Cell, 1983 May, 33(1), 249 - 60 A novel repair enzyme: UVRABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region; Sancar A et al.; The uvrA, uvrB, and uvrC proteins of Escherichia coli were purified from strains that greatly overproduce these proteins . Using the purified proteins, the UVRABC nuclease was reconstituted in vitro . The reconstituted enzyme acted specifically on DNA damaged with UV, cis-platinum, and psoralen plus near UV . When UV-irradiated DNA was used as substrate, the enzyme made two cuts on the damaged DNA strand, one on each side of the damaged region . The enzyme hydrolyzed the eighth phosphodiester bond on the 5' side of pyrimidine dimers . On the 3' side of pyrimidine dimers, the UVRABC nuclease cut the fourth or the fifth phosphodiester bond 3' to pyrimidine dimers . The oligonucleotide with the damaged bases that is generated by these two cuts was released during treatment with the enzyme . We have also obtained evidence suggesting that the enzyme acts by the same mechanism on PydC photoproducts which are thought to be of primary importance in UV-induced mutagenesis. Cell, 1983 May, 33(1), 241 - 8 Transcription of the S10 ribosomal protein operon is regulated by an attenuator in the leader; Lindahl L et al.; Previous studies have shown that ribosomal protein L4 specifically inhibits the expression of its own operon, the 11-gene S10 operon . To elucidate the mechanism for this regulation, we have examined the effect of protein L4 on transcription of the S10 operon . Hybridization and gel electrophoresis studies indicate that in the presence of excess L4 only RNA molecules about 140 bases long are transcribed from the S10 operon . These short RNA molecules contain the leader, but not structural gene, sequences . Our results suggest that protein L4 stimulates premature termination (attenuation) of transcription about 30 bases upstream from the start of the first structural gene of the S10 operon . The attenuation appears to be independent of the regulation of translation of the operon . We suggest that attenuation of transcription plays a primary role in the autogenous regulation of the S10 operon. Cell, 1983 May, 33(1), 231 - 40 Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation; Randall LL; Accessibility of nascent chains of periplasmic proteins to externally added proteinase K was used as the criterion for translocation of polypeptides across the cytoplasmic membrane of E . coli during the process of export . It is concluded for maltose-binding protein and ribose-binding protein that nascent chains carrying the signal sequence are not accessible to the proteinase while chains that have been matured span the membrane and are degraded . Translocation of polypeptides is a late event relative to extent of elongation, occurring only after maltose-binding protein has reached molecular weight 33,000 (80% of its entire length) and after ribose-binding protein has been fully elongated (molecular weight 29,000) . The data presented here are inconsistent with postulated mechanisms of export requiring a strict coupling of translocation to elongation of nascent polypeptide chains . In contrast, the data support the idea that entire domains of polypeptides are transferred after their synthesis . This is the case whether the translocation of a protein is initiated post-translationally or begins before synthesis of the entire protein is completed. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1983 May, 254(3), 397 - 402 Colicine production and colicine sensitivity typing of nephropathogenic serotypes of Escherichia coli; Nowicki B; Usefulness of colicine sensitivity typing and colicine production typing methods of E . coli strains from UTI was different . Colicine sensitivity typing appeared to be useless because of too high proportion of different types and low reproducibility of results . The advantage of the colicinogenotyping was the possibility of conducting more detailed analysis of strains belonging to one serotype as well as of differentiating a part of NT and Aut strains . Reduction of Abbott-Shannon's set to 6 indicator strains did not limit the practical value of the set for differentiation of E . coli strains . The great number of different colicinogenic types among strains belonging to serotype 06 pointed to nonhomogenecity of this serotype . The colicinogenic pattern 2 being more frequent in UTI was colicine V producer in 93,2% . Serotype 06 and 075 were colicine V producers in smaller percentages than other NPG and NT strains . In the light of our investigations it seems that correlation between the resistance of E . coli to colicines, colicine production and pathogenicity was doubtful. Can J Biochem Cell Biol, 1983 May, 61(5), 287 - 92 Primary structure determination of Escherichia coli heat-stable enterotoxin of porcine origin; Lazure C et al.; The chemical characterization of Escherichia coli heat-stable enterotoxin (ST) is described . The toxin was isolated and purified to homogeneity from the E . coli strain F11 (PI55) of porcine origin . Following quantitative amino acid analysis, the enterotoxin was found to contain 18 amino acids including 6 cysteines, but was devoid of Ser, Val, Met, Ile, Lys, His, and Arg residues . All cysteine residues were found to be involved in disulfide bridges, even though their positions could not be localized . The enterotoxin thus has a molecular weight of 1979 and was shown to be an octadecapeptide with the following sequence: H2N-Asn-Thr-Phe-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys-Tyr- COOH . Its relationship to other known enterotoxins is discussed. Tsitologiia, 1983 May, 25(5), 572 - 80 {Tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a protein in human lymphocytes binding satellite DNA}; Svetlova MP et al.; Interactions between a satellite DNA fragment (SH3, 1.8 kb) cloned in pBR325 plasmid and DEAE-protein fractions from human lymphocytes treated with phorbol-12-myristate-13-acetate (TPA) have been studied by filtration technique through nitrocellulose filters . The 0.3 M fraction was found to have a protein which binds SH3 DNA specifically . Cytogenetic studies have shown an increased frequency of chromosome endoreduplications which may be due to the binding of TPA-induced protein to centromeres. Hoppe Seylers Z Physiol Chem, 1983 May, 364(5), 593 - 606 Synthesis of the mitogenic S-{2,3-bis(palmitoyloxy)propyl}-N-palmitoylpentapeptide from Escherichia coli lipoprotein; Wiesmuller KH et al.; The N-terminal pentapeptide of the lipoprotein from the outer membrane of Escherichia coli was obtained by coupling S-{2,3-bis(palmitoyloxy)-(2RS)-propyl}-N-palmitoyl-(R)-cysteine to O-tert-butylseryl-O-tert-butyl-seryl-asparaginyl-alanine tert-butyl ester followed by deprotection with trifluoroacetic acid . The tetrapeptide was built up from alanine tert-butyl ester with N-9-fluorenylmethyloxycarbonyl protected amino acids . S-{2,3-Bis(palmitoyloxy)propyl}-N-palmitoylcysteine was obtained from N,N'-dipalmitoylcystine di-tert-butyl ester via reduction to the thiol, and S-alkylation with racemic 3-bromo-1,2-propanediol followed by esterification with palmitic acid in the presence of dicyclohexylcarbodiimide/dimethylaminopyridine and deprotection with trifluoroacetic acid . The compounds were characterized unequivocally by 13C-NMR and mass spectra . The diastereomers of S-{2,3-bis(palmitoyloxy)propyl}-N-palmitoylcysteine tert-butyl ester with opposite configuration at the propyl-C-2 atom could be separated on a silica-gel column. J Comput Tomogr, 1983 May, 7(2), 179 - 82 Computed tomographic characteristics of pleural empyema; Shin MS et al.; The computed tomographic characteristics of the pleural empyema were illustrated in three patients . A typical empyema is characterized by lower thorax location, oblong and round contour in cross-section, homogeneous content with or without air spaces or air-fluid levels, regularly thin wall opposing the chest wall (a rim sign), irregularly thick wall opposing the lung parenchyma, and smooth inner and outer margins . By application of such computed tomographic criteria, a pleural empyema can be readily distinguished from a peripheral lung abscess. Radiobiologiia, 1983 May-Jun, 23(3), 318 - 22 {Role of a change in oxygen concentration in modifying in vitro reproductive cell death . 3 . Modification of radiosensitivity by compounds reducing oxygen}; Korystov IuN; The data are discussed concerning the protection of cells in vitro by the oxygen reducing agents . The analysis has demonstrated that the protective effect of sulfites can only be related to hypoxia they create . Hypoxia also contributes considerably to the protective effect of thiols (maximum DMF approximately 3) . However the protective effect of thiols cannot wholly be attributed to hypoxia . The oxygen--independent component of the protective effect of thiols (maximum DMF approximately 1.5) is conditioned by the metabolic changes increasing the enzymic repair of potential damages. Plasmid, 1983 May, 9(3), 286 - 97 Escherichia coli minichromosomes containing the pSC101 partitioning locus are stably inherited; Hinchliffe E et al.; The par region of pSC101, required in cis to promote its stable inheritance, was joined, in combination with the tetr determinant of pBR325, to large and small minichromosomes . These hybrid minichromosomes were examined for stability and found to be no more stable than their parent minichromosomes . Indeed, one recombinant plasmid, pEH21, showed reduced stability, which was not attributable to a reduced copy number . Neither pEH21 nor pEH22, a plasmid composed of the same DNA arranged differently, was stabilized by the presence of a Par+ pSC101 derived replicon in the same cell . We conclude that the par region of pSC101 does not stabilize minichromosomes. Arch Biochem Biophys, 1983 May, 223(1), 319 - 24 Effect of ppGpp on the accuracy of protein biosynthesis; Dix DB et al.; The maintenance of accuracy in protein biosynthesis in amino acid-starved rel+ strains of Escherichia coli has been attributed to an effect of ppGpp on the accuracy of aa-tRNA selection by the ribosome . It has been determined that concentrations of ppGpp characteristic of those found in amino acid-starved cells have no effect on the rate of reaction of poly(U)-programmed ribosomes with either the cognate (Phe) or the near-cognate (Leu2) ternary complexes . Neither the rate of GTP hydrolysis, which signals selection of the ternary complex, nor the rate of peptide formation, which signals the acceptance of the aa-tRNA after proofreading, is affected by the nucleotide . The results indicate that the effect of ppGpp in maintaining the accuracy of protein biosynthesis in cells starved for an amino acid is not due to a direct effect on the rate constants for substrate selection by the ribosome. Proc Soc Exp Biol Med, 1983 May, 173(1), 27 - 31 Influence of the genotype of mice on the effect of interferon on phagocytic activity of macrophages; Degre M et al.; The influence of genotype on interferon effect on phagocytic activity of unstimulated mouse peritoneal macrophages (MPM) was studied in in vitro experiments . Treatment of MPM from BALB/c and ICR mice with mouse fibroblast interferon (MuIFN-beta) enhanced the ingestion of non-opsonized Escherichia coli . This effect was dose-dependent and neutralized by anti-interferon globulin . MPM from C57B1/6 mice were not stimulated by the same treatment . Treatment of MPM with pH 2-sensitive immune interferon (MuIFN-gamma) depressed the ingestion independently on the genotype of mice. Proc Natl Acad Sci U S A, 1983 May, 80(10), 2907 - 11 A computer model analysis of the active-site coupling mechanism in the pyruvate dehydrogenase multienzyme complex of Escherichia coli; Hackert ML et al.; A computer modeling system developed to analyze experimental data for inactivation of the Escherichia coli alpha-ketoglutarate dehydrogenase complex (KGDC) accompanying release of lipoyl moieties by lipoamidase and by trypsin {Hackert, M.L., Oliver, R.M . & Reed, L.J . (1983) Proc . Natl . Acad . Sci . USA 80, 2226-2230} was used to analyze analogous data for the E . coli pyruvate dehydrogenase complex (PDC) . The model studies indicate that the activity of PDC, as found for KGDC, is influenced by redundancies and random processes, which we describe as a multiple random coupling mechanism . In both complexes more than one lipoyl moiety services each pyruvate dehydrogenase (EC 1.2.4.1) or alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) (E1) subunit, and an extensive lipoyl-lipoyl interaction network for exchange of electrons and possibly acyl groups must also be present . The best fit between computed and experimental data for PDC was obtained with a model that has four lipoyl domains with four or, more probably, eight lipoyl moieties servicing each E1 subunit . The lipoyl-lipoyl interaction network for PDC has lipoyl domain interactions similar to those found for KGDC plus the additional possibility of interaction of a lipoyl moiety and its paired mate on each dihydrolipoamide acetyltransferase (EC 2.3.1.12) (E2) subunit . The two lipoyl moieties on an E2 subunit in PDC appear to be functionally indistinguishable, each servicing the acetyltransferase site of that E2 subunit and a dihydrolipoamide dehydrogenase (EC 1.6.4.3) (E3) subunit if the latter is bound to that particular E2 subunit . The observed difference between inactivation of PDC by lipoamidase and by trypsin appears to be due to dead-end competitive inhibition by lipoyl domains that have been modified by excision of lipoyl moieties by lipoamidase. Proc Natl Acad Sci U S A, 1983 May, 80(10), 2889 - 93 Shape determinations of ribosomal proteins in situ; Nierhaus KH et al.; Ribosomes are heterogeneous for neutrons because RNA and proteins have different neutron-scattering-length densities . This heterogeneity is an obstacle to the shape determination of single ribosomal components within the ribosome . Therefore, we homogenized (matched) the neutron-scattering-length densities of RNA and proteins . 23S and 5S RNA from the large ribosomal subunit were isolated from cells grown in a medium containing 76% 2H2O . The total protein fraction of the large ribosomal subunit was isolated from cells grown in a medium containing 84% 2H2O . When these constituents were used for total reconstitution of 50S subunits, neutron scattering measurements of the reconstituted particles revealed excellent matching near 100% 2H2O . A three-step reconstitution procedure was developed that allowed the reconstitution of 50S subunits from deuterated RNA, deuterated total (i.e., unfractionated) proteins, and single protonated proteins . The reconstituted particles contain one protonated protein or two in a matched ribosomal matrix and were used for shape determination or distance measurement of mass centers of gravity, respectively . The signal/noise ratio is high enough to allow measurement in solutions containing nearly 100% 2H2O at concentrations of only 300-500 A260 nm units/ml . Our experiments have proved the feasibility of our biochemical strategy . The shape determinations of ribosomal proteins in situ gave radii of gyration for L1, L3, L4, and L23 of 26 +/- 2, 22 +/- 2, 20 +/- 2, and 13 +/- 2 A, respectively. Mutat Res, 1983 May-Jun, 117(3-4), 259 - 69 The optimal design of batteries of short-term tests for detecting carcinogens; Heinze JE et al.; The optimal design of a battery of short-term tests for detecting carcinogens has been explored by constructing tables describing the theoretical accuracy of various batteries of tests . The analysis has revealed that the expected accuracy of the testing battery is greatly influenced by the design of the battery, particularly by the number of tests in the battery and by the number of tests which must give a positive result before the compound is considered positive in the battery . The recommended optimal design is not affected by the expected accuracy of the tests comprising the battery, at least not for reasonably accurate short-term tests . Two types of analyses of published data have been presented in support of these theoretical considerations . The first is a statistical analysis (chi-square test) showing that most short-term tests independently detect carcinogens and noncarcinogens, thus fulfilling a key assumption for the use of batteries of short-term tests to improve the detection of carcinogens and noncarcinogens . The second comes from the results of a recent international collaborative study showing that the recommended design of testing batteries would optimize the identification of carcinogens and noncarcinogens by five of the most accurate tests in the study. Surgery, 1983 May, 93(5), 653 - 9 Mechanisms of the adjuvant effect of hemoglobin in experimental peritonitis . VI . Effects of stroma-free hemoglobin and red blood cell stroma on mortality and neutrophil function; Dunn DL et al.; Hemoglobin is known to increase the lethality of experimental E . coli peritonitis, but its mechanism of action has not been defined . Some evidence from this laboratory previously suggested that hemoglobin could interfere with local leukocyte function in the peritoneal cavity . In the present study we compared the effect of impure bovine hemoglobin, highly purified stroma-free hemoglobin, and red blood cell stroma on the mortality rate of rats with Escherichia coli peritonitis, as well as on human neutrophil function in vitro . Both hemoglobin preparations markedly increased the mortality rate at low bacterial concentrations . Red blood cell stromal elements increased the rate to a minimal degree and only at high bacterial concentrations . The character of the E . coli-induced peritoneal leukocytosis was not significantly affected by concomitant administration of either stroma-free hemoglobin or red blood cell stroma . Although crystalline bovine hemoglobin inhibited in vitro leukocyte chemotaxis, centrifugation of this preparation to remove debris abrogated this effect . Stroma-free hemoglobin had no effect on in vitro leukocyte chemotaxis . Red blood cell stromal elements in high concentration diminished phagocytosis and bacterial killing by human neutrophils, but stroma-free hemoglobin had no effect on these neutrophil functions . The results confirm that hemoglobin is a potent adjuvant in experimental E . coli peritonitis, but contrary to our previous conclusion does not owe this effect to direct interference either with neutrophil influx into the peritoneal cavity during bacterial peritonitis or with human leukocyte function in vitro . Such prior conclusions appear to have been based on insoluble material including stromal elements in the hemoglobin preparations used . Red cell stromal elements derived from as little as 1 ml of packed red cells can significantly interfere with the phagocytosis and killing of E . coli. Proc Natl Acad Sci U S A, 1983 May, 80(9), 2467 - 71 Amino acid sequence of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli; Konigsberg WH et al.; We propose a primary structure for the catalytic subunit of aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli based on amino acid sequences of fragments obtained by cyanogen bromide cleavage, by tryptic digestion of the succinylated polypeptide, and by chymotryptic and proteinase C digestion of the intact catalytic chain . The protein contains 310 amino acids and has a calculated molecular weight of 33,944 . The negatively and positively charged residues are distributed uniformly, and there is no indication of charge clustering in the linear sequence. Mutat Res, 1983 May, 109(2), 169 - 82 A screening procedure for the efficient recognition of Escherichia coli K-12 mutants; Orrego C et al.; A method is described for the rapid screening of large numbers of E . coli colonies for detection of auxotrophs and mutants defective in sugar or succinate metabolism . The procedure permits recognition of forward mutations in a large number of functions and is applicable to the isolation of temperature-sensitive mutants in essential and nonessential genes. J Virol, 1983 May, 46(2), 454 - 65 Expression of adenovirus E1a and E1b gene products and the Escherichia coli XGPRT gene in KB cells; Babiss LE et al.; The recombinant plasmid pSV2-gpt, which contains the Escherichia coli XGPRT gene under the control of a simian virus 40 early promoter, was modified to contain the type 2 adenovirus (Ad2) XhoI-C (0 to 15.5 map units) restriction endonuclease fragment . Plasmid (pLB206) DNA was introduced into human KB cells by Ca2+-mediated DNA transfection, and transformants were selected in medium containing xanthine, aminopterin, and mycophenolic acid, as a consequence of expression of the dominant, selectable XGPRT gene . A series of 13 gpt+ cell lines were isolated and tested for their ability to complement Ad5 deletion mutants in E1a (H5dl312) and E1b (H5dl315) . Four classes of gpt+ KB cell lines were identified, including clones constitutively expressing both E1a and E1b, only E1a, or only E1b or not expressing either E1a or E1b . DNA and RNA filter transfer hybridization analysis substantiated the conclusions that those cell lines capable of complementing viral host range mutants contained the appropriate viral DNA sequences and cytoplasmic polyadenylated RNA species . DNA filter transfer hybridization studies also revealed that the transfected vector DNA was stably integrated into chromosomal DNA in the KB transformants and the number of integrated sites ranged from 1 to 3 . The gpt+ KB cell line that only expressed E1b gene functions only contained viral E1b gene sequences; those cell lines that expressed neither E1a nor E1b gene function contained only small or no regions of Ad2 DNA . When weaned off the selective medium, transformed KB cell lines stably maintained their inserted DNA in the absence of selective pressure and could easily be adapted to growth in suspension culture. J Cell Biol, 1983 May, 96(5), 1471 - 4 Nascent polypeptide chains exit the ribosome in the same relative position in both eucaryotes and procaryotes; Bernabeu C et al.; We located the polypeptide nascent chain as it leaves cytoplasmic ribosomes from the plant Lemna gibba by immune electron microscopy using antibodies against the small subunit of the enzyme ribulose-1,5-bisphosphate carboxylase . Similar studies with Escherichia coli ribosomes, using antibodies directed against the enzyme beta-galactosidase, show that the polypeptide nascent chain emerges in the same relative position in plants and bacteria . The eucaryotic ribosomal exit site is on the large subunit, approximately 75 A from the interface between subunits and nearly 160 A from the central protuberance, the presumed site for peptidyl transfer . This is the first functional site on both the eucaryotic and procaryotic ribosomes to be determined. J Cell Biol, 1983 May, 96(5), 1464 - 9 Construction of a synthetic messenger RNA encoding a membrane protein; Rubenstein JL et al.; We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription . For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J . K., and C . J . Gallione, 1981, J . Virol., 39:519-528) . This DNA served as the template in an in vitro transcription reaction utilizing E . coli RNA polymerase . The RNA product was capped using the vaccinia guanylyltransferase . A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV-infected cells . This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion . This technique should aid in identifying features needed by proteins for insertion into membranes. J Bacteriol, 1983 May, 154(2), 980 - 3 ST:LT:CFA/II plasmids in enterotoxigenic Escherichia coli belonging to serogroups O6, O8, O80, O85, and O139; Penaranda ME et al.; Colonization factor antigen II-producing enterotoxigenic Escherichia coli serotypes possessed at least one large plasmid . Loss of colonization factor antigen II correlated with either complete or partial loss of the large plasmid . Complete loss of the plasmid always correlated with complete loss of enterotoxin production . Three of five deletion events also correlated with the loss of toxin production. J Bacteriol, 1983 May, 154(2), 756 - 62 Genetic and biochemical analyses of Escherichia coli strains having a mutation in the structural gene (poxB) for pyruvate oxidase; Chang YY et al.; Mutants of Escherichia coli K-12 deficient in pyruvate oxidase were isolated from an aceEF (pyruvate dehydrogenase-deficient) strain by selection for a complete absence of growth on medium lacking acetate . Extracts of two of the mutants were shown to contain normal levels of pyruvate oxidase antigen, although the enzymatic activities of the extracts were reduced or absent . The poxB locus was mapped by using closely linked transposon insertions to min 18.7 of the E . coli linkage map between the cmlA and aroA loci, a location far removed from that of the regulatory gene, poxA. J Bacteriol, 1983 May, 154(2), 708 - 18 Membrane cytochromes of Escherichia coli grown aerobically and anaerobically with nitrate; Hackett NR et al.; Redox titration has been coupled to spectroscopic techniques, enzyme fractionation, and the use of mutants to examine the cytochrome composition of the membranes from cells grown aerobically and anaerobically with nitrate . A combination of techniques was found to be necessary to resolve the cytochromes . At least six b-type cytochromes were present . Besides cytochromes bfdh and bnr, components of the formate dehydrogenase-nitrate reductase pathway, cytochromes b556, b555, b562, and o, characteristic of aerobic respiratory pathways, were present . The midpoint oxidation-reduction potentials of the aerobic b-type cytochromes suggested that the sequence of electron transfer is: cytochrome b556 leads to b555 leads to b562 leads to O2. J Bacteriol, 1983 May, 154(2), 656 - 62 Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry; Skarstad K et al.; The cell cycle kinetics of Escherichia coli B/r A and B/r K cells were studied by flow cytometry . Three-dimensional histograms of cell cultures show the number of cells as a function of cellular DNA and protein contents and give detailed pictures of the cell cycle distribution with regard to these parameters . Histograms of slowly growing chemostat cultures showed that cell cycle periods B and C + D increase with a decreasing growth rate and that the B period occupies an increasing fraction of the cycle . The DNA replication patterns of B/r A and K were found to be quite similar . At extremely low growth rates (doubling time {T} = 17 h), B/r A cells had a B period of 0.8 T, a C period of 0.1 T, and a D period of 0.1 T, and B/r K cells (T = 16 h) had a B period of 0.6 T, a C period of 0.15 T, and a D period of 0.25 T . Mass increase, i.e., essentially protein synthesis, was seen in all three periods of the cell cycle . For B/r A cells, the average rate of mass increase was 11 times greater in the D period than in the B period, whereas for B/r K cells the rate of mass increase was twice as great in the D period as in the B period . The DNA and cell size distributions of batch cultures in exponential growth were found to vary with time, indicating that such cultures are not suitable for studies of cell cycle kinetics. J Bacteriol, 1983 May, 154(2), 569 - 72 Genetic mapping of the Escherichia coli leader (signal) peptidase gene (lep): a new approach for determining the map position of a cloned gene; Silver P et al.; The gene for leader peptidase, termed lep, was mapped to the region between purI and nadB at min 54 to 55 on the Escherichia coli chromosome . Mapping involved (i) cloning the gene into the plasmid pBR322, (ii) transforming the plasmid into a polA strain where it cannot replicate autonomously, (iii) selecting by ampicillin resistance the rare cell in which the plasmid had recombined into the chromosome, and (iv) mapping the chromosomal site of drug resistance (and thus plasmid integration) by Hfr matings and P1 transduction . The map position was confirmed by an assay of the enzyme content of cells bearing an F' factor which covered that region of the chromosome. J Bacteriol, 1983 May, 154(2), 554 - 60 Role for fadR in unsaturated fatty acid biosynthesis in Escherichia coli; Nunn WD et al.; Escherichia coli K-12 mutants constitutive for the synthesis of the enzymes of fatty acid degradation (fad) synthesize significantly less unsaturated fatty acid (UFA) than do wild-type (fadR+) strains . The constitutive fadR mutants synthesize less UFA than do fadR+) strains both in vivo and in vitro . The inability of fadR strains to synthesize UFAs at rates comparable to those of fadR+ strains is phenotypically asymptomatic unless the fadR strain also carries a lesion in fabA, the structural gene for beta-hydroxydecanoyl-thioester dehydrase . Unlike fadR+ fabA(Ts) mutants, fadR fabA(Ts) strains synthesize insufficient UFA to support their growth even at low temperatures and, therefore, must be supplemented with UFA at both low and high temperatures . The low levels of UFA in fadR strains are not due to the constitutive level of fatty acid-degrading enzymes in these strains . These results suggest that a functional fadR gene is required for the maximal expression of UFA biosynthesis in E . coli. J Bacteriol, 1983 May, 154(2), 1009 - 12 traJ independence in expression of traT on F; Rashtchian A et al.; Studies of F plasmids in the same cell as a transfer-repressed IncFII R plasmid showed a 100 to 1,000-fold decrease in transfer and a 75-fold decrease in surface exclusion, but no detectable change was shown in the amount of TraTp synthesized . Moreover, a mutation in traJ on F which caused a 10(4)-fold reduction in transfer caused only a 3.6-fold decrease in TraTp . These two findings suggest that a significant amount of traT expression on F is independent of traJ . Furthermore, we showed, using immunoprecipitation of TraTp, that normal amounts of this protein could be present in the cell without producing normal levels of surface exclusion. Infect Immun, 1983 May, 40(2), 701 - 7 Purification and characterization of heat-stable enterotoxin from bovine enterotoxigenic Escherichia coli; Saeed AM et al.; Heat-stable enterotoxin (ST) from Escherichia coli pathogenic for cattle was mass produced in a chemically defined medium . The toxin was concentrated and purified by sequentially applying batch adsorption chromatography on Amberlite XAD-2 resin, acetone fractionation, and preparative isoelectric focusing in a flatbed granulated gel . Reverse-phase high-performance liquid chromatography was used to purify the toxin further and to eliminate contaminating ampholytes . The toxin was purified more than 2,000-fold and had a minimal effective dose of less than 0.5 ng . It was biologically active after heating to 100 degrees C for 30 min and was not hydrolyzed by trypsin, pronase, and subtilisin, but it was inactivated by treatment with 0.1 M 2-mercaptoethanol or 4 X 10(-5) M dithiothreitol, suggesting that disulfide bonds are essential for retaining its biological activity . The amino acid analysis revealed 18 amino acid residues per molecule, which is in agreement with the composition of ST from a human strain of enterotoxigenic E . coli . The amino acid composition of our ST matched the published coding sequence of the last 18 codons of Tn1618, a transposon isolated from the bovine enterotoxigenic E . coli strain B41 and shown to be present also in some strains of porcine enterotoxigenic E . coli . These findings further support the existence of a form of ST common to bovine, porcine, and human strains of enterotoxigenic E . coli. Infect Immun, 1983 May, 40(2), 653 - 8 Cloning and molecular characterization of the B subunit of Escherichia coli heat-labile enterotoxin; Clements JD et al.; We have constructed a plasmid containing the gene for production of the B subunit of the heat-labile enterotoxin (LT-B) from a human isolate of Escherichia coli, strain H10407 . The 0.8-kilobase gene fragment encoding synthesis of LT-B was cloned onto plasmid pBR322 after sequential digestion of the enterotoxin plasmid of strain H10407 with restriction endonucleases PstI and HindIII . LT-B was isolated by agarose affinity chromatography from cell lysates of recombinant clones expressing the B subunit . The B subunit was isolated in its oligomeric form, was structurally identical to native B subunit when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dissociated to monomeric B in the presence of 5 M guanidine, was immunologically identical to native B subunit in an enzyme-linked immunosorbent assay, and contained no demonstrable A subunit in any of the assays. Cancer Res, 1983 May, 43(5), 1951 - 6 Cellular uptake and inhibition of DNA synthesis by dihydroxyanthraquinone and two analogues; Nishio A et al.; Three analogues of aminoalkylamino-substituted anthraquinone derivatives, namely, 1,4-dihydroxy-5,8-bis(((2-{(2-hydroxyethyl)amino}ethyl)amino))-9,10-anthracenedione (DHAQ), 1-hydroxy-5,8-bis(((2-{(2-hydroxyethyl)amino}ethyl)amino))-9,10-anthracenedione (HAQ), and 1,4-bis(((2-{(2-hydroxyethyl)amino}ethyl)amino))-9,10-anthracenedione (AQ), were chosen with respect to the number of hydroxyl groups on the aromatic ring . DHAQ showed about 100 times more potent antiproliferative activity on cultured mouse L-cells than did AQ; HAW showed intermediate activity . This antiproliferative activity was correlated with their inhibitory effect on DNA synthesis in culture . When their inhibitory effect on DNA synthesis was conducted in a permeabilized L-cell assay, all compounds were inhibitory; the order of potency was DHAQ greater than HAQ greater than AQ . The same order of potency was also observed in calf thymus DNA and Escherichia coli DNA polymerase I system . Their inhibitory effect in the latter system was correlated with the drug:DNA molar ratio, and not with drug:enzyme ratio . Comparative uptake of the drugs by intact L-cells showed the highest uptake of DHAQ followed by those of HAQ and AQ . The large differences in their uptake by intact cells became minimal when cells were rendered permeable to exogenous materials or when nuclei were used . Hence, these studies revealed that the hydroxyl group on the aromatic ring of the compounds influenced their biological activity not only by potentiating drug-target interaction but also by drug uptake into cells. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1983 May, 254(3), 379 - 87 Transport of aminoglycosides in Escherichia coli; Dalhoff A; Uptake of nine aminoglycosides was studied in E . coli K12 and mutants being defective in the outer membrane proteins OmpF and OmpC or in ATPase (uncA) . OmpF/OmpC as well as uncA mutants did not take up the aminoglycosides; transport in wild type cells was cAMP dependent . The specific binding of phages using the OmpF and OmpC proteins as receptors was strongly reduced by the aminoglycosides or by other polycationic peptides . Thus it may be assumed that the initial step of aminoglycoside transport is their electrostatic binding to the anionic porins, especially to the ompF porin, followed by a facilitated permeation through the outer membrane . As the synthesis of this porin is under cAMP control, aminoglycoside transport is cAMP dependent as well . The fact that the uncA mutant did not take up the aminoglycosides to any appreciable extent indicates that the cytoplasmic membrane is involved in aminoglycoside transport too. J Gen Microbiol, 1983 May, 129 (Pt 5), 1345 - 55 The reaction with oxygen of cytochrome oxidase (cytochrome d) in Escherichia coli K12: optical studies of intermediate species and cytochrome b oxidation at sub-zero temperatures; Poole RK et al.; Optical changes in d- and b-type cytochromes, following initiation of the reaction of cytochrome oxidase d with O2, have been studied in cells and derived membrane particles from oxygen-limited cultures of Escherichia coli K12 . At successively higher temperatures between -132 and -88 degrees C, the first scan after photolysis of the Co-liganded, reduced oxidase in the presence of O2 and a slow increase in absorbance at 675 to 680 nm due to an unidentified chromophore . A similar sequence occurs when a single sample is scanned repetitively at -91 degrees C . At higher temperatures, oxidation of at least two spectrally distinct cytochromes b occurs . Selective photolysis of the cytochrome d-CO complex with a He-Ne laser shows that neither of these cytochromes is the CO-binding cytochrome o436 . In all oxidation states examined, no absorbance in the 720 to 860 nm region was observed; it is concluded that both cytochromes d and o436 lack redox-active copper that has an environment similar to the copper(s) in mitochondrial cytochrome c oxidase . The amount of cytochrome d650 (but not the amount of reduced cytochrome o436) formed after photolysis is directly proportional to the oxygen concentration in the sample at the time of freeze trapping . The results are discussed in relation to the composition and mechanism of action of cytochrome d. J Gen Microbiol, 1983 May, 129 (Pt 5), 1335 - 44 The 650 and chromophore in Escherichia coli is an 'oxy-' or oxygenated compound, not the oxidized form of cytochrome oxidase d: an hypothesis; Poole RK et al.; The form of cytochrome d in Escherichia coli and Azotobacter vinelandii that shows an absorption maximum at 648 to 652 nm ('cytochrome d650') is generally regarded as the oxidized form of this terminal oxidase . Membranes from E . coli grown under oxygen-limited conditions, when treated with ferricyanide, do not reveal cytochrome d650, whereas a sharp symmetrical band at 652 nm results from the reaction of the reduced enzyme with O2 at either room temperature or after flash photolysis of the CO-liganded form at -130 degrees C . Electron paramagnetic resonance spectroscopy of cytochrome d650 trapped at -130 degrees C shows that its spectrum is indistinguishable from the CO-liganded form and does not reveal resonances of high spin ferric haem previously attributed to cytochrome d . An hypothesis is proposed in which cytochrome d650 is an early intermediate in the reaction of reduced cytochrome d650 and oxyhaemoglobin is presented and the hypothesis discussed in relation to earlier work, in which the indirect interconversions of reduced cytochrome d and d650 have been explained by proposing the existence of an 'invisible' form . It is suggested that this form could be the oxidized enzyme. Vopr Med Khim, 1983 May-Jun, 29(3), 8 - 11 {The modern system for selection of cryptic plasmids based on replicon pMBI}; Kalinin VN et al.; Cytotoxicity of hybrid plasmide pGalI was associated with the genome site measuring for 1.1.mD and flanked by the recognizing sites of Eco RI restrictase . The cytotoxic effect of pGalI might be limited not only by plasmide pMB9 but also by other plasmides constructed on the base of pMBI replicone . Limitation of the cytotoxicity occurred due to a decrease in amount of the pGalI copies . Capacity of the plasmides studied to limit the pGalI cytotoxicity enabled to clone them as cryptic plasmides under conditions of both simultaneous with pGalI transformation of E . coli and the subsequent selection of clones with the Gal+ phenotype. Vopr Med Khim, 1983 May-Jun, 29(3), 22 - 5 {Recognition site in the restriction endonuclease Eco CK}; Khudiakov IuE et al.; 5'-Terminal nucleotide was degenerated in the fragments produced by means of hydrolysis with vestrictase Eco CK . Analysis of the nucleotide sequence, carried out in four DNA fragments after the enzymatic hydrolysis' enabled to detect the only one common 13 residues nucleotide sequence, which is apparently involved in the recognizing site for vestrictase Eco CK: (A/T) (A/T) N (A/T) CGCNCNNNG . This sequence was not found in several DNA molecules with well-known primary structure, stable to the action of this enzyme. Gene, 1983 May-Jun, 22(2-3), 281 - 7 A partial restriction map of the proA-purE region of the Escherichia coli K12 chromosome; Hadley RG et al.; EcoRI restriction mapping data for fragments larger than 0.7 kb and contained in a 350-kb region of the Escherichia coli K-12 chromosome are presented . 75% of these fragments have been located relative to proA, B, argF, lac, proC, purE, and various insertion sequence elements normally present in this region . BglII and BamHI maps for the regions near argF and purE are also provided. Gene, 1983 May-Jun, 22(2-3), 277 - 80 Revised sequence of the tetracycline-resistance gene of pBR322; Peden KW; A revised sequence of the tetracycline-resistance gene of pBR322 is reported . The change, the presence of an additional CG base pair at position 526, adjusts the published sequence to allow an open reading frame from nucleotides 86-1273 (new number) and increases the size of the plasmid to 4363 bp . The predicted polypeptide encoded by this region would contain 396 amino acid residues and have a calculated Mr of 41518 . A polypeptide of the predicted size has been reported previously when pBR322 is used as template in the maxicell system. Gene, 1983 May-Jun, 22(2-3), 267 - 75 A new hybrid plasmid capable of transforming Escherichia coli and Anacystis nidulans; Friedberg D et al.; We have constructed a hybrid plasmid, pDF30, by combining the 8-kb pDF3 plasmid derived from the cyanobacterium Anacystis nidulans 6311 with the Escherichia coli vector pBR325 . pDF30 transforms, replicates and confers chloramphenicol resistance (Cmr) and ampicillin resistance (Apr) on both A . nidulans and E . coli . The level of resistance to ampicillin in A . nidulans transformants, although low, is above background resistance and an Apr activity was demonstrated in cell-free extracts of A . nidulans that harbored pDF30 . pDF30 is stably maintained within E . coli and can be isolated intact from A . nidulans after several months of subculture under chloramphenicol selection and by this criterion is stable in vivo . The unique restriction sites for XhoI, SalI and EcoRI can be used for molecular cloning of chromosomal genes in E . coli and A . nidulans . Until now pDF3 is phenotypically cryptic in both A . nidulans and E . coli . Examination of the polypeptides encoded on and expressed by pDF30 in E . coli minicells revealed only gene products from pBR325 but none from pDF3. Gene, 1983 May-Jun, 22(2-3), 255 - 65 New runaway-replication-plasmid cloning vectors and suppression of runaway replication by novobiocin; Uhlin BE et al.; Two new cloning vectors (pBEU28 and pBEU50) with temperature-controlled runaway-replication properties are described . pBEU28 is similar to aphA+ (KanR) plasmid pBEU2 but lacks a 1.8-kb duplication which is responsible for plasmid instability . pBEU50 is an analog of pBR313 and pBR322 in that it carries bla+(AmpR), which can be used for selection, and tet+(TetR) which can be inactivated by cloning at HindIII and BamHI restriction sites . Sublethal concentrations of novobiocin were exploited to suppress runaway replication and to restore the viability of the plasmid carriers . By this method copB deletion mutants of two temperature-controlled, conditional runaway-replication plasmids were detected and isolated . The unconditional runaway-replication property of these plasmids leads us to hypothesize that there are at least two controls of plasmid R1 copy number and that the copB-dependent control is temperature-sensitive in the conditional runaway replication mutants . The novobiocin suppression of the runaway replication permitted us to clone dnaN+ on pBEU28 and to identify its presence at 42 degrees C with a dnaN59 transformation recipient which was temperature-sensitive due to a defect in the dnaN gene. Gene, 1983 May-Jun, 22(2-3), 245 - 53 Cloning of mutH and identification of the gene product; Grafstrom RH et al.; A specialized lambda transducing phage carrying the mutH gene and several deletion derivatives of this phage were characterized by restriction enzyme analysis . This analysis localized the mutH gene to a small region of bacterial DNA on the transducing phage and facilitated the subsequent cloning of this gene into the multicopy plasmid pBR322 . The mutH gene is contained entirely on a 1.5-kb HindIII fragment as judged by the ability of plasmids carrying this fragment to complement mutH- alleles on the bacterial chromosome . Using recombinant plasmids containing the 1.5-kb HindIII fragment, we identified an Mr 25000 protein as the product of the mutH gene in an in vitro transcription-translation system as well as in maxicells . Various deletion derivatives of the mutH-containing plasmids that exhibit a Mut- phenotype also have lost the Mr 25000 protein. Gene, 1983 May-Jun, 22(2-3), 237 - 43 Transposition of DNA fragments flanked by two inverted Tn1 sequences: translocation of the plasmid RP4::Tn1 region harboring the Tcr marker; Dobritsa AP et al.; We have demonstrated the possibility of transposition of the plasmid RP4::Tn1 fragment (21.2 kb) carrying the tetracycline resistance (Tcr) gene and flanked by two Tn1 copies . The new transposon, designated Tn1756, bears lethal genes that kill host cells . Therefore, its transposition can only be revealed in the presence of lethality-compensating helper regions of the plasmid RP4 . Thus, RP4::Tn1 consists of two transposons, Tn1755 (Tn1-Kmr-Tn1) and Tn1756 (Tn1-Tcr-Tn1), sharing the Tn1 sequences . Both of these transposons are capable of recA-independent translocation to other plasmids . Therefore, transposition of DNA fragments flanked by two inverted Tn1 sequences does not depend on Tn1 orientation. Gene, 1983 May-Jun, 22(2-3), 191 - 201 Cloning of the promoters of an Escherichia coli rRNA gene . New experimental system to study the regulation of rRNA transcription; Boros I et al.; The promoters of the rrnB gene of Escherichia coli have been cloned on a multicopy, pBR322-derived plasmid by deleting most of the structural part of rrnB and fusing the terminators of the gene immediately to the promoters . Several further deletions were constructed to vary the promoter-terminator distance, destroy or damage selectively any of the promoters or terminators, and vary the distance between the two pairs of P1 P2 and P3 P4 promoters . All these transcription signals were shown to function on the plasmids in vitro and in vivo . The truncated in vivo transcription products initiated at the P1 and P2 promoters of the recombinant plasmids were found to be stable, and the accumulated transcripts could be easily distinguished from the chromosome-coded rRNA . This provides a convenient experimental system to study the regulation of rRNA biosynthesis. Gene, 1983 May-Jun, 22(2-3), 175 - 80 Cloning of the gene for Escherichia coli glutamyl-tRNA synthetase; Sanfacon H et al.; The structural gene for the glutamyl-tRNA synthetase of Escherichia coli has been cloned in E . coli strain JP1449, a thermosensitive mutant altered in this enzyme . Ampicillin-resistant and tetracycline-sensitive thermoresistant colonies were selected following the transformation of JP1449 by a bank of hybrid plasmids containing fragments from a partial Sau3A digest of chromosomal DNA inserted into the BamHI site of pBR322 . One of the selected clones, HS7611, has a level of glutamyl-tRNA synthetase activity more than 20 times higher than that of a wild-type strain . The overproduced enzyme has the same molecular weight and is as thermostable as that of a wild-type strain, indicating that the complete structural gene is present in the insert . These characteristics were lost by curing this clone of its plasmid with acridine orange, and were transferred with high efficiency to the mutant strain JP1449 by transformation with the purified plasmid . A physical map of the plasmid, which contains an insert of about 2.7 kb in length, is presented. Gene, 1983 May-Jun, 22(2-3), 139 - 56 Molecular cloning of a functional dam+ gene coding for phage T4 DNA adenine methylase; Schlagman SL et al.; Phages T2 and T4 induce synthesis of a DNA-adenine methylase which is coded for by a phage gene, dam+ . These enzymes methylate adenine residues in specific sequences which include G-A-T-C, the methylation site of the host Escherichia coli dam+ methylase . Methylation of G-A-T-C to G-m6A-T-C protects the site against cleavage by the MboI restriction nuclease . We have taken advantage of this property to enrich and screen for transformants which contain a cloned, functional T4 dam+ gene . These recombinant molecules consist of a 1.85-kb HindIII fragment inserted into the plasmid pBR322; both orientations of the fragment express the methylase gene, suggesting that transcription is from a T4 promoter . We have tested the 1.85-kb insert for sensitivity to a variety of restriction nucleases and have found single sites for EcoRI, BalI, XbaI, and at least two sites for BstNI (EcoRII) . The relative positions of these restriction sites have also been determined . Physical mapping was carried out by Southern blot hybridization with 32P-labeled (nick-translated clone) probe . These experiments showed that the insert corresponds to a HindIII fragment located on the physical map of T4 between positions 16.2 and 18.1 kb from the T4rIIA-rIIB junction . E . coli dam- possesses several phenotypic differences from the wild-type dam+ parent, including an increased sensitivity to 2-aminopurine (2-AP) . We found that T4 dam+ clones could relieve dam- cells of their increased sensitivity to 2-AP. Genetika, 1983 May, 19(5), 714 - 9 {Cloning of the gene controlling catabolite repression with the participation of cyclic adenosine monophosphate in Escherichia coli K-12}; Lisenkov AF et al.; The crp gene coding for cyclic adenosine monophosphate receptor protein has been cloned on the vehicle pBR325 using restriction endonuclease PstI and the recipient strain C600 crp . The pCAP2 hybrid plasmid obtained has a molecular weight 7.0 MD and in the pBR325 with the insertion into a PstI site . Bacterial clones carrying pCAP2 restore Crp+ phenotype, as judged by the capacity of bacteria for utilization of various carbohydrates and by the activity of catabolite sensitive enzymes. Genetika, 1983 May, 19(5), 708 - 13 {Molecular cloning of the genetic determinants responsible for enterotoxin synthesis in Escherichia coli}; Markov KI et al.; Recombinant plasmids responsible for production of heat stable and heat labile enterotoxins have been obtained . Genetic determinants controlling enterotoxin production have been localized. Plasmid, 1983 May, 9(3), 262 - 72 Cloning and polypeptide analysis of the leading region in F plasmid DNA transfer; Ray A et al.; A segment of the F plasmid DNA, located between the origin of transfer and the primary F replication region, is the first to enter the recipient cell during conjugation . PstI, SalI, and SmaI restriction endonuclease sites have been mapped within this leading region in conjugational DNA transfer and chimeric plasmids carrying overlapping fragments of the region have been constructed . Analyses of polypeptides of Mr 27,800, 23,100, 14,400, and 11,000 to be encoded by sequences within the leading region. Plasmid, 1983 May, 9(3), 247 - 61 R46 encodes a site-specific recombination system interchangeable with the resolution function of TnA; Dodd HM et al.; Transposition of Tn4 onto the IncN plasmic R46 generates unstable DNA molecules . The R46::TnA recombinant plasmids undergo further DNA rearrangements which depend on the orientation in which the TnA element is inserted into the plasmid, and deletions and inversions of R46 and TnA sequences have been observed . Both types of rearrangement have the same specific endpoints, one within TnA and one located between the R46 coordinates, 36.0 and 37.0 . The results are consistent with the operation of a recA-independent, site-specific recombination system utilizing, at least in part, the transposon cointegrate resolution system of TnA, together with R46-encoded functions . Data are presented that indicate that R46 encodes analogs of both the res site of TnA and its tnpR gene, although little homology between this element and the plasmid is apparent . Models for the TnA-induced generation of site-specific deletions and inversions upon transposition of TnA to R46 are presented. Plasmid, 1983 May, 9(3), 227 - 39 Transfer-defective and tetracycline-sensitive mutants of the incompatibility group HI plasmid R27 generated by insertion of transposon 7; Taylor DE; Transposon Tn7 insertion was used to obtain either transfer-defective (Tra-) or tetracycline-sensitive (Tc-) mutants of the HI incompatibility group (IncHI) plasmid R27 . The 600 apparent R27::Tn7 derivatives fell into three classes: Tra-, Tc-, and Tra- Tc- . Mutants of R27 defective in the thermosensitive mode of transfer characteristic of IncH plasmids were obtained with transfer frequencies of less than 1 X 10(-8) transconjugants per recipient after 18 hr at 26 degrees C . These mutants, which were generated at a frequency of 1 per 100 insertions, were nonleaky and nonrevertible . Tc- mutants of R27, generated at a frequency of 0.5 per 100 insertions, were also nonrevertible . Loss of tetracycline resistance was associated with an increased frequency of transfer (average 3.6 X 10(-3) transconjugants per donor per hour at 30 degrees C) compared with transfer of the wild-type R27 plasmid (1.6 X 10(-8) per donor per hour) . Tn7 insertions which generated Tc- or Tra- mutants of R27 had no effect on entry exclusion of other H group plasmids . The molecular weights of Tra- and Tc- R27::Tn7 derivatives were approximately 120.5 MDa, corresponding to the sum of R27 (112 MDa) and Tn7 (8.5 MDa) . A third class of Tn7 insertion derivatives (Tra- Tc-) was obtained; however, strains expressing this phenotype were plasmid free, and appeared to have Tn7 integrated at a chromosomal site . Restriction digestion with XbaI and subsequent hybridization with ColE1::Tn7 were used to compare R27::Tn7 derivatives and to locate Tn7 insertion sites . Loss of tetracycline resistance was associated with Tn7 insertion into a 24-kb XbaI fragment of R27 . Although loss of plasmid transfer in several R27::Tn7 derivatives was accompanied by insertion of Tn7 into a 14-kb XbaI fragment of the plasmid, these mutants had also undergone a small increase in the size of the 24-kb XbaI fragment of R27. Am J Physiol, 1983 May, 244(5), R718 - 23 Changes in beta-adrenergic receptors in dog livers during endotoxic shock; Liu MS et al.; The effects of endotoxin administration on beta-adrenergic receptors in dog liver plasma membranes were studied using {3H}dihydroalprenolol as a radioactive ligand . The Scatchard analysis revealed a one-component binding characteristic both in control and endotoxin-injected dogs . The Kd (dissociation constant) value was increased by 60% (4.2 +/- 0.5 nM for control vs . 6.7 +/- 0.5 nM for endotoxic; P less than 0.01) and the Bmax (maximum binding capacity) was decreased by 38% (600 +/- 60 and 370 +/- 70 fmol/mg protein for control and endotoxic, respectively; P less than 0.01) 2 h following endotoxin administration . The competitive inhibition studies show that the apparent Kd values for (-)-isoproterenol, (-)-epinephrine, and (-)-norepinephrine were increased by 14, 51, and 5 times, respectively, 2 h postendotoxin . In addition, endotoxin in vitro had a dose-dependent inhibitory effect on the specific binding of {3H}dihydroalprenolol, and it also reduced the number of beta-receptors . These data demonstrate that endotoxin, both in vivo and in vitro, decreased the binding affinity and the number of beta-adrenergic receptors in dog liver plasma membranes . A modification of the beta-adrenergic receptors in dog livers induced by endotoxin administration may play an important role in the development of hepatic glucose dyshomeostasis during shock. Proc Natl Acad Sci U S A, 1983 May, 80(9), 2539 - 43 Orientation of a human leukocyte interferon molecule on its cell surface receptor: carboxyl terminus remains accessible to a monoclonal antibody made against a synthetic interferon fragment; Arnheiter H et al.; An 125I-labeled monoclonal antibody made against a synthetic 56-residue fragment of human leukocyte interferon (IFN) alpha 1 recognizes human, Escherichia coli-derived IFN alpha A bound to the surface of Madin-Darby bovine kidney cells . A major fraction of the antibody recognizes IFN specifically bound to the cells, because the number of bound antibody molecules corresponds to the number of cell-bound IFN molecules (as measured with radiolabeled ligand) and because the fraction of the IFN unspecifically bound to the cells is less than 10% of the total bound IFN . A synthetic carboxyl-terminal 16-residue IFN peptide, though not inhibiting binding of IFN to cells, inhibits binding of antibody to IFN . A recombinant IFN alpha A molecule with a carboxyl-terminal 13-residue deletion, though still able to compete for binding of IFN to cells, is not recognized by the antibody . Scatchard plot analysis of the binding data revealed apparent dissociation constants of 6.0 x 10(-10) M for the antibody-IFN interaction and of 4.0 x 10(-11) M for the IFN-cell receptor interaction . The antibody inhibits the binding of IFN to cells only weakly and neutralizes the antiviral activity of the ligand only when in a large molar excess . We conclude that the carboxyl-terminal 10-16 residues that are predicted from the cloned IFN cDNAs and that are present in some natural IFNs are not involved in binding to cells but are antigenic and hence exposed on the molecules' surface . That the carboxyl terminus is not directly involved in binding to cells is consistent with the observation that some IFNs with carboxyl-terminal deletions are biologically active. Proc Natl Acad Sci U S A, 1983 May, 80(9), 2462 - 6 Nucleotide sequence of the structural gene (pyrB) that encodes the catalytic polypeptide of aspartate transcarbamoylase of Escherichia coli; Hoover TA et al.; The deoxyribonucleotide sequence of pyrB, the cistron encoding the catalytic subunit of aspartate transcarbamoylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2), has been determined . The pyrB gene encodes a polypeptide of 311 amino acid residues initiated by an NH2-terminal methionine that is not present in the catalytically active polypeptide . The DNA sequence analysis revealed the presence of an eight-amino-acid sequence beginning at Met-219 that was not detected in previous analyses of amino acid sequence . This octapeptide sequence provides an additional component of the disordered loop in the equatorial domain of the catalytic polypeptide . It had been found previously that the catalytic polypeptide is expressed from a bicistronic operon that also produces the regulatory polypeptide encoded by pyrI . A single transcriptional control region precedes the structural gene of the catalytic polypeptide and a simple 15-base-pair region separates its COOH terminus from the structural gene of the regulatory polypeptide . The chain-terminating codon of the catalytic polypeptide may contribute to the ribosomal binding site for the regulatory polypeptide and thus assist coordinate expression of the two cistrons. J Bacteriol, 1983 May, 154(2), 992 - 4 Effects of aerobic and anaerobic shock on catabolite repression in cyclic AMP suppressor mutants of Escherichia coli; Lee JH et al.; Cultures of Escherichia coli K-12 grown on glucose or gluconate under aerobic conditions exhibited catabolite repression of beta-galactosidase synthesis . Depression occurred when these cultures were subjected to anaerobic shock . These states of repression and depression were found to be associated with low and high differential rates of cyclic AMP synthesis, respectively . This observation is consistent with the view that cyclic AMP plays a central role in the catabolite repression phenomenon . We report here, however, that identical stages of repression and derepression occur in mutant strains possessing cya crp(Csm) genotypes and therefore unable to synthesize cyclic AMP . These results suggest that cyclic AMP is not the sole regulator involved in catabolite repression. J Bacteriol, 1983 May, 154(2), 984 - 7 Isolation, by affinity chromatography, of mutant escherichia coli cells with novel regulation of lamB expression; Ferenci T et al.; Affinity chromatography was used as a positive genetic selection technique for the isolation of cells exhibiting high levels of surface receptor expression . Starting from a large population of Escherichia coli with no maltodextrin receptor due to a deletion of malT, the positive regulator gene required for receptor synthesis, cells were chromatographically enriched that could bind to starch-Sepharose, an immobilized ligand of the receptor . One such isolate showed over 25% of wild-type-induced levels of receptor in the absence of malT and levels higher than that of the wild type in a malT+ background . In contrast to wild-type cells, receptor expression in the isolate was insensitive to control by cAMP . The maltodextrin receptor synthesized by the mutant was identical to wild-type protein in terms of ligand affinity and electrophoretic mobility and was dependent on lamB, the structural gene for the receptor . The directed evolution of this novel form of lamB expression was dependent on at least two mutations in the isolate. J Bacteriol, 1983 May, 154(2), 854 - 8 Energetics of calcium efflux from cells of Escherichia coli; Tsujibo H et al.; Intact cells of a H+-translocating ATPase-deficient strain of Escherichia coli were starved of endogenous energy reserves and passively loaded with 45CaCl2 . Energy-dependent efflux of calcium was observed upon addition of glucose or respiratory substrates . Addition of cyanide or uncouplers prevented efflux . It is concluded that calcium efflux in intact cells is coupled to the proton motive force via secondary calcium-proton exchange. J Bacteriol, 1983 May, 154(2), 719 - 27 Membrane cytochromes of Escherichia coli chl mutants; Hackett NR et al.; The cytochromes present in the membranes of Escherichia coli cells having defects in the formate dehydrogenase-nitrate reductase system have been analyzed by spectroscopic, redox titration, and enzyme fractionation techniques . Four phenotypic classes differing in cytochrome composition were recognized . Class I is represented by strains with defects in the synthesis or insertion of molybdenum cofactor . Cytochromes of the formate dehydrogenase-nitrate reductase pathway are present . Class II strains map in the chlC-chlI region . The cytochrome associated with nitrate reductase (cytochrome bnr) is absent in these strains, whereas that associated with formate dehydrogenase (cytochrome bfdh) is the major cytochrome in the membranes . Class III strains lack both cytochromes bfdh and bnr but overproduce cytochrome d of the aerobic pathway even under anaerobic conditions in the presence of nitrate . Class III strains have defects in the regulation of cytochrome synthesis . An fdhA mutant produced cytochrome bnr but lacked cytochrome bfdh . These results support the view that chlI (narI) is the structural gene for cytochrome bnr and that chlC (narG) and chlI(narI) are in the same operon, and they provide evidence of the complexity of the regulation of cytochrome synthesis. J Bacteriol, 1983 May, 154(2), 580 - 90 Alignment of genetic and restriction maps of the photosynthesis region of the Rhodopseudomonas capsulata chromosome by a conjugation-mediated marker rescue technique; Taylor DP et al.; The restriction map of a 46-kilobase fragment of the Rhodopseudomonas capsulata chromosome was aligned with the genetic map of the photosynthesis region of that chromosome by a marker rescue technique . Marker rescue was effected by mobilization of vectors bearing fragments of R . capsulata DNA from Escherichia coli to a set of R . capsulata mutants . Plasmids pDPT51 and pDPT55 were constructed to mediate the intergeneric mobilization of pBR322 derivatives, and a mutant of R . capsulata with improved intergeneric recipient activity was isolated . Four previously unmapped genes affecting bacteriochlorophyll synthesis and two genes affecting photochemical reaction center synthesis have been located by marker rescue . Some of the fragments of R . capsulata DNA are capable of vector-independent complementation, implying that promoters are located on these fragments . Other fragments complement only in one orientation of insertion in the vector, implying transcription from promotors on the vectors and thereby fixing the direction of transcription for those fragments . Still other fragments of DNA show rescue only via recombination between homologous plasmid-borne DNA fragments and chromosomal mutations . The physical dimensions of the genetic map are 3.0 megadaltons per map unit, which agrees with previous estimates based on the size of the R . capsulata gene transfer agent. J Bacteriol, 1983 May, 154(2), 1005 - 8 Positive-selection cloning vehicle useful for overproduction of hybrid proteins; Cheng SC et al.; Plasmid pSCC31 contains the EcoRI endonuclease gene downstream from lambda pL . It does not yield transformants upon introduction into Escherichia coli unless the structural integrity of the endonuclease is destroyed . This makes it useful as a positive-selection cloning vehicle which can be employed for regulated overproduction of hybrid proteins. Infect Immun, 1983 May, 40(2), 647 - 52 Conformity between heat-labile toxin genes from human and porcine enterotoxigenic Escherichia coli; Dallas WS; The genes encoding the heat-labile toxin of Escherichia coli were isolated by recombinant DNA methods from enterotoxigenic E . coli recovered from a human and a piglet with diarrhea . With restriction endonucleases, a fine-structure map was made for the toxin genes . Both genes were found to be highly homologous within the toxin-coding DNA, but the surrounding DNA sequences were found to be quite divergent . Analysis of in vitro-derived mutants demonstrated that the cistrons for the toxin proteins were located at the same sites on each restriction enzyme map. Proc Natl Acad Sci U S A, 1983 May, 80(9), 2666 - 70 Escherichia coli extract-catalyzed recombination in switch regions of mouse immunoglobulin genes; Kataoka T et al.; We have shown that Escherichia coli extracts catalyze recombination between mouse immunoglobulin mu and alpha genes inserted separately in lambda phage vectors carrying different genetic markers . Most of the recombination sites in the inserts are located in the switch regions of the heavy chain genes, as previously found in the expressed genes of myeloma cells . The recombination took place at relatively high frequency (10(-4)) . The recombinational system in E . coli or lambda phage seems to prefer short nucleotide sequences similar to those used in the class switch recombination. Bioorg Khim, 1983 May, 9(5), 633 - 40 {RNA polymerase-rifamycin . A molecular model of inhibition}; Chertov OIu et al.; By fluorimetric titration of Rifs (E . coli B) and Rifr (E . coli rpoB255) RNA polymerases with rifamycin, the mutant polymerase was demonstrated to bind rifamycin . A comparison of spatial structures of rifamycin and dinucleotide fragment of RNA in the hybrid with DNA revealed their similarity . Taking into account this structural similarity and also the fact that two phosphodiester bonds can be formed by RNA polymerase in the presence of rifamycin, a model for the inhibition mode was proposed . According to this model, rifamycin occupies the place of two terminal nucleotides of synthesized, but not translocated pentanucleotide in the transcribing complex . Asp-516 of the wild type beta-subunit was assumed to form a hydrogen bond with the rifamycin C(23) hydroxyl group . On the base of this model, reduced "cycling" synthesis of tetra-, penta-.. . up to decanucleotides by the Rifr RNA polymerase, in comparison with Rifs, was predicted. Biochem Int, 1983 May, 6(5), 653 - 61 Expression of the cDNA for alpha 1-acid glycoprotein, a rat plasma protein, in Escherichia coli; Birch HE et al.; Polyadenylated RNA was isolated from acute-phase liver and transcribed into cDNA . After homopolymer tailing this was cloned into the PstI site of pBR322 which was used to transform E . coli RR1 cells . Clones containing cDNA corresponding to acute-phase proteins were identified by differential hybridization with 32P-labelled normal and acute-phase cDNA . A clone synthesizing about 10 ng of alpha 1-acid glycoprotein per E . coli colony was detected using a solid-phase based immunochemical procedure . The identification of the clone was verified by competition assay with authentic alpha 1-acid glycoprotein isolated from serum and by restriction analysis of the cDNA inserted into pBR322. Vopr Virusol, 1983 May-Jun, (3), 270 - 3 {Polyadenylation of influenza virion RNA in an in vitro system}; Samokhvalov EI et al.; A method for isolation of terminal polyriboadenylate transferase from E . coli cells (MPE 600) is presented . The specific activity of the enzyme yield at the terminal stage was 933 units per 1 mg of protein . Analysis of polyadenylated in vitro virion RNA of influenza virus A/USSR/90/17 strain in polyacrylamideagarose gel (2.2%-0.6%) in the presence of 6 M urea showed all the 8 fragments of genome RNA to be adenylated, their sizes being retained with regard to the distribution in gel of the initial RNA fragments . In vitro polyadenylated virion RNA was an effective matrix in reverse transcription reaction with RNA-dependent DNA-polymerase using oligo (dT) as a primer . Complementary DNAs obtained in this way may be the starting material for synthesis of double-stranded DNAs and subsequent construction of recombinant DNAs containing influenza virus genetic information. J Bacteriol, 1983 May, 154(2), 809 - 18 Characterization and complementation of pMB1 copy number mutant: effect of RNA I gene dosage on plasmid copy number and incompatibility; Moser DR et al.; A 16-base-pair insertion has been identified as the mutation responsible for the high-copy-number phenotype of the plasmid copy number mutant pFH118 . The mutation is located near the plasmid origin of replication in a region of the genome that encodes two overlapping RNA transcripts . One of these transcripts, RNA I, acts as a negative regulator of plasmid replication . The second transcript is the precursor to the primer for the initiation of DNA synthesis . We demonstrate through complementation that the pFH118 DNA overproduction phenotype is a consequence of the reduced effectiveness of the mutant RNA I at inhibiting plasmid replication and not a consequence of an altered target site on the primer precursor . In addition, a series of plasmids containing multiple RNA I-coding genes was constructed for investigating the effects of RNA I gene dosage on plasmid copy number and incompatibility . The results of this study strongly support the inhibitor dilution model of plasmid copy control with RNA I as the plasmid-specified inhibitor responsible for both copy number control and incompatibility. J Bacteriol, 1983 May, 154(2), 787 - 92 rpoB mutation in Escherichia coli alters control of ribosome synthesis by guanosine tetraphosphate; Little R et al.; An isogenic pair of relA+ and relA strains of Escherichia coli B/r with a mutation in the RNA polymerase subunit gene rpoB (Rifr) was isolated in which the relationship between guanosine tetraphosphate (ppGpp) concentration and stable RNA (rRNA, tRNA) gene activity was altered . The RNA polymerase in the rpoB strains was found to be about 20-fold more sensitive to ppGpp with respect to its stable RNA promoter activity than was the wild-type enzyme . The existence of such mutants is consistent with the idea that ppGpp interacts with the RNA polymerase enzyme and thereby alters its promoter selectivity, i.e., reduces its affinity for the stable RNA promoters . Under most conditions, the rpoB mutants had a reduced rate of growth and about a 10-fold-reduced intracellular concentration of ppGpp compared with the rpoB wild-type strains . The reduction of the level of ppGpp in the rpoB mutants during exponential growth was presumably a reflection of an indirect effect of the rpoB mutation on the control of relA-independent ppGpp metabolism. J Bacteriol, 1983 May, 154(2), 676 - 85 Localization and functional analysis of transposon mutations in regulatory genes of the TOL catabolic pathway; Franklin FC et al.; Mutant derivatives of the TOL plasmid pWW0-161, containing Tn5 insertions in the xylS and xylR regulatory genes of the catabolic pathway, have been identified and characterized . The two genes are located together on a 1.5- to 3.0-kilobase segment of TOL, just downstream of genes of the enzymes of the meta-cleavage pathway . As predicted by a current model for regulation of the TOL catabolic pathway, benzyl alcohol dehydrogenase, a representative enzyme of the upper (hydrocarbon leads to carboxylic acid) pathway, was induced by m-methylbenzyl alcohol in xylS mutant bacteria but not in a xylR mutant, whereas catechol 2,3-oxygenase, a representative enzyme of the lower (meta-cleavage) pathway, was induced by m-toluate in a xylR mutant but not in the xylS mutants . Unexpectedly, however, catechol 2,3-oxygenase was not induced by m-methylbenzyl alcohol in xylS mutants but was induced by benzyl alcohol and benzoate . These results indicate that expression of the TOL plasmid-encoded catabolic pathway is regulated by at least three control elements, two of which (the products of the xylS and xylR genes) interact in the induction of the lower pathway by methylated hydrocarbons and alcohols and one of which responds only to nonmethylated substrates. J Bacteriol, 1983 May, 154(2), 650 - 5 Analysis of ColE1 expression in vitro after chromosome fragmentation; Chen HZ et al.; The RNA and protein products synthesized from ColE1 DNA were observed before and after cutting the DNA with different restriction enzymes . Synthesis was carried out in the DNA-directed coupled transcription translation system . The S-30 extracts used to catalyze synthesis were prepared from a recB mutant in which the linear DNA fragments resulting from restriction enzyme cleavage were spared from the usual degradation by exonucleolytic attack . By correlating the observed in vitro synthesized products with the location of the cleavage sites in the plasmid chromosome, it was possible to identify specific gene products . The col gene catalyzes the synthesis of numerous peptides in addition to the 56-kilodalton colicin protein encoded by this gene . Most of the subsidiary products appear to arise as the result of premature termination by a mechanism(s) which remains to be determined . A unique RNA and protein were characterized as products of the imm gene . The RNA has an estimated mass of 150 kilodaltons, and the protein has an estimated mass of 13 kilodaltons . From the DNA sequence of the chromosome, it was concluded that the transcripts from the imm and col genes must crisscross each other over a region of about 75 base pairs . Such a pattern of transcription might lead to interference of transcription of one gene by the other gene . Consistent with this hypothesis, it was found that imm gene transcription increased severalfold in vitro when the chromosome was cleaved in a way that eliminated transcription originating at the col gene promoter . Surprisingly, the increase in transcription by this mechanism did not result in a significant increase in the synthesis of the imm gene-encoded protein. Ann Microbiol (Paris), 1983 May-Jun, 134A(3), 247 - 54 {Structures and hemagglutinating functions of pili K99 with glucose-dependent or constitutive biosynthesis and of pili F41}; Ollier JL et al.; The K99 and F41 pili produced by most bovine enterotoxigenic Escherichia coli were composed of polypeptide subunits with molecular weights of 18,300 and 29,000 . The glucose dependency of K99 pili biosynthesis in some strains of E . coli was evidenced by results of SDS-polyacrylamide gel electrophoresis . Differences in electrophoretic mobility in "Noble" agar between K99 pili from glucose-dependent and independent pathways were unrelated to differences in subunit molecular weight . The K99 and F41 pili displayed two kinds of mannose-resistant haemagglutination properties: 1) strong and thermostable; and 2) weaker than "1" and properties: 1) strong and thermostable; and 2) weaker than "1" and thermolabile . The reproducibility of results concerning "2" was poor and depended on the age of extracts and erythrocytes . It was therefore proposed that only "1" be retained, with sheep and guinea-pig erythrocytes for F41 pili and horse erythrocytes for K99 pili. J Bacteriol, 1983 May, 154(2), 598 - 603 pH dependence of the Coxiella burnetii glutamate transport system; Hackstadt T et al.; The transport of glutamate, apparently a primary energy source for Coxiella burnetii, has been examined . C . burnetii is shown to possess a pH-dependent active transport system for L-glutamate with an apparent Kt of 61.1 microM and Vmax of 8.33 pmol/s per mg at pH 3.5 . Both L-glutamine and L-asparagine competitively inhibited transport of glutamate, but D-glutamate, L-aspartate, L-glutamate-gamma-methyl ester, methionine sulfoximine, or alpha-ketoglutarate did not compete . This transport system is both temperature and energy dependent . Uptake of glutamate is highly sensitive to uncouplers of oxidative phosphorylation such as 2,4-dinitrophenol and carbonyl cyanide-m-chlorophenyl hydrazone that decrease the proton motive force across the cytoplasmic membrane . ATPase inhibitors such as dicyclohexylcarbodiimide or metabolic poisons such as KCN, NaF, or arsenite were much less effective as inhibitors of glutamate transport . Uptake of glutamate did not appear to be coupled to Na+ symport as in Escherichia coli since no monovalent cation requirement could be demonstrated . Instead, the Vmax of glutamate transport showed good correlation with the transmembrane pH gradient (delta pH) . From these results, we propose that L-glutamate transport by C . burnetii is energized via a proton motive force. Virology, 1983 Apr 30, 126(2), 466 - 79 Analysis of the genome of fish lymphocystis disease virus isolated directly from epidermal tumours of pleuronectes; Darai G et al.; Virions of fish lymphocystis disease virus (FLDV), a member of the iridovirus family, were isolated directly from lymphocystis disease lesions of individual flatfishes and purified by sucrose and subsequent cesium chloride gradient centrifugation to homogeneity as judged by electron microscopy . The isolated FLDV DNAs appear to be heterogeneous in size . Contour length measurements of 43 DNA molecules gave an average length of 49 +/- 23 microns, corresponding to 93 +/- 44 X 10(6) D . Molecular weight estimations of FLDV DNA by restriction enzyme analysis resulted in only 64.8 X 10(6) D indicating an excess length of the DNA of about 50% . FLDV DNA was sensitive to lambda 5'-exonuclease and to E . coli 3'-exonuclease III without preference of any one terminal DNA restriction fragment . Denaturation and reannealing experiments of FLDV DNA resulted in the formation of circular DNA molecules of 34.25 microns contour length (= 65.22 X 10(6) D) . This result suggests that FLDV DNA contains directly repeated sequences at both ends and that it is terminally redundant . FLDV DNA is methylated in cytosine . FLDV DNA did not hybridize with frog virus DNA indicating that the two iridoviruses are not closely related to each other . Restriction enzyme analysis and Southern blot hybridizations revealed that FLDV isolates can be classified into two different strains: FLDV strain 1 occurs in flounders and plaice, whereas strain 2 is usually found in lesions of dabs. Biochem Biophys Res Commun, 1983 Apr 29, 112(2), 723 - 8 Metal content of DNA polymerase I purified from overproducing and wild type Escherichia coli; Ferrin LJ et al.; DNA polymerase I purified from both E . coli strain B, and from an overproducing E . coli stain lysogenized with a lambda pol A phage were analyzed for metal content . After gel filtration to remove loosely bound metals, DNA polymerase I from both strains contained less than or equal to 0.2 gm atoms Zn2+/mole enzyme and 0.09 to 0.7 Mg2+/mole enzyme . Substoichiometric amounts of Fe, Co, Ni (less than or equal to 0.2 gm atoms), and Mn (less than or equal to 0.1 gm atoms) were detected . Since the metal content does not correlate with enzymatic activity, we conclude that DNA polymerase I is not a metalloenzyme. Nucleic Acids Res, 1983 Apr 25, 11(8), 2479 - 94 CAP binding to B and Z forms of DNA; Fried MG et al.; We have examined the interaction between the cyclic AMP receptor protein (CAP) and a small DNA fragment containing its specific recognition sequence by circular dichroism spectroscopy . The binding of CAP to this fragment induces a B to "C-like" change in the CD spectrum, which is different from that observed for non-specific binding . A one-to-one (CAP dimer to DNA) binding stoichiometry was deduced from spectroscopic titration data, as was a non-specific binding site size of 17 bp/dimer . In addition, we have compared the non-specific binding affinity of CAP for the B and Z forms of synthetic DNA copolymers . A slight preference for the B form was found . These results do not support the recent specific suggestion that CAP binds to a left-handed form of DNA (1), but indicate more generally that an optically detectable conformational change takes place in DNA on binding CAP. Nucleic Acids Res, 1983 Apr 25, 11(8), 2465 - 77 Molecular structure and function of the bacteriocin gene and bacteriocin protein of plasmid Clo DF13; van den Elzen PJ et al.; In this paper we present the complete nucleotide sequence of the bacteriocin gene of plasmid Clo DF13 . According to the predicted aminoacid sequence the bacteriocin, cloacin DF13, consists of 561 aminoacids and has a molecular weight of 59,293 D . To obtain insight into the structure and function of specific parts of the cloacin molecule, we constructed a hydration profile and we predicted the secondary structure of the protein . According to our predictions, the N-terminus of cloacin DF13 (corresponding to the first 150-180 aminoacids) is relatively hydrophobic and is rich in glycine residues . The data obtained support previous findings that the N-terminal part of cloacin DF13 is involved in translocation of this protein across the cell membrane . The C-terminal part of the cloacin protein is rich in positively charged aminoacids; this might reflect the RNase activity located within this domain . A comparison of the bacteriocin genes and corresponding proteins of Clo DF13 and Col E1 did not reveal any homology at the level of either the nucleotide or the aminoacid sequence . The codon usage of both genes, however, exhibits striking similarities . The sequence data obtained during this study enabled us to present the nucleotide sequence of the entire cloacin operon . The structure of this operon and the regulation of expression of the genes, located within this operon, is discussed. Nucleic Acids Res, 1983 Apr 25, 11(8), 2237 - 55 Compilation and analysis of Escherichia coli promoter DNA sequences; Hawley DK et al.; The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled . The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria . A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations . In addition, we have tabulated 98 promoter mutations . Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence. J Mol Biol, 1983 Apr 25, 165(4), 655 - 67 Kinetics of base misinsertion by DNA polymerase I of Escherichia coli; Fersht AR et al.; A simple kinetic analysis of the values of kcat and KM for base insertion and misinsertion during DNA replication is presented and applied to the problem of base misinsertion by DNA polymerase I of Escherichia coli . The role of minor tautomeric forms of deoxynucleoside triphosphates (dNTPs) in purine x pyrimidine mismatching has been examined and it has been shown that the misinsertion frequency via this route should be close to the tautomerization constant in solution and is independent of any effect of the polymerase on the tautomerization of a dNTP when bound . Kinetic data on purine x pyrimidine mismatching indicate that the dNTP in a polymerase-DNA-mismatched-dNTP complex is predominantly in the major tautomeric form . The mutagenic effect of Mn2+ in DNA replication is shown to be mediated by decreasing the values of kcat/KM for the insertion of correct dNTPs, whilst the values of this rate constant for misinsertion are relatively unaffected or increased. J Biol Chem, 1983 Apr 25, 258(8), 5189 - 95 Isolation and characterization of the protease-activated form of pyruvate oxidase . Evidence for a conformational change in the environment of the flavin prosthetic group; Recny MA et al.; Pyruvate oxidase is a tetrameric enzyme consisting of four identical subunits . The specific activity of the enzyme may be increased more than 20-fold by limited proteolytic digestion by alpha-chymotrypsin in the presence of pyruvate and thiamin pyrophosphate . This "activation" phenomenon is due to the specific cleavage of an Mr = approximately 2000 peptide from each subunit . The Mr = 2000 "activation peptide" (alpha) may be readily separated from the activated enzyme by high performance liquid chromatography under nondenaturing conditions . The alpha peptide is not required to maintain the modified tetramer in the activated state . Cleavage of the alpha peptide from each monomer is directly correlated with a substantial change in the visible spectrum of the flavin, characteristic of a shift from a hydrophobic to a more hydrophilic environment . Proteolytic cleavage by alpha-chymotrypsin in the absence of thiamin pyrophosphate irreversibly inactivates the enzyme by cleavage at a different site, producing an Mr = approximately 9000 "inactivation peptide" (beta) . The beta peptide remains noncovalently associated with the inactivated tetramer . Cleavage of the beta peptide does not alter the spectrum of the flavin, even though the beta peptide contains the alpha peptide sequence . These results suggest that cleavage and release of the alpha peptide opens up the flavin active site and may be directly responsible for the observed stimulation of enzymatic activity. J Biol Chem, 1983 Apr 25, 258(8), 4839 - 41 Phosphorus 31 nuclear magnetic resonance study of tryptophanase . Pyridoxal phosphate-binding site; Schnackerz KD et al.; The pyridoxal phosphate-dependent enzyme tryptophanase has been investigated using 31P nuclear magnetic resonance at 72.86 MHz . In the native enzyme, the pyridoxal-P 31P chemical shift was found to be 3.55 ppm and independent of pH, indicating that the dianionic phosphate group of the cofactor is not accessible to solvent . Binding of the competitive inhibitor, beta-phenyl-DL-serine, results in the formation of the transaldimination complex . This complex is fixed to the enzyme via the dianionic phosphate group of the cofactor: again the observed shift is independent of pH . In both cases, restricted rotational freedom of the phosphate group around the C-O bond linking the phosphate ester to the pyridine moiety of the cofactor could be asserted from line width data . Addition of the competitive inhibitor, L-alanine, to tryptophanase produces the quinonoid intermediate . The phosphate group of this complex has lost its specific interaction (probably a salt bridge) with the protein, as indicated by the pH dependence of the chemical shift. J Mol Biol, 1983 Apr 25, 165(4), 701 - 10 Two-dimensional crystal packing of matrix porin . A channel forming protein in Escherichia coli outer membranes; Dorset DL et al.; Two-dimensional crystalline porin sheets were obtained by reconstitution of monodisperse protein trimers and phospholipids (dimyristoylphosphatidylcholine) by detergent dialysis, analogous to the reconstitution method used for functional tests (Schindler & Rosenbusch, 1981) . Three different packing arrangements were observed: two were hexagonal (with p3 symmetry and lattice constants of 9.3 nm and 7.9 nm), and one rectangular (a = 7.9 nm, b = 13.9 nm) . The different crystals could be correlated to phospholipid-to-protein weight ratios of 0.16 to 0.72 . At the higher ratio, large hexagonal lattices predominated . Higher lipid ratios did not reveal other crystal forms . The packing arrangement of the large hexagonal form appears very similar to the hexagonal habit of three-dimensional crystal forms (Garavito et al., 1983) . The shape of the stain-penetrated triplet indentations appeared conserved in the crystal forms to a resolution of 2.2 nm . The mass distribution between triplets, however, were significantly different . They are likely to correspond primarily to lipids . Mass determinations of unstained porin by scanning transmission electron microscopy showed that unit cells consisted of single trimers . The mass found (100,000 daltons) is in good agreement with the value obtained by sedimentation equilibrium analysis. J Biol Chem, 1983 Apr 25, 258(8), 5050 - 5 Multiple covalent modifications of Trg, a sensory transducer of Escherichia coli; Kehry MR et al.; The sensory transducers of Escherichia coli are integral membrane proteins that mediate the tactic response of cells to chemical stimuli . Adaptation to environmental stimuli is correlated with methylation of the transducer proteins . Two transducer genes, tsr and tar, exhibit extensive homologies while no homology has been detected between a third transducer, trg, and those genes . The Tsr and Tar proteins have been shown to contain multiple sites for methylation as well as two sites for another modification that requires an active cheB gene product and is designated the CheB-dependent modification . In this study, covalent modifications of the Trg protein were characterized by analysis of tryptic peptides . We found that methylation occurred at several sites on the Trg protein and that the protein contained at least three sites for CheB-dependent modification, two of which were located on a tryptic peptide that contains both methionine and lysine . This tryptic peptide is analogous to the methionine- and lysine-containing methyl-accepting peptides isolated from the Tsr and Tar proteins and like those peptides may contain several methyl-accepting sites . We estimated the pKa of the group created by the CheB-dependent modification on the methionine- and lysine-containing peptide of Trg to be between pH 2.2 and 5.8 . This result supports the idea that the CheB-dependent modification is an enzymatic deamidation of glutamine to glutamic acid. J Mol Biol, 1983 Apr 25, 165(4), 669 - 82 Contribution of 3' leads to 5' exonuclease activity of DNA polymerase III holoenzyme from Escherichia coli to specificity; Fersht AR et al.; The effects of deoxynucleoside monophosphates on the 3' leads to 5' exonuclease activity of DNA polymerase III holoenzyme have been correlated with their effects on the fidelity of DNA replication . In particular, dGMP inhibits the proofreading activity of the enzyme and decreases the fidelity in those cases where a "following nucleotide effect" is also noted . This is strong evidence for proofreading . However, the absence of the effects of proofreading inhibitors or following nucleotides need not be evidence against the occurrence of proofreading: a theoretical analysis shows that these effects may not be observed even though there is active proofreading . This is suggested to be the case with the phage T4 enzyme system . The proofreading activity of Pol III appears to be directed primarily towards removing purine x pyrimidine-mediated rather than purine x purine-mediated misincorporations . recA protein inhibits the proofreading activity of Pol III on synthetic templates containing mismatched 3' termini . This is paralleled by a decrease in the fidelity of DNA replication in vitro . The inhibition is increased in the presence of dGMP or dAMP but there is no further increase in the infidelity of replication . The presence of both dNMPs and recA protein does not enable Pol III to copy past pyrimidine photodimers. J Biol Chem, 1983 Apr 25, 258(8), 4895 - 900 Binding of eucaryotic elongation factor Tu to nucleic acids; Slobin LI; The binding of eucaryotic elongation factor Tu (eEF-Tu) to nucleic acids was investigated . eEF-Tu binds to a variety of different nucleic acids with high affinity, showing a strong preference for 18 S and 28 S rRNA over transfer RNA and for ribose-containing polymers over polydeoxyribonucleotides . The factor binds at multiple sites on 28 S rRNA without strong cooperativity . eEF-Tu binds strongly to poly(G) and poly(U) but weakly, if at all, to poly(A) and poly(C) . Experiments employing an airfuge demonstrate that eEF-Tu can form a quaternary complex containing the factor, 28 S rRNA, aminoacyl-tRNA, and GTP . The existence of two distinct RNA binding sites on eEF-Tu suggests that rRNA may play a role in the recognition of eEF-Tu.aminoacyl-tRNA.GTP complexes by polysomes . Support for this suggestion comes from experiments which show that poly(G) inhibits the factor-dependent binding of aminoacyl-tRNA to mRNA-programmed 80 S ribosomes . In addition, it is shown that eEF-Tu possesses an intrinsic GTPase activity which is stimulated significantly by 28 S rRNA, poly(G), and poly(U) . The binding of eEF-Tu to poly(G) lowers the activation energy for eEF-Tu GTPase from 74.3 to 65.9 kJ . mol-1 and approximately doubles the Vmax of the enzymatic reaction . The results are discussed in relation to the binding of eEF-Tu to ribosomes during protein synthesis. Vet Rec, 1983 Apr 23, 112(17), 402 - 3 Prognosis for cows with severe clinical coliform mastitis; Golodetz CL et al.; A retrospective study was performed at the New York State college of veterinary medicine, ambulatory clinic to determine the prognosis for cows with peracute or acute coliform mastitis . Eighty-eight cows were identified by their clinical signs and positive culture of coliform organisms . In 59.1 per cent of the affected cows the quarters returned to a milk-like secretion approximately one month following treatment . The 59.1 per cent was composed of 37.5 per cent (of the initial 88 cows), which milked in the affected gland the following lactation, 11.4 per cent which were culled, and 10.2 per cent which had not yet calved and begun their next lactation . The remainder of the affected cows failed to return to milk in the affected quarter that lactation . This percentage consisted of 17.0 per cent which were culled for hypogalactia, 6.8 per cent which died during the attack, 4.6 per cent in which the affected quarters were rendered inactive with an irritant, 1.1 per cent in which the outcome was undetermined since they had not begun their next lactation and 11.4 per cent which remained in the herd . Of the 11.4 per cent, half produced milk in the affected quarter the following lactation, and half failed to return to milk. Biochim Biophys Acta, 1983 Apr 22, 723(1), 91 - 103 The effect of pH, ubiquinone depletion and myxothiazol on the reduction kinetics of the prosthetic groups of ubiquinol:cytochrome c oxidoreductase; De Vries S et al.; (1) The kinetics of the reduction by duroquinol of the prosthetic groups of QH2:cytochrome c oxidoreductase and of the formation of ubisemiquinone have been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow . (2) The formation of the antimycin-sensitive ubisemiquinone anion parallels the reduction of both high-potential and low-potential cytochrome b-562 . (3) The rates of reduction of both the {2Fe-2S} clusters and cytochromes (c + c1) are pH dependent . There is, however, a pH-dependent discrepancy between their rate of reduction, which can be correlated with the difference in pH dependencies of their midpoint potentials . (4) Lowering the pH or the Q content results in a slower reduction of part of the {2Fe-2S} clusters . It is suggested that one cluster is reduced by a quinol/semiquinone couple and the other by a semiquinone/quinone couple . (5) Myxothiazol inhibits the reduction of the {2Fe-2S} clusters, cytochrome c1 and high-potential cytochrome b-562 . (6) The results are consistent with a Q-cycle model describing the pathway of electrons through a dimeric QH2:cytochrome c oxidoreductase. Biochim Biophys Acta, 1983 Apr 20, 756(3), 335 - 40 Structure of the core regions in lipopolysaccharides from Escherichia coli K12 W2252-11U-, the Ter-15 mutant, and Ter-15 (F'-lac) and Ter-15 (F+) cells; Ohkawa T; From Escherichia coli K12 W2252-11U-cells, the Ter-15 mutant, the Ter-15 (F'-lac) and the Ter-15 (F+) cells, lipopolysaccharides were isolated and the primary structure of its core oligosaccharides was elucidated . When the F'-lac episome is transferred to the Ter-15 mutant by conjugation, the structure of the glucose III(1 leads to 3)glucose II(1 leads to 3)glucose I residue and the galactose I(1 leads to 2)-linked to the glucose I residue in the core oligosaccharide from the Ter-15 mutant changes into the structure of the glucose IV(1 leads to 6)glucose III(1 leads to 2)glucose II(1 leads to 3)glucose I residue and the galactose I (1 leads to 6)-linked to the glucose I residue in the core oligosaccharide from the Ter-15 (F'-lac) cells, but the core oligosaccharide in the Ter-15 (F+) cells is the same structure with that of the core oligosaccharide from the Ter-15 mutant when F+ episome is transferred to the Ter-15 mutant . Also, the core oligosaccharide from the Ter-15 (F'-lac) cells shows the same structure with that of the core oligosaccharide from E . coli K12 W2252-11U- cells (the parent cells) . As the result, the ability to produce the structure of the core oligosaccharide in E . coli K12 W2252-11U- cells is recovered in the Ter-15 (F'-lac) cells by the dominant expression of lac gene or its containing DNA segment in F'-lac episome. FEBS Lett, 1983 Apr 18, 154(2), 343 - 6 Stoichiometry of the H+-ATPase of Escherichia coli cells during anaerobic growth; Kashket ER; The H+/ATP stoichiometry of the H+-ATPase was investigated in Escherichia coli cells growing under anaerobic conditions at pH 6 and 7 . The protonmotive force was determined from the intracellular accumulation of benzoate and tetraphenylphosphonium ions, as well as the accumulation of lactose in this lac operon inducible, but beta-galactosidase negative strain . The phosphorylation potential was calculated from the cellular concentrations of ATP, ADP and inorganic phosphate . By comparing the phosphorylation potential and the proton motive force under these steady state conditions, the H+/ATP stoichiometry was determined to be 3, similar to the value previously found in the same cells growing under aerobic conditions. Tijdschr Diergeneeskd, 1983 Apr 15, 108(8), 319 - 21 {A comparison of the effectiveness of furazolidone and apramycin in the control of colibacillosis in weaned piglets}; Jansen WA; To compare the effectiveness of furazolidone and apramycin (Apralan) in the treatment of oedema disease in pigs, a trial was made on a commercial farm on which colibacillosis was a recurrent problem . Medicated feed containing 100 ppm of apramycin and 400 ppm of furazolidone respectively was given for three weeks after weaning . 112 Piglets were distributed over 10 battery houses at random . Results are summarized in Figure 1. J Mol Biol, 1983 Apr 15, 165(3), 523 - 39 alpha-Ketoglutarate dehydrogenase complex may be heterogeneous in quaternary structure; Wagenknecht T et al.; The quaternary structure of the alpha-ketoglutarate dehydrogenase complex (KGDC) from Escherichia coli has been investigated by electron microscopy . KGDC consists of an octahedral cube-shaped structural core, lipoyl transsuccinylase (E2), to which 12 polypeptide chains each of alpha-ketoglutarate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) are non-covalently bound . The analysis was greatly simplified by analyzing subcomplexes of KGDC prepared by assembly of the purified component enzymes in vitro; the subcomplexes consisted of the E2 component to which only a few E1 or E3 subunits were attached . We find that both the E1 and E3 bind on the surface of the E2 molecule approximately midway between the 4-fold and 2-fold symmetry axes of E2 . There are 24 such positions per E2 molecule but, based upon the observed stoichiometries of the component enzymes, it is clear that at least half of these sites are unoccupied in KGDC . If KGDC possesses symmetry, then a mechanism must exist for selecting a symmetrically distributed subset of the potential binding sites for the E1 and E3 . However, analysis of images of subcomplexes in which two E1 or E3 subunits are present suggests that binding to the E2 occurs through random selection of the potential binding sites . If native KGDC is assembled by such a mechanism, then KGDC would not have a unique quaternary structure, but instead would consist of a family of structural isomers having up to approximately 125,000 members . Consideration of independent structural and biochemical data regarding the mechanism of action of the E2 indicates that the kind of structural heterogeneity being proposed is consistent with a functional KGDC. Eur J Biochem, 1983 Apr 15, 132(1), 139 - 45 Proteins of the 30-S subunit of Escherichia coli ribosomes which interact directly with natural mRNA; Broude NE et al.; Ultraviolet irradiation (254 nm) of the complexes of MS2 phage RNA (mRNA) and the 30-S subunits of Escherichia coli ribosomes prepared at 0 degrees C and 37 degrees C in the presence and absence of initiation factor 3 (IF-3) causes cross-linking of mRNA with proteins S3, S4, S5, S7, S9, S18 and IF-3 . Hence, these proteins interact directly with mRNA within the complex 30-S-subunit . mRNA . Addition of IF-3 results in an increase of the rate of complex formation and decrease of its dissociation rate . The addition of IF-3 changes the relative amounts of cross-linked proteins (mainly S3, S4 and S18) . Decreasing the temperature from 37 degrees C to 0 degrees C not only decelerates the complex formation rate but also changes the relative amount of cross-linked proteins, indicating the influence of the conditions on the structure of the complex. Biochem Biophys Res Commun, 1983 Apr 15, 112(1), 80 - 7 DNA replication by novel macromolecular complexes involving DNA polymerase III holoenzyme activity; Kobayashi Y et al.; A fraction (P1) which showed DNA polymerase III holoenzyme activity was obtained by partial purification including Polymin P fractionation from extracts of E . coli K-12 wild type (pol A+, pol B+) cells . The P1 fraction was composed of three macromolecular complexes, 11 S, 18 S and 24 S, all of which possessed holoenzyme activity . The activity of the P1 fraction was maximal at about 70 mM NaCl . The synthesis of long-chain poly(dT) with a poly(dA) oligo(dT)10 primer was dependent on the presence of ATP, but not on the presence of spermidine, suggesting that the single-stranded DNA binding protein (SSB) was present in the fraction . The intermediate lengths of the products in the absence of ATP and NaCl also suggest the functioning of DNA polymerase III'. Biochem Biophys Res Commun, 1983 Apr 15, 112(1), 66 - 72 Origin of the labile sulfide in the iron-sulfur proteins of Escherichia coli; White RH; A method has been devised for measuring the abundance of sulfur-34 in the hydrogen sulfide released upon the acidification of Escherichia coli cells . Evidence is presented, based on the rate at which the hydrogen sulfide is released from the cells as well as the total amount released, that this hydrogen sulfide originates from the iron-sulfur proteins present in the cells . The sulfur-34 abundance in this hydrogen sulfide which was isolated from cells grown with {sulfane-34S} thiocystine, a compound which can differentially label in vivo the sulfur-34 abundance of cysteine and hydrogen sulfide, shows cysteine sulfur and not hydrogen sulfide to be the origin of the sulfide sulfur of iron-sulfur proteins in aerobically grown E . coli. Biochem Biophys Res Commun, 1983 Apr 15, 112(1), 320 - 6 Chemical synthesis of a highly potent and heat-stable analog of an enterotoxin produced by a human strain of enterotoxigenic Escherichia coli; Aimoto S et al.; A shorter analog of a heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli SK-1, consisting of 14 amino acid residues including 6 half-cystine residues, was synthesized by conventional methods . The peptide was evaluated for ability to induce intestinal secretion in suckling mice and for stability at high temperature under various conditions . The peptide was 2-5 times more potent than native toxin and was still toxic after heat-treatment at 120 degrees C for 30 min. Biochim Biophys Acta, 1983 Apr 15, 739(3), 265 - 75 A cellular factor involved in the formation of a DNA-synthesizing complex from DNA polymerase I in Escherichia coli; Scharff R et al.; A factor ('E') has been identified which stabilizes an endogenous DNA-synthesizing complex involving DNA polymerase I . The complex is separated from free DNA polymerase by polyacrylamide gel electrophoresis . The factor will reform the complex after it has been dissociated and will convert a preparation of DNA polymerase I to complex . The factor and the DNA-synthesizing complex both appear to be localized at the cell membrane. Biochem J, 1983 Apr 15, 212(1), 105 - 12 The use of valinomycin, nigericin and trichlorocarbanilide in control of the protonmotive force in Escherichia coli cells; Ahmed S et al.; Valinomycin, nigericin and trichlorocarbanilide were assessed for their ability to control the protonmotive force in Escherichia coli cells . Valinomycin, at high K+ concentrations, was found to decrease the membrane potential delta phi and indirectly to decrease the pH gradient delta pH . Nigericin was found to have two modes of action . At low concentrations (0.05-2 microM) it carried out K+/H+ exchange and decreased delta pH . At higher concentrations (50 microM) it carried out a K+-dependent transfer of H+, decreasing both delta phi and delta pH . In EDTA-treated cells only the latter mode of action was evident, whereas in a mutant sensitive to deoxycholate both types of effect were observed . Trichlorocarbanilide is proposed as an alternative to nigericin for the specific control of delta pH, and it can be used in cells not treated with EDTA. Eur J Biochem, 1983 Apr 15, 132(1), 189 - 94 Membrane topology of ATP synthase from bovine heart mitochondria and Escherichia coli; Montecucco C et al.; The polypeptides exposed to lipids in the membranous F0 sector of the mitochondrial and Escherichia coli ATP synthases were labelled with radioactive photoreactive lipids . Highly resolving gel electrophoretic conditions were used in order to separate all the eighteen components forming the bovine heart mitochondrial enzyme . The hydrophobic labelling was performed on fully active and inhibitor-sensitive ATP synthases . In the mitochondrial enzyme prepared according to Serrano et al . (1976) {J . Biol . Chem . 251, 2453-2461} seven polypeptides of Mr 30500; 11500; 10500; 10000; 9500; 8500 and 4500 were labelled . The major amount of radioactivity was associated with the 30500-Mr component, which is thought to be the adenine nucleotide carrier . In the preparation of Galante et al., (1979) which almost completely lacks this component {J . Biol . Chem . 254, 12372-12378} nine polypeptides of Mr 25000; 21000; 11500; 10500; 10000; 9500; 9200; 8500 and 4500 were labelled . In the ATPase synthase from E . coli the major amount of labelling was associated with subunit b and only a minor portion with subunit c. Virology, 1983 Apr 15, 126(1), 168 - 82 Studies on the replication of Escherichia coli phage lambda DNA . I . The kinetics of DNA replication and requirements for the generation of rolling circles; Better M et al.; Escherichia coli phage lambda DNA has been isolated from infected bacteria using a new technique by which virtually all phage DNA is recovered . Isolated DNA is examined by electron microscopy . Addition of phi X174 RF1 molecules as a counting standard enables us to determine the average number of lambda DNA molecules present in an infected cell . In this study, we have followed the kinetics of lambda DNA replication and examined rolling circle replication . The most important findings are the following: (1) Rolling circle replication is initiated at roughly the same time as is theta replication, indicating that the rolling circle is not solely a late-replicating form . (2) theta replication stops at about 16 min after infection . (3) Early in infection the number of DNA molecules per cell doubles every 2-3 min until theta replication stops, at which point most DNA synthesis consists of growth of the tails of about three rolling circles per cell . (4) Neither the timing of rolling circle replication nor the number of molecules is affected by the activity of the lambda red genes . (5) . The red genes are responsible for the production of oligomeric circles late in infection. J Mol Biol, 1983 Apr 15, 165(3), 419 - 42 Characteristics of the nascent and non-nascent small DNA molecules found in Escherichia coli; Denhardt DT et al.; We have purified nascent DNA molecules from Escherichia coli pulse-labeled with 5-bromo{6-3H}deoxyuridine by repeated chromatography on nitrocellulose and isopycnic centrifugation in CsCl . The nascent molecules were labeled with 32P either at their 5' ends using polynucleotide kinase or at their 3' ends using terminal transferase . Compared to the non-nascent DNA of normal density, the nascent dense DNA contained a higher proportion of molecules terminated at their 5' ends with ribonucleotides . Exposure of the dense DNA to alkali generated 5' OH termini quantitatively equivalent to the number of molecules bearing 5' ribonucleotides . Experiments designed (1) to detect structures at the 5' ends of phosphatase-treated nascent DNA molecules that caused them to be resistant to hydrolysis by spleen exonuclease or (2) to detect polypeptides that were associated covalently with small DNA molecules and could be iodinated with the Bolton-Hunter reagent did not yield positive results . We conclude that many, if not all, of the intermediates in E . coli DNA replication are initiated with one or more ribonucleotides . The nascent molecules are outnumbered by small non-nascent DNA molecules in the cell, many of which appear to become slightly longer when cells are pulsed with thymidine . Many of the non-nascent DNA molecules behave as if they were self-complementary or crosslinked. Biochemistry, 1983 Apr 12, 22(8), 1883 - 8 Proton exchange on carbons 2 and 3 of serine during their conversion into methyl groups of methionine and thymine in Escherichia coli; White RH; The conversion of the C-2 and C-3 carbons of serine and their attached protons into the methyl groups of methionine and thymine was studied in vivo with exponentially growing cultures of Escheric