Cell, 1982 Sep, 30(2), 627 - 36 Nucleotide sequence of the gene encoding the RNA subunit (M1 RNA) of ribonuclease P from Escherichia coli; Reed RE et al.; The gene encoding the RNA subunit (M1 RNA) of RNAase P (EC 3.1.26.5) from Escherichia coli has been isolated, and its complete nucleotide sequence, including flanking regions, has been determined . The promoter region, similar to others near genes under stringent control, and the site of transcription termination have been identified . The transcript from the gene (M1 RNA) can be drawn in a secondary structure that has approximately 60% G-C base pairs . One hairpin loop of this hypothetical structure has five contiguous nucleotides complementary to invariant nucleotides in the TpsiCG loop of all E . coli tRNAs . The M1 gene, when subcloned in the plasmid pBR325, can be amplified . It directs production of functional M1 RNA . In an E . coli strain thermosensitive for RNAase P function, the size of the gene transcript is the same as in wild-type E . coli, but less mature M1 RNA is made in the mutant cells. J Bacteriol, 1982 Sep, 151(3), 1425 - 32 Control of RNA synthesis in Escherichia coli after a shift to higher temperature; Ryals J et al.; Parameters of RNA synthesis were measured after a temperature upshift in a pair of Escherichia coli B/r strains that are isogenic except for having relA and relA+ loci, to examine the cause for a reported anomaly in the correlation between guanosine tetraphosphate (ppGpp) and stable RNA (rRNA, tRNA) synthesis under such conditions . Two main results were: (i) the specific stable RNA gene activity (stable RNA per total RNA synthesis) correlated in the conventionally expected fashion with the level of ppGpp but was obscured by a nonspecific increase in the RNA chain elongation rate due to the higher temperature; (ii) the temperature upshift caused a transient reduction in the RNA polymerase activity (transcribing per total enzyme) that accounts for the previously observed oscillating RNA synthesis rate after a temperature shift. J Bacteriol, 1982 Sep, 151(3), 1261 - 8 Control of rRNA and tRNA syntheses in Escherichia coli by guanosine tetraphosphate; Ryals J et al.; The expression of stable RNA (rRNA and tRNA) genes and the concentration of guanosine tetraphosphate (ppGpp) were measured in an isogenic pair of relA+ and relA derivatives of Escherichia coli B/r . The cells were either growing exponentially at different rates or subject to amino acid starvation when they were measured . The specific stable RNA gene activity (rs/rt, the rate of rRNA and tRNA synthesis relative to the total instantaneous rate of RNA synthesis) was found to decrease from 1.0 at a ppGpp concentration of 0 (extrapolated value) to 0.24 at saturating concentrations of ppGpp (above 100 pmoles per optical density at 460 nm unit of cell mass) . The same relationship between the rs/rt ratio and ppGpp concentration was obtained independent of the physiological state of the bacteria (i.e., independent of the growth rate or of amino acid starvation) and independent of the relA allele . It can be concluded that ppGpp is an effector for stable RNA gene control and that stable RNA genes are not controlled by factors other than the ppGpp-mediated system . The results were shown to be qualitatively and quantitatively consistent with data on in vitro rRNA gene control by ppGpp, and they were interpreted in the light of reported ideas derived from those in vitro experiments. J Bacteriol, 1982 Sep, 151(3), 1210 - 21 A surface polysaccharide of Escherichia coli O111 contains O-antigen and inhibits agglutination of cells by O-antiserum; Goldman RC et al.; The repeating pentasaccharide of O-antigen from Escherichia coli O111 contains galactose, glucose, N-acetylglucosamine, and colitose, the latter representing the major antigenic determinant . Phenol extraction of this strain was previously shown to release two fractions (I and II) containing O-antigen carbohydrate, and both fractions were believed to be lipopolysaccharide . We have now characterized fractions I and II and conclude that only fraction II represents lipopolysaccharide . Fraction II contains phosphate, 2-keto-3-deoxyoctonate, beta-hydroxymyristic acid, and potent endotoxin activity, whereas fraction I was deficient in all of these properties of the lipid A and core oligosaccharide regions of lipopolysaccharide . Fractions I and II each represented 50% of the total cellular O-antigen, and both were present on the cell surface . Both fractions were metabolically stable, and no precursor-product relationship existed between them . Fraction II had a number-average molecular weight of 15,800, corresponding to an average of 12 O-antigen repeats per molecule . In contrast, fraction I had a number-average molecular weight of 354,000, corresponding to an average of 404 O-antigen repeats per molecule . Before heat treatment, cells of E . coli O111 are poorly agglutinated by O-serum; although this indicates the presence of a capsule, the corresponding K-antigen was never detected . We conclude that fraction I, when present on the cell surface, inhibits agglutination of unheated cultures of E . coli O111 by O-serum because: (i) a variant strain which lacks fraction I was agglutinated by O-serum without prior heating; (ii) erythrocytes coated with purified fraction I behaved like bacteria containing fraction I in showing inhibition of O-serum agglutination; and (iii) heat treatment released fraction I and rendered bacterial cells agglutinable in O-serum. Infection, 1982 Sep-Oct, 10(5), 324 - 6 Intestinal colonization and antibody response; Soderstrom T et al.; Rapid bacterial colonization of the gastrointestinal tract takes place immediately after birth . Only a few of the Escherichia coli strains colonizing the gut of healthy full-term neonates expressed MS pili . On the other hand, most E . coli strains isolated carried MR pili resembling the P-fimbriae which are a known virulence factor for pyelonephritogenic E . coli . The presence of serum antibodies against pili and K antigens of E . coli after vaccination did not influence the capacity of E . coli to colonize and persist in the intestine of experimental animals. Acta Virol, 1982 Sep, 26(5), 305 - 11 Photobiology of furocoumarins . Various types of crosslinking with DNA and their interference with the development of lambda phage; Hradecna Z et al.; It was shown that the multiplication of phage lambda was strongly suppressed by furocoumarins after irradiation with near ultraviolet light of 365 nm wavelength . Using xanthotoxin or angelicin there was a marked inhibition of the phage DNA injection and replication but adsorption was unaffected . This inhibition was attributed to various types of DNA crosslinking produced in the phage heads . Type I crosslink corresponded to covalent binding between adjacent sites in opposite strands of the double helix . Crosslink type II (hairpin crosslink) required a highly condensed DNA and corresponded to covalent binding between adjacent sites on double-helical segments of a folded DNA molecule . The relationships of the type I crosslinks to the DNA replication and of the type II crosslinks to DNA injection are being discussed . Like type II crosslinks, the nucleic acid--protein crosslinks hinder injection. Infect Immun, 1982 Sep, 37(3), 1170 - 80 Use of specific antibody to demonstrate glycocalyx, K99 pili, and the spatial relationships of K99+ enterotoxigenic Escherichia coli in the ileum of colostrum-fed calves; Chan R et al.; The attachment of enterotoxigenic Escherichia coli (ETEC) strain B44 (O9:K30:K99:F41:H-) to the ileal epithelium of newborn colostrum-fed calves was studied by electron microscopy . Stabilization of the bacterial glycocalyx (K30) and pili (K99) by fixation of tissue sections in specific antibody and staining with ruthenium red were used so that the bacterial surface structures could be clearly visualized and their spatial relationship to the intestinal brush border defined . When sections of ileum from infected calves were neither fixed in antibody nor stained with ruthenium red, the ETEC cells colonizing the small intestine were separated from each other and from the brush border by an electron-translucent halo; neither the glycocalyx nor the pili could be clearly resolved . When ruthenium red staining was used, the halo was partially filled by a net of electron-dense fibers composed of pili and condensed glycocalyx which extended to the brush border . Tissue sections reacted with anti-K30 antibody before staining with ruthenium red revealed microcolonies of ETEC surrounded by a discrete electron-dense glycocalyx 0.3 to 1.0 micrometers thick and in tight contact with the epithelial cell surface . When ileal tissue was treated with K99 antibody, the K99 pili were visible as discrete fibers extending from the bacterial cell surface through the glycocalyx . We discuss the role of these cell surface components in pathogenic adhesion and in the formation of protected microcolonies at the surface of the infected ileal epithelium. J Bacteriol, 1982 Sep, 151(3), 1109 - 17 Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli; Mengin-Lecreulx D et al.; The cellular pool levels of most of the cytoplasmic precursors of peptidoglycan synthesis were determined for normally growing cells of Escherichia coli K-12 . In particular, a convenient method for analyzing the uridine nucleotide precursor contents was developed by associating gel filtration and reverse-phase high-pressure liquid chromatography techniques . The enzymatic parameters of the four synthetases which catalyze the stepwise addition of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine to uridine diphosphate-N-acetylmuramic acid were determined . It was noteworthy that the pool levels of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine were much higher than the Km values determined for these substrates, whereas the molar concentrations of the uridine nucleotide precursors were lower than or about the same order of magnitude as the corresponding Km values . Taking into consideration the data obtained, an attempt was made to compare the in vitro activities of the D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine adding enzymes with their in vivo functioning, expressed by the amounts of peptidoglycan synthesized . The results also suggested that these adding activities were not in excess in the cell under normal growth conditions, but their amounts appeared adjusted to the requirements of peptidoglycan synthesis . Under the different in vitro conditions considered, only low levels of L-alanine adding activity were observed. Cancer Res, 1982 Sep, 42(9), 3526 - 31 Purification and properties of inorganic pyrophosphatase of rat liver and hepatoma 3924A; Yoshida C et al.; Inorganic pyrophosphatase (EC 3.6.1.1) has been purified to electrophoretic homogeneity from the soluble portion of the cytoplasm of rat Hepatoma 3924A and rat liver . It has a specific activity of 600 to 700 mumol inorganic orthophosphate liberated per min per mg protein at 25 degrees, a value in the same range as the highly purified enzymes from yeast and Escherichia coli . By all criteria applied, the hepatoma inorganic pyrophosphatase is identical with the liver enzyme . It is a dimer with subunits with molecular weights of approximately 30,000 to 33,000 and has a pH optimum of 7.4, a Km for pyrophosphate of 5 microM, and a Ka for Mg2+ of 0.3 mM with a pyrophosphate concentration of 0.2 mM . It is not inhibited by high Mg2+ concentrations up to 20 mM . Other metal ions such as Zn2+ and Ca2+ do not activate . Mn2+ activates to less than 10% that of Mg2+ at 0.6 mM and has no effect at 1 mM or higher . In the presence of optimal (4 mM) Mg2+ concentration, Ca2+, Mn2+, Hg2+, and F- at 0.2 mM inhibited strongly, but Zn2+ at 1 mM was not inhibitory . The enzyme had no phosphatase activity toward any of the purine or pyrimidine nucleoside mono-, di-, and triphosphates or toward p-nitrophenyl phosphate, beta-glycerophosphate, glucose 6-phosphate, or glucose 1-phosphate . Bromo- or iodoacetate at high concentration had no inhibitory effect, but p-chloromercuribenzoate and p-chloromercuriphenylsulfonate inhibited strongly at low concentration . The purified enzyme was very unstable but was protected markedly at or above the pH optimum of 7.4 by cysteine, dithiothreitol, and glutathione. Biochemistry, 1982 Aug 31, 21(18), 4271 - 5 Metabolism of L-{sulfane-34S}thiocystine by Escherichia coli; White RH; The metabolism of L-thiocystine {bis(2-amino-2-carboxyethyl) trisulfide} by Escherichia coli was studied by using L-{sulfane-35S}thiocystine . This compound was found to serve as a source of sulfur for E . coli grown on a defined medium free of other sulfur sources and to incorporate its labeled sulfur into cysteine as well as the other sulfur-containing cellular components . For determination of the extent of the synthesis of new cysteine in these cells, cells were grown with {3,3-2H2}serine and L-{sulfane-34S}thiocystine, and the extent of incorporation of both deuterium and 34S into the cellular cysteine was measured by gas chromatography-mass spectrometry . The results show that approximately 50% of the cysteine which is incorporated into cellular macromolecules is derived from the thiocystine without cleavage of the carbon-sulfur bond, the remaining portion being newly biosynthesized from serine and 34S-enriched H2S . These results suggest that the first step in the metabolism of thiocystine by E . coli involves the beta elimination of pyruvate . This type of reaction is characteristic of the cleavage reactions catalyzed by beta-cystathionase. Biochemistry, 1982 Aug 31, 21(18), 4438 - 42 Proximity of reactive cysteine residue and flavin in Escherichia coli pyruvate oxidase as estimated by fluorescence energy transfer; Koland JG et al.; Pyruvate oxidase of Escherichia coli possesses a reactive cysteine residue believed to be associated with the thiamin pyrophosphate (TPP) binding site . This residue is not reactive in the presence of TPP . Exposure of the enzyme to cysteine-directed fluorescent reagents results in the formation of fluorescent protein conjugates . Although these reagents do not react solely with the TPP-protectable cysteine residue, the fluorescence emission spectrum of a probe attached to this residue can be obtained by a difference technique . It was determined that the fluorescence emission of probes at the TPP-protectable site is very low due to energy transfer to the FAD coenzyme and that this fluorescence is greatly enhanced upon reduction or extraction of the flavin . Application of fluorescence energy transfer theory enabled the determination of an upper limit for the distance between the probes at the TPP-protectable site and the flavin adenine dinucleotide (FAD) (roughly 20 A) . Thus, the TPP binding site and the FAD coenzyme are likely in close proximity. Biochemistry, 1982 Aug 31, 21(18), 4332 - 7 recA protein from Escherichia coli . a very rapid and simple purification procedure: binding of adenosine 5'-triphosphate and adenosine 5'-diphosphate by the homogeneous protein; Cotterill SM et al.; The recA protein from Escherichia coli may be rapidly purified to homogeneity by a simple procedure involving only selective precipitation and one gel filtration step . The binding of ATP to the homogeneous protein has been measured by nonequilibrium dialysis . At pH 8.1 and 25 degrees C, the stoichiometry of the recA X ATP complex is 1:1 and the dissociation constant 24 microM . The binding of ADP to the enzyme and its complexes with single-stranded (ss) DNA and double-stranded (ds) DNA has been measured by equilibrium dialysis . In the absence of DNA, the binding is similar to that observed for ATP . The addition of ssDNA weakens the binding 3-fold . The addition of dsDNA causes a significant drop in the stoichiometry, suggesting an asymmetric distribution of active sites in the complex. Biochim Biophys Acta, 1982 Aug 30, 698(2), 116 - 27 Interaction of elongation factor EF-Tu with gamma-amides of GTP and beta-amides of GDP bearing the azidoaryl group or the chloroethylaminoaryl group placed at the terminal phosphate; Babkina GT et al.; New types of azidoaryl analogs of GTP: gamma-(4-azido)anilide of GTP (I), gamma-(n-(4-azidobenzyl)-N-methyl)amide of GTP (II) and of GDP: beta-(4-azido)anilide of GDP (III), beta-(N-(4-azidobenzyl)-N-methyl)amide of GDP (IV) have been synthesized by treatment of the nucleotide in aqueous solution with N-cyclohexyl-N-beta-(4-methylmorpholinium)-ethylcarbodiimide p-toluene sulfonate and the respective amine . The analog of GTP bearing at the gamma-phosphate an alkylating 2-chloroethylamino group: gamma-(4-N-(2-chloroethyl)-N-methylaminobenzyl)amide of GTP (V) was prepared by the method described previously for the preparation of the analog of ATP (Knorre, D.G., Kurbatov, V.A . and Samukov, V.V . (1976) FEBS Lett . 70, 105-108) . Azidoaryl analogs of GTP and GDP as well as the chloroethylaminoaryl analog of GTP compete with GDP in the formation of the binary complex EF-Tu.GDP with the respective Ki values 3.9.10(-7) M (I), 2.9.10(-8)M (II), 6.9.10(-7)M (III), 5.0.10(-7)M (IV) and 3.8.10(-8)M (V) relative to GDP . The dissociation constants of the complexes of the radioactively-labeled GTP analogs I, II and V with elongation factor Tu were calculated to be 8.5.10(-6)M, 3.4.10(-7)M and 4.6.10(-8)M, respectively, or approx . 1740-, 70- and 9-times greater than that of GDP . GTP analogs I, II and V were found to substitute GTP in the stimulation of EF-Tu-dependent binding of aminoacyl-tRNA to the ribosome-mRNA complex. Science, 1982 Aug 27, 217(4562), 845 - 8 Endotoxin-stimulated opioid peptide secretion: two secretory pools and feedback control in vivo; Carr DB et al.; Small doses of endotoxin evoked a dramatic biphasic response of opioid peptide secretion into blood in sheep . The first phase began within minutes and coincided with a brief hypertensive response to endotoxin well before the appearance of fever or hypotension . The ratio of beta-endorphin to beta-lipotropin fell abruptly at the onset of the second phase of release, suggesting early depletion of a pool rich in beta-endorphin and subsequent emergence of a pool rich in unprocessed precursor . The concentration of cerebrospinal fluid opioids increased tenfold during the second phase . Naloxone administration augmented endotoxin-induced opioid secretion in both early and late phases, suggesting a short-loop feedback regulation of stress-induced endorphin secretion. Nature, 1982 Aug 26, 298(5877), 863 - 5 DNA precursors in chemical mutagenesis: a novel application of DNA sequencing; Topal MD et al.; Recently, we have shown that deoxyribonucleoside residues in the cellular DNA precursor pool are generally more susceptible to methylation than are residues within the DNA duplex . The N-1 position of adenosine, for example, was found to be at least 13,000 times more susceptible to methylation by N-methyl-N-nitrosourea (MNU) than the same site in the DNA . These results suggest that potential sites for alkylation in the double-strand duplex are relatively inaccessible to direct alkylation in vivo . Many of these sites are probably protected from alkylation not only by their position in the interstices of the DNA helix, but also by further in vivo 'packaging' of the DNA in chromatin . We have now used DNA sequencing to demonstrate the incorporation properties of products of the reaction of MNU with dATP and of deoxy-N4-hydroxycytidine triphosphate during DNA replication in vitro by phage T4 DNA polymerase and the 'Klenow' fragment of Escherichia coli pol I . The results suggest that DNA precursor nucleotides due to their greater availability for alkylation, may offer routes for the introduction of alkylated residues into double-stranded DNA. J Biol Chem, 1982 Aug 25, 257(16), 9770 - 80 Studies on the mechanism of Escherichia coli DNA polymerase I large fragment . Chain termination and modulation by polynucleotides; Detera SD et al.; Homopolymer replication systems and measurement of precise product chain length have been used to elucidate two new points about the mechanism of Escherichia coli DNA polymerase I large fragment: chain termination as a function of product chain length is multiphasic, and polynucleotides exert a secondary effect in the mechanism of this enzyme . During replication of (dT)800 or (dA)800 with short oligonucleotides as primer, DNA polymerase I large fragment was processive, catalyzing hundreds of dNMP incorporations during each cycle of binding to the template-primer, incorporation, and termination . Our observations indicated, however, that polynucleotides could terminate chain elongation and that this effect probably occurred through interaction at a secondary binding site on the enzyme . Thus, in the presence of higher levels of template-primer, early termination occurred and the relatively short product molecules could be resolved by gel electrophoresis . Incubation conditions were adjusted so that the number of product molecules at each chain length was equal to the actual number of termination events, and, therefore, the statistical chance for termination as a function of product chain length could be calculated . These termination probability values depended upon specific incubation conditions, such as dNTP level and whether the primer was a ribo- or deoxyribonucleotide, and interestingly, the values changed as the chain length of the product increased . For the first 5 to 10 dMP residues added to the primer, termination probability declined with each dNMP addition, but then remained constant for the addition of the next 20 to 40 dNMP residues . These results are discussed in the context of a kinetic model representing two stages of synthesis during the formation of each product molecule. J Biol Chem, 1982 Aug 25, 257(16), 9598 - 604 Purification and properties of chloroplast leucyl-tRNA synthetase from a higher plant: Phaseolus vulgaris; Souciet G et al.; The chloroplast leucyl-tRNA synthetase from Phaseolus vulgaris was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, hydroxylapatite, and phosphocellulose . Finally, the pure enzyme was obtained after affinity chromatography on Blue Sepharose CL-6B using specific elution with a pure Escherichia coli tRNALeu isoacceptor . The specific activity of chloroplast leucyl-tRNA synthetase (1550 units/mg) is the highest ever obtained for a higher plant aminoacyl-tRNA synthetase . The purified enzyme has an optimal pH of 9.0 and Km values are, respectively, 2.6 X 10(-6) M for unfractionated E . coli tRNA, 0.85 X 10(-6) M for E . coli tRNA5Leu, 1.8 X 10(-4) M for ATP, and 1.4 X 10(-5) M for L-leucine . Chloroplast leucyl-tRNA synthetase is a large monomer which has a Mr of 122,000 as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate and urea and by gel filtration . Determination of Stokes radius, diffusion coefficient, and frictional ratio suggests that the enzyme structure is rather compact . The amino acid composition shows a relatively large proportion of apolar residues . Specific antibodies were raised in rabbits against the pure chloroplast leucyl-tRNA synthetase. Nucleic Acids Res, 1982 Aug 25, 10(16), 5033 - 42 Sequence of the N2 neuraminidase from influenza virus A/NT/60/68; Bentley DR et al.; The complete sequence of the neuraminidase gene of influenza virus A/NT/60/68 (N2 subtype) was determined following cloning of full length complementary DNA into pBR322 . Comparison of the predicted amino acid sequence with a closely related neuraminidase from A/Udorn/72 suggests that point mutations over an extensive region of the primary sequence can contribute to antigenic drift, although the region between amino acid residues 308 and 371 may be particularly significant. Nucleic Acids Res, 1982 Aug 25, 10(16), 5043 - 57 PpGpp regulates the binding of two RNA polymerase molecules to the tyrT promoter; Travers AA et al.; Bacterial promoters differ in the number of RNA polymerase molecules that bind to form a filterable polymerase-promoter complex . We show that two holoenzyme molecules interact with the tyrT promoter, probably as a dimer . This interaction is inhibited by ppGpp . By contrast a single holoenzyme monomer suffices for complex formation at the lacUV5 promoter . We propose that In vivo promoter selection by monomeric and dimeric forms of the enzyme could coordinate the synthesis of stable RNA with that of mRNA and could also account in part for the switch in transcriptional selectivity during the stringent response. Nucleic Acids Res, 1982 Aug 25, 10(16), 5015 - 31 Transcription initiation sites within an IS2 insertion in a Gal-constitutive mutant of Escherichia coli; Hinton DM et al.; Insertion of the insertion sequence Is2(I) directly before the galE gene of the galactose operon results in a Gal minus phenotype (1, 2) . The Gal-constitutive allele galc200 (and its deletion derivative galc200 delta 31) arise from such a Gal minus mutant by the insertion of LS2(II) DNA within the LS2(I) sequence (3) . We have transcribed in vitro a DNA template representing the IS2-galE region of galc200 delta 31 . Gal-directed transcription initiates at two sites within the IS2(I) sequence, 51 and 52 bp from the IS2-galE junction . The promoter for these transcripts, Pgal200 delta 31, is composed of a novel joint between a -10 region from the IS2(I) DNA and a -35 region contributed by the IS2(II) insertion . No promoters intrinsic to the 121 bp of the IS2(II) sequence also present on the template were detected . The relevance of Pgal200 delta 31 to the Galc phenotype of galc200 and to general mechanisms for the constitutive expression of genes adjacent to IS2 is discussed. Nucleic Acids Res, 1982 Aug 25, 10(16), 4985 - 5002 The maize chloroplast genes for the beta and epsilon subunits of the photosynthetic coupling factor CF1 are fused; Krebbers ET et al.; We have cloned and sequenced the maize chloroplast genome fragment Eco RI e which contains the 2.2 kb transcript previously reported (Link, G . and Bogorad, L . (1980) Proc . Nat . Acad . Sci . 77 6821-6825) to lie next to the maize gene for the large subunit of ribulose bisphosphate carboxylase (LS) and to be transcribed divergently . Immunochemical and sequencing data show that the gene codes for the beta subunit of the maize chloroplast coupling factor complex (CF1) . The derived amino acid sequence is highly homologous to that of the corresponding E . coli protein (Saraste et al . (1981) Nucleic Acids Res . 9 5287-5296) . The last base of the codon for the terminal lysine residue of the beta subunit of CF1 is the first base of the codon for the initiating methionine of an open reading frame whose derived amino acid composition and size closely match that reported for the epsilon subunit (Binder et al . (1978) J . Biol . Chem . 253 3094-3100) . The close coupling of the two genes may serve to in sure their stoichiometric production. Nucleic Acids Res, 1982 Aug 25, 10(16), 4913 - 22 Molecular cloning and characterisation of the two DNA components of tomato golden mosaic virus; Bisaro DM et al.; We report the molecular cloning of the tomato golden mosaic virus (TGMV) genome in the E . coli plasmid pAT 153 . The results of this work conclusively show that TGMV DNA consists of two components (designated A and B) of almost, but not exactly, the same size . Four different recombinant plasmids are described, two containing component A in opposite orientation and two containing component B in opposite orientation . Southern blot analysis revealed little sequence homology between A and B and showed both components to be equally represented in viral and intracellular DNA forms . Detailed restriction maps of the cloned DNAs are presented, and a comparison of these with digests of intracellular viral dsDNA indicates that the former are full-length faithful copies of TGMV DNA . This is the first report of the cloning of a geminivirus genome. J Biol Chem, 1982 Aug 25, 257(16), 9605 - 11 Role of the divalent metal cation in the pyruvate oxidase reaction; Blake R 2nd et al.; Purified pyruvate oxidase requires a divalent metal cation for enzymatic activity . The function of the divalent metal cation was studied for unactivated, dodecyl sulfate-activated, and phosphatidylglycerol-activated oxidase . Assays performed in the presence of Mg2+, CA2+, Zn2+, Mn2+, Ba2+, Ni2+, Co2+, Cu2+, and Cr3+ in each of four different buffers, phosphate, 1,4-piperazinediethanesulfonic acid, imidazole, and citrate, indicate that any of these metal cations will fulfill the pyruvate oxidase requirement . Extensive steady state kinetics data were obtained with both Mg2+ and Mn2+ . All the data are consistent with the proposition that the only role of the metal is to bind to the cofactor thiamin pyrophosphate (TPP) and that it is the Me2+-TPP complex which is the true cofactor . Values of the Mg2+ and Mn2+ dissociation constants with TPP were determined by EPR spectroscopy and these data were used to calculate the Michaelis constant for the Me2+-TPP complexes . The results show that the Michaelis constants for the Me2+-TPP complexes are independent of the metal cation in the complex . Fluorescence quenching experiments show that the Michaelis constant is equal to the dissociation constant of the Mn2+-TPP complex with the enzyme . It was also shown that Mn2+ will only bind to the enzyme in the presence of TPP and that one Mn2+ binds per subunit . Steady state kinetics experiments with Mn2+ were more complicated than those obtained with Mg2+ because of the formation of an abortive Mn2+-pyruvate complex . Both EPR and steady state kinetics data indicated complex formation with a dissociation constant of about 70 mM. J Biol Chem, 1982 Aug 25, 257(16), 9518 - 24 Structure of catabolite gene activator protein at 2.9-A resolution . Incorporation of amino acid sequence and interactions with cyclic AMP; McKay DB et al.; The amino acid sequence of the Escherichia coli catabolite gene activator protein has been fit into a 2.9-A resolution electron density map . Each subunit of the dimer consists of two structurally distinct domains . The larger NH2-terminal domain is seen to bind cyclic AMP and forms all of the contacts between the subunits . The cyclic AMP is completely buried between the interior of the "beta roll" structure of the large domain and a long alpha helix; it makes important hydrogen-bonding interactions with residues from both subunits . The guanidinium group of a buried Arg makes an internal salt link with the phosphate of cyclic AMP . The 6-amino group of adenine interacts simultaneously with both subunits . This interaction with both subunits and the fact that cyclic GMP and cyclic IMP do not activate catabolite gene activator protein suggest that the binding of cyclic AMP may alter the relative orientation of the two subunits, which in turn would change the structure of a DNA binding site that is presumed to span the two smaller domains . The distribution and nature of side chains in the small domain do not rule out the possibility that catabolite gene activator protein binds to left-handed B-DNA. Nucleic Acids Res, 1982 Aug 25, 10(16), 4973 - 83 NMR evidence for the existence of two native conformations of 5S RNA; Kime MJ et al.; NMR spectra of the non-exchangeable protons in 5S RNA from E . coli show the existence of two distinct conformers of the molecule which meet the operational definition of "A form" or native 5S RNA . Both are easily distinguished spectroscopically from denatured, "B form" 5S RNA . The conditions which interconvert the two A form conformers strongly suggest that the transition between them gives rise to the low temperature optical melting transition first reported in 5S RNA by Kao and Crothers (1). J Biol Chem, 1982 Aug 25, 257(16), 9872 - 7 Cloning, partial sequencing, and expression of glyceraldehyde-3-phosphate dehydrogenase gene in chick embryonic heart muscle cells; Arnold HH et al.; Two recombinant plasmids containing structural gene sequences of chick embryonic heart glyceraldehyde-3-phosphate dehydrogenase (GAP dehydrogenase) were constructed and characterized . The plasmids pGAP 30 and pGAP 36 have inserts of 1200 and 950 base pairs, respectively . The identity of the clones was established by hybrid-arrested and hybrid-selection translation assays, and by immunoprecipitation of hybrid-selected translation product with GaP dehydrogenase antiserum . Hybridization of labeled pGAP 30 DNA to size-fractionated chick heart poly(A) RNA occurred at the region on the gel corresponding to the mobility of GAP dehydrogenase mRNA . Base sequence analysis of plasmid pGAP 30 and the comparison of the amino acid sequence derived from it with that of pig muscle GAP dehydrogenase revealed that the amino acid sequence of GAP dehydrogenase is strictly conserved between the chick and pig muscle tissues . Expression of GAP dehydrogenase mRNA in developing chick heart cells in cultures was monitored by in situ hybridization . The GAP dehydrogenase mRNA was present in 5-h-old dividing myoblasts, in contrast to mRNAs specific for contractile proteins, which appear late in myoblast development paralleling morphogenetic differentiation of myoblasts into myocytes (Jakowlew, S . B., Khandekar, P., Datta, K., Narula, S . K., Arnold, H . H., and Siddiqui, M . A . Q . (1982) J . Mol . Biol . 156, 673-682). J Biol Chem, 1982 Aug 25, 257(16), 9822 - 9 Ribosome structure . Localization of 7-methylguanosine in the small subunits of Escherichia coli and chloroplast ribosomes by immunoelectron microscopy; Trempe MR et al.; The minor nucleoside 7-methylguanosine occurs in Escherichia coli 16 S ribosomal RNA at a single site . High pressure liquid chromatographic analysis shows that a single residue of 7-methylguanosine is also present in chloroplast 16 S ribosomal RNA, presumably at an analogous position in the sequence . Antibodies to 7-methylguanosine were induced in rabbits and shown to be highly specific for the intact methylated base . These antibodies were reacted with 30 S ribosomal subunits from E . coli and from the chloroplasts of Alaskan peas . These two types of ribosome have been shown to be topographically similar (Trempe, M . R., and Glitz, D . G . (1981) J . Biol . Chem . 256, 11873-11879) . Electron microscopy of the subunit-antibody complexes showed similar subunit-IgG monomers and antibody-linked subunit dimers . In greater than 95% of the complexes observed for each type of ribosome, antibody contact was consistent with a single binding site, which places 7-methylguanosine near the junction of the upper one-third and lower two-thirds of the subunit and maximally distant from the platform . The analogous localization in both E . coli and chloroplast 30 S ribosomal subunits lends support to their proposed common evolutionary origin. J Biol Chem, 1982 Aug 25, 257(16), 9226 - 9 An alternate method for synthesis of double-stranded DNA segments; Rossi JJ et al.; Recent progress in the chemical synthesis of DNA has now made it possible to rapidly synthesize single-stranded DNAs over 40 bases in length . We have taken advantage of these longer DNAs in assembling and cloning a 132-base pair gene segment coding for amino acids 126 through the stop codon of human leukocyte interferon alpha 2 . The method used involves DNA polymerase I-mediated repair synthesis of synthetic oligonucleotide substrates having short stretches of complementary sequence at their 3' termini . In the presence of DNA polymerase I and the four deoxyribonucleoside triphosphates, those primer-templates are converted to full length double-stranded DNAs . The economy in chemical synthesis using this approach is substantial with a greater than 40% reduction in the amount of chemical synthesis required as compared with the conventional approach . We describe in detail this methodology for the biochemical assembly of long gene segments from synthetic oligodeoxyribonucleotides. Biochim Biophys Acta, 1982 Aug 20, 681(2), 177 - 90 A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis; Van Wielink JE et al.; An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed . From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results . In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with alpha-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, -75 and 187 mV, respectively . In addition, two very small contributions to the alpha-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished . On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively . In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c1 (lambda m (25 degrees C) = 553.5 nm; E'0 = 238 mV) and four cytochromes b (lambda m (25 degrees C) = 558.6, 561.2, 562.1, 566.1 nm and E'0 = -83, 26, 85, -60 mV). Nature, 1982 Aug 19, 298(5876), 718 - 23 The molecular basis of DNA-protein recognition inferred from the structure of cro repressor; Ohlendorf DH et al.; Recognition by cro repressor protein of its specific DNA binding sites appears to occur via multidentate hydrogen bonds between amino acid side chains of the protein and base-pair atoms in the major groove of right-handed B-form DNA . Most of the sequence-specific interactions between cro and DNA, as well as a number of sequence-independent ones, are mediated by a two-alpha-helical unit which appears to be common to many proteins that regulate gene expression. Biochemistry, 1982 Aug 17, 21(17), 3914 - 21 Photoreactivating enzyme from Escherichia coli: isolated enzyme lacks absorption in its actinic wavelength region and its ribonucleic acid cofactor is partially double stranded when associated with apoprotein; Cimino GD et al.; Isolated photoreactivating enzyme (PRE) from Escherichia coli exhibits some optical density at wavelengths greater than 300 nm . After correcting for the effects of light scattering, however, we find no true absorption in the spectral region that is required for enzymatic activity (320-450 nm) . At shorter wavelengths, there is an absorption maximum near 260 nm that is due primarily to an RNA cofactor . Heating to 60 degrees C and subsequently cooling to 4 degrees C release the RNA cofactor from association with apoprotein and result in hyperchromicity . Circular dichroism indicates that the RNA associated with native enzyme is partially double stranded . At low ionic strength (mu = 0.01), heating to 15 degrees C or protease treatment at 4 degrees C results in irreversible loss of part of the double strandedness . We show that the difference spectrum at 4 degrees C between the absorption spectra of native enzyme and heat-treated enzyme can be fit by a superposition of reference spectra for denaturation of A-U and G-C base pairs derived from model polynucleotides . The coefficients of the linear combination of reference spectra were used to calculate the fraction of A-U and G-C base pairs . We find that both A-U and G-C base pairs are present in equal concentrations and that about 20% are in a double-stranded conformation in the native enzyme. J Immunol Methods, 1982 Aug 13, 52(3), 323 - 31 The detection of endotoxin by in vitro production of endogenous pyrogen: comparison with limulus amebocyte lysate gelation; Duff GW et al.; The sensitivities of leukocyte endogenous pyrogen (EP) production and limulus amebocyte lysate (LAL) gelation to endotoxin from E . coli (minimum i.v . pyrogenic dose 4 ng/kg in rabbits) were determined . Concentrations of 0.5-1.0 ng/ml could be detected by LAL . The minimum endotoxin concentrations which generated detectable EP from 2 X 10(6) monocytes was 10-fold lower (0.05-0.1 ng/ml) . At an endotoxin concentration of 0.4 ng/ml the minimum number of monocytes required for detectable EP production was 5 X 10(5) . It is concluded that the LAL gelation test cannot safely be used to exclude significant endotoxin contamination in a cellular system where EP production is being measured . The same conclusion applies even more forcibly to the in vitro production of lymphocyte activating factor (LAF, interleukin-1), since it appears that LAF and EP are identical and sub-pyrogenic amounts of EP are easily detectable in the LAF assay. Nucleic Acids Res, 1982 Aug 11, 10(15), 4703 - 13 Production and characterization of monoclonal antibody against 10S DNA polymerase alpha from calf thymus; Masaki S et al.; One hybridoma cell line that produces an antibody directed against 10S DNA polymerase alpha purified from calf thymus was obtained . The monoclonality of the antibody was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectrofocusing and antibody subclass determination . The antibody specifically recognized the 10S DNA polymerase alpha and 6.5S DNA polymerase alpha-2 from calf thymus, but not 6.5S DNA polymerase alpha-1 . The antibody precipitated both polypeptides of 140-150,000 and 46-50,000 dalton of 10S DNA polymerase alpha . The antibody also recognized the DNA polymerase alpha purified from human cells, but did pig DNA polymerase alpha only partially . The antibody did not crossreact with rat DNA polymerase alpha, calf DNA polymerase beta, virus DNA polymerase and E . coli DNA polymerase I . This antibody will be a useful tool for studying the mechanism of DNA replication in eukaryotic cells. Nucleic Acids Res, 1982 Aug 11, 10(15), 4795 - 801 Mitochondrial L-rRNA from Aspergillus nidulans: potential secondary structure and evolution; Kochel HG et al.; The alignment of gene sequences coding for A . nidulans mitochondrial L-rRNA and E . coli 23S rRNA indicates a strong conservation of primary and potential secondary structure of both rRNA molecules, except that homologies to the 5'-terminal 5.8S-like region and the 3'-terminal 4.5S-like region of bacterial rRNA are not detectable on mtDNA . The structural organization of the A . nidulans mt L-rRNA gene corresponds to that of yeast omega + strains: both genes are interrupted by a large intron sequence (1678 and 1143 bp, respectively) and by another smaller insert (91 and 66 bp) at homologous positions within domain V . An evolutionary tree derived from conserved L-rRNA gene sequences of yeast nuclei, E . coli, maize chloroplasts and six mitochondrial species exhibits a common root of organelle and bacterial sequences separating early from the nuclear branch. Nucleic Acids Res, 1982 Aug 11, 10(15), 4783 - 94 Nucleotide sequence of Aspergillus nidulans mitochondrial genes coding for ATPase subunit 6, cytochrome oxidase subunit 3, seven unidentified proteins, four tRNAs and L-rRNA; Netzker R et al.; The complete nucleotide sequence of a 14 kb segment of A . nidulans mtDNA reveals a rather compact organization of genes transcribed from the same strand and coding for two functionally known proteins, seven unidentified polypeptides (URFs), 24 tRNAs and two rRNAs . One of the URFs is located in the intron of the L-rRNA gene and codes for a basic protein of 410 residues . The other URFs are in spacer regions and code for hydrophobic proteins . URFa is homologous to human URF4, and URFb produces a polypeptide of 48 residues resembling the human URF6L product (hydrophobic N-terminus, basic C-terminus) . The ATPase subunit 6 genes from mitochondria and E . coli appear to share a common ancestor . The codon frequencies of identified genes and URFs are similar, and codons ending with G or C are rarely used . The structures of tRNAs specific for arginine, asparagine, tyrosine and histidine are deduced from gene sequences. Nucleic Acids Res, 1982 Aug 11, 10(15), 4525 - 42 A cloned immunoglobulin cDNA fragment enhances transposition of IS elements into recombinant plasmids; Amster O et al.; Evidence is presented indicating that a novel DNA sequence arrangement generated by in vitro recombination may elicit high frequency transpositions of IS elements . A 109 bp Bam HI fragment of the cDNA for the immunoglobulin kappa light chain from MOPC 321 myeloma was cloned into the Bam HI site of pBR313 . The cloned fragment extends from the codon for Gly 57 to the V-J junction . Insertions of IS1 or IS5 were identified in 6 of 50 plasmid DNAs isolated from freshly transformed clones . Additional transposition events were detected after subculturing for several growth cycles . Three independent insertions of IS1 occurred in the promoter region of the TcR operon . All IS5 and the remaining IS1 insertions were located in the TcR region upstream to the cloned DNA sequence . Sequences homologous to the ends of IS1, or corresponding to the consensus sequence at the target site of IS5 are present near the estimated sites of insertion of IS1 or IS5 respectively . Bacteria harboring recombinant plasmids carrying the cloned DNA in either orientation grew at a reduced rate relative to cells harboring pBR313, suggesting that fused gene products made from the two types of plasmid were inhibitory to cell growth . IS insertions, which relieved this inhibitory effect and thereby provided a selective advantage, were found exclusively in plasmids carrying the cloned DNA in only one of the two orientations . The fact that IS elements were not observed in the other type of recombinant plasmid indicates that selective pressure alone is not sufficient to account for the frequent IS insertions observed and that sequences at a distance from the site of IS insertion may be critical in the regulation of transposition frequency. Nucleic Acids Res, 1982 Aug 11, 10(15), 4467 - 82 Chemical synthesis and cloning of a gene for human beta-urogastrone; Smith J et al.; A DNA duplex coding for the 53 amino acids of human beta-urogastrone has been synthesised . Computer assisted design of the gene included restriction endonuclease sites for plasmid insertion, a termination codon and two triplets coding for lysine at the 5'-end of the structural gene . The synthesis involved preparation of 23 oligodeoxyribonucleotides by phosphotriester procedures coupled to rapid HPLC techniques . The gene was constructed in two halves by enzymatic ligation of the oligonucleotides and cloned into a specially constructed chimeric plasmid vector . Escherichia coli K12 MRC8 was transformed by the plasmid and clones containing the full gene sequence were isolated and characterised. J Biol Chem, 1982 Aug 10, 257(15), 9173 - 80 Preparation of psoralen-cross-linked R-loops and generation of large deletions by their repair in vivo; Chatterjee PK et al.; A technique for site-selective introduction of psoralens into DNA has been developed . Irradiation of Escherichia coli 16 S rRNA with aminomethyltrimethyl psoralen (AMT) at 390 nm leads to the incorporation of AMT monoadducts in double-stranded regions of the RNA, but no cross-links . The AMT-containing RNA hybridizes efficiently to a supercoiled plasmid, pCS3, a derivative of pBR313 containing an insert of part of the E . coli rrnC cistron including the 3' 60% of the 16 S rDNA . When the resulting hybrids are re-irradiated at 360 nm, psoralen cross-links form between the rRNA and the rDNA, resulting in covalent R-loops . These were partially purified and used to transform E . coli after various nuclease treatments, employing an ampicillin selection to make cell survival dependent on successful repair of the psoralen-containing region . S1 nuclease-treated AMT-containing covalent R-looped plasmids show efficient transformation, and 9 mutants with large deletions were isolated by random screening of 100 colonies . These fell into 3 specific classes, each of which appears to start some place within the AMT-cross-linked rDNA . The structure of one mutant has been analyzed by DNA sequencing which shows a deletion which extends approximately 6800 nucleotides from nucleotide 868 of 16 S rDNA to nucleotide 315 on the tetracycline gene . Some hints of symmetry exist around the point of the deletion, but the pattern is not a striking one . This technique should be useful in making covalent D-loops as well as R-loops . It offers a number of potential applications for site-specific DNA modification and studies of DNA repair. J Biol Chem, 1982 Aug 10, 257(15), 9043 - 8 Amplification and modification of dihydrofolate reductase in Escherichia coli . Nucleotide sequence of fol genes from mutationally altered plasmids; Smith DR et al.; Recombinant plasmids carrying the structural gene for Escherichia coli dihydrofolate reductase (fol) were mutagenized in vitro and in vivo and were used to transform a suitable recipient strain . Twenty-three transformants were isolated that were able to grow in the presence of high levels of the folate analog trimethoprim, and, in each strain, the resistance determinant was shown to be carried on the plasmid . Three of the strains produced dihydrofolate reductase with an increased Ki value for trimethoprim . DNA sequence analysis showed that the plasmids in these strains had mutations in fol which altered a conserved region of the polypeptide that forms part of the dihydrofolate-binding site . Two other strains had approximately 3-fold elevated dihydrofolate reductase levels, apparently resulting from plasmid copy number mutations . The remaining 18 strains had dihydrofolate reductase levels that were 10-30 times higher than those of the starting strain . Surprisingly, three of these strains had no discernible changes either in plasmid copy number or in the nucleotide sequence of the plasmid fol gene . Sequence analysis of the plasmids in 12 more of the strains revealed mutations in the promoter region adjacent to the fol gene . Most of these mutations occurred in the conserved sequences known as the Pribnow box and the -35 region and increased the homology of these sequences with the consensus E . coli promoter sequence . Strains carrying these plasmids produced a significant fraction of their total cell protein as wild type dihydrofolate reductase and should therefore be useful as sources of the purified enzyme. J Biol Chem, 1982 Aug 10, 257(15), 8902 - 6 Altered cation coupling to melibiose transport in mutants of Escherichia coli; Niiya S et al.; The alpha-galactoside transport system of Escherichia coli utilizes either H+ of Na+ as a coupling cation for melibiose transport . Mutants were isolated which showed altered cation recognition for melibiose transport . The transport carrier of the mutants has lost the ability to accept H+ but can utilize either Na/ or Li+ for co-transport with melibiose . Such mutants might be predicted if the melibiose carrier were viewed as a descendant of an evolutionary transition between the H+-substrate co-transport systems of primitive cells and the Na+-substrate systems of more complex organisms . A second mutation was found in these mutants which involves the Na+ (Li+)-H+ exchange carrier . The maximal rate of Li+-H+ exchange in one of the mutants was 11 times higher than the parent and the Km for Li+ by this carrier was one-sixth that of the parent. J Biol Chem, 1982 Aug 10, 257(15), 8795 - 8 Regulation of tryptophan operon expression by attenuation in cell-free extracts of Escherichia coli; Das A et al.; Expression of the tryptophan (trp) operon of Escherichia coli was shown to be regulated by attenuation in an in vitro DNA-dependent protein-synthesizing system . In extracts prepared from a temperature-sensitive tryptophanyl-tRNA synthetase mutant, plasmid-directed trpE enzyme synthesis was inhibited 2- to 3-fold by addition of purified wild type tryptophanyl-tRNA synthetase . When the extract used from a strain bearing a trpTts mutation that reduces charging of tRNATrp in vivo, a 2- to 3-fold increase in trpE enzyme synthesis was observed when an excess of uncharged wild type tRNATrp was added . Analysis of attenuation by measurement of trp mRNA synthesis was facilitated by constructing a plasmid (pAD1) containing the rpoC transcription terminator inserted early in trpE . Transcription proceeding past the trp attenuator of this plasmid terminates at this new terminator sequence and results in the production of a approximately 400-nucleotide long read-through transcript . Using this plasmid and extracts prepared from the tryptophanyl-tRNA synthetase mutant, a 4- to 8-fold decrease in relative read-through transcription was observed in response to exogenously added wild type tryptophanyl-tRNA synthase . Kinetic analyses of trp mRNA synthesis and studies using plasmid template DNAs bearing trp attenuator mutations indicate that translation of the leader peptide coding region of the transcript regulates transcription termination at the trp attenuator. J Biol Chem, 1982 Aug 10, 257(15), 8788 - 94 Protein composition of the bovine mitochondrial ribosome; Matthews DE et al.; The protein complement of the bovine mitochondrial ribosome has been analyzed by two-dimensional electrophoresis in polyacrylamide gels to determine the number and molecular weights of the ribosomal proteins . Salt-washed ribosomal subunits are found to contain a total of 85 ribosomal proteins, 84 of which are electrophoretically distinct between the two subunits . These proteins are also electrophoretically distinguished from those of cytoplasmic ribosomes . This large number of proteins does not appear to be due to contamination by cytoplasmic ribosomal proteins or by adherent nonribosomal proteins . The molecular weights of these proteins are considerably larger than those of Escherichia coli ribosomal proteins, and are similar to those of bovine cytoplasmic ribosomal proteins . The sum of the molecular weights of the 85 proteins agrees well with that predicted by physical chemical measurements of the total mass of protein in the two subunits . Bovine mitochondrial ribosomes thus contain about twice as much protein as RNA, a highly unusual composition in comparison to the other kinds of ribosomes which have been characterized to date . In addition, it appears that the ribosomal proteins themselves are less basic than the proteins of most other ribosomes. J Biol Chem, 1982 Aug 10, 257(15), 9030 - 4 Identification of the mglA gene product in the beta-methylgalactoside transport system of Escherichia coli using plasmid DNA deletions generated in vitro; Rotman B et al.; Three genes (mglA, mglB, and mglC) required for active transport of substrate by the methylgalactoside permease were identified in a hybrid ColE1-DNA plasmid isolated from a clone (pLC3-14) of the Clarke-Carbon bank of Escherichia coli genes . A 4.6-kilobase DNA fragment obtained from pLC3-14 was cloned into the plasmid vector pBR322 . The presence of the three mgl genes in the resultant plasmid, pMG3, was verified by genetic complementation and biochemical analysis of mgl mutants transformed with pMG3 DNA . Derivatives of pMG3 containing deletions in each mgl gene were constructed; restriction endonuclease mapping and functional analysis of these plasmids allowed us to physically locate the mgl genes within the inserted plasmid DNA and also to identify a heretofore unknown protein component of the transport system . Expression of these plasmids in vivo resulted in the specific synthesis of three major proteins of apparent molecular weight of 19,000, 36,000, and 52,000 . The 36,000-dalton protein is the galactose-binding protein previously identified as the mglB product . The 19,000-dalton protein maybe the product of mglD, a regulatory gene mapping outside of the mgl gene cluster . The 52,000-dalton protein is a new permease component which we have identified here as the mglA product based on the observation that pMG6, a plasmid with a 0.6-kilobase mglA deletion, failed to encode for this protein but produced a truncated polypeptide showing a reduction in molecular weight comparable to the extent of the deletion . In bacteria bearing an mglA+, B-, C+ plasmid (Pmg4), the 52,000-dalton protein is located to a large extent (73%) in the membrane fraction. Biochim Biophys Acta, 1982 Aug 10, 705(3), 348 - 56 Studies on yeast sulfite reductase . IV . Structure and steady-state kinetics; Kobayashi K et al.; Yeast NADPH-sulfite reductase (hydrogen sulfide:NADP+ oxidoreductase, EC 1.8.1.2) is a complex hemoflavoprotein . The Strokes radius was determined to be 80 A by gel filtration and the molecular weight was estimated to be 604,000 . The minimal molecular weight calculated from flavin and heme content was 306,000, indicating that this enzyme contains two FAD, two FMN and two hemes per molecule . The enzyme consists of two types of subunit, alpha and beta, having molecular weights of 116,000 and 167,000, respectively . The subunit structure is suggested to be alpha 2 beta 2 . The secondary structure, the amino acid composition and the isoelectric point were also investigated . The Km values for sulfite and NADPH were 17 microM and 10 microM, respectively . Sulfite and NADPH affected the reaction velocity to give parallel Lineweaver-Burk plots, indicating the involvement of the 'ping-pong' mechanism in the overall reaction . NADP+ inhibited the reaction competitively with NADPH and noncompetitively with sulfite . Inhibition by sulfide was partially noncompetitive with both substrates but very weak . These results are discussed and compared with sulfite reductase from Escherichia coli. Biochim Biophys Acta, 1982 Aug 10, 705(3), 321 - 9 Conformational studies of Escherichia coli pyruvate oxidase; O'Brien TA et al.; Pyruvate oxidase (pyruvate:oxygen oxidoreductase (phosphorylating), EC 1.2.3.3) is a peripheral membrane enzyme from Escherichia coli which utilizes the cofactors thiamin pyrophosphate (TPP) and flavin-adenine dinucleotide (FAD) to catalyze the decarboxylation of pyruvate to acetic acid and carbon dioxide . The specific activity of the oxidase is enhanced 25-fold when assayed in the presence of certain lipids and detergents . Previous studies have demonstrated that the affinity of pyruvate oxidase for phospholipids and detergents is substantially increased when the flavin is reduced . In this paper, several techniques are utilized to probe both the nature of the active site and the conformational changes in the protein which are concomitant with flavin reduction and with the binding of lipids to the enzyme . Analysis of the circular dichroism spectrum in the far ultraviolet region indicates that neither the binding of lipid activators to the oxidase nor reduction of the enzyme-bound flavin by pyruvate has a significant effect on the average secondary structure of the enzyme . High-resolution electron microscopy demonstrates that at low enzyme concentrations, i.e., assay conditions, incubation of the reduced flavoprotein in the presence of an amphiphilic activator does not alter the quaternary structure of pyruvate oxidase . The results indicate that the conformational changes in the protein due either to reduction of the flavin or to the binding of lipid activators are localized. J Biol Chem, 1982 Aug 10, 257(15), 8799 - 805 Subunit structure of Escherichia coli exonuclease VII; Vales LD et al.; Exonuclease VII has been purified 7,500-fold to 87% homogeneity from Escherichia coli K12 using a new purification procedure . The enzyme has been shown to be composed of two nonidentical subunits of 10,500 and 54,000 daltons . This has been confirmed by restoration of exonuclease VII activity after renaturation of denatured and purified subunits . The structure of the native enzyme consists of one large subunit and four small subunits . We have previously isolated exonuclease VII mutant strains containing defects which map at two distinct loci . Subunit-mixing experiments utilizing wild type enzyme and temperature-sensitive enzyme produced by an xseB mutant strain have shown that the xseB gene codes for the small subunit of the enzymes. J Biol Chem, 1982 Aug 10, 257(15), 8781 - 7 Extraction of proteins from the large subunit of bovine mitochondrial ribosomes under nondenaturing conditions; Schieber GL et al.; The 55 S mammalian mitochondrial ribosome (referred to hereafter as "mitoribosome") is protein-rich, containing nearly twice as much protein as the Escherichia coli ribosome . In order to produce soluble mitochondrial proteins and protein-deficient subribosomal particles for use in functional and structural studies, the proteins of bovine mitoribosomes were extracted by washing in a series of buffers containing increasing concentrations of LiCl as the only chaotropic agent . LiCl disruption is used in order to preserve the solubilized proteins in a substantially "native" configuration . The extraction mixtures were characterized by sucrose density gradient analysis and the compositions of the stripped protein and residual pellet fractions were determined by two-dimensional polyacrylamide gel electrophoresis . In order to analyze the behavior or individual proteins, the intensity of Coomassie blue stain for each protein was normalized against the intensity of stain for the same protein in a control sample . Buffers with 1, 2, and 4 M LiCl each extract a specific subset of mitoribosomal proteins, while another group of proteins remains in the residual pellet fraction . Although very few proteins are detected in only one condition, most proteins are specifically enriched in one fraction . This LiCl procedure, therefore, produces fractionated groups of mitoribosomal proteins which can be used directly as a source for those proteins in which they are enriched, or they can be used as a starting point in further purification procedures . In contrast to results with E . coli ribosomes, several mitoribosomal proteins remain core-associated, indicating a different structural organization in these ribosomes. J Biol Chem, 1982 Aug 10, 257(15), 8600 - 3 Oxygen transfer from bleomycin-metal complexes; Murugesan N et al.; Both Fe(III) and Cu(II) complexes of bleomycin (BLM), but not N-acetyl BLM . Fe(III), mediated the transfer of oxygen from iodosobenzene to organic substrates . In analogy with results obtained using certain cytochrome P-450 analogs, cis-stilbene was converted cleanly to the respective oxide, while no more than traces of trans-stilbene oxide were formed from trans-stilbene under identical conditions . The possible relevance of these observations to the degradation of DNA by bleomycin was also studied . In both the presence and absence of O2, BLM . Cu(II) . C6H5IO effected DNA degradation, as judged by the release of {3H}thymine from radiolabeled Escherichia coli DNA . These findings provide a valuable new assay system for the study of bleomycin analogs and suggest the possibility that bleomycin may function as an "oxygen transferase" in its degradation of DNA in situ. Biochemistry, 1982 Aug 3, 21(16), 3757 - 65 Kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli; Stone SR et al.; The kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli has been investigated by using progress curve, initial velocity, product inhibition, and dead-end inhibition studies as well as isotope effects . The results indicate that the reaction conforms to a random mechanism involving two dead-end complexes, viz., enzyme-DHF-THF and enzyme-NADP-DHF . At higher concentrations, DHF causes substrate inhibition by combining at the NADPH binding site on the enzyme . The steady-state velocity data can be analyzed adequately on the basis that rapid-equilibrium conditions apply . However, this can be only an approximate description of the reaction since the isotope effects observed with NADPD demonstrate clearly that catalysis cannot be rate limiting at pH 7.4 . The choice of conditions for analysis of progress-curve data is discussed in the Appendix. Biochemistry, 1982 Aug 3, 21(16), 3809 - 17 Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analogue of puromycin . Evidence for the functional site specificity of labeling; Nicholson AW et al.; The photoincorporation of p-azido{3H}puromycin {6-(dimethylamino)-9-{3'-deoxy-3'-{(p-azido-L-phenylalanyl)amino}-beta-D-ribofuranosyl}purine} into specific ribosomal proteins and ribosomal RNA {Nicholson, A . W., Hall, C . C., Strycharz, W . A., & Cooperman, B . S . (1982) Biochemistry (preceding paper in this issue)} is decreased in the presence of puromycin, thus demonstrating that labeling is site specific . The magnitudes of the decreases in incorporation into the major labeled 50S proteins found on addition of different potential ribosome ligands parallel the abilities of these same ligands to inhibit peptidyltransferase . This result provides evidence that p-azidopuromycin photoincorporation into these proteins occurs at the peptidyltransferase center of the 50S subunit, a conclusion supported by other studies of ribosome structure and function . A striking new finding of this work is that puromycin aminonucleoside is a competitive inhibitor of puromycin in peptidyltransferase . The photoincorporation of p-azidopuromycin is accompanied by loss of ribosomal function, but photoincorporated p-azidopuromycin is not a competent peptidyl acceptor . The significance of these results is discussed . Photolabeling of 30S proteins by p-azidopuromycin apparently takes place from sites of lower puromycin affinity than that of the 50S site . The possible relationship of the major proteins labeled, S18, S7, and S14, to tRNA binding is considered. Infect Immun, 1982 Aug, 37(2), 710 - 9 Cell-mediated cytotoxicity against rat fibroblasts induced by Actinomyces viscosus; Gaegauf-Zollinger R et al.; Cell-mediated cytotoxicity against syngeneic fetal rat fibroblasts that require in vitro exposure of effector cells to Actinomyces viscosus Ny1 fractions was investigated by measuring the uptake of radioactivity by fibroblasts during a 2-h pulse with {14C}aminoisobutyric acid after 1 to 3 days of coculture with splenic effector cells . By using splenocytes from inbred RIC-Sprague-Dawley rats as effector cells and syngeneic embryonic rat fibroblasts as target cells, strong cell-mediated cytotoxicity dependent on the in vitro exposure to an A . viscosus Ny1 fraction was observed, but only within a small range of effector-to-target cell ratios (3:1 to 10:1) . Concanavalin A and lipopolysaccharide from Escherichia coli induced a comparable cytotoxicity, indicating that the effect might be connected with the mitogenic activity of the A . viscosus NY1 fraction . Splenocytes from rats immunized with A . viscosus Ny1 and from control rats induced similar levels of cytotoxicity in 72-h cytotoxicity assays . In shorter assays (24 h), however, splenocytes from immune animals induced low cytotoxicity, which was, however, significantly higher than that induced by splenocytes from control animals . We conclude that both antigen- and mitogen-dependent cell mediated effector mechanisms are operative in this system and that the two normally overlapping effects can be experimentally separated . This new system describes a fibroblast impairment in the presence of splenocytes and bacterial components and may provide a useful model for studying pathogenic mechanisms operative in periodontal disease. J Infect Dis, 1982 Aug, 146(2), 220 - 6 Protection against chronic pyelonephritis in rats by suppression of acute suppuration: effect of colchicine and neutropenia; Bille J et al.; Previous experiments in rats have suggested that renal scarring after acute, obstructive pyelonephritis due to Escherichia coli results from parenchymal damage due to acute inflammation and suppuration . To assess the role of acute infiltration by polymorphonuclear leukocytes (PMNLs) in the pathogenesis of chronic pyelonephritis (CPN), rats were either treated with colchicine to depress leukocyte motility or rendered neutropenic with a single dose of cyclophosphamide . Colchicine given during acute pyelonephritis reduced kidney inflammation and protected against CPN two months later . Similarly, neutropenia reduced acute inflammation and protected against chronic parenchymal destruction and scarring . Protection against renal scarring in both colchicine-treated and neutropenic rats occurred despite higher renal bacterial counts during acute pyelonephritis . These experiments provide further evidence that CPN (renal scarring) results from kidney damage that occurs during early acute obstructive pyelonephritis . This damage appears to result from infiltration of the kidney by PMNLs rather than direct damage from bacterial infection. Chem Biol Interact, 1982 Aug, 41(2), 217 - 33 Alkaline opening of imidazole ring of 7-methylguanosine . 1 . Analysis of the resulting pyrimidine derivatives; Chetsanga CJ et al.; Column chromatography and spectroscopy have been employed in analyzing pyrimidine derivatives obtained from alkaline-treated 7-methylguanosine (7-meGuo) . High performance liquid chromatography (HPLC) revealed that the alkaline generated products consist predominantly of two forms of ring opened 7-methylguanine (rom7Gua) in equal amounts . Material from both Dowex 50 and Sephadex LH-20 columns was readily resolvable into two HPLC peaks . The species in one peak appears to be composed of formylated and that in the other of deformylated rom7Gua . The presence of a deformylated species is supported by the absence of radioactivity in one of the two peaks obtained when ring opened {8-14C}-guanosine was analyzed by HPLC . The formylated species was retained on the liquid chromatography column for 8 min with a 3% methanol, 0.01 M NH4H2PO4 (pH 5.1) solvent and for 6 min with a 6% methanol, 0.01 N NH4H2PO4 (pH 5.1) solvent system; the deformylated species was retained for 6.3 min with the first solvent and 4.5 min with the second solvent . Subsequent to Dowex 50 chromatography in an ammonium formate solvent, abut 90% of the material was formylated . When stored at 24 degrees C for 72 h in a solvent without formate ions, the material was shown by HPLC to consist of equal amounts of the formylated and deformylated species . These results indicate that the two species of rom7Gua are in equilibrium . The rom7Gua excised from DNA by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species. J Bacteriol, 1982 Aug, 151(2), 983 - 8 Colicin A receptor: role of two Escherichia coli outer membrane proteins (OmpF protein and btuB gene product) and lipopolysaccharide; Chai T et al.; ompF cells were completely resistant to colicin A, whereas btuB cells were partially resistant . The OmpF protein, in the presence of added lipopolysaccharide, inactivated colicin A . This inactivation was enhanced by added btuB gene product, btuB gene product with lipopolysaccharide did not inactivate colicin A . These data, together with the observation that vitamin B12 protected btuB+ cells from the killing effect of colicin A, suggest that the colicin A receptor in Escherichia coli K-12 is composed of the OmpF protein, the btuB gene product, and lipopolysaccharide. J Bacteriol, 1982 Aug, 151(2), 976 - 82 Mutations in genes cpxA and cpxB of Escherichia coli K-12 cause a defect in acetohydroxyacid synthase I function in vivo; Sutton A et al.; Mutations in Escherichia coli genes cpxA and cpxB together cause a temperature-sensitive defect in isoleucine and valine syntheses that is related specifically to acetohydroxyacid synthase I . This enzyme catalyzes the first pair of homologous reactions required for the synthesis of these two amino acids . At both permissive and nonpermissive temperatures, mutant cells containing ilvB (the structural gene for acetohydroxyacid synthase I) cloned in a derivative of plasmid pBR322 synthesized comparable amounts of ilvB mRNA and contained several times the enzyme activity normally required to sustain exponential growth, yet these cells remained temperature sensitive for growth in the absence of isoleucine and valine . These observations suggest that the primary effect of the cpx mutations is to block enzyme function in vivo . The enzyme was unstable in mutant cells at growth temperatures above 37 degrees C, but this instability appeared to be a secondary effect on the cpx mutations. J Bacteriol, 1982 Aug, 151(2), 918 - 23 Regulation of tryptophanyl-tRNA synthetase formation; Hall CV et al.; A previously constructed trp-S-lacZ fusion encoding a hybrid protein with beta-galactosidase activity was subcloned from a multicopy plasmid onto a lambda vector . Single-copy lysogens of lambda trpS-lacZ were used to determine whether trpS was regulated in a manner similar to that of other aminoacyl-tRNA synthetases . trpS regulation was found to resemble that of the majority of synthetases, in that expression of the lysogen-encoded hybrid beta-galactosidase varied with growth rate; beta-galactosidase activity increased 2.5-fold as the generation time decreased from 150 to 37 min . This regulatory response was confirmed by DNA/RNA hybridization experiments, which also suggested that this form of metabolic regulation occurred at the transcriptional level . No alteration in the level of hybrid beta-galactosidase was observed, however, when cells were starved for tryptophan. J Bacteriol, 1982 Aug, 151(2), 788 - 99 Nitrate reductase in Escherichia coli K-12: involvement of chlC, chlE, and chlG loci; Stewart V et al.; We examined the properties of mutants of E . coli which are defective with respect to nitrate reductase activity . chlE::Mu cts and chlG::Mu cts mutants were all chlorate resistant, and the strains that we examined all synthesized nitrate reductase apoenzyme . We concluded that the chlE and chlG loci, like the chlA, chlB, and chlD loci, are involved in the synthesis of insertion of molybdenum cofactor . We identified at least four distinct phenotypic classes of chlC::Tn10 mutants, all of which were fully or partially sensitive to chlorate . Two of these classes may represent lesions in the structural genes for nitrate reductase subunits A and C . Two other classes may be altered in the regulation of the expression of nitrate reductase or other anaerobic enzymes . We propose the mnemonic nar for naming individual genes within the chlC locus. J Bacteriol, 1982 Aug, 151(2), 657 - 67 Suppression of Escherichia coli dnaA46 mutations by integration of plasmid R100.1 . derivatives: constraints imposed by the replication terminus; Louarn J et al.; We have studies the phenotypic suppression of a dnaA46 mutation by plasmid integration at preselected chromosomal sites after introducing homologous sequences (Mu prophages) onto both the chromosomes and the suppressive plasmid . The plasmids used were all derived from plasmid R100.1 . We found that the conditions required to get viable suppressive integration varied as the plasmid integration site moved from the origin to the terminus of chromosome replication . Two constraints were observed . Both appeared to be linked to the new characteristics acquired by chromosome replication from the integrated plasmid . One constraint was that strains with integrative suppression near the terminus terC were viable only in minimal medium . The rich medium sensitivity of these strains was correlated with a loss of regulation of initiation . The other constraint was a requirement for a specific orientation in certain regions of the chromosome . The two branches defined by normally initiated replication, between oriC and terC, were also symmetrical with respect to these plasmid orientation constraints . In studying the possible reasons for a plasmid orientation constraint, we found that, of the two forks initiated in bidirectional replication from the integrated plasmid, one was capable of moving across the terC region with a higher movability than the other. J Bacteriol, 1982 Aug, 151(2), 553 - 9 Chromosomal genes for ColV plasmid-determined iron(III)-aerobactin transport in Escherichia coli; Braun V et al.; Four chromosomal genes, tonA (fhuA), fhuB, tonB, and exbB, were required for the transport of iron(III)-aerobactin specified by the plasmids ColV-K311, ColV-K229, ColV-K328, and ColV-K30 . These genes also determine the transport system in Escherichia coli for the iron ionophore ferrichrome . Aerobactin and ferrichrome are both iron ligands of the hydroxamate type, but they are of different structure . The ColV plasmids determine an outer membrane protein that serves as a receptor for cloacin . Cloacin-resistant mutants were devoid of iron(III)-aerobactin transport but were unimpaired in ferrichrome transport . We conclude that for iron(III)-aerobactin transport two outer membrane proteins, the TonA and the cloacin receptor protein, have to interact functionally or structurally or both. J Bacteriol, 1982 Aug, 151(2), 529 - 33 New pleiotropic alkaline phosphatase-negative mutants of Escherichia coli K-12; Heyde M et al.; Escherichia coli K-12 mutants showing reduced alkaline phosphatase activity were isolated as 5-fluorouracil-plus-adenosine-resistant derivatives of a upp pho (either phoS or phoT) strain . One class of these mutants displayed a temperature-sensitive alkaline phosphatase-negative phenotype, a pleiotropic defect for growth on some substrates, an increased sensitivity to toxic compounds (e.g., EDTA, mitomycin, and chloramphenicol), and alterations in the expression of some membrane proteins . It phenotypically differed from previously described mutants . The mutation was located at min 8.5 close to the phoA gene and defines a new genetic locus we called napA (for negative alkaline phosphatase pleiotropic phenotype) . As these mutants have lost the ability to grow on lactose and galactose, Lac+ and Gal+ revertants were isolated that simultaneously recovered the parental phenotype. J Bacteriol, 1982 Aug, 151(2), 1046 - 50 Comparison of DNA sequences required for the function of citrate utilization among different citrate utilization plasmids; Shinagawa M et al.; The relatedness of DNA sequences encoding citrate utilization was examined by hybridization with a cloned DNA fragment from a citrate utilization (Cit) plasmid, pOH30221, and DNA of other Cit plasmids . This revealed that there are at least two groups of Cit plasmids: the Inc W Cit plasmids, which show homology with the probe, and the Inc H1 plasmids, which do not. J Bacteriol, 1982 Aug, 151(2), 1013 - 21 Aminoacyl-tRNAs from Physarum polycephalum: patterns of codon recognition; Hatfield D et al.; Isoacceptors of Physarum polycephalum Ala-, Arg-, Glu-, Gln-, Gly-, Ile-, Leu-, Lys-, Ser-, Thr-, and Val-tRNAs were resolved by reverse-phase chromatography and isolated, and their codon recognition properties were determined in a ribosomal binding assay . Codon assignments were made to most isoacceptors, and they are summarized along with those determined in other studies from Escherichia coli, yeasts, wheat germ, hymenoptera, Xenopus, and mammals . The patterns of codon recognition by isoacceptors from P . polycephalum are more similar to those of animals than to those of plants or lower fungi. Cancer, 1982 Aug 1, 50(3), 451 - 6 Depression of functional and antigenic plasma antithrombin III (AT-III) due to therapy with L-asparaginase; Liebman HA et al.; Eleven patients with leukemia and lymphoma were treated with 14 courses of E . coli L-asparaginase . Abnormalities of the coagulation screening tests and decreased fibrinogen levels were observed in all patients during treatment . Significant depressions of functional (mean 32%) and antigenic (mean 48%) antithrombin III were observed by day 14 of therapy . There was no laboratory evidence of intravascular coagulation during 11/14 courses of L-asparaginase . Crossed immunoelectrophoresis of plasma obtained at the antithrombin nadir did not demonstrate an abnormal pattern which can be associated with an abnormal antithrombin III or an increase in antithrombin III-coagulation factor complexes . The major underlying mechanism of this depression is believed to be decreased hepatic synthesis, and the low levels of antithrombin III may be associated with an increased risk of thrombosis. Gastroenterology, 1982 Aug, 83(2), 441 - 54 A clinicopathologic study of enterocyte-adherent Escherichia coli: a cause of protracted diarrhea in infants; Rothbaum R et al.; Fifteen infants (age 3-28 wk) suffered from severe diarrhea with acute dehydration and poor growth . Persistent watery stools and suboptimal nutrition necessitated central venous alimentation with prolonged hospitalization . Repeated stool and small intestinal fluid cultures yielded the classical enteropathogenic Escherichia coli serotype 0119:B14 . In all patients, biopsy of the jejunum or rectal mucosa, or both, showed moderate to severe damage, irregular atrophy of surface epithelium, and subnuclear vacuolization of crypt epithelium . Ultrastructural studies revealed bacteria adherent to mucosal cells with flattening of microvilli, loss of the cellular terminal web, and cupping of the plasma membrane around individual bacteria . Heavily colonized cells had marked intracellular damage . Assays for heat-labile, heat-stable, and vero cell toxins were negative for these Escherichia coli isolates . Oral neomycin and nutritional support resulted in clearing of Escherichia coli 0119:B14 from stool and small bowel with improvement in histologic characteristics . Damage to enterocytes and villi by adherent nontoxigenic Escherichia coli 0119:B14 results in protracted diarrhea in infants. Proc Natl Acad Sci U S A, 1982 Aug, 79(16), 5038 - 41 Isolation of a genomic clone partially encoding human hypoxanthine phosphoribosyltransferase; Jolly DJ et al.; Mouse cells deficient in the enzyme hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) have been transfected with total human DNA, and cells producing human enzyme were isolated by growth in selective medium . DNA from several such cell lines has been used to generate secondary transfectants that make human HPRT . Blots of the DNA of these secondary cells have been hybridized with total human DNA probes or with cloned human Alu sequences, and one of several common bands has been cloned in pBR322 . Colonies of transformed Escherichia coli containing human sequences were detected by their homology with human DNA, and subclones of resulting recombinant plasmids were prepared . Two subclones free of Alu sequences were found to contain human sequences that hybridized to human X chromosome DNA . One of these, pBR1.5, also hybridized to a single RNA band on gel blots of human and secondary transfectant cytoplasmic poly(A)+RNA but not to RNA from the parent mouse cell line . These results indicate that these clones represent human HPRT gene fragments . This has been confirmed by using pBR1.5 as a probe to isolate an authentic and expressible human HPRT cDNA clone from a library prepared by H . Okayama and P . Berg. J Pharmacol Exp Ther, 1982 Aug, 222(2), 441 - 6 Prevention of endotoxin-induced pulmonary hypertension in primates by the use of a selective thromboxane synthetase inhibitor, OKY 1581; Casey LC et al.; Endotoxin-induced pulmonary hypertension can be attenuated by nonsteroidal anti-inflammatory drugs and is associated with increased plasma levels of thromboxane (Tx) B2, prostaglandin (PG) F2, PGE and PGI2 . Because nonsteroidal anti-inflammatory drugs block prostacyclin production and may also shift arachidonic acid into the lipoxygenase pathway, we have evaluated a selective Tx synthetase inhibitor (OKY 1581) as a means for preventing endotoxin-induced pulmonary hypertension . An LD70 dose of Escherichia coli endotoxin (6 mg/kg) was given i.v . to two groups of unanesthetized baboons . Group I received endotoxin alone and Group II was pretreated with i.v . OKY 1581 (2 mg/kg) 10 min before the endotoxin . OKY 1581 produced a significant decrease in the basal plasma TxB2 from 0.432 +/- 0.82 to 0.147 +/- 0.032 ng/ml (P less than .01), but no significant change in plasma 6-keto PGF1 alpha . After the administration of the endotoxin, Group I developed pulmonary hypertension (from 11 +/- 1 to 19 +/- 2 mm Hg . P less than .005) and an 8-fold increase in plasma TxB2 (P less than .02), whereas Group II did not develop pulmonary hypertension or an increase in plasma TxB2 . However, Group II had a 26-fold increase in plasma 6-keto PGF1 alpha (P less than .05) . From these studies, we conclude that: 1) OKY 1581 is an effective Tx synthetase inhibitor in vivo; 2) endotoxin-induced pulmonary hypertension is mediated largely by increased Tx; and 3) the inhibition of Tx synthetase results in shunting of endoperoxides into the prostacyclin pathway. Biochimie, 1982 Aug-Sep, 64(8-9), 809 - 13 Inhibition of DNA synthesis is not sufficient to cause mutagenesis in Chinese hamster cells; Rossman TG et al.; Experiments were designed to test whether the inhibition of DNA synthesis in Chinese hamster V79 cells would result in increased mutagenesis by a mechanism similar to "SOS repair" in E . coli . Treatment of cells for 16 hours with excess of the deoxynucleosides TdR, UdR, AdR and GdR was mutagenic, whereas treatment with hydroxyurea demonstrated no mutagenic effect . The mutagenicity of TdR could be reversed by the addition of CdR . In E . coli, inhibition of DNA synthesis by a short exposure to hydroxyurea resulted in the induction of lambda prophage and increased mutagenesis . These results show that whereas the presence of a stalled replication fork in E . coli can result in mutagenesis via induction of the "SOS system", the same phenomenon does not seem to occur in Chinese hamster V79 cells . The mutagenic mechanism of high concentrations of deoxynucleosides in Chinese hamster V79 cells is likely to be due to replication errors which result from alterations in deoxynucleotide pools. Biochimie, 1982 Aug-Sep, 64(8-9), 805 - 7 A brief consideration of the SOS inducing signal; Roberts JW et al.; SOS functions are induced in E . coli by treatments that damage cellular DNA or interrupt its synthesis . The biochemical basis of induction is activation of the specific proteolytic activity of recA protein, which then inactivates the lexA repressor . We discuss the development of the inducing signal in the cell. Biochimie, 1982 Aug-Sep, 64(8-9), 709 - 12 How Escherichia coli sets different basal levels in SOS operons; Huisman O et al.; The recA and sfiA genes of Escherichia coli are SOS operons regulated negatively by the LexA repressor . The steady state level of expression of recA is 10-fold higher than that of sfiA, as measured by means of recA::lac and sfiA::lac operon fusions . To study the molecular basis of this difference, we have compared the expression of these two operons in strains in which the concentration of LexA repressor was normal (lexA+), zero (spr amber mutation) or higher than normal (plasmid pJL45, carrying the lexA gene linked to the lac promoter) . The results indicate (i) that the recA promoter is about 4 times stronger than the sfiA promoter (as measured in the spr strains), (ii) that neither operon has a physiologically significant level of lexA-independent expression (pJL45 strains), and (iii) that the recA operator has about 2.5 times lower affinity than the sfiA operator for LexA repressor (comparison of lex+ and spr strains) . Considering our previous results that the sfiA operon (high operator affinity of LexA) is derepressed very rapidly after inducing treatments and that the recA operon (low operator affinity) is repressed very rapidly when induction is stopped, we conclude that differences in operator affinity do not affect inducibility but serve only to set the basal levels of the different SOS functions. Biochimie, 1982 Aug-Sep, 64(8-9), 637 - 41 Mutagenesis by alkylating agents: coding properties for DNA polymerase of poly (dC) template containing 3-methylcytosine; Boiteux S et al.; After treatment of poly(dC) by the simple alkylating agent (3H)dimethylsulfate, 90 per cent of the radioactivity cochromatographied with 3-methylcytosine and 10 per cent with 5-methylcytosine which is the normally occurring methylated base . In order to study the influence of 3-methylcytosine on DNA replication, untreated and DMS-treated poly(dC) were used as templates for E . coli DNA polymerase I . The alkylation of poly(dC) inhibits DNA chain elongation, and does not induce any mispairing under high fidelity conditions . The alteration of DNA polymerase I fidelity by manganese ions allows some replication of 3-methylcytosine which mispairs with either dAMP or dTMP . Our results suggest that 3-methylcytosine could be responsible, at least partially, for the killing and the mutagenesis observed after cell treatment by alkylating agents. Biochimie, 1982 Aug-Sep, 64(8-9), 603 - 5 Repair of AP sites in DNA; Verly WG; Escherichia coli endonuclease VI is a deoxyribonuclease specific for AP (apurinic or apyrimidinic) sites; it cleaves the phosphodiester bond immediately neighbouring the AP site on its 5' side leaving 3'-hydroxyl and 5'-phosphate ends . DNA with AP sites can be repaired in vitro with endonuclease VI, DNA polymerase I and ligase; the repair mechanism is described . E . coli has other AP endonucleases; some of them are not specific for AP sites and some of them cut 3' to the AP sites . Most of the rat liver AP endonuclease activity is in chromatin . Some is however found in other cell compartments and it has been speculated that these enzymes might be precursors of the chromatin enzyme . The chromatin AP endonuclease is specific for AP sites; it cuts 5' to the AP site . DNA with AP sites can be repaired in vitro with enzymes purified from chromatin; AP endonuclease, 5'-3 exonuclease, DNA polymerase beta and ligase. Biochimie, 1982 Aug-Sep, 64(8-9), 585 - 9 Control of the SOS regulatory system by the level of RecA protease; Little JW; The SOS regulatory system of E . coli controls the cellular response to DNA damage and other treatments which interfere with normal DNA replication . This system can exist in two states--a repressed state, in which a set of genes (SOS genes) is repressed by the LexA repressor; and an induced state, in which the RecA protein is activated as a specific protease which cleaves LexA repressor, leading to derepression of the SOS genes . This article reviews evidence that the state of the SOS regulatory system is controlled by the level of RecA protease activity . This level is controlled in turn by a reversible activation by one or more cofactors . In vitro studies indicate that ATP or dATP and single-stranded polynucleotide are both required to activate the protease; the identity of the in vivo cofactors ("inducing signals") is not yet certain . New experiments are also described which characterize the in vivo cleavage of LexA repressor . These data support the model that the level of RecA protease controls the state of the regulatory system. Biochimie, 1982 Aug-Sep, 64(8-9), 581 - 3 Inducible DNA repair enzymes involved in the adaptive response to alkylating agents; Lindahl T et al.; Two different DNA repair enzymes are induced in E . coli by methylating agents: a methyltransferase acting on 0(6)-methylguanine residues, and a DNA glycosylate for N-methylated purines . The properties of the homogeneous methyltransferase are described. Biochimie, 1982 Aug-Sep, 64(8-9), 571 - 5 Mutation rate: some biological and biochemical considerations; Echols H; This article discusses ideas about the ways in which the high fidelity of DNA replication is achieved: base selection, exonucleolytic editing, and postreplicative proofreading . I also review possible mechanisms for the enhanced mutation rate associated with SOS induction . The concept of environmental control of mutation rate and other modes of genetic variation is also considered from the point of view that SOS induction is an example of "genetic revolution". Biochimie, 1982 Aug-Sep, 64(8-9), 565 - 9 Regulation and autoregulation by lexA protein; Brent R; The lexA protein represses many genes in E . coli . When the cell's DNA is damaged, lexA protein is inactivated and these genes are induced . Three aspects of lexA protein's repressor function are reviewed: how it regulates genes of the SOS response, how it regulates its own synthesis, and how it recognizes its operator sites . Recent studies of lexA protein's repressor function have suggested that the concentration of intact lexA protein after induction may determine the detailed control of the SOS response. Biochimie, 1982 Aug-Sep, 64(8-9), 559 - 64 Conservation and diversification of genes by mismatch correction and SOS induction; Bourguignon-Van Horen F et al.; Regulation of genetic stability is discussed in terms of interactions between constitutive and inducible DNA repair processes with specific emphasis on the results of our experimental studies of mismatch correction and SOS induction in Escherichia coli. Proc Natl Acad Sci U S A, 1982 Aug, 79(15), 4585 - 8 Synthesis by the DNA primase of Drosophila melanogaster of a primer with a unique chain length; Conaway RC et al.; The primase associated with the DNA polymerase alpha from embryos of Drosophila melanogaster catalyzes the synthesis of ribo-oligonucleotide primers on single-stranded M13 DNA or polydeoxythymidylate templates, which can be elongated by DNA polymerase action {Conaway, R . C . & Lehman, I . R . (1982) Proc, Natl . Acad . Sci, USA 79, 2523--2527} . The primers synthesized in a coupled primase-DNA polymerase alpha reaction with an M13 DNA template are of a unique size (15 residues); those synthesized with poly(dT) range from 8 to 15 nucleotides . Primer synthesis is initiated at multiple but nonrandom sites . Like the DNA primase of Escherichia coli and the comparable activity in intact nuclei of polyoma-infected mouse cells, the DNA primase of D . melanogaster can substitute deoxynucleotides for ribonucleotides during primer synthesis. J Hyg (Lond), 1982 Aug, 89(1), 149 - 54 Rapid single-tube confirmatory test for Escherichia coli; Smith JL et al.; A single-tube confirmatory test that allows a result to be obtained in 4 h has been developed from the single-tube confirmatory test recomended by the Joint Committee of the Public Health Laboratory Service and the Standing Committee of Analysts (PHLS/SCA, 1980) . A variety of river, lake and reservoir samples were examined for the presence of E . coli using either most probable number (MPN) or membrane filtration (MF) technique, and the PHLS/SCA recommended confirmatory medium (LTMB) was evaluated against traditional methods . To improve the performance of LTMB, the medium was modified and this modified medium when used in 0.1 ml volumes and incubated for 4 h at 44 degrees C provided 99% agreement with traditional methods. J Bacteriol, 1982 Aug, 151(2), 952 - 61 Transfer of the phosphatidyl moiety of phosphatidylglycerol to phosphatidylethanolamine in Escherichia coli; Yokota K et al.; Phosphatidylglycerol was pulse-labeled with radioactive lipid precursors in a serine auxotroph of Escherichia coli . Most of the radioactivity of phosphatidylglycerol labeled in a serine-depleted medium was transferred to phosphatidylethanolamine during a chase in the presence of L-serine, but not in its absence . Metabolism of fatty acyl moieties labeled with {1-14C}acetate, acylated glycerol moieties labeled with {2-3H}glycerol, and phosphate moieties labeled with 32Pi, followed by a chase in the presence of cerulenin, showed that the intact phosphatidyl moiety of phosphatidylglycerol was transferred to phosphatidylethanolamine . The composition of phosphatidylethanolamine molecular species was unaltered and not perturbed by the transfer of the phosphatidyl moiety of phosphatidylglycerol . The increase of phosphatidylethanolamine with a concomitant decrease of phosphatidylglycerol was not coupled with the postulated turnover of phosphatidylglycerol to membrane-derived oligosaccharides and lipoprotein . It is suggested that phosphatidylglycerol is capable of providing its phosphatidyl moiety for the production of phosphatidylethanolamine in response to the relief of serine limitation by addition of L-serine. J Bacteriol, 1982 Aug, 151(2), 777 - 82 L-serine degradation in Escherichia coli K-12: a combination of L-serine, glycine, and leucine used as a source of carbon; Newman EB et al.; Escherichia coli K-12 strain CU1008 cannot use L-serine as the sole carbon source, but it could use L-serine as an auxiliary carbon source with glucose, L-alanine, or pyruvate and could derive energy from L-serine to support oxygen uptake . CU1008 grew with L-serine if it was also provided with glycine and leucine . These may act by increasing the available activity of L-serine deaminase; other explanations are also explored. J Gen Microbiol, 1982 Aug, 128 (Pt 8), 1731 - 3 Mutants of Escherichia coli K12 sensitive to acidic pH; Bielecki J et al.; Four mutants of Escherichia coli K12 were isolated on the basis of their sensitivity to pH 5.4 . Under non-permissive conditions their growth was reversibly inhibited . At pH 7.0 these mutants showed a highly pleiotropic phenotype, which included altered phage and detergent sensitivities and leakage of periplasmic proteins . The findings suggest a defect in the outer membrane, perhaps in lipopolysaccharide . Two mutants mapped in or near the rfa locus, while the other two were removed from this region. J Gen Microbiol, 1982 Aug, 128 (Pt 8), 1685 - 96 A re-examination of the cytochromes of Escherichia coli using fourth-order finite difference analysis: their characterization under different growth conditions and accumulation during the cell cycle; Scott RI et al.; The cytochromes of Escherichia coli K12 have been studied using low-temperature (77 K) difference spectrophotometry . Numerical (i.e . fourth-order finite differences) analysis resolved the alpha band of reduced minus oxidized spectra of whole cells from aerobically growth cultures into five components, with absorption maxima at 548, 551.5, 555.5, 560 and 563 nm . Using the same differencing intervals, numerical analysis of cells grown under oxygen-limited conditions revealed only two components, at 555.5 and 559 nm . Similar analysis of cells grown anaerobically with fumarate as electron acceptor revealed four absorption maxima, at 548, 550.5, 555.5 and 559 nm . Membrane particles from aerobically grown cells showed the same absorption bands as intact cells; preliminary evidence was obtained for a component of the 555.5 nm band that could be relatively easily washed from the membrane . The contribution of cytochrome o or its CO-liganded form to the alpha region could not be determined by numerical analysis . We conclude, in contrast to a previous application of numerical analysis to the cytochromes of E . coli, that growth under anaerobic or oxygen-limited conditions results in the appearance of cytochromes spectrally distinct from those in aerobically grown cells . An attempt has been made to reconcile the presence of multiple components detected in the alpha region by numerical analysis of aerobically grown cells with the diverse components described by others . Quantification of cytochromes revealed by numerical analysis in aerobically grown cells separated into size (and thus age) classes by zonal centrifugation showed that the major components accumulated continuously, probably exponentially, throughout the cell cycle. J Dairy Res, 1982 Aug, 49(3), 381 - 5 Effect of Escherichia coli endotoxin-mediated inflammation of one mammary quarter of the bovine udder on diapedesis into other quarters; Schultze WD et al.; Implantation of 1 microgram Escherichia coli endotoxin into one papillary duct of a bovine udder frequently resulted in a mild inflammatory process in the treated quarter which was measured by use of the Wisconsin mastitis test (WMT) on quarter fore milk samples . Inflammation of a fore quarter was usually (18/24) accompanied by a transient elevation in WMT score from the homolateral hind quarter, but rarely (1/25) from the contralateral quarter . Inflammation in a hind quarter rarely (2/26) affected the homolateral fore quarter and never (0/25) the contralateral quarter . The pattern of responses suggests tha absorption and initial removal of endotoxin from the mammary gland occurs via the lymphatic rather than the blood system. Can J Microbiol, 1982 Aug, 28(8), 945 - 50 Biosynthetic ornithine and arginine decarboxylases: correlation of rates of synthesis with activities in Escherichia coli during exponential growth and following nutritional shift-up; Boyle SM et al.; Whether guanosine tetraphosphate (ppGpp) has a role in the regulation of the putrescine biosynthetic enzyme, ornithine decarboxylase, in Escherichia coli is controversial . Different laboratories have reported either direct or indirect correlations between ppGpp levels and ornithine decarboxylase activity using different in vivo conditions . In this report, using conditions in vivo to modulate ppGpp levels, experiments are described which bear on the controversy . The rates of synthesis and biological activities of the biosynthetic ornithine and arginine decarboxylases (ODC and ADC) were measured in E . coli K-12 during experimental growth and during nutritional shift-up . There were good correlations between changes in their respective activities and the rates of synthesis of these enzymes during steady state or shift-up . ODC activity or rate of synthesis changed directly in concert with ppGpp levels, while ADC activity or rate of synthesis changed inversely with ppGpp levels . These observations support the contention that ppGpp does not inhibit ODC activity. Aust N Z J Med, 1982 Aug, 12(4), 255 - 8 Ontogeny of the secretory immune system in man; Gleeson M et al.; Immunoglobulin and albumin concentrations and Escherichia coli antibody levels were determined in a prospective study of saliva taken from 63 healthy infants during the first year of life . Albumin and IgG were present in high concentrations at birth (57 +/- 6 and 35 +/- 8 mg/l respectively) and decreased in parallel to low values at two months of age . IgA was detected by three weeks of age . The IgA concentration fluctuated until six months of age, after which constant values were observed (14 +/- 3 mg/l) . Low levels of salivary IgM (2.4 +/- 1.2 mg/l) were demonstrated in 37% of infants at four weeks of age . No E . coli antibody was detected . There was no significant difference between breast-fed and formula-fed infants . These findings suggest: (i) that mucosal membrane permeability is not restricted to the gut; (ii) that changes in mucosal permeability are non specific and not restricted to the uptake of specific food protein or ingestion of maternal milk; (iii) maternal IgG may contribute to mucosal defence in the neonate; (iv) fluctuations in the concentration of IgA may reflect a balance between intense antigenic stimulation in the gut in the first weeks of life and immune regulatory mechanisms and (v) ingestion of maternal milk does not modify the pattern of ontogeny. J Biochem (Tokyo), 1982 Aug, 92(2), 423 - 32 Phosphoenolpyruvate carboxylase of Escherichia coli . Hydrophobic chromatography using specific elution with allosteric inhibitor; Izui K et al.; The adsorption of Escherichia coli phosphoenolpyruvate carboxylase {EC 4.1.1.31} to butyl-, hexyl-, and octyl-Sepharose gels was investigated . The enzyme was nearly completely adsorbed to the latter two gels both in the absence and presence of high concentrations of ammonium sulfate . At intermediate concentrations--0.1 M in the case of hexyl-Sepharose--virtually no adsorption was observed . Upon application of an increasing or decreasing concentration gradient of the salt, the enzyme was eluted at various concentrations of the salt depending on chain length of the immobilized alkyl groups . The adsorption to hexyl-Sepharose at 0.7 M ammonium sulfate was markedly decreased by L-aspartate, the allosteric inhibitor, whereas it was increased by acetyl-CoA, one of the allosteric activators . Evidence was obtained suggesting that these changes in adsorption were due to conformational alterations of the enzyme elicited by these effectors . The enzyme seemed to have been adsorbed at its hydrophobic regions which were distinct from the allosteric site for long-chain fatty acids . The specific elution with L-aspartate in the presence of 0.82 M ammonium sulfate could successfully be applied to purification of the enzyme . By this hydrophobic interaction chromatography, the enzyme was purified about 55-fold over its partially purified preparation with a recovery of 73% . The obtained enzyme preparation was almost homogeneous as judged from sodium dodecylsulfate-polyacrylamide gel electrophoresis. J Biochem (Tokyo), 1982 Aug, 92(2), 391 - 7 Autonomous control of the level of methylation of methyl-accepting chemotaxis protein; Hayashi H et al.; Methylation and demethylation of the methyl-accepting chemotaxis protein (MCP) was studied in vitro . The in vitro MCP methylating system showed the following characteristics . 1 . Multiple bands of methylated MCP were produced . 2 . The reaction could be separated into two phases: the initial phase, in which the level of methylation increased as a result of multiple methylation; and the stationary phase, in which methylation and demethylation took place at the same velocity without apparent change in the level of methylation . 3 . Pulse-chase analysis of the reaction showed that MCP was demethylated preferentially at the relatively highly methylated state . 4 . The behavior of MCP in this system was similar to that of MCP stimulated in vivo with attractant . Based on the above results, an autonomous control mechanism of the level of MCP methylation is discussed. Eur J Biochem, 1982 Aug, 126(2), 299 - 309 Optical characteristics of all individual proteins from the small subunit of Escherichia coli ribosomes; Venyaminov SY et al.; The procedure of isolation and renaturation of all ribosomal proteins from the 30-S subunit of Escherichia coli ribosomes is described . Absorption spectra of these proteins in the near-ultraviolet region have been measured and molar absorption coefficients have been determined on the basis of nitrogen content . Molar absorption coefficients have been calculated for 20 proteins with a known amino acid sequence and the calculated values have been compared with the experimentally determined ones . The absorption spectra obtained allow an easy, precise and highly reproducible spectrophotometric determination of the concentration of individual ribosomal proteins . Circular dichroic spectra of 21 individual proteins from the 30-S subunit of E . coli ribosomes were measured in the range 184-310 nm . The secondary structure of the proteins studied was calculated from the spectra in the range 190-240 nm . Almost all proteins (except proteins S12, S17, S18 and S19) are characterized by a high content of secondary structure . Circular dichroic spectra in the near-ultraviolet region (240-310 nm) indicate that the side groups of aromatic amino acids are fixed in the tertiary structure of the proteins studied . Some internal characteristics (independent of the measurement conditions) of the circular dichroic spectrum in the far-ultraviolet region were proposed as a measure of the resemblance to the native state of ribosomal proteins; these characteristics may be useful for comparison of protein preparations obtained by different methods in different laboratories. Eur J Biochem, 1982 Aug, 126(1), 57 - 60 Substrate specificity of CTP synthetase from Escherichia coli; Scheit KH et al.; The stoichiometry of the enzymatic reaction catalyzed by CTP synthetase from Escherichia coli was analyzed by high-performance liquid chromatography . The results revealed that for every mole of UTP transformed to CTP, one mole of ATP was converted to ADP . The substrate specificity of CTP synthetase from E . coli was investigated by means of UTP analogs . Chemical modification of UTP involved either the uracil, ribose or 5'-triphosphate part . None of the UTP analogs studied proved to be a substrate . The capacity of the UTP analogs to inhibit CTP synthetase was investigated . From the UTP derivatives employed only 2-thiouridine 5'-triphosphate was found to inhibit the enzyme competitively with reasonable affinity: Ki/Km(UTP) = 1 . This study indicated that the three main structural elements of the UTP molecule: uracil, ribose and 5'-triphosphate moiety, contribute to substrate specificity . The behaviour of a limited number of CTP analogs as product-like inhibitors supported this view. Eur J Biochem, 1982 Aug, 126(1), 211 - 6 Location and nucleotide sequence of frdB, the gene coding for the iron-sulphur protein subunit of the fumarate reductase of Escherichia coli; Cole ST et al.; The frdB gene, encoding the iron-sulphur protein subunit of fumarate reductase, has been located and its complete nucleotide sequence determined . The identity of the gene was confirmed by protein chemical studies and determination of the NH2-terminal sequence of the FrdB protein . The frdB gene is situated distal to and partially overlapped by frdA which codes for the flavoprotein subunit of the reductase . Its reading frame contains 244 codons and predicts a protein of Mr 27092 . In composition, the FrdB protein is strikingly similar to the corresponding subunit of the related flavoenzyme, succinate dehydrogenase . Analysis of the protein's primary structure revealed several features characteristic of iron-sulphur proteins. Cell, 1982 Aug, 30(1), 37 - 44 Formation of nascent heteroduplex structures by RecA protein and DNA; Wu AM et al.; E . coli RecA protein promotes homologous pairing in two distinguishable phases: synapsis and strand exchange . With circular single strands (plus strand only) and linear duplex DNA, polarized or unidirectional strand exchange appeared to cause heteroduplex joints to form and grow from a unique end of the duplex DNA . However, a variety of other pairs of substrates appeared to form joint molecules without regard to the polarity of the strands involved . This paradox has been resolved by observations that show that synapsis is fast, nonpolar and sensitive to inhibition by ADP, whereas strand exchange is slow, directional and relatively insensitive to inhibition by ADP . Thus a heteroduplex joint initiated at one end of the duplex DNA grows by continued strand exchange, whereas a joint initiated at the other end dissociates and is unable to start again because accumulating ADP inhibits synapsis . RecA protein appears to form a nascent protein-DNA structure, the RecA synaptic structure, in which at least 100-300 bp in the duplex molecule are held in an unwound configuration and in which the incoming strand is aligned with its complement. Cell, 1982 Aug, 30(1), 311 - 9 Regulation of a membrane component required for protein secretion in Escherichia coli; Oliver DB et al.; We have previously described a gene, secA, which may code for a component of the secretion machinery of E . coli . Temperature-sensitive mutations in this gene lead to the cytoplasmic accumulation of precursors to a number of secreted proteins . In this paper, we describe the use of antibody to the SecA protein to characterize the cellular location and regulation of the protein . The antibody was elicited in response to a SecA-LacZ hybrid protein, produced by a strain carrying a secA-lacZ gene fusion . The secA gene product is a 92 kd polypeptide that is present in small amounts in the cell and that fractionates as a peripheral cytoplasmic membrane protein . The synthesis of the SecA protein is greatly derepressed (at least tenfold) when secretion in E . coli is blocked either in a secAts mutant or in the presence of a MalE-LacZ hybrid protein . We suggest that components of the secretion machinery of E . coli, such as the SecA