal population levels of the E . coli strains attained 3 and 7 days after colonization . Neither oral nor intraperitoneal immunization with the E . coli strains before colonization decreased the incidence of bacterial translocation to the mesenteric lymph nodes or reduced the number of viable E . coli cells per mesenteric lymph node . There also was no relation in individual mice between serum antibody titers and the numbers of viable E . coli cells translocating to the mesenteric lymph nodes . Thus, prior vaccination with E . coli in this study did not decrease the incidence or reduce the numbers of viable E . coli translocating to the mesenteric lymph nodes in gnotobiotic mice monoassociated with E . coli. Proc Natl Acad Sci U S A, 1980 Dec, 77(12), 7117 - 21 Nucleotide sequence and expression of Escherichia coli trpR, the structural gene for the trp aporepressor; Gunsalus RP et al.; The nucleotide sequence of trpR of Escherichia coli was determined . This gene codes for a polypeptide (Mr 12,356) that is 108 amino acid residues in length . NH2-terminal, COOH-terminal, and total amino acid analyses of purified aporepressor agree with the deduced amino acid sequence and establish the translation start and stop codons of the structural gene . The transcription start site for trpR mRNA synthesis in vitro was shown to be 56 base pairs prior to the translation start site . The nucleotide sequence on either side of the transcription start site is homologous to the trp operon operator . Purified trp aporepressor, when activated by L-tryptophan, protects restriction sites in this region, the presumed trpR operator, from cleavage by the respective restriction endonucleases . Bound RNA polymerase protects the same restriction sites . These findings and the additional observation that trp repressor inhibits transcription initiation in vitro establish that there is a functional overlap of operator and promoter sequences in the regulatory region of the trpR operon . These findings indicate that expression of trpR is autoregulatory. Proc Natl Acad Sci U S A, 1980 Dec, 77(12), 7084 - 8 Feedback regulation of ribosomal protein gene expression in Escherichia coli: structural homology of ribosomal RNA and ribosomal protein MRNA; Nomura M et al.; Certain ribosomal proteins (r proteins) in Escherichia coli, such as S4 and S7, function as feedback repressors in the regulation of r-protein synthesis . These proteins inhibit the translation of their own mRNA . The repressor r proteins so far identified are also known to bind specifically to rRNA at an initial stage in ribosome assembly . We have found structural homology between the S7 binding region on 16S rRNA and a region of the mRNA where S7 acts as a translational repressor . Similarly, there is structural homology between one of the reported S4 binding regions on 16S rRNA and the mRNA target site for S4 . The observed homology supports the concept that regulation by repressor r proteins is based on competition between rRNA and mRNA for these proteins and that the same structural features and of the r proteins are used in their interactions with both rRNA and mRNA. Gene, 1980 Dec, 12(1-2), 33 - 9 The nucleotide sequence of tufB and four nearby tRNA structural genes of Escherichia coli; An G et al.; In order to approach some of the problems suggested by the presence of two genes for EF-Tu in Escherichia coli we have determined the nucleotide sequence of tufB and its genetic surroundings . Comparison with the known amino acid sequence of EF-Tu (Arai et al., 1980) and with the tufA nucleotide sequence (Yokota et al., 1980) allows some conclusions to be made regarding possible relationships between tufA and tufB . We also report the sequence of four tRNA structural genes (thrU, tyrU, glyT and thrT) that lie immediately to the 5' side of tufB. Gene, 1980 Dec, 12(1-2), 25 - 31 The nucleotide sequence of the cloned tufA gene of Escherichia coli; Yokota T et al.; The 4 kb (8.5 % lambda units) EcoRI fragment harboring the tufA gene of Escherichia coli was cloned using plasmid pTUA1 (Shibuya et al., 1979) and its structure was analyzed . The nucleotide sequence of about 1500 base pairs, covering the C-terminal portion of elongation factor EF-G (fus gene), the intercistronic region between fus and tufA, the entire structural gene for tufA with the GUG initiation and UAA termination codons, and the 3' flanking region of tufA, was determined . Comparison of the tufA nucleotide sequence with the tufB sequence (An and Friesen, 1980) and the known amino acid sequence of EF-Tu (Arai et al., 1980) revealed that the products of genes tufA and tufB are identical except for one amino acid at the C-terminal, i.e., glycine for tufA and serine for tufB . Nucleotide differences between tufA and tufB were found at 13 positions . Among them, one in the initiation codon and the other one in the C-terminal amino acid codon had replacements at the first letter of the codons . The other eleven changes were in the third codon positions, which did not affect the amino acid coding . The pattern of codon usage in tufA and tufB is highly nonrandom, and remarkably similar to that in ribosomal protein genes, with the codons for the most abundant species of isoaccepting tRNAs being preferentially utilized (Post et al., 1979; Post and Nomura, 1980). Neurosurgery, 1980 Dec, 7(6), 551 - 9 Double compartment hydrocephalus--a new clinical entity; Foltz EL et al.; We are reporting eight patients who demonstrated double compartment hydrocephalus, i.e., supratentorial and infratentorial hydrocephalus in clinical sequence and separately . One infant with veil occlusion of the aqueduct was operated on to remove the veil and then later demonstrated panhydrocephalus . Six patients had been treated months to years earlier by the performance of a ventriculoperitoneal shunt for aqueductal hydrocephalus and then developed characteristic cerebellar-brain stem deficits from 4th ventricle enlargement . The work-up included computed tomographic scan, air study, isotope cerebrospinal fluid flow study, and direct 4th ventricle pressure studies . Operation with removal of a veil occlusion of the upper 4th ventricle aqueduct produced immediate recovery in five of six patients . The conversion of aqueductal stenosis to veil occlusion is postulated as the mechanism of "primary" veil obstruction found in infants . This new clinical entity is more common than realized . We report one patient with compartmental 4th ventricular hydrocephalus. J Infect Dis, 1980 Dec, 142(6), 892 - 8 Detection of enterotoxigenic Escherichia coli by DNA colony hybridization; Moseley SL et al.; A method fo detecting large numbers of isolates of enterotoxigenic Escherichia coli is described in which the genes encoding th enterotoxins are detected, rather than the toxins themselves . Radiolabeled fragments of DNA encoding the heat-labile (LT) or heat-stable (ST) toxins were used as hybridization probes for homologous DNA sequences in E . coli colonies grown and lysed in situ on nitrocellulose filters . The LT probe detected all of 31 E . coli strains producing ST and LT or only LT, while the ST probe detected 12 of 17 strains producing only ST and three of 26 strains producing ST and LT . These results suggest that the LTs produced by different isolates of E . coli are homologous and that human isolates of E . coli produce at least two heterologous STs detectable in the infant mouse assay . The hybridization method also detected the presence of enterotoxigenic E . coli in bacterial growth in directly spotted stools from patients with acute diarrhea. J Hyg (Lond), 1980 Dec, 85(3), 347 - 58 Bacteriostasis of Escherichia coli by milk . V . The bacteriostatic properties of milk of West African mothers in the Gambia: in-vitro studies; Dolby JM et al.; PIP: Bacteriostatic activity was measured in 244 specimens of milk from 78 mothers collected during 1977 throughout lactation of up to 1 year; the activity varied from very good to fair and only 7 were inactive . There was a wider range of activity than was found previous in milk from English mothers . Activity usually fell slowly during lactation but some of the Gambian mothers produced milk of very high activity, like that of colostrum, into the 2nd week of lactation, and 2 mothers did so at 6 and 9 months; others motherss produced good-activity milk throughout lactation . The bacteriostatic activity varied little with the season but slight decreases from that expected were found after the high incidence of infant diarrhea towards the end of the rainy season . The bacteriostatic activity of most of the milk tested could be prevented by iron salts but that of colostrum and some of the milks with high activity could not . Only these highly active colostra and milks were inhibitory in vitro when the inoculum was increased from 104 to 106 organisms/ml . These and less active milks were able to inhibit the smaller, standard inoculum for longer than 3 hours with the addition of bicarbonate and extra iron-binding protein at the concentrations likely to be present in vivo . Both commensal and pathogenic E . coli were inhibited to a similar degree by these milks and there was no evidence of serotype specificity . J Hyg (Lond), 1980 Dec, 85(3), 327 - 30 Investigation into an outbreak of food poisoning; Hayden BJ et al.; During an outbreak of food poisoning at a church camp, 16 of the 25 people attending were affected . Despite a thorough search for a bacterial pathogen none was identified . An examination of the Escherichia coli serotypes present suggest that E . coli O159 . H9 may have been the organism causing the outbreak. J Biochem (Tokyo), 1980 Dec, 88(6), 1879 - 82 Augmentation of cyclopropane fatty acid synthesis under stringent control in Escherichia coli; Taguchi M et al.; An abrupt increase of cyclopropane fatty acid (CFA) occurred concomitant with a decrease of the corresponding unsaturated fatty acids in CP78 (rel+) of Escherichia coli at the onset of the stationary growth phase, whereas such variations were slight in CP79 (rel-) . When the cells were starved for isoleucine, the CFA content increased in CP78 but not in CP79 . The rate of 14C-incorporation from {methyl-14C}methionine into CFAs increased in CP78 abut two-fold due to the starvation . The apparent level of CFA synthase also increased due to the starvation . These results that the CFA formation is augmented under stringent control. J Biochem (Tokyo), 1980 Dec, 88(6), 1733 - 8 State of Tyr49 in a mutant tryptophan synthase alpha-subunit substituted at position 49; Ogasahara K et al.; The states of tyrosine residues in an alpha-subunit of wild-type tryptophan synthase from Escherichia coli and a mutant protein which has tyrosine in place of glutamic acid at position 49, were examined by absorption spectrum, spectrophotometric titration of phenolic hydroxyl ionization, and deuteration kinetics of phenolic hydrogen monitored by fluorescence measurement . The difference absorption spectrum of the mutant protein against the wild-type protein at pH 7.0 and 25 degrees C had peaks at 289, 282, and 276.5 nm . These positions corresponded to those in the absorption spectrum of L-tyrosine derivatives in a non-aqueous solvent at 77 K and these bands were well-resolved even at 25 degrees C as if the tyrosine residue were fixed at lower temperature . The titration curve of the mutant protein at 3 degrees C differed from that of the wild-type protein only above pH 12.7, where the difference molar extinction coefficient at 295 nm reached a plateau, indicating that ionization of Tyr49 took place at an abnormally high pH . These results suggest that Tyr49 is buried in the hydrophobic interior and fixed in a certain orientation . The deuterium exchanges of phenolic hydrogen at pH 7.0 and 13 degrees C in the wild-type and mutant proteins consisted of a single and two first order processes, respectively, all three having smaller rate constants than that of free tyrosine, indicating that these tyrosine residues are buried . It is concluded that Tyr49 in the mutant protein is not on the surface of the molecule. Eur J Biochem, 1980 Dec, 113(1), 67 - 74 4-Thiouridine triggers both growth delay induced by near-ultraviolet light and photoprotection; Thomas G et al.; 4-Thiouridine, a rare nucleoside present in Escherichia coli tRNAs, has been recently proposed to be the major chromophore leading to near-ultraviolet (315-400-nm)-induced growth delay . Here this is established by the isolation of mutants exhibiting a reduced growth delay . The selection procedure involves several successive cycles of 365-nm illumination of the cells in the stationary phase, followed by growth for two or three generations . After the eighth cycle, the level of 4-thiouridine in the culture decreases to 20% of the original level and all individual clones tested show a 4-thiouridine deficiency . One mutant exhibiting a complete lack of 4-thiouridine in its tRNAs has been characterized . In the dark the growth characteristics of the mutant and of the parental strain are indistinguishable . In contrast after near-ultraviolet illumination the nuv mutation abolishes the growth delay and considerably reduces the photoprotection efficiency. Metabolism, 1980 Dec, 29(12), 1262 - 6 Glucose tolerance an insulin release in L-asparaginase treated rabbits; Lavine RL et al.; Male New Zealand White Rabbits were injected intravenously with either a single dose of 10,000 IU Escherichia coli L-asparaginase/kg body weight containing 80 mg of D-mannitol/10,000 IU E . coli L-asparaginase or 80 mg D-mannitol kg/body weight alone . Elevated fasting glucose (G) and elevated fasting immunoreactive insulin (IRI) levels were observed in the L-asparaginase treated rabbits at 1 wk . They peaked at 3 wk and declined thereafter . However, fasting G and IRI levels remained significantly elevated at the end of the study (9-15 wk after injection) compared to preinjection levels and levels of the controls . Glucose and IRI levels 0.5 hr post and intravenous glucose load (1 g/kg body weight) also became elevated post L-asparaginase and followed a time course similar to that of the fasting G and IRI levels . These 0.5-hr levels also remained significantly elevated at the end of the study . These data show that a single dose of 10,000 IU/kg body weight produced a hyperinsulinemic diabetes in New Zealand White Rabbits that appears to persist in a mild form for at least 9-15 week. J Med Chem, 1980 Dec, 23(12), 1299 - 305 Analogues of chloramphenicol: circular dichroism spectra, inhibition of ribosomal peptidyltransferase, and possible mechanism of action; Bhuta P et al.; Circular dichroism spectra of series of chloramphenicol derivatives la-r were measured in water at pH 7 . Compounds 1a-o exhibit two positive Cotton effects at 310--340 and 240--260 nm, respectively, and a weaker negative Cotton effect at 280--300 nm . In analogues 1c, 11, and 1m there is only a minimum between the two positive Cotton effects . Derivatives 1p--r possess a strong negative Cotton effect at ca . 280 nm . Compounds 1a--r were examined as inhibitors of the puromycin reaction with Escherichia coli 70S ribosome-poly(U)-N-AcPhe-tRNA complex . Analogues 11, 1n, lo, and lq are potent competitive inhibitors of puromycin comparable to or better than chloramphenicol (1b) . Compounds 1k and 1m are less active, whereas 1d--g and 1j are only moderately effective . The rest of the analogues have marginal or no activity . The results are compared with previous biological data and discussed in terms of a retro-inverso relationship of chloramphenicol (1b) to the aminoacyl moiety of puromycin (aminoacyl-tRNA) and to a hypothetical transition state of peptide bond formation. Am J Physiol, 1980 Dec, 239(6), E391 - 5 Free fatty acid utilization by skeletal muscle after endotoxin administration; Romanosky AJ et al.; The effect of endotoxin administration on free fatty acid (FFA) utilization by skeletal muscle was investigated . Albumin bound {1-14C}palmitate was continuously infused into anesthetized dogs . Blood samples from the carotid artery and profunda femoris veins were obtained and blood flow through the thigh muscles was determined . After endotoxin, skeletal muscle blood flow decreased by 29%, reflecting the average decrease in cardiac output . Arterial FFA concentration in endotoxin-treated animals was significantly reduced relative to saline-treated animals . Whole-animal FFA turnover decreased up to 37% after endotoxin . FFA uptake was linearly correlated with arterial FFA concentration both preceding and following endotoxin and the slopes of the regression lines were identical . FFA uptake by skeletal muscle was significantly reduced during the early postendotoxin time period . Skeletal muscle FFA oxidation after endotoxin also tended to decrease . It is concluded that the changes in skeletal muscle FFA metabolism are most likely due to decreased arterial FFA levels after endotoxin rather than a direct effect of endotoxin on skeletal muscle. Mutat Res, 1980 Dec, 79(4), 319 - 25 Differential mutagenicities of 6 N-nitroso-N-alkylurea derivatives in Escherichia coli strains with different DNA-repair capacities; Yoshikawa K et al.; Reverse mutations to prototrophy by, and killing effects of, 6 N-nitroso-N-alkylurea derivatives such as NMU, NEU, NPU, NBU, NIBU and NAU were studied with E . coli strains H/r30R (wild-type), Hs30R (uvrA-), O16 (polA-) and NG30 (recA-) . Both strains polA- and recA- were far more sensitive to killing by the 6-compounds than were the wild-type and uvrA- strains, and there was no difference in the sensitivity between the latter 2 strains . NMU was mutagenic in all 4 strains; wild-type, uvrA- and polA- strains were almost equally mutable but the compound was slightly mutagenic in the recA- strain . The other 5 compounds, NEU, NPU, NBU, NIBU and NAU, caused hardly any mutation in the recA- strain . NEU, NPU and NBU were equally mutagenic in the wild-type, uvrA- and polA- strains; however, NIBU and NAU were more mutagenic in the uvrA- than in the wild type or polA- . From the differential mutagenicities and lethalities induced by the 6 N-nitroso-N-alkylureas into the 4 tester strains, mutational patterns of the compounds can be classified into 3 types. J Nucl Med, 1980 Dec, 21(12), 1158 - 61 Effects of fever and hyperthermia on thyroid function; Shafer RB et al.; Thyroid hormones in man are affected by acute and chronic febrile states . To define these acute changes, we used a previously described rabbit model . Serum levels of T3, rT3, and T4 were measured at 0, 2, 4, 6, and 24 hr following injection of 1 microgram E . coli endotoxin, and during heat-induced hyperthermia . All rabbits receiving endotoxin developed fever with peaks at one hour (delta T = 1.1 degrees C) and three hours (delta T = 1.4 degrees C); they then defervesced to base levels at 6 hr . Similar temperature elevations occurred with heat-induced hyperthermia . Results show that endotoxin-induced fever produces changes similar to those reported during infections to man, and more rapidly than previously recognized . These include a prompt decrease in T3, reciprocal rise in rT3, and an initially reduced T4 that rebounds above basal levels . These findings may represent suppressed TSH release, alteration of peripheral monodeiodination of T4 from T3 to rT3, or enchanced clearance of T3 . Heat-induced hyperthermia, except for slight decrease in T4 at 6 and 24 hr, had little effect on thyroid hormones. J Bacteriol, 1980 Dec, 144(3), 904 - 16 Characterization of promoter-cloning plasmids: analysis of operon structure in the rif region of Escherichia coli and isolation of an enhanced internal promoter mutant; An G et al.; Using the promotor-cloning vehicle described by An and Friesen (J . Bacteriol . 140:400-410, 1979), Escherichia coli chromosomal deoxyribonucleic acid fragments derived from the lambda drifd18 transducing phage were cloned in one of several unique restriction endonuclease sites adjacent to tetracycline(tet) genes that lack their own promotor . One of these plasmids has been used to isolate nine variants having mutations that lie in a putative internal promoter which is located between rplL and rpoB . Deoxyribonucleic acid sequence analysis revealed that, in all nine mutants, a single base change, C to T, in the ribonucleic acid polymerase recognition site led to a large increase in promoter activity . Analysis of a variety of plasmids in which tet is fused to various promoters yielded the following results: (i) rplK and rplA, genes for ribosomal protein L11 and L1, respectively, were cotranscribed from a common promoter located upstream from rplK; (ii) there was a strong promoter in the region between the rplKA operon and rplJ, the gene for ribosomal protein L10; (iii) an attenuator region was located between rplL, the gene for ribosomal protein L12, and rpoB, the gene for ribonucleic acid polymerase subunit beta; (iv) transcription terminated immediately after rpoC, the gene for ribonucleic acid polymerase subunit beta'; (v) a gene coding for unknown protein U, which is located between tufB and the rplKA operon, had its own promoter; (vi) the tufB gene was separated from all of the genes described above and had its own promoter. J Bacteriol, 1980 Dec, 144(3), 1205 - 7 Suppression of polarity in the gal operon by ultraviolet irradiation; Fassler JS et al.; The polarity of nonsense mutations in the galE gene of Escherichia coli can be suppressed by rho mutations (O . Reyes et al., J . Bacteriol . 126:1108-1112, 1976) . We show here that this polarity can also be suppressed by ultraviolet irradiation . The effect is analogous to that already observed for polar nonsense mutations in phi X174 and S13 and suggests that ultraviolet irradiation suppression of polarity may be a general phenomenon. J Bacteriol, 1980 Dec, 144(3), 1182 - 5 OmpC and LamB proteins can serve as substitute receptors for host range mutants of coliphage TuIa; Moreno F et al.; Coliphage TuIa, which uses the OmpF protein as a receptor, can adapt by mutation to using instead the OmpC or LamB protein, or both . Most of the phage mutants retained the ability to use the OmpF protein, when present, but one class of mutants lost this ability and could only use the OmpC protein. J Bacteriol, 1980 Dec, 144(3), 1176 - 8 Simplified fluctuation test to distinguish liquid holding recovery from cell multiplication in ultraviolet-irradiated Escherichia coli; Aragao BR et al.; Improved plating techniques and a fluctuation test have been used to distinguish between liquid holding recovery of ultraviolet-irradiated cells and cell multiplication in Escherichia coli . Among tested strains, only recA mutants showed true recovery. J Bacteriol, 1980 Dec, 144(3), 1024 - 33 Protein identifications on O'Farrell two-dimensional gels: locations of 55 additional Escherichia coli proteins; Phillips TA et al.; The resolution of proteins from whole-cell homogenates by two-dimensional gel electrophoresis is sufficiently reproducible and precise to permit different laboratories to exchange information about them . To the previous total of 81 we add the locations of 55 Escherichia coli proteins determined with the aid of purified proteins and mutant strains supplied by many investigators . The criteria used to establish the identifications of protein spots include migration with marker proteins, altered position or amount in appropriate mutant or plasmid-carrying strains, physiological behavior, and peptide map pattern. J Gen Microbiol, 1980 Dec, 121(2), 381 - 6 The susceptibility of strains of Mycobacterium tuberculosis to catalase-mediated peroxidative killing; Jackett PS et al.; At low pH and with continuous low concentrations of hydrogen peroxide generated in situ, catalase was able to replace peroxidase in the peroxidase/hydrogen peroxide/iodide microbicidal system . The system was effective against Escherichia coli and Mycobacterium tuberculosis . Iodide could not be replaced by chloride . The system was effective in lactate buffer, but not in citrate/phosphate buffer . Strains of M . tuberculosis with high and low virulence were equally susceptible . The observations are discussed in the context of an involvement of host-cell catalase in a possible intracellular killing mechanism against M . tuberculosis. Genetics, 1980 Dec, 96(4), 1007 - 17 On the evolution of new metabolic functions in diploid organisms; Hall BG; Evolution of lactose utilization via the ebg system of Escherichia coli requires both structural gene (ebgA) and regulatory gene (ebgR) mutations . Because evolution of new metabolic functions in diploids might be subject to constraints not present in haploid organisms, merodiploid strains carrying a wild-type and an evolved ebgA allele, or a wild-type and an evolved ebgR allele were constructed . I show that heterozygosity at ebgA does not significantly affect the selective advantage of the evolved ebgA allele; whereas heterozygosity at ebgR eliminates the selective advantage of the evolved ebgR allele . Is is suggested that, in diploid organisms, evolution of new functions for systems under negative control would be very difficult. J Biochem (Tokyo), 1980 Dec, 88(6), 1785 - 92 A radioimmunoassay for guanosine-5'-diphosphate-3'-diphosphate and adenosine-5'-triphosphate-3'-diphosphate; Hamagishi Y et al.; A radioimmunoassay for guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and adenosine-5'-triphosphate-3'-diphosphate (pppApp) has been developed . The assay method is based on competition of an unlabeled highly phosphorylated nucleotide with 3H-labeled highly phosphorylated nucleotide for binding sites on a specific antibody . Antibodies to ppGpp and pppApp were obtained by immunizing rabbits with the antigen prepared by conjugating ppGpp with human serum albumin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and with the antigen prepared by conjugating 8-(6-aminohexyl)amino-adenosine-5'-triphosphate-3'-diphosphate with human serum albumin using glutaraldehyde, respectively . Antibody-bound 3H-labeled highly phosphorylated nucleotides were separated from the free 3H-labeled highly phosphorylated nucleotides by selective adsorption on dextran-coated charcoal . Displacement plots were linear over a concentration range of 5-1,000 pmol/assay tube in a log-probit percentage plot . Application of this method to biological systems offers improved accuracy and convenience compared with the previous 32PO4-labeling technique. J Bacteriol, 1980 Dec, 144(3), 1048 - 60 Genetic and biochemical properties of Escherichia coli mutants with defects in serine chemotaxis; Hedblom ML et al.; In Escherichia coli, taxis to certain chemoeffectors is mediated through an intrinsic membrane protein called methyl-accepting chemotaxis protein I (MCP I), which is the product of the tsr gene . Mutants were selected that are defective in taxis toward all MCP I-mediated attractants (alpha-aminoisobutyrate, L-alanine, glycine, and L-serine) but are normal to MCP I-mediated repellents and to chemoeffectors mediated by other MCPs . The mutants could be divided into two classes based on their ability to respond to various concentrations of L-serine . Two MCP I-mediated L-serine systems appear to function in the wild type: one of high and one of lower affinity . The mutations responsible for the serine taxis defects map at about 99 min on the E . coli chromosome and are not complemented by episomes carrying mutations in the tsr gene; this suggests that they are defective in tsr function . Low concentrations of L-{14C}serine specifically bound to wild-type membranes with a Km of 5 microM; in contrast, there was greatly decreased binding to vesicles prepared from the new mutants or from the tsr mutant AW518 . Binding of labeled serine to wild-type vesicles was inhibited by MCP I-mediated attractants, but not by MCP II-mediated attractants . The data suggest that MCP I may function as the L-serine chemoreceptor in E . coli. J Gen Microbiol, 1980 Dec, 121(2), 277 - 92 Molecular cloning of the pyruvate dehydrogenase complex genes of Escherichia coli; Guest JR et al.; The three components of the pyruvate dehydrogenase complex of Escherichia coli are encoded by three linked genes, ace E (pyruvate dehydrogenase, E1), aceF (dihydrolipoamide acetyltransferase, E2) and lpd (lipoamide dehydrogenase, E3, situated close to the nadC (quinolinate phosphoribosyltransferase) and aroP (general aromatic amino acid permease) genes with the gene order: nadC-aroP-aceE-aceF-lpd . Several types of transducing phages, lambda nadC and lambda lpd, carrying the nadC and lpd genes were isolated from populations of artificially constructed transducing phages containing R.HindIII or R.EcoRI fragments of bacterial DNA, by selecting for their ability to complement the metabolic lesions of the corresponding mutants . The cloned fragments were extended to include a functional ace operon by in vivo methods involving prophage insertion into the nadC-lpd region and aberrant excision to yield lambda nadC-lpd and lambda lpd-ace phages . These contained overlapping segments of bacterial DNA capable of expressing the aceE, aceF and lpd genes . A physical map of a 20 kilobase pairs (kb) segment of bacterial DNA encoding the entire nadC-lpd region, bounded by R.HindIII and R.EcoRI targets and possessing several internal restriction targets, R.HindIII (3) and R.EcoRI (2), was constructed . Using a combination of nutritional and enzymological studies with dilysogens and genetic analysis with ace mutants the approximate positions of the genes specifying the pyruvate dehydrogenase complex were traced to a 9.5 kb segment of the restriction map . The cloned lpd gene was expressed in the complete absence of a functional ace operon and when the major lambda promoters were repressed . This confirms that the lpd gene can be independently transcribed from its own promoter. Zh Mikrobiol Epidemiol Immunobiol, 1980 Dec, (12), 21 - 4 {Formation of specifically transducing lambda dg-particles . II . Relationship between the transduction rate, the qualitative composition of the transductants, and the dose of the inducing agent}; Amirov EIa; The formation of specifically transducing lambda dg-particles in E.coli strain W3110 (lambda) due to the effect of different doses of UV irradiation was studied . A sharp increase in both the absolute number of transductants and the relative transduction frequency was found to occur as the dose of UV irradiation increased . On achieving definite doses this relationship became almost direct . At higher doses similarity was observed in the kinetics of a decrease in the absolute number of transductants and in active phage titers . As the dose increased further, a sharp increase in the percentage of heterogenoue transductants occurred . The conclusion that the formation of specifically transducing lambda dg-particles is due to the effect of the inducing factor on the DNA of lysogenic cells has been made. Zh Mikrobiol Epidemiol Immunobiol, 1980 Dec, (12), 17 - 20 {Formation of specifically transducing lambda alpha g-particles . I . Relationship between the quantitative composition of transductants and the type of inductive activity}; Amirov EIa; To study the formation of specifically transducing particles, different physical and chemical prophage-inducing factors (spontaneous induction, UV and roentgen irradiation, treatment with mitomycin C and nalidixic acid) . Each kind of induction used in this study was shown to bring about the formation of transducing particles . The kind of inducing action influenced the qualitative composition of transductants . DNA ruptures caused by inducing factor may be supposed to play the main role in the formation of specifically transducing lambda dg-genomes. Gene, 1980 Dec, 12(3-4), 267 - 75 Single-strand and double-strand cleavage at half-modified and fully modified recognition sites for the restriction nucleases Sau3a and Taqi; Streeck RE; The influence of cytosine methylation on the cleavage of DNA by the restriction nucleases Sau3A and TaqI has been investigated . Bovine satellite DNA fragments containing a GATCGA sequence, i.e . a Sau3A site overlapping with a TaqI site have been used in this study . The methylation of these fragments has been determined by sequence analysis . It has been found that a TaqI site (TCGA) methylated at cytosine in both DNA strands is still sensitive to double-strand cleavage . A Sau3A site (GATC), however, is rendered resistant to double-strand cleavage by methylation of a single cytosine . Fragments containing the "half-modified" Sau3A site are nicked in the unmethylated DNA strand . It has been shown by sequence analysis of nicked DNA that the single-strand break occurs at the same position which is cleaved in unmodified DNA. Gene, 1980 Dec, 12(3-4), 201 - 14 Functional analysis of hybrid plasmids carrying genes for lambda site-specific recombination; Strizhov N et al.; A number of hybrid plasmids, carrying lambda genes involved in site-specific integrative recombination, have been constructed in vitro . Analysis of protein synthesis in Escherichia coli minicells has shown that Int protein is synthesized only when int gene is expressed constitutively . The plasmids RSF2124::lambda-CD, RSF2124::lambda-Cint-c57, and pInt lambda were able to integrate into the chromosome of E.coli at the attB . The integration of hybrid plasmids into the genome of bacteria has also been shown for polA1 strains restricting the autonomous replication of ColE1 type plasmids . Genetic markers of hybrid plasmids are maintained in polA1 bacteria for at least 50 generations under nonselective conditions . The Southern blotting experiments using {32P}pBR322 DNA and EcoRI fragments of E . coli polA1 chromosome carrying integrated plasmid pInt lambda demonstrated that in this strain hybrid plasmids can be observed only when integrated into the attB of the chromosome according to Campbell's model of integration . In the cells, where autonomous replication of plasmids is possible, they can be observed both in extrachromosomal and integrated states . The integration of the ColE1 replication origin into the chromosome of bacteria is not lethal for the cells . Only attP and the int gene of lambda are necessary for the integration of hybrid plasmids under conditions of effective int gene expression . If the level of Int protein synthesis is high enough, the prophage excision can be observed in the absence of Xis product . The six-fold decrease of Int protein concentration in the cell (in case of pInt lambda 2 as compared to pInt lambda 1) is critical both for integration and excision. Can J Microbiol, 1980 Dec, 26(12), 1508 - 11 Mannose utilization in Escherichia coli requires cyclic AMP but not an exogenous inducer; Fraser AD et al.; It has been clarified whether the utilization of mannose by Escherichia coli requires adenosine 3',5'-cyclic monophosphate (cyclic AMP) . Using an adenylyl cyclase deficient mutant (CA8306B) and a cyclic AMP receptor protein (CRP) deficient mutant (5333B) we have shown that the utilization of mannose is dependent on the cyclic AMP - CRP complex . 2-Deoxyglucose (DG) is a nonmetabolizable glucose analog specific for the phosphotransferase system (PTS) which transports mannose (termed here PTSM) . Growth of CA8306B on glycerol is unaffected by addition of the analog, whereas growth of the strain on glycerol plus cyclic AMP ceases immediately upon addition of DG . These results suggest that the formation of PTSM is dependent on cyclic AMP . In addition, CA8306B grown on glycerol plus cyclic AMP can immediately utilize mannose when transferred to a medium containing mannose as a sole carbon source, whereas the same strain grown on glycerol without cyclic AMP cannot utilize mannose when so transferred . The results suggest that the formation of PTSM does not require an exogenous inducer. Proc Natl Acad Sci U S A, 1980 Dec, 77(12), 7176 - 80 Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903; Grindley ND et al.; The kanamycin resistance transposon Tn903 consists of a unique region of about 1000 base pairs bounded by a pair of 1050-base-pair inverted repeat sequences . Each repeat contains two Pvu II endonuclease cleavage sites separated by 520 base pairs . We have constructed derivatives of Tn903 in which this 520-base-pair fragment is deleted from one or both repeats . Those derivatives that lack both 520-base-pair fragments cannot transpose, whereas those that lack just one remain transposition proficient . One such transposable derivative, Tn903 delta I, has been selected for further study . We have determined the sequence of the intact inverted repeat . The 18 base pairs at each end are identical and inverted relative to one another, a structure characteristic of insertion sequences . Additional experiments indicate that a single inverted repeat from Tn903 can, in fact, transpose; we propose that this element be called IS903 . To correlate the DNA sequence with genetic activities, we have created mutations by inserting a 10-base-pair DNA fragment at several sites within the intact repeat of Tn903 delta 1, and we have examined the effect of such insertions on transposability . The results suggest that IS903 encodes a 307-amino-acid polypeptide (a "transposase") that is absolutely required for transposition of IS903 or Tn903. Proc Natl Acad Sci U S A, 1980 Dec, 77(12), 7029 - 33 Cell-free coupled transcription-translation system for investigation of linear DNA segments; Yang HL et al.; Heretofore the DNA-directed coupled transcription-translation system, most useful in gene expression analysis, has been limited to the use of circular or long linear DNAs . Linear DNAs are degraded in this system by an exonucleolytic activity that can be eliminated by making the synthetic extracts from a suitable recB mutant of Escherichia coli . Using these extracts, we have examined the gene expression of a variety of linear DNAs . In particular, the complex pattern of expression of ribosomal protein genes and RNA polymerase genes in the rpoBC-rplLJ region has been analyzed by comparing the protein products obtained when using lambda rifd18 DNA with the product obtained when using the same DNA segmented with various restriction enzymes . The results obtained confirm the conclusions of others obtained by much more elaborate in vivo techniques . It seems highly likely that this cell-free system will have extensive applications in the area of analysis of gene expression. Proc Natl Acad Sci U S A, 1980 Dec, 77(12), 7005 - 9 Organization of unc gene cluster of Escherichia coli coding for proton-translocating ATPase of oxidative phosphorylation; Kanazawa H et al.; The proton-translocating ATPase (F1-F0) of oxidative phosphorylation (ATP phosphohydrolase, EC 3.6.1.3) is coded for by a set of structural genes comprising the unc operon in Escherichia coli . We have analyzed several new transducing phages and plasmids carrying various lengths of the DNA segments of the unc operon by complementation assay using 14 new unc- mutants and representatives of previously described strains which were made available to us . Transducing phages carrying parts of the unc gene cluster were isolated: lambda uncA-9 and lambda glmS phages converted only some of the unc- mutants to the Unc+, as determined by complementation assays . A new hybrid plasmid (pMCR533) carrying part of the unc operon was constructed by inserting the HindIII fragment of lambda asn-5 DNA (a phage carrying the entire unc operon) into the unique HindIII site of pBR322 . This plasmid transformed eight unc- strains to Unc+, including uncB402 and uncA401, but did not complement uncD11 or four other strains . Two minichromosomes which carry the E . coli replication origin were also tested: plasmid pNH05 transformed the uncB402 but not the uncA401 strain to Unc+, whereas plasmid pMCF1 transformed none of the mutants tested . Analysis of the DNAs from these transducing phages and plasmids with restriction endonucleases suggested that all of the structural genes for the F1-F0 complex are localized within a DNA segment of approximately 4.5 megadaltons containing two EcoRI sites . The approximate locations of the unc- mutations were mapped on this DNA segment. Gene, 1980 Dec, 12(1-2), 51 - 61 Relief of polarity caused by transposon Tn5: application in mapping a cloned region of the Escherichia coli uvrB locus essential for UV resistance; Pannekoek H et al.; The structure and function of recombinant plasmid pNP5, which consists of vector pMB9 and a 2.5 kb EcoRI fragment harbouring the Escherichia coli uvrB gene, has been investigated . Insertional inactivation with the transposons Tn1 (Apr) or Tn5 (Kmr) has been used to determine the region on pNP5 DNA that is essential for UV resistance in uvrB deletion strains . This region spans approx . 1.8 kb and is separated by at least 280 bp from the pMB9 promoter to which it has been fused . Furthermore, a procedure is described to eliminate the polarity exerted by the transposon Tn5 . A combination of in vitro digestion of pNP5::Tn5 DNA with restriction endonuclease XHoI, followed by ligation and subsequent in vivo propagation of the resulting plasmid DNA yields predominantly pNP5 molecules with a site-specific nonpolar mutation . The method allows an investigation of cloned complex genetic units, such as operons. Gene, 1980 Dec, 12(1-2), 171 - 4 The nucleotide sequence recognized by the BstEII restriction endonuclease; Lautenberger JA et al.; The restriction site for the BstEII endonuclease is characterized by the heptamer sequence: {formula: see text} with five nucleotide long cohesive termini. Gene, 1980 Dec, 12(1-2), 165 - 70 The ilvG gene is expressed in Escherichia coli K-12; Berg CM et al.; Three genes code for isozymes of acetohydroxy acid synthase (AHAS) in Escherichia coli K-12 . To test the previously published supposition that one of them, ilvG, is silent in ilvO+ strains, we isolated mutants which had deletions of various lengths in the ilvGEDA operon . Some of these mutants have severely reduced levels of AHAS activity . We conclude that ilvG is expressed in ilvO+ strains but is deleted in these mutants . In addition, we find that AHAS II, the ilvG gene product, is sensitive to feedback inhibition by valine . We hypothesize that ilvO- mutations are ilvG frameshift mutations which render AHAS II valine resistant and enhance transcription of distal genes. J Virol, 1980 Dec, 36(3), 878 - 82 Cloned human polyomavirus JC DNA can transform human amnion cells; Howley PM et al.; The genome of the human polyomavirus JC (Mad-1 strain) was molecularly cloned in Escherichia coli by using the plasmid vector pBR322 . Recombinant DNA molecules were constructed with the entire JC genome inserted either at its unique EcoRI site at 0.0 map units or at its unique BamHI site at 0.51 map units . Viral DNA from each of these recombinant plasmids was capable of transforming human amnion cells, and cell lines established from transformed foci were positive for JC tumor antigen as assayed by indirect immunofluorescence. Eur J Biochem, 1980 Dec, 113(1), 33 - 8 Opening of potassium channels in Escherichia coli membranes by thiol reagents and recovery of potassium tightness; Meury J et al.; The retention of high potassium levels in Escherichia coli is not dependent on intact energy metabolism, since without the presence of a carbon source or in the presence of energy inhibitors significant K+ gradients can be maintained . In contrast, with 0.5 mM N-ethylmaleimide, K+ depletion is immediate and complete . As a final result, intracellular K+ is approximately three times more concentrated than the K+ in the medium . Increase of K+ in the medium is immediately followed by K+ uptake whereas in the unpoisoned state only an increase in the osmotic pressure of the medium would result in an increase of the K+ pool . The intracellular K+ undergoes continuous turnover in the poisoned cells whereas in intact cells turnover is strictly dependent on the presence of a metabolizable carbon source . After removal of the thiol reagent the cell recovers its capacity to concentrate potassium . The recovery process is inhibited by energy inhibitors or by incubation at low temperature but not by chloramphenicol . It is only slightly slowed down by carbon or sulfur starvation . The leak provoked by N-ethylmaleimide is similar in wild-type E . coli cells when a derepressed kdp uptake system working in the micromolar range of the K+ concentration is responsible for the intracellular pool of K+ and when, in a medium millimolar K+ concentration range, the trkA and trkD systems are predominant. Eur J Biochem, 1980 Dec, 113(1), 223 - 7 'Swell dialysis' demonstrates that adenylate cyclase in Trypanosoma brucei is regulated by calcium ions; Voorheis HP et al.; A new technique, 'swell dialysis', is described that allows the study of cellular enzymes in situ if they are located in the cytoplasm or on the cytoplasmic face of any membrane . The technique is potentially applicable to any population of individual eukaryotic cells that lack a cell wall . In this study swell dialysis has been applied to an examination of the regulation of adenylate cyclase in blood-stream forms of Trypanosoma brucei (427-12/ICI-060) . Adenylate cyclase in this protozoon is shown to be controlled by Ca2+ . There is a 16-fold stimulation of the cyclase at 100 microM Ca2+ over basal activity without added Ca2+ . The ability of Ca2+ to stimulate adenylate cyclase is lost upon rupture of the cell, which is reminiscent of the loss of control of the adenylate cyclase in Escherichia coli by sugars of the phosphotransferase system when cell breakage occurs . The physiological function of the Ca2+ activation of adenylate cyclase in T . brucei has not been established but a possible role in the change of surface coat in bloodstream forms should be considered. Ann Intern Med, 1980 Dec, 93(6), 802 - 5 An immunofluorescence test to detect serum antibodies to Giardia lamblia; Visvesvara GS et al.; We used an indirect immunofluorescence test with Giardia lamblia trophozoites as antigen to detect anti-G . lamblia antibodies in serum . Seventy-one patients and control subjects were studied in a blinded protocol . Titers in 29 of 30 patients with symptomatic giardiasis (1:16 to 1:1024) did not overlap titers in 19 healthy control subjects (1:2 to 1:4); titers in 15 patients with hookworm, Entamoeba histolytica, or intestinal bacterial overgrowth were 1:16 or less Absorption of giardiasis patients' sera with G . lamblia trophozoites but not with E . histolytica, Trichomonas vaginalis, or Escherichia coli reduced the titers to, or nearly to, control values . Titers in individual sera were 93.9% reproducible within a fourfold or less dilution . Our results indicate that G . lamblia, an intestinal parasite often regarded as noninvasive, induces a systemic antibody response . The indirect immunofluorescence test for anti-G . lamblia antibodies is specific and reproducible; it may be useful in epidemiologic and immunologic studies of giardiasis. J Bacteriol, 1980 Dec, 144(3), 923 - 32 Location of two relaxation nick sites in R6K and single sites in pSC101 and RSF1010 close to origins of vegetative replication: implication for conjugal transfer of plasmid deoxyribonucleic acid; Nordheim A et al.; A nick-labeling method has been used to localize the relaxation complex nick sites in three plasmids (pSC101, RSF1010, and R6K) that differ markedly in their host range, deoxyribonucleic acid replication, and conjugal transfer properties . Single specific relaxation sites were located in pSC101 and RSF1010, but surprisingly two distinct sites could be identified in the bi-origin plasmid R6K . In all cases, relaxation nick sites, which are thought to be origins of plasmid conjugal transfer, were shown to be located near origins of vegetative replication . This result suggests a functional interaction between these two types of deoxyribonucleic acid loci, and we speculate here that application events initiated at origins of replication may constitute an integral part of the process of conjugal transfer of small plasmids among bacteria . Consistent with this proposal is the finding that inhibition of vegetative replication of the pSC101 and ColE1 plasmids results in a severe inhibition of their conjugal transfer ability. J Bacteriol, 1980 Dec, 144(3), 1208 - 11 The ebg operon consists of at least two genes; Hall BG et al.; The ebg operon of Escherichia coli includes a second gene designated ebgB . The ebgB gene product is a 79,000-molecular-weight protein and is expressed coordinately with the ebgA gene product, ebg beta-galactosidase . Insertion of the transposable elements Tn5 and Tn9 into ebgA eliminates the expression of ebgB, suggesting that ebgB is distal to ebgA . Ultraviolet light mapping confirms that gene order . The function of the ebgB gene product is unknown. Sci Rep Res Inst Tohoku Univ {Med}, 1980 Dec, 27(1-4), 1 - 9 Partial purification of a stimulatory factor of RNA polymerase B in nonhistone proteins; correlation with nuclear protein kinase; Kikuchi H et al.; A stimulatory factor of DNA-dependent RNA polymerase B (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) in nonhistone proteins was partially purified from rat liver nuclei on a column of daunomycin-CH Sepharose 4B and of phosphocellulose . In the process of purification, the stimulatory factor was separated from the main fraction of nuclear protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) . This factor enhanced specifically the activity of RNA polymerase B on rat liver DNA as template and did not affect RNA polymerase A and Escherichia coli RNA polymerase at all . The polynucleotide elongation rate was increased by the addition of this factor. Zh Mikrobiol Epidemiol Immunobiol, 1980 Dec, (12), 86 - 91 {Stimulation of antibody formation in mice by endogenous and exogenous interferon}; Zheleznikova GF et al.; The effect of 2 types of interferon, virus-induced and endotoxin-induced, on the dynamics of antibody production in the immunization of mice with sheep red blood cells (SRBC) or E . coli vaccine was studied . Both types of interferon stimulated the production of antibodies against SRBC and, less effectively, against E . coli . This effect was found to depend on the dose of interferon (in a limited range) . The immunostimulating titer of sera was comparable with their antivirus titer . These 2 types of interferon stimulated both primary and secondary response of mice to the injection of SRBC. Proc Natl Acad Sci U S A, 1980 Dec, 77(12), 7410 - 4 Anti-AMP antibody precipitation of multiply adenylylated forms of glutamine synthetase from Escherichia coli: a model relating both concentration and density of antigenic sites with the antibody-antigen interaction; Hohman RJ et al.; Sheep antibodies directed against an AMP-bovine serum albumin conjugate exhibit highly specific binding toward AMP . These antibodies bind to the AMP moiety of adenylylated glutamine synthetase {L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2} from Escherichia coli and to no other antigenic determinant on the protein . E coli glutamine synthetase can exist in variously modified (isomeric) forms that differ with respect to the number (0-12) and the distribution of identically adenylylated subunits {Ciardi, J . E., Cimino, F . & Stadtman, E . R . (1973) biochemistry 12, 4321-4330} . Using this enzyme, together with the AMP-specific antibodies, we have investigated the effects of the total concentration, population density, and topographical distribution of multiple identical antigenic determinants on the antigen-antibody interaction . Stopped flow fluorescence measurements show that the rate and extent of initial binding of the antibodies to the antigen are a function of the total concentration of AMP groups and are independent of the number of AMP groups der dodecamer . However, the rate of lattice formation increases with increasing epitope density, and the maximal amount of glutamine synthetase precipitated is directly proportional to the average number of adenylylated subunits per dodecamer . The data suggest that partially adenylylated enzyme preparations are composed of subpopulations of glutamine synthetase molecules that differ in their tendency to form precipitable aggregates, due presumably to differences in the topographical distribution of antigenic determinants on the surface of the enzyme . The enzyme species that form soluble immune complexes do so possibly due to intramolecular crosslinkage of the bivalent antibodies with adenylylated subunits to the exclusion of intermolecular crosslinkage. Proc Natl Acad Sci U S A, 1980 Dec, 77(12), 7152 - 6 Two interferon mRNAs in human fibroblasts: in vitro translation and Escherichia coli cloning studies; Weissenbach J et al.; Two mRNA species that produce biologically active interferon were isolated from human fibroblasts and studied by size fractionation and cloning in Escherichia coli plasmid pBR322 . The major fibroblast interferon (Hu IFN-beta 1) is coded for by the smaller of the two mRNAs, an 11S species, 900 nucleotides long, which in cell-free systems yields a 20,000 Mr protein . The second interferon mRNA species (Hu IFN-beta 2) is 14S, about 1300 nucleotides long, and codes for another protein of 23,000-26,000 Mr . The two interferon mRNAs do not cross-hybridize . Both are induced by poly(rI.rC), but IFN-beta 2 mRNA is induced to about 10% in cells by cycloheximide treatment alone whereas under these conditions IFN-beta 1 is not induced. Mol Biochem Parasitol, 1980 Dec, 2(2), 93 - 108 Kinetoplast DNA and RNA of Trypanosoma brucei; Simpson AM et al.; Kinetoplast DNA (kDNA) and kinetoplast RNA (kRNA) were isolated from bloodstream and procyclic culture forms of two clonal strains of Trypanosoma brucei . No differences were observed in kDNA (maxicircle) restriction profiles between bloodstream or procyclic culture forms of the same strain . Some differences were observed in kDNA maxicircle restriction sites between the two strains . Buoyant density analysis of Pst I digested kDNA showed the release of a minor low density band representing unit length linearized maxicircle DNA . Pst I or Bam H1-linearized maxicircle DNA was isolated by the Hoechst 33258 dye--CsCl method and a restriction enzyme map of the maxicircle was constructed . Closed monomeric minicircles released from kDNA networks by sonication sedimented with a molecular size of around 1100 base pairs . A substantial minor length heterogeneity was evident in acrylamide gel electrophoresis of once cut minicircles . Several minicircle sequence classes and two Hind III maxicircle fragments representing approx . 50% of the maxicircle were cloned in the bacterial plasmid, pBR322, in Escherichia coli . A purified kinetoplast-mitochondrion fraction was isolated from procyclic culture forms by the Renografin flotation method . The major kRNA components were two small RNAs which comigrated with Leishmania tarentolae 9 and 12 S kRNAs in denaturing gels . These RNAs hybridized to the maxicircle component of the kDNA, specifically to the smaller cloned Hind III maxicircle fragment . This cloned fragment had substantial sequence homology with the cloned maxicirc