J Biol Chem, 1976 Feb 25, 251(4), 975 - 81 On the fidelity of DNA replication . Enzyme activities associated with DNA polymerases from RNA tumor viruses; Seal G et al.; DNA polymerase from RNA tumor viruses ("reverse transcriptase") has been analyzed for activities which have been associated with other DNA polymerases . Homogeneous DNA polymerase from avian myeoblastosis virus catalyzes pyrophosphate exchange and pyrophosphorolysis . Pyrophosphate exchange is dependent on a template and is base-specific . With avian myeloblastosis virus DNA polymerase, ribonucleotide templates are more efficient for synthesis while deoxyribonucleotide templates are more effective for pyrophosphate exchange . Synthesis, pyrophosphate exchange, and pyrophosphorolysis were inhibited by the chelating agent 1,10-phenanthroline, suggesting that enzyme-bound zinc is required for each of these reactions . The pyrophosphate exchange reaction was also demonstrated with the DNA polymerase from a mutant of Rous sarcoma virus that possesses a temperature-sensitive DNA polymerase . The pyrophosphate exchange reaction with the mutant polymerase is temperature-sensitive which demonstrates that pyrophosphate exchange is indeed catalyzed by the viral DNA polymerase and that the same mutation effects both DNA polymerase and pyrophosphatase activity . Unlike Escherichia coli DNA polymerase I, the DNA polymerase from avian myeloblastosis virus fails to degrade polydeoxyribonucleotides or to convert deoxynucleoside triphosphates into monophosphates . This lack of hydrolytic activities in avian myeoblastosis DNA polymerase should facilitate kinetic studies on the mechanism of DNA synthesis by this enzyme. J Biol Chem, 1976 Feb 25, 251(4), 962 - 7 Calcium transport driven by a proton gradient and inverted membrane vesicles of Escherichia coli; Tsuchiya T et al.; Calcium transport into inverted vesicles of Escherichia coli was observed to occur without an exogenous energy source when an artificial proton gradient was used . The orientation of the proton gradient was acid inside and alkaline outside . Either phosphate or oxalate was necessary for transport, as was found for respiratory-driven or ATP-driven uptake (Tsuchiya, T., and Rosen, B.P . (1975) J . Biol . Chem . 250, 7687-7692) . Phosphate accumulation was found to occur in conjunction with calcium accumulation . Calcium transport driven by an artificial proton gradient was stimulated by dicyclohexylcarbodiimide, an inhibitor of the Mg2+ATPase (EC 3.6.1.3) . Valinomycin, which catalyzes electrogenic potassium movement, stimulated calcium accumulation, while nigericin, which catalyzes electroneutral exchange of potassium and protons, inhibited both artificial proton gradient-driven transport and respiratory-driven transport . Other properties of the proton gradient-driven system and the previously reported energy-linked calcium transport system are similar, indicating that calcium is transported by the same carrier whether energy is supplied through an artificial proton gradient or an energized membrane state . These results suggest the existence of a calcium/proton antiport. J Biol Chem, 1976 Feb 25, 251(4), 1207 - 16 31P NMR of phosphate and phosphonate complexes of metalloalkaline phosphatases; Chlebowski JF et al.; 31P NMR spectra of phosphate and phosphonate complexes of Escherichia coli alkaline phosphatase have been obtained by Fourier transform NMR methods . One equivalent of P1i, bound to Zn(II) alkaline phosphatase, pH 8, gives rise to a single 31P resonance 2 ppm downfield from that for Pi, and assignable to the noncovalent complex, E-P . Inorganic phosphate in excess of 1 eq per enzyme dimer gives rise to a resonance at the position expected for free Pi . At pH 5.1, a second resonance appears 8.5 ppm downfield from that for free Pi, and is assignable to the covalent complex, E-P . The large downfield shift suggests that the enzyme phosphoryl group is highly strained with an O-P-O bond angle of under 100 degrees. J Biol Chem, 1976 Feb 25, 251(4), 1202 - 6 Metallophosphoryl and Apophosphoryl Alkaline Phosphatases; Chlebowski JF et al.; The noncovalent phosphate (E-P) and covalent phosphory (E-P) complexes of Zn(II), Cd(II), and apoalkaline phosphatases of Escherichia coli have been studied by stopped flow kinetic methods and 32P-labeling techniques . With 2,4-dinitrophenylphosphate as substrate, preincubation of the Zn(II) enzyme with Pi at pH 8 slows the pre-steady state burst rate, but does not affect the burst magnitude of 1 mol of ROH per enzyme dimer . Preincubation of the enzyme with Pi at pH 5.5 reduces the burst magnitude by one-half, as well as reducing the burst rate . Reduction of the burst magnitude as a function of the pH of the preincubation with Pi follows the same function as that previously established for the formation of E-P . Hence, ROP phosphorylates the enzyme by displacing phosphate from E-P during a pre-steady state reaction, while E-P turns over at the steady state velocity. Biochemistry, 1976 Feb 24, 15(4), 818 - 23 Mechanism of aminoacylation of tRNA . Proof of the aminoacyl adenylate pathway for the isoleucyl- and tyrosyl-tRNA synthetases from Escherichia coli K12; Fersht AR et al.; The following observations show that the formation of isoleucyl-tRNA catalyzed by the isoleucyl-tRNA synthetase from Escherichia coli K12 involves the initial rapid formation of an isoleucyl adenylate complex followed by the slow, rate-determining, transfer of the isoleucyl moiety to tRNA . (1) The rate constant for the transfer of {14C}Ile from the E-{14C}Ile approximately AMP complex to tRNA is the same as the turnover number for the steady-state isoleucylation of tRNA at pH 7.78 (1.5 s-1) and pH 5.87 (0.34 s-1) . (2) On mixing a solution of isoleucyl-tRNA synthetase and tRNA with {14C}Ile and ATP the steady-state rate of isoleucylation is attained in the first turnover of the enzyme, with little or no "burst" or charging that would indicate a slow step after the transfer step . (3) The pyrophosphate exchange reaction in the presence of tRNA is 40 times faster than the overall rate of isoleucylation of tRNA . (4) Similarly, rapid quenching experiments indicate that isoleucyl adenylate is formed prior to the transfer step . The possibility that isoleucyl adenylate formation is a parallel reaction caused by a second active site on the enzyme is ruled out both by the stoichiometry in this rapid quenching experiment and also the overall stoichiometry of isoleucyl-tRNA formation . At saturating reagent concentrations the major species in solution is the E-tRNA-Ile approximately AMP complex . Similar observations are found for the tyrosyl-tRNA systhetase except that at saturating reagent concentrations the rate constants for both tyrosyl adenylate formation and transfer are similar so that both processes contribute to the rate-determining step. Biochemistry, 1976 Feb 24, 15(4), 804 - 11 The role of 16S rRNA in ribosomal binding of IF-3; Pon CL et al.; The binding of initiation factor IF-3 to Escherichia coli 30S ribosomal subunits has been found to be inhibited by rRNA ligands such as ethidium bromide, polyamines, and monovalent alkali metals . The order of effectiveness of the polyamines (spermine greater than spermidine greater than putrescine) and alkali metals (Li+ greater than Na+ greater than K+) in inhibiting the ribosomal binding of IF-3 parallels their degree of affinity for the RNA . Furthermore, the binding of IF-3 to 30S subunits chemically modified by photooxidation with rose bengal, nitration with tetranitromethane, and reaction with kethoxal, monoperphthalic acid, and p-chloromercuribenzoic acid was studied . Results obtained after the direct treatment of the 30S subunits with the above chemical reagents or upon reconstitution of 30S particles having a modified rRNA or ribosomal proteins indicate that the IF-3 binding site is preferentially lost when the rRNA becomes modified . It was found that IF-3 could bind normally to 30S subunits lacking protein S1 or proteins S11, S12, S19, and S21 (and perhaps S14) which had been cross-linked to IF-3 in other laboratories. Biochemistry, 1976 Feb 24, 15(4), 734 - 40 Synchronous digestion of SV40 DNA by exonuclease III; Wu R et al.; We have established an optimal condition for the synchronous digestion of SV40 DNA with Escherichia coli exonuclease III . Electron microscopy and polyacrylamide gel electrophoresis were used to obtain accurate measurements on the lengths of DNA before and after exonuclease III digestion . Based on this finding, a new method for determining the sequence of long duplex DNA can be realized . It involves (a) the synchronous digestion of the DNA from the 3' ends with exonuclease III, followed by (b) repair synthesis with labeled nucleotides and DNA polymerase, and (c) sequence analysis of the repaired DNA. Eur J Biochem, 1976 Feb 16, 62(2), 313 - 22 Molecular-weight determination of animal-cell RNA by electrophoresis in formamide under fully denaturing conditions on exponential polyacrylamide gels; Spohr G et al.; A method for electrophoretic analysis of RNA under fully denaturing conditions on exponential gradient polyacrylamide gels is described . Full denaturation, and strand separation of DNA - RNA hybrids and double-stranded RNA is obtained in dry formamide only if electrophoresis is carried out at 45 degrees and 55 degrees C, respectively . In such conditions, the effects of secondary structure of RNA, important in aqueous medium, are suppressed and a linear correlation is obtained between the logarithm of the molecular weight of an RNA and its final position in the gel over the entire molecular weight range of 10(4) - 10(7) . Based on absolute molecular weight standards, obtained from sequenced rRNA of Escherichia coli and tRNA and extrapolating to higher molecular weights the size of animal cell was reexamined . Precursor tRNA from HeLa cells migrates according to a molecular weight of 4.1 x 10(6) . Nascent precursor mRNA has molecular weights of up to 5 x 10(6) in the case of duck erythroblasts and of up to 10(7) in HeLa cells . This seems to represent the largest size of non-viral animal-cell RNA molecules. Eur J Biochem, 1976 Feb 16, 62(2), 285 - 92 Enzyme-linked immunoassay . Conjugation of rabbit anti-(human immunoglobulin G) antibody with beta-D-galactosidase from Escherichia coli and its use for human immunoglobulin G assay; Kato K et al.; Rabbit immunoglobulin G (IgG) was reduced by incubating with 10 mM 2-mercaptoethylamine and then treated with excess amounts of N,N'-o-phenylenedimaleimide . As a result, maleimide residues were introduced into rabbit IgG molecules . Rabbit IgG containing maleimide residues could be coupled to beta-D-galactosidase from Escherichia coli which has sulfhydryl groups in the molecule . The resulting rabbit IgG (antibody)-enzyme complex may be useful for immunoassay of antigens . As an example, human IgG was assayed by the sandwich method using the rabbit anti-(human IgG) IgG-beta-D-galactosidase complex and amounts of human IgG as small as 3 fmoles were measurable. Biochem J, 1976 Feb 15, 154(2), 311 - 8 An unusual precursor of 50S ribosomes in a mutant of Escherichia coli; Markey F et al.; Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that leads to the accumulation of large amounts of ribonucleoprotein ("47S") particles during exponential growth . These particles are precursors to 50S ribosomes, but are distinct from precursors detected by pulse-labelling of the parent strain and also from ribosome precursors that accumulate during inhibition of growth by CoC12 . Either ribosome assembly in the mutant differs from that in the wild-type strain, or 47S particles represent a hitherto unstudied stage in the synthesis of 50S ribosomes. J Am Vet Med Assoc, 1976 Feb 15, 168(4), 317 - 8 Use of a formalin-treated, live Escherichia coli vaccine in the prevention of neonatal enteric colibacillosis in swine; Ward GE et al.; A field trial was conducted to evaluate the effectiveness of an Escherichia coli vaccine in prevention of neonatal enteric colibacillosis in swine . Sows (272 total) were allotted to control (151 sows) and vaccinated groups (121 sows) . Sows in the vaccinated groups were given a single intramuscular injection of a formalin-treated, live E coli vaccine 10 to 20 days prior to farrowing . The effects of the vaccination were to: (1) reduce mortality from 2.14 to 0.93 (P less than 0.001) pigs per litter; (2) reduce number of pigs with diarrhea, from 7.28 to 3.12 (P less than 0.004) per litter; and (3) increase number of pigs weaned, from 7.62 to 8.2 (P less than 0.005) per litter . The advantages of vaccination were most apparent in the barns with less than adequate sanitation and ventilation. Biochem J, 1976 Feb 15, 154(2), 285 - 94 Kinetic characterization of the membrane-bound cytochromes of Escherichia coli grown under a variety of conditions by using a stopped-flow dual-wavelength spectrophotometer; Haddock BA et al.; A study was made of the rapid oxidation kinetics of the cytochromes of Escherichia coli . The b-type cytochromes were kinetically heterogeneous, with one species (presumably cytochrome o) oxidized so rapidly that it could fully support observed oxidation rates . Cytochrome d but not cytochrome a1 was also kinetically competent to support respiration . However, in cells grown anaerobically in the presence of NO3-, cytochrome d exhibited slow oxidation kinetics and a red-shift in its reduced-minus-oxidized difference spectrum. Biochim Biophys Acta, 1976 Feb 13, 422(2), 302 - 8 Conformations of lysine-sensitive aspartokinase; Shaw JF et al.; 1 . The technique of differential thermal and proteolytic inactivation has been employed as a conformational probe for the lysine-sensitive aspartokinase (EC 2.7.2.4) of Escherichia coli B . 2 . L-Amino acid inhibitors of this enzyme each induce a characteristic enzyme conformation . This is evidenced by rates of thermal and proteolytic inactivation and Arrhenius activation energies for thermal inactivation which are characteristic of the amino acid present . 3 . Phenylalanine and leucine binding are mutually exclusive as evidenced by competitive behavior in thermal inactivation experiments, suggesting a hydrophobic amino acid binding site with broad specificity . 4 . The phenylalanine-dependent conformation and the leucine-dependent conformation differ considerably . In comparison with the native enzyme, the former is more labile to proteolysis by trypsin whereas the latter is more stable . First-order rate constants for thermal inactivation of the phenylalanine- and leucine-dependent conformations are, respectively, about one-half and one-tenth that of the native enzyme . 5 . Items 3 and 4 taken together suggest that the conformations are ligand induced and do not arise via ligand stabilization of spontaneously arising conformers. J Bacteriol, 1976 Feb, 125(2), 518 - 23 Selection and properties of Escherichia coli mutants defective in the synthesis of cyclopropane fatty acids; Taylor F et al.; Mutants of Escherichia coli K-12 defective in the synthesis of cyclopropane fatty acids (CFA) have been selected and isolated by a L-{methyl-3H}methionine suicide procedure . Two mutants were isolated . Stationary-phase cultures of both mutants contain less than 0.7% of the CFA content found in the parental strain . The CFA deficiency is attributed to a deficiency of CFA synthetase activity . Extracts of both mutants contain less than 10% of the CFA synthetase activity found in extracts of the parental strain . Experiments in which parental and mutant extracts were mixed indicate that the lack of activity in the mutant strains is not due to an inhibitor of CFA synthetase present in the mutant extracts . We have not yet detected a physiological phenotype for these mutants . These strains grow normally at various temperatures in a variety of media . We have tested survival (colony-forming ability) in response to (i) prolonged incubation in stationary phase, (ii) exposure to drying, and (iii) exposure to detergents, heavy metals, low pH, high salt concentration, and a variety of other environmental conditions . The survival of both mutants is identical to that of the parental strain under all conditions tested . The compositions (excepting the CFA deficiency) and metabolic turnover rates of the phospholipids of both mutant strains are indistinguishable from those of the wild-type strain . The transport of several amino acids also seems normal in these mutants. Biochemistry, 1976 Feb 10, 15(3), 593 - 9 Changes in the expression of the chloroplast genome of Euglena gracilis during chloroplast development; Chelm BK et al.; The transcription program from the chloroplast genome of Euglena gracilis Z during light-induced chloroplast development has been characterized by hybridization of total cell RNA to 3H-labeled chloroplast DNA . Pancreatic DNase activated, purified Euglena chloroplast DNA was enzymatically labeled by Escherichia coli DNA polymerase I with {3H}TTP as a substrate . The {3H}DNA 'hybridization probe" was characterized by the kinetics of its renaturation with purified chloroplast DNA, and the thermal stability of {3H}DNA-DNA, and {3H}DNA-RNA hybrids . The {3H}DNA was hybridized in trace amounts to total cellular RNA extracted from Euglena cells 0, 4, 8, 12, 24, 48, and 72 h after the onset of chloroplast development . A large percentage (17%) of the chloroplast genome was found to be transcribed in dark adapted cells . Development is marked by an initial decrease in the fraction of the genome transcribed followed by an increase to 23% transcribed at the end of 72 h of light growth . Chloroplast RNA transcripts were also characterized by the kinetics of their hybridization to chloroplast DNA . The chloroplast specific RNA population is composed of three abundance classes, and the R0t1/2 for each class varies during the early stages of chloroplast development. Biochemistry, 1976 Feb 10, 15(3), 666 - 71 Behavior of colicins E1, E2, and E3 attached to sephadex beads; Lau C et al.; Colicins E1, E2, and E3 were covalently attached to Sephadex G-25 beads by cyanogen bromide activation . These immobilized colicins were still active in binding to specific receptors on sensitive and tolerant cells but not to resistant cells which lack such receptors . Bound colicin E3 also retained its ability to inhibit protein synthesis in vitro . Leakage of free colicin from these coated beads was negligible . Assays sensitive to free colicin activity of 1 part in 10(7) of the bound toxin failed to detect any soluble activity . The viability of different cell types bound specifically onto these colicin-Sephadex beads was assayed by using autoradiography based on labeled amino acid uptake . Immobilized E1 killed 90% of bound sensitive cells while less than 10% of sensitive cells bound to E2 and E3 were killed in this assay . These observations agree very well with previously suggested mechanisms which propose that E1, whose target site appears to be at the membrane level, can kill sensitive cells by binding to the cell surface, but that for E2 and E3 penetration of part or all of the molecule is necessary for killing action to be observed. Biochemistry, 1976 Feb 10, 15(3), 569 - 75 Nepsilon-acetyllysine transfer ribonucleic acid: a biologically active analogue of aminoacyl transfer ribonucleic acids; Johnson AE et al.; Unfractionated Escherichia coli tRNA has been aminoacylated with lysine and preferentially acetylated at the epsilon-amino nitrogen of lysine by reaction with N-acetoxysuccinimide . After treatment with peptidyl-tRNA hydrolase, 90% of the aminoacylated tRNA molecules were Nepsilon-acetyl-Lys-tRNA . Post-ribosomal supernatant enzymes would not deacylate Nepsilon-acetyl-Lys-tRNA in the presence of AMP and PPi, even though such mixed enzymes could acylate, with lysine, tRNA which had been exposed to the acetylation reaction conditions . Poly(rA) stimulated the binding of Nepsilon-acetyl-Lys-tRNA to E . coli ribosomes . At the ribosome and tRNA concentrations used, Nepsilon-acetyl-Lys-tRNA was bound nearly as well as Lys-tRNA at 30 mM Mg2+; at 10 mM Mg2+, the analogue was bound one-half as well as Lys-tRNA . Both Lys-tRNA and Nepsilon-acetyl-Lys-tRNA reacted only slightly with puromycin at either 10 or 30 mM Mg2+ . When Lys-tRNAE . coli or Nepsilon-acetyl-Lys-tRNAE . coli were added to rabbit reticulocyte cell-free protein synthesizing incubations, the incorporation of either amino acid into protein was complete within 5 min . The final incorporation level of the analogue was 82% that of the unmodified lysine . After protein synthesized in the presence of Nepsilon-acetyl-{14C}Lys-tRNA had been digested enzymatically to single amino acids, ion-exchange chromatography and paper electrophoresis showed that nearly all of the radioactivity was present as Nepsilon-acetyllysine . Gel filtration of the post-ribosomal supernatant revealed that most of the Nepsilon-acetyllysine radioactivity cochromatographed with tetrameric hemoglobin. Biochemistry, 1976 Feb 10, 15(3), 536 - 43 Manganese(II) and substrate interaction with unadenylylated glutamine synthetase (Escherichia coli w) . I . Temperature and frequency dependent nuclear magnetic resonance studies; Villafranca JJ et al.; A comprehensive study of solvent interaction with unadenylylated glutamine synthetase (E1.7) has been conducted using the enzyme isolated from Escherichia coli W . The longitudinal, (1/T1p)b, and transverse, (1/T2p)b, proton relaxation rates were measured with various enzyme samples as a function of frequency (6-48 MHZ) and temperature (1-40 degrees C) . With Mn(II) bound at the "tight" metal ion site approximately two water molecules are rapidly exchanging with bulk solvent . This number is reduced to approximately one in the presence of glutamine . All data were successfully analyzed according to the Solomon-Bloembergen-Morgan (SBM) scheme for dipolar relaxation of water protons interacting with enzyme-bound Mn(II) . The correlation time for this process varies from 1 to 3 X 10(-9) for the complexes described above . Significant contributions to the correlation time arise from both 1/taum, the exchange rate for water molecules bound at the metal site, and from 1/taus, the electron spin relaxation rate for Mn(II) with the latter rate showing a frequency dependence at the magnetic field strengths used in this study . A study of Mn(II) binding to E1.7 at 25 degrees C revealed two classes of metal ion sites, a "tight" set of one per subunit with KD=5.0 X 10(-7) M and a "weak" set of one per subunit with KD=4.5 X10(-5)M . In the presence of glutamine the affinity of the first site for Mn(II) was unchanged but the KD value for the weak site changed to 3 X 10(-6)M . In E1.7 samples with Mn(II) bound at both the tight and weak metal ion sites the data are interpretable with two rapidly exchanging water molecules interacting with each bound Mn(II)ion . With saturating amounts of glutamine or of ADP or of glutamine plus ADP plus arsenate, the proton relaxation rates progressively decreased suggesting that the substrates or inhibitors used were interacting with the bound Mn(II) ions resulting in diminished solvent accessibility to these bound ions . These results are interpretable in terms of ligand substitution into the coordination sphere of the bound Mn(II) ions . Indeed this is probably the case for Mn(II) at the weak metal ion site since Hunt et al . ((1975), Arch . Biochem . Biophys . 166, 102) showed that Mn(II) can bind as the Mn(II)-ADP complex to the second metal ion site . Results of proton relaxation rate data on E1.7 with Mn(II) bound at both the tight and weak metal ion sites led to the conclusion that these metal ion sites are greater than 6 A apart . In comparison with proton relaxation rate data on fully adenylylated glutamine synthetase (E11.8) as studied by Villafranca and Wedler ((1974), Biochemistry 13, 3286), the first "tight" metal ion site in E11.8 has three rapidly exchanging water molecules . Mn(II) has a weaker binding constant to E11.8 (KD approximately 5 X 10(-6)M) at the pH value used in both studies and a suggestion is made that an additional protein ligand is binding to Mn(II) in glutamine synthetase when the subunits are not adenylylated. J Biol Chem, 1976 Feb 10, 251(3), 898 - 901 Syntheses of elongation factors Tu and G are under stringent control in Escherichia coli; Furano AV et al.; We have compared the synthesis of the elongation factors Tu and G at 30 and 36.5 degrees in three strains of Escherichia coli: NF314 (rel+valS+), NF536 (rel+valSts), and NF537 (rel-valSts) . At the elevated temperature, the latter two strains continue to grow but suffer a partial deprivation of valyl-tRNA . As a consequence, the syntheses of stable RNA, Tu, and G are decreased in NF536; the syntheses of stable RNA, Tu, and G are increased in NF537 . These results indicate that the syntheses of Tu and G are directly or indirectly under the influence of the rel gene. J Biol Chem, 1976 Feb 10, 251(3), 883 - 92 Regulation of carbohydrate uptake and adenylate cyclase activity mediated by the enzymes II of the phosphoenolpyruvate: sugar phosphotransferase system in Escherichia coli; Saier MH Jr et al.; The uptake of various carbohydrates and the synthesis of adenosine 3':5'-monophosphate (cyclic AMP) are subject to inhibition by sugar substrates of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli . The induced synthesis of the sugar-specific components of the phosphotransferase system was studied and correlated with the induction of regulatory interactions controlling glycerol uptake and net cyclic AMP synthesis . Activities of the Enzyme II complexes specific for glucose, fructose, and mannitol were measured both in vivo and in vitro . These activities were induced 8- to 40-fold by growth in the presence of the appropriate substrate-inducers . Cross inducer specificities were noted . Maximal inhibition of glycerol uptake and cyclic AMP synthesis by a sugar substrate of the phosphotransferase system required induction of the Enzyme II complex specific for that sugar and was abolished by mutations which destroyed Enzyme II activity . The inducer specificities of the regulatory systems and of the Enzymes II were found to be the same . A mutation which depressed the cellular activity of Enzyme I of the phosphotransferase system did not relieve sensitivity to inhibition . The results suggest that adenylate cyclase and several carbohydrate permeases are subject to coordinate regulation by a mechanism which depends on the catalytic activities of the protein components of the phosphotransferase system. J Biol Chem, 1976 Feb 10, 251(3), 651 - 7 Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli . 9 . Enzymatic joining of chemically synthesized deoxyribopolynucleotide segments corresponding to nucleotide sequence 57-94; Panet A et al.; The DNA duplexes representing nucleotide sequences 61-89 and 57-94 have been synthesized, isolated pure, and fully characterized . Synthesis of the duplex with the nucleotide sequence 61-89 involved the DNA ligase-catalyzed joining of chemically synthesized deoxyoligonucleotide segments 14 to 18 shown in Fig . 1A, while for the longer duplex (sequences 57-94) seven deoxyribooligonucleotides (segments 13 to 19, Fig . 1B) were used in one-step enzymatic joining . The joining of the short tetranucleotide (segment 16) to the segment 17 required the presence of the adjacent segment 14, even if the latter did not contain a 5'-phosphate group, to allow its joining to segment 16 . However, in the synthesis of both of the DNA duplexes, the yields were comparatively low (30 to 40%) and could not be significantly increased although a variety of conditions was tried . The main cause in both cases evidently was the sluggish joining of segment 14 to 16 and of segment 16 to segment 17 . Although the original plan for the total synthesis of this part of the gene for the tRNA precursor involved the DNA duplex consisting of segments 14 to 18, this duplex could not be quantitatively phosphorylated at the two 5'-OH ends for subsequent joining to the adjoining parts of the gene . The DNA duplex consisting of segments 13 to 19, which possesses both terminal 5'-OH groups at protruding single-stranded ends, was readily phosphorylated and used successfully in the total synthesis of the gene as described in an accompanying paper. J Biol Chem, 1976 Feb 10, 251(3), 642 - 59 Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli . 8 . Enzymatic joining of the chemically synthesized segments to form DNA duplexes corresponding to nucleotide sequences 23-60 and 23-66; Loewen PC et al.; Polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxyribopolynucleotide segments (Fig . 1) comprising the nucleotide sequence 23-66 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been systematically investigated . Joining was studied using all possible combinations of 3, 4, and 5 and larger numbers of segments at a time . The extent of joining varied widely (0 to about 90%) in three component systems . The "self-structure" of some of the components evidently inhibited the joining . Addition of a fourth segment in general enhanced the extent of joining and optimal yields were obtained in systems containing six or more segments . A comparison of the T4-induced ligase and the E . coli polynucleotide ligase for joining of the chemically synthesized segments showed the E . coli enzyme to be inferior to the T4-induced ligase . Satisfactory syntheses of the duplexes {IIa} and {IIb} comprising, respectively, eight and seven segments were achieved in single steps . Of the two terminal segments carrying 5'-OH groups in the duplexes, only one (segment 7) was used in the prephosphorylated form . The duplexes were isolated pure and characterized by enzymatic degradations and by electrophoresis. J Biol Chem, 1976 Feb 10, 251(3), 634 - 41 Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli . 7 . Enzymatic joining of the chemically synthesized segments to form a DNA duplex corresponding to the nucleotide sequence 1-26; Sekiya T et al.; Duplex {I}, which represents the nucleotide sequence 1-26 of the double-stranded DNA corresponding to the precursor for a tyrosine suppressor tRNA, has been synthesized by the enzymatic joining of five chemically synthesized deoxyribooligonucleotide segments . The synthesis was accomplished in two different ways . In a one-step synthesis, all of the five segments were used together: segments 2, 3, and 5 carried 5'-33P-labeled phosphate groups while segment 4 carried a 32P-phosphate group . An alternative, two-step method involved the joining of 5'-32P-phosphorylated segment 2 to segment 4 (carrying 5'-OH group or 5'-32P- or 33P-labeled phosphate group) in the presence of segment 3 followed by the joining of {5-32P}segment 5 in a second step . The duplex {I}' (segments 2 to 5) thus obtained was phosphorlated at the 5'-ends with polynucleotide kinase and then joined to segment 1 to give duplex {I} quantitatively . The preparative methods described have the desired flexibility for performing the subsequent operations necessary for the total synthesis of the structural gene for the tyrosine suppressor tRNA precursor. J Biol Chem, 1976 Feb 10, 251(3), 624 - 33 Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli . 6 . Synthesis of the deoxyribopolynucleotide segments corresponding to the nucleotide sequence 100-126; Agarwal KL et al.; Chemical syntheses of the tridecanucleotide, d(G-C-T-T-C-C-C-G-A-T-A-A-G), the dodecanucleotide, d(G-C-T-C-C-C-T-T-A-T-C-G), the decanucleotide, d(G-G-A-G-C-A-G-G-C-C), the nonanucleotide, d(T-A-C-T-G-G-C-C-T), and the hexanucleotide, d(G-G-A-A-G-C), are described . Together, these syntheses represent the nucleotide sequence 100-126 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor . The synthesis of the dodecanucleotide d(G-C-T-C-C-C-T-T-A-T-C-G), was accomplished by the condensation of the previously described protected nonanucleotide, d{(MeOTr)ibG-anC-T-anC-anC-anC-T-T-bzA}, with the trinucleotide block d{pT-anC-mbG(Ac)} . Synthesis of the other segments involved stepwise condensations to the 3'-OH group of growing oligonucleotide chains, starting with suitably protected deoxyribonucleosides and using protected mono-, di-, and trinucleotides as the incoming blocks . The final products, after deprotection, were purified by anion exchange chromatography and characterized . The synthesis of this part of the DNA was planned so that only a hexanucleotide segment is used to go up to the 3'-end (nucleotide 126) of the DNA and, therefore, it is amenable to elongation by chemical methods when the nucleotide sequence of the several nucleotides beyond this end becomes known. J Biol Chem, 1976 Feb 10, 251(3), 609 - 23 Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli . 5 . Synthesis of the deoxyribopolynucleotide segments representing the nucleotide sequence 71-103; Jay E et al.; Chemical syntheses of the pentadecanucleotide, d(G-G-T-G-G-G-G-T-T-C-C-C-G-A-G), the undecanucleotides, d(G-G-T-G-G-G-G-T-T-C-C) and d(C-C-C-C-A-C-C-A-C-G-G), the decanucleotide, d(G-T-A-A-T-G-C-T-T-T), and the nonanucleotides, d(A-T-T-A-C-C-C-G-T) and d(A-G-T-A-A-A-A-G-C) are described . The deoxyribopolynucleotides together represent the DNA duplex corresponding to the nucleotide sequence 71-103 (from the 3'-end) of the gene for the tyrosine suppressor tRNA . Synthesis of the guanine-rich undecanucleotide d(G-G-T-G-G-G-G-T-T-C-C) was performed by the use of a new protecting group for the guanine ring, the methylbutyryl group . The heptanucleotide d{(MeOTr)mbG-mbG-T-mbG-mbG-mbG-mbG}, prepared by the new method, was condensed with the tetranucleotide d{panC-anC-T-T(Ac)} . All of the condensations described followed previously developed chemical principles and started with the N- and 5'-protected deoxyribonucleosides . Successive condensations at the 3'-end with protected mononucleotides, preformed di-, tri-, or tetranucleotides gave products which were separated by anion exchange chromatography and characterized by chemical and enzymatic methods. J Biol Chem, 1976 Feb 10, 251(3), 599 - 608 Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli . 4 . Synthesis of deoxyribopolynucleotide segments corresponding to the nucleotide sequence 47-78; Agarwal KL et al.; The chemical syntheses of the dodecanucleotide, d(G-C-C-G-C-T-C-G-G-G-A-A), the decanucleotides, d(T-T-T-A-G-A-G-T-C-T), d(G-C-T-C-C-C-T-T-T-G), d(C-G-G-C-C-A-A-A-G-G), and the nonanucleotide, d(G-A-G-C-A-G-A-C-T), are described . The deoxypolynucleotides together represent the DNA duplex corresponding to the nucleotide sequence 47-78 of the gene for the tyrosine suppressor tRNA . Chemical syntheses used protected mono- and oligonucleotides and stepwise condensation methods . The detailed plans used are given in Diagrams 1 to 4 in the text . Two additional notable features in the present syntheses were: (a) solvent extraction procedures for the preparation of oligonucleotides as large as pentanucleotides and (b) the demonstration that a heptanucleotide containing 5'-phosphate group can be used successfully in condensation with the 3'-OH group of another oligonucleotide . The synthetic polynucleotides were purified and characterized at protected and unprotected levels. J Biol Chem, 1976 Feb 10, 251(3), 571 - 86 Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli . 2 . Chemical synthesis of the deoxypolynucleotide segments corresponding to the nucleotide sequence 1-31; van de Sande JH et al.; Chemical synthesis of the undecanucleotides d(T-G-G-G-G-G-A-A-G-G-A), d(C-C-C-C-A-C-C-A-C-C-A), and d(T-C-G-A-A-T-C-C-T-T-C), and the nonanucleotides, d(T-T-C-G-A-A-C-C-T) and d(T-T-C-G-A-A-G-G-T) are described . The deoxyribopolynucleotides together represent the DNA duplex corresponding to the nucleotide sequence 1-31 (from the 3'-end) of the gene for the tyrosine suppressor tRNA . The synthesis, which basically used the previously developed chemical methods, started with 5' and N-protected deoxyribonucleosides . Successive condensations at the 3'-end were performed using suitably protected mononucleotides or preformed di- and trinucleotides . The condensing agents used were mesitylenesulfonyl chloride or triisopropyl benzenesulfonyl chloride . The required condensation products were isolated partly by solvent partition methods or, in the case of longer chains, by anion exchange chromatography . The completely deprotected deoxypolynucleotides were further purified by anion exchange chromatography in the presence of 7 M urea and characterized by chemical and enzymatic methods. J Biol Chem, 1976 Feb 10, 251(3), 565 - 70 Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli . 1 . General introduction; Khorana HG et al.; With the ultimate objective of the total synthesis of a tRNA gene including its transcriptional signals, an Escherichia coli tyrosine suppressor tRNA gene was chosen . The arguments in favor of this choice are presented . A plan for the total synthesis of the 126-nucleotide-long DNA duplex corresponding to a precursor (Altman S., and Smith, J . D . (1971) Nature New Biol . 233, 35) to the above tRNA is formulated . The plan involves: (a) the chemical synthesis of 26 deoxyribooligonucleotide segments, (b) polynucleotide ligase-catalyzed joining of several segments at a time to form a total of four DNA duplexes with appropriate comlementary single-stranded ends, and (c) the joining of the duplexes to form the entire DNA duplex . Ten accompanying papers describe the experimental realization of this objective. Biochemistry, 1976 Feb 10, 15(3), 487 - 93 Isolation of viral specific RNA from SV40 infected cells by viral DNA chemically linked to a cellulose matrix; Shih TY et al.; SV40 DNA fragments chemically attached to neutral cellulose powder with a water-soluble carbodiimide have been used to isolate late lytic viral specific RNA from virus infected cells . Exhaustive hybridization to SV40 DNA reveals that virtually all of the isolated RNA molecules contain SV40 specific sequences . Comparison with SV40 cRNA prepared with purified Escherichia coli RNA polymerase and a SV40 DNA I template suggests that the purity of the isolated SV40 specific RNA is very close to 100% . The background level for the nonspecific binding of RNA to a purified cellulose matrix is very low . Retention of nonspecific RNA by SV40 DNA-cellulose is only 1.5% of the viral specific RNA isolated under saturating conditions for the column . Sedimentation in neutral sucrose suggests that the major 16S viral specific RNA has been isolated largely intact. J Biol Chem, 1976 Feb 10, 251(3), 676 - 94 Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli . 12 . Synthesis of a DNA duplex corresponding to a sequence of 23 nucleotide units adjoining the C-C-A end; Ramamoorthy et al.; In continuing the work on the total synthesis of the gene for an Escherichia coli tyrosine suppressor tRNA (accompanying papers) and as a part of a study of the mechanism of transcription of this gene, a 23-nucleotide unit-long DNA corresponding to the previously determined (Loewen, P., Sekiya, T., and Khorana, H . G . (1974) J . Biol . Chem . 249, 217) sequence has been synthesized . The synthesis was carried out by dividing the total duplex into the following five deoxyribooligonucleotide segments, all of which were chemically synthesized: (a) the undecanucleotide, d(A-G-T-G-A-T-G-G-T-G-G); (b)the undecanucleotide, d(T-C-A-C-T-T-T-C-A-A-A); (c) the undecanucleotide, d(G-G-A-C-T-T-T-T-G-A-A); (d) the dodecanucleotide, d(A-G-T-C-C-C-T-G-A-A-C-T); and (e) the heptanucleotide, d(A-G-T-T-C-A-G) . All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling the 5'-ends with a 32P-phosphate group . Synthesis of the double-stranded DNA duplex was completed by joining 5'-phosphorylated segments 1, 3, and 4 in the presence of segments 2 and 5 using T4-polynucleotide ligase . The DNA duplex was characterized. J Biol Chem, 1976 Feb 10, 251(3), 667 - 75 Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli . 11 . Enzymatic joining to form the total DNA duplex; Kleppe R et al.; The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized . Duplex {I} (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H . G.(1976) J . Biol . Chem . 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex {II} (Loewen, P . C., Miller, R . C., Panet, A., Sekiya, T., and Khorana, H . G . (1976) J . Biol . Chem . 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with {gamma-33P}ATP at the 5'-OH ends . Duplex {III} (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H . G . (1976) J . Biol . Chem . 251, 651-657) (nucleotide sequence 57-94 (Fig . 2)) was also phosphorylated at 5'-ends with {gamma-33P}ATP and was joined to duplex {IV} (Caruthers, M . H., Kleppe, R., Kleppe, K., and Khorana, H . G . (1976) J . Biol . Chem . 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90 . The joined product, duplex {III + IV} (nucleotide sequence 57-126) was characterized . The latter duplex was joined to the duplex {I + II} to give the total duplex . The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends. J Biol Chem, 1976 Feb 10, 251(3), 658 - 66 Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli . 10 . Enzymatic joining of chemically synthesized segments to form the DNA duplex corresponding to the nucleotide sequence 86-126; Caruthers MH et al.; The polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxypolynucleotides (segments 19 to 26), comprising the nucleotide sequence 86-126 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been investigated . Joining was studied using various combinations of 3, 4, or larger number of segments at a time . The extent of joining was in general low (0 to 40%) for the three-component as well as for the four-component systems . Joining of the five- and six- component systems was more satisfactory with yields from 25 to about 60% . The three duplexes {IVa} to {IVc}were prepared in single step reactions in yields of about 50% and were characterized . Duplex {IVd} could not be prepared in a single step reaction because of the failure of 5'-phosphorylated segment 26 to join to the rest of the duplex . Using a carefully annealed mixture of segments 24, 25, and phosphorylated segment 26, the joining of the latter to segment 24 could be realized in about 25% yield, much activated intermediate being concurrently present. J Biol Chem, 1976 Feb 10, 251(3), 587 - 98 Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli . 3 . Synthesis of deoxyribopolynucleotide segments corresponding to the nucleotide sequence 27-51; Minamoto K et al.; Chemical syntheses of the four deoxyribodecanucleotides, d(T-C-G-A-A-G-T-C-G-A), d(C-G-T-C-A-T-C-G-A-C), d(T-G-A-C-G-G-C-A-G-A), and d(C-T-A-A-A-T-C-T-G-C) are described . These polynucleotides form, respectively, segments 7 to 10 in the plan adopted for the total synthesis of the DNA corresponding to the precursor for the Escherichia coli tyrosine tRNA . The syntheses used the principles of stepwise addition of protected mono- and oligonucleotides to the 3'-hydroxyl end of growing oligonucleotide chains . Detailed schemes used in the present syntheses are shown in Diagrams 1 to 4 in the text . The final products were subjected to extensive chromatography and were characterized as pure by chemical and enzymatic procedures. J Biol Chem, 1976 Feb 10, 251(3), 808 - 12 Purification and characterization of a DNA-dependent ATPase from Escherichia coli; Richet E et al.; A DNA-dependent ATPase has been isolated and purified from an Escherichia coli cell-free extract . The ATPase has the following characteristics: preferential dependence on single-stranded DNA, specificity for ATP hydrolysis, Km value of 1.4 X 10-4 M for ATP, and molecular weight of approximately 69,000 . The ATPase can be shown to bind to single stranded DNA . The resemblance between this ATPase and that isolated from vaccinia cores is discussed. Biochemistry, 1976 Feb 10, 15(3), 544 - 53 Manganese (II) and substrate interaction with unadenylylated glutamine synthetase (Escherichia coli w) . II . Electron paramagnetic resonance and nuclear magnetic resonance studies of enzyme-bound manganese(II) with substrates and a potential transition-state analogue, methionine sulfoximine; Villafranca JJ et al.; The enhancement of the longitudinal proton relaxation rate of solvent water protons which occurs when Mn(II) is bound to the "tight" metal ion site of unadenylylated glutamine synthetase (GS) was used to determine the binding constant of L-methionine (SR)-sulfoximine to GS-Mn(II) complexes . The binary enhancement for GS-Mn(II) is 22 at 24 MHz, 25 degrees C . The enhancement is lowered in the presence of the sulfoximine and the computed dissociation constant is 30 muM with epsilont, the enhancement for the ternary complex, equal to 3.0 . Titration curves for the sulfoximine were also obtained in the presence of Mg-ADP, Mg-ADP plus Pi, and Mg-ATP . The dissociation constants were 9, 5, and 0.8 muM, respectively . The progressive tightening of the dissociation constants is symptomatic of conformational changes at the active site as the total subsite occupied by ATP is filled . The number of rapidly exchanging water molecules drops from 2 to approximately 0.1 when saturating concentrations of L-methionine (SR)-sulfoximine and nucleotide are present . The kinetically determined KI value of approximately 4 muM for the sulfoximine is about three orders of magnitude tighter than thee Km' value of approximately 3 mM for L-glutamate . The previously mentioned dissociation constants obtained by enhancement titrations are also orders of magnitude tighter than Km' . These data suggest that L-methionine (SR)-sulfoximine is a "transition-state" analogue for the glutamine synthetase reaction . ... Science, 1976 Feb 6, 191(4226), 468 - 9 Characterization of a cell-lethal product from the photooxidation of tryptophan: hydrogen peroxide; McCormick JP et al.; Near-ultraviolet (300 to 400 nanometers) irradiation of saturated, oxygenated solutions of tryptophan in the absence of added sensitizer gives rise to substances that have various biological effects on isolated cells, including mutagenicity and selective lethality to recombination-deficient bacterial mutants . One of these biologically active products has been identified as H2O2, on the basis of spectrometric, chromatographic, chemical, and biological properties . Now H2O2 has been shown to account for the biological activities mentioned above. Biochim Biophys Acta, 1976 Feb 5, 418(3), 315 - 20 Hydrodynamic determination of polynucleotide chain discontinuities . Improved molecular weight correlations for denatured DNA; Mingot F et al.; Evidence is presented of the constancy in the conformation of denatured DNA in 2% formaldehyde in SSC (0.15 M NaCl/0.015 M trisodium citrate (pH 7.0)) over a wide range of molecular weights . It is also shown that denatured RNA behaves in the same way as DNA . The range of molecular weights studied runs from 0.02 to 16 X 10(6) . In accordance with these results, biparametric expressions are proposed for molecular weight calculations from sedimentation or viscosity data of denatured DNA or RNA, when determined in 2% formaldehyde in SSC . Testing of the expressions with standard DNA and RNA preparations showed good correlation. Biochim Biophys Acta, 1976 Feb 5, 418(3), 249 - 56 Informational complexity of the nuclear and chloroplast genomes of Chlamydomonas reinhardi; Howell SH et al.; DNA - DNA reassociation kinetics analyzed by hydroxylapatite chromatography have been used to determine the informational content of the Chlamydomonas reinhardi nuclear and chloroplast genomes . The kinetics indicate that nuclear DNA, with the exception of ribosomal cistrons, renatures as a single component with an informational complexity 25 times that of the Escherichia coli genome . The chloroplast genome has less than 0.3% of the informational complexity of the nuclear genome, but is present in about 50 copies in the vegetative cell . Chloroplast DNA shows about a 10-12% zero-time binding component. Biochim Biophys Acta, 1976 Feb 5, 418(3), 266 - 76 Partial purification and properties of a DNA-binding protein from nuclei of cells infected with polyoma virus; Yeh WS et al.; A DNA-binding protein has been purified from nuclei of 3T3 cells infected with polyoma virus . The assay used to detect this activity measures the amount of double-stranded DNA retained on a nitrocellulose membrane filter in the presence of binding protein . The interaction between DNA and protein is salt dependent and occurs optimally at 0.8 M NaCl . The isolated protein can bind to both circular and linear duplex DNA . Incubation of the binding protein with PM2 or polyoma DNA results in the formation of a fast sedimenting DNA structure in neutral sucrose gradients . The isolated binding protein is also capable of producing a considerable stimulation of both Escherichia coli (Pol I) and T4 DNA polymerase activities when either single-stranded or intact, native T7 DNA is used as the template . The binding protein itself is free of detectable DNA polymerase or nuclease activity. Eur J Biochem, 1976 Feb 2, 62(1), 189 - 92 The reconstitution of Anacystis nidulans DNA-dependent RNA polymerase from its isolated subunits; Herzfeld F et al.; The DNA-dependent RNA polymerase of the blud-green alga Anacystis nidulans was reconstituted from its isolated subunits in the absence of urea . Applying this technique the kinetics and the subunit requirements of the reconstitution process were analyzed . The results reveal differences with respect to the reconstitution of Escherichia coli polymerase . Reconstitution proceeds much more slowly in the case of the A . nidulans enzyme . Reconstitution here is absolutely dependent on the presence of the subunit sigma . On the other hand, the largest of the subunits of Mr=190000 can be fully substituted by a specific degradation product of this subunit of Mr=175000 . Heterologous reconstitution between subunits of E . coli and A . nidulans polymerase does not result in active enzyme hybrids, showing a divergent evolution of the structure of this enzyme in these procaryotic organisms. Mol Gen Genet, 1976 Feb 2, 143(3), 339 - 44 R factor-mediated tetracycline resistance in Escherichia coli K12 . Dominance of some tetracycline sensitive mutants and relief of dominance by deletion; Foster TJ; Strains of Escherichia coli K12 heterozygous for the R100-1 tetracycline resistance region were constructed . They carried the wild-type Tetr genes in the chromosome and single site Tets mutations on plasmids . Some heterozygotes could not express tetracycline resistance fully after induction . The mutant tet allele was thus partially dominant . When heterozygotes carrying the dominant tet mutant were plated on agar containing 20 mg/ml tetracycline, mutants which grew normally occurred at a frequency of 1-4 X 10(-4) . Analysis of these dominance relief mutants showed that in 53/56 isolates the dominant tet allele was lost forming either Tra+ or Tra- deletion mutants of the plasmid . The mutation frequency was not affected either by the host chromosomal recA mutation or by the temperature of growth of the culture. Mol Gen Genet, 1976 Feb 2, 143(3), 319 - 25 Characterisation of plasmids coding for the restriction endonuclease EcoRI; Smith HR et al.; The properties of two plasmids coding for the CcoRI restriction and modification enzymes are described . Both plasmids are non auto-transferring (NTP) but can be mobilised by transfer factors . Strains carrying NTP13 produce colicin E1 and the EcoRI enzymes . This plasmid has a molecular weight of 6 X 10(6) daltons and is present as approximately 12 copies per chromosome . The second plasmid, NTP14, was detected after mobilisation of the EcoRI plasmid with the R factor RI-19 . NTP14 codes for ampicillin resistance, synthesis of the EcoRI enzymes and colicin E1 . The molecular weight of NTP14 is 10.7 X 10(6) daltons and there are about 14 copies per chromosome . DNA-DNA reassociation experiments were performed to determine the interrelationships of NTP13, NTP14, ColE1 and the R factor R1-19 . NTP13 and NTP14 continue to replicate when cellular protein synthesis is inhibited by the addition of chloramphenicol. Mol Gen Genet, 1976 Feb 2, 143(3), 297 - 9 Biosynthesis of Escherichia coli RNA polymerase subunits upon release of rifampicin inhibition; Engbaek F et al.; Upon release of rifampicin inhibition of Escherichia coli cells, the initiation of transcription will resume . The sequential resumption of the synthesis of proteins after release of rifampicin inhibition reflects the genetic order and size of the corresponding transcriptional units . We have used this approach to analyze whether the genes for alpha and sigma are on the same transcriptional unit as the genes for beta and beta', employing a method, which allowed us to measure the amounts of RNA polymerase subunits, alpha, beta, beta' and sigma in crude extracts . We have found that the alpha and sigma subunits are synthesized concurrently with the beta subunit in the rifampicin restart experiment, which suggests that the genes for alpha and sigma belongs to different transcriptional units. Mol Gen Genet, 1976 Feb 2, 143(3), 243 - 52 Expression of a valine-resistant acetolactate synthase activity mediated by the ilv O and ilv G genes of Escherichia coli K-12; Favre R et al.; A strain carrying the ilv0603 mutation has been isolated in E . coli K-12 and its characteristics were found to be very similar to those previously reported by Ramakrishnan and Adelberg (1965a) for other ilv0 mutants . The strain carrying the ilv0603 mutation is resistant to valine inhibition (Valr) and we show that this resistance depends on the expression of a newly recognized gene, ilvG, which is located at min 75, between ilvE and ilvD on the E . coli K-12 map . The ilvG gene causes the expression of a Valr acetolactate synthase, which is detectable only when the ilv0603 mutation is also present in cis on the same chromosome . Under these conditions the Valr acetolactate synthase activity is eluted, on a hydroxylapatite column, at an ionic strength slightly lower than that required for elution of the remaining acetolactate synthase activity (sensitive to valine inhibition) . The Valr peak is missing in a strain carrying an ilvG (amber) mutation. Mol Gen Genet, 1976 Feb 2, 143(3), 233 - 41 RNA polymerase mutants of Escherichia coli . III . A temperature-sensitive rifampicin-resistant mutant; Kawai M et al.; Temperature-sensitive mutants of Escherichia coli that are unable to grow at high temperature can be obtained among those selected for resistance to streptovaricin or rifampicin at low temperature (Yura et al., 1973) . One of these mutants (KY5323) that was supposed to carry a single mutation affecting both rifampicin resistance and temperature sensitivity was further investigated . Using purified RNA polymerase preparations obtained from the mutant and the wild type, it was found that the activity for RNA chain elongation is more sensitive to heat treatment than that for RNA chain initiation or DNA binding, and that the mutant enzyme is significantly more labile than the wild-type enzyme with respect to RNA chain elongation, when heat treatment is carried out at high salt concentration . These results, taken together with those of the enzyme reconstitution experiments, strongly suggest that the beta subunit of the polymerase is directly involved in both RNA chain initiation and elongation reactions . Enzyme reconstitution experiments using isolated subunits derived from the mutant and the wild-type polymerases demonstrate that the alteration of beta subunit is primarily responsible for both rifampicin resistance and thermolability of the mutant enzyme . In addition, the results suggested the apparent alteration of both beta and alpha subunits in this mutant . Extensive transduction experiments provided genetic evidence that are consistent with the view that the strain KY5323 carries a second mutation affecting the beta subunit, beside the primary mutation affecting the beta subunit . The hypothetical beta subunit mutation seems to modify quantitatively the rifampicin resistance caused by the beta subunit mutation. Eur J Biochem, 1976 Feb 2, 62(1), 117 - 23 The involvement of protein L11 in the joining of the 30-S initiation complex to the 50-S subunit; Naaktgeboren N et al.; Ribosomal protein L11 participates in the coupling of the 30-S initiation complex with the 50-S subunit . P37 cores, lacking L7, L8, L12, L33, L10 and L11 were reconstituted with L7 and L10 . These particles are unable to join successfully to the 30-S initiation complex, whereas reconstitution of the same cores in the presence of L7, L10 and L11 restores 60-80% of the original coupling activity . P0 cores lacking only L7, L8, L12 and L33 are able to carry out one round of initiation, addition of L7 resulting in complete restoration of full activity . The data obtained with these P37 core particles resemble those obtained with untreated 50-S particles carrying thiostrepton, which prevents the binding of initiation factor IF-2 into the 70-S initiation complex . It is postulated that L11 induces a niche on the ribosomal surface to facilitate the proper binding of the IF-2 X GTP X fMet-tRNA complex . This binding of IF-2 enables the 30-S initiation complex to join to the 50-S subunit, because of the associative ability of IF-2 . If joining is impaired than both the level of fMet-tRNA binding and of the IF-2-mediated GTP hydrolysis is lowered. J Pharmacol Exp Ther, 1976 Feb, 196(2), 469 - 77 Studies on the binding of 5,5-diphenyl-hydantoin to nucleic acids in vitro and to rat brain subcellular fractions in vitro and in vivo; Boykin ME et al.; The binding of 5,5-diphenylhydantoin (DPH) to nucleic acids (bovine brain RNA, rat liver ribosomal and tRNA, Torula utilis RNA, and calf thymus and Escherichia coli DNA was studied using ultraviolet spectroscopy, gel chromatography and thermal transition profiles . Within the sensitivity of these methods, it was found that there is essentially little or no interaction between DPH and nucleic acids in vitro as has been reported previously . Little, if any evidence of DPH intercalation with DNA was noted during thermal transition studies . DPH does not interfere with DNA reassociation . Further studies into the nature of the in vivo subcellular distribution of 14C-DPH in rat brain revealed accumulation of the radioactivity primarily in the soluble fractions . The nuclear fraction and the microsomes, containing high DNA and RNA tissue ratios, demonstrated the greatest particulate association with radioactivity at 2 and 12 hours, respectively . This association with particulate fractions was not demonstrated after gel chromatography . These data do not support a hypothesis relating DPH binding to nucleic acids in vitro or in vivo to a possible mechanism of action of the drug. Proc Natl Acad Sci U S A, 1976 Feb, 73(2), 415 - 9 Studies on nucleic acid reassociation kinetics: empirical equations describing DNA reassociation; Britten RJ et al.; The rate of appearance of duplex DNA renaturation, measured with single strand specific nuclease, deviates significantly from a second order reaction . Measurements reported in paper I of this series indicate an inhibition in the rate of reassociation of single strand tails on partially reassociated molecules by a factor of at least two . Equations are derived that describe the observed form of reassociation kinetics as measured with hydroxyapatite and with single strand specific nuclease . The free parameter that describes the extent of inhibition of nucleation with single strand tails in these equations has been evaluated by least squares methods and agrees with the experimentally measured value. J Lab Clin Med, 1976 Feb, 87(2), 251 - 9 Autoantigens in human sweat: purification and characterization of the D-group antigens; Silpananta P et al.; We have found several heteropolysaccharides in human thermal sweat which are both autoantigenic and isoantigenic for a majority of healthy adults . They are present in trace amounts and are difficult to isolate . We have facilitated extraction through the use of a DEAE-Sephadex batch technique . One of the major antigenic fractions, the D group, has been purified and characterized . It contains 88.5 per cent carbohydrate as uronic acid, hexosamine, methyl pentose, and glucose . The protein content was 12.5 per cent. J Infect Dis, 1976 Feb, 133(2), 153 - 6 Enteropathogenic Escherichia coli: lack of correlation of serotype with pathogenicity; Goldschmidt MC et al.; Forty-eight strains of Escherichia coli isolated from children with diarrhea were classified according to nine enteropathogenic serotypes . The strains were examined for production of enterotoxin and for invasiveness by study of bacteria or bacteria-free filtrates in conventional animal and tissue culture models . Filtrates of only three strains (6%) consistently dilated rabbit ileal loops, while all 48 strains yielded negative results in suckling mice, adrenal cells, and guinea pig eyes . When filtrates of the three strains that dilated the rabbit ileum were heated at 60 C for 30 min, the reaction in rabbit ileal loops was negative; this finding indicated the production of a heat-labile enterotoxin . This study shows the lack of correlation between classical enteropathogenic serotypes of E . coli and presently known virulence properties in animal models . The results raise doubts about the value of serotyping E . coli isolates from sporadic cases of diarrhea . When it is suspected that an E . coli isolate is enteropathogenic, it may be important to perform more than one laboratory assay. J Bacteriol, 1976 Feb, 125(2), 713 - 8 Analytical isoelectric focusing of R factor-determined beta-lactamases: correlation with plasmid compatibility; Matthew M et al.; R factor-determined beta-lactamases have been investigated by analytical isoelectric focusing . The enzymes such as those specified by the R6K and RP4 plasmids (TEM-type enzymes) are notably homogenous in biochemical tests (Hedges et al., 1974), but two subclasses can be distinguished by isoelectric focusing . Three subclasses can be distinguished among the oxacillin-hydrolyzing enzymes, in good agreement with the classification based upon biochemical characteristics (Dale and Smith, 1974) . The TEM-type beta-lactamases are promiscuously distributed among plasmids of a wide variety of compatibility groups, whereas the various oxacillin-hydrolyzing enzymes show some degree of correlation with compatibility. J Bacteriol, 1976 Feb, 125(2), 670 - 8 Adaptation of membrane lipids to alcohols; Ingram LO; The effects of alcohols of different chain lengths on the fatty acid composition of Escherichia coli K-12 have been examined . My results indicate that these cells radically change their fatty acid composition when grown in the presence of alcohols . These changes represent an adaptive membrane alteration compensating for the direct physicochemical interaction of alcohols with the membrane . Similar adaptive responses of membrane lipids are proposed as a possible biochemical basis for tolerance to alcohol and related drugs. J Bacteriol, 1976 Feb, 125(2), 540 - 44 Evidence for the involvement of ribonucleic acid in the production of F pili; Fives-Taylor P et al.; The effects of rifampin and streptolydigin, inhibitors of ribonucleic acid (RNA) synthesis, on the production of F pili by Escherichia coli were studied by electron microscopy . The inhibition of RNA synthesis reduces the number of new pili produced by depiliated cells, but does not affect their length or the number of pili present at the time of inhibition or the retraction of pili . We suggest that the rifampin-sensitive step may be linked to the establishement of a site for pili production . Evidence is provided that chloramphenicol inhibits retraction . We suggest that retraction requires some protein whose pool size is limited. J Bacteriol, 1976 Feb, 125(2), 423 - 8 H2-dependent anaerobic growth of Escherichia coli on L-malate: succinate formation; Macy J et al.; Escherichia coli grew anaerobically on L-malate only in the presence of H2; 91% of the L-malate utilized was converted to succinate . Anaerobically isolated membrane vesicles catalyzed the reduction of fumarate with H2 and contained a b-type cytochrome . Cytochrome c552 was present in the "periplasmic space." J Biochem (Tokyo), 1976 Feb, 79(2), 289 - 92 Studies of enzyme-catalyzed modification of proteins . I . Tyrosinase-catalyzed modification of asparaginase; Tokushige M et al.; Asparaginase {EC 3.5.1.1.} of Escherichia coli, an anti-tumor enzyme, was inactivated in a time-dependent fashion by mushroom tyrosinase {EC1.14.18.1.} . The inactivation did not proceed, however, when heat-inactivated tyrosinase was used . Exculusion of the atmospheric oxygen or addition of diethyldithiocarbamate, a copper selective chelating agent, prevented the inactivation . The difference absorption spectrum of tyrosinase-inactivated asparaginase versus intact asparaginase exhibited the appearance of marked absorption peaks at 300 and 350 nm . These results indicate that the tyrosyl residue(s) of asparaginase, which is essential for the activity is enzymatically modified by tyrosianes. Nucleic Acids Res, 1976 Feb, 3(2), 371 - 80 Comparative base compositions of chloroplast and cytoplasmic tRNAPhe's from Euglena gracilis; Hecker LI et al.; The nucleoside compositions of chloroplast and cytoplasmic tRNAPhe's from Euglena gracilis have been determined . The modified nucleoside compositions of these two tRNAs indicate that tRNAPheChl is more similar to procaryotic (E . coli) tRNAPhe than to either the Euglena cytoplasmic tRNAPhe or other eucaryotic cytoplasmic tRNAPhe's. J Natl Cancer Inst, 1976 Feb, 56(2), 333 - 8 Altered immunologic responsiveness in mastocytoma-bearing mice; Kamo I et al.; Immune responses to sheep erythrocytes were enhanced in mice bearing small mastocytomas soon after injection of a few tumor cells . In contrast, mice with larger tumors after transfer of a greater number of mastocytoma cells and those in the later stages of tumor development after transfer of small numbers of tumor cells showed moderately suppressed immune responses . Transfer of spleen cells from mastocytoma-bearing mice to irradiated recipients resulted in more antibody-forming cells as compared to transfer of splenocytes from normal donor mice . The addition of graded numbers of mastocytoma cells to a constant amount of normal spleen cells transferred to irradiated mice also resulted in enhanced responses and increased spleen weights in the recipients . This increase, in direct proportion to the number of mastocytoma cells transferred, also occurred when Escherichia coli lipopolysaccharide (a T-cell independent antigen) was used to immunize animals given spleen cells from normal mice and mastocytoma cells . Mastocytoma cell-free homogenates or X-irradiated tumor cells also heightened immune responses in recipient mice, which indicated that viable cells were not needed for the effect . Such homogenates, as well as the tumor cells per se, stimulated increased lactate dehydrogenase (LDH) activity in the sera of recipient mice . However, tumor cells passaged in tissue culture for several months, those derived from mice bearing a mastocytoma cell line with a low LDH-stimulatory activity, or UV-irradiated mastocytoma cells with a high LDH-stimulatory activity did not induce enhanced plaque-forming cell responses. J Med Chem, 1976 Feb, 19(2), 342 - 4 Racemic diastereoisomers of 1-amino-2-hydroxycyclopentanecarboxylic acid; Gaitanopoulos DE et al.; The synthesis and characterization of the two diastereoisomeric forms of 1-amino-2-hydroxycyclopentanecarboxylic acid have been accomplished . A previously reported synthesis produced a racemic mixture of the threonine analog trans-2-hydroxy-1-aminocyclopentanecarboxylic acid (trans with respect to the hydroxy and carboxyl group) . The alternate allothreonine analog was produced by conversion of cyclopentene oxide to trans-2-methoxycyclopentanol, followed by oxidation to 2-methoxycyclopentanone and conversion to a hydantoin . Fractional crystallization of the hydantoin sample, followed by hydrolysis, produced cis-2-hydroxy-1-aminocyclopentanecarboxylic acid (cis with respect to the hydroxy and carboxyl group) in high purity . Neither of the isomeric forms significantly inhibited the growth of the bacterial strains examined nor were they effective in inhibiting Jensen sarcoma cells in tissue culture. J Pediatr Surg, 1976 Feb, 11(1), 23 - 30 Endotoxin clearance after intralipid infusion; Tovar JA et al.; Healthy 6-8-wk-old New Zealand white rabbits were injected with chromium-chloride- or sodium-chromate-labeled E . coli endotoxin after rapid infusion (10 ml/kg in 1 hr) or slow and repeated infusions (40 ml/kg daily for 7 consecutive days) of 10% Intralipid . Endotoxin clearance rates and RES organ uptakes were determined and the results were compared with those of the controls treated with correspondingly equal volumes of 5% D/W instead of fat . In the acute experiment, the clearance rates were similar in all animals during the first 15 min following endotoxin injection . After this phase, however, experimental animals had faster endotoxin clearance and eventually higher organ uptakes than the controls . In the chronic experiment, there was no significant difference in endotoxin clearance rates or total and per-gram organ uptakes between experimental and corresponding control animals infused with 5% D/W instead of fat . Experimental animals, particularly those having received multiple infusions of fat emulsion, showed deposition of polarizable brown pigment inside and outside the reticuloendothelial cells in the liver, spleen, and bone marrow . None of the controls had these pigments in their organs. Jpn J Med Sci Biol, 1976 Feb, 29(1), 25 - 37 Further studies on the lethal effect of long-chain fatty acids on mycobacteria; Kondo E et al.; Mycobactericidal activity of long-chain fatty acids was confirmed by in vitro exposure of BCG . The killing effect was accompanied by inhibition of the membrane-bound acid phosphatase activity . Such active fatty acids were those having a stronger hemolytic activity (e.g., C12:0, C14:0, C18:1, C18:2) . Heat-killed BCG cells or their cell walls adsorbed the toxic fatty acids, whereas the fatty acid-insensitive E . coli cells did not . It was suggested that the mycobactericidal action of long-chain fatty acids is due to their detergent-like action on the cytoplasmic membrane, and that the determinant factor for the fatty acid-sensitivitiy of bacteria is the property of the cell wall by which fatty acids are adsorbed so that the active site is brought into contact with the inner membrane. Cell, 1976 Feb, 7(2), 205 - 12 A temperature-sensitive suppressor enabling the manipulation of the level of individual proteins in intact cells; Oeschger MP et al.; A temperature-sensitive suppressor strain of E . coli has been isolated and characterized . The properties of the mutant indicate a strong potential for its use in biochemical and genetic work . In particular, the mutant makes possible the variation of the intracellular concentration of selected protein, permitting an evaluation of its role in cell growth and biochemistry . The mutant also permits the selective radiochemical labeling of proteins in vivo for in vitro identification and analysis . The utilization of the mutant for these and other applications is discussed. Agents Actions, 1976 Feb, 6(1-3), 251 - 5 In vivo and in vitro macrophage activation by systemic adjuvants; Bruley-Rosset M et al.; Six systemic adjuvants of immunity were tested for their ability to induce macrophage activation . Four of them: living BCG, hydrosoluble extracts from BCG (HIU II) and from M.smegmatis (IPM), and lipopolysaccharide from E.coli (LPS), when administered to normal mice render macrophages non-specifically cytotoxic for tumor cells in vitro . The intensity of this phenomenon varied according to the route and time of adjuvant administration . In contrast, lentinan extracted from Lentinus edodes, and levamisole which is a synthetic chemical compound, depressed macrophage cytotoxic potential . BCG, IPM and LPS were shown to have a direct action on macrophages . After in vitro exposure to these agents, the cytotoxic potential of normal macrophages was greatly increased . Levamisole was unable to stimulate this macrophage function directly in vitro . On the other hand, such a macrophage activation has been induced in vitro when normal macrophages were cultivated in the presence of MIF coming from the supernatant of human lymphoblastoid cell lines. Arzneimittelforschung, 1976 Feb, 26(2), 187 - 9 {Spontaneous peripheral proteolysis/2nd communication: Effect of peripheral proteolysis (author's transl)}; Greuer W et al.; With the modified Astrup-plate method a spontaneous peripheral proteolysis was often found in chronical infections of the skin and mucous membranes . The origin of this proteolysis and its effects on the bacterial growth and wound healing were discussed. J Biochem (Tokyo), 1976 Feb, 79(2), 305 - 11 Dessociation and reconstitution of colicin E3 and immunity substance complex; Hirose A et al.; It has recently been found that so-called native colicin E3, which has been used for studies of its mode of action, is a complex of two kinds of proteins . The complex could be dissociated into the two components in SDS . These components were isolated by gel filtration of 1% SDS followed by treatment with Sowex-2 to remove bounds SDS . One component, characterized by its low molecular weight, prevented colicin E3-induced inhibition of poly(U)-dependent protein synthesis and was designated as immunity substance . The other component (protein A), which was of high molecular weight, had 100-fold higher in vitro ribosome-inactivating activity than native colicin E3, but had lower bacteriocidal activity . Colicin E3 was reconstituted from the two isolated protein components . The reconstituted colicin E3, when compared with protein A, showed a decrease in in vitro activity (inhibition of poly(U)-dependent protein synthesis), but had higher bacteriocidal activity in vivo . Thus complex formation of protein A with immunity substance should play and important role in the bacteriocidal action, but protein A itself might inactivate ribosomes in the interior of the sensitive cells. Jpn J Antibiot, 1976 Feb, 29(2), 189 - 96 {Clinical studies with amoxicillin (Pasetocin) (author's transl)}; Ito K et al.; This paper describes briefly the results obtained with AMPC (Pasetocin) administered by oral route to 21 patients with infection of the respiratory tract . 1 . There was no difference in therapeutic effect on infection of the respiratory tract between daily dosages of 1,000 mg and 750 mg (the former was orally given in 4 divided doses every 6 hours a day and the latter in 3 divided doses after meal) . The response of the patients to AMPC was remarkable or satisfactory . 2 . Acute aggravation symptoms associated with chronic infection of the respiratory tract showed similar improvement to single acute infection of the respiratory tract . 3 . The patients presenting acute aggravation symptoms received treatment for chronic stage consisting of consecutive oral administration of AMPC (500 approximately 750 mg a day) and their prognosis was favorable . Side effects attributable to prolonged treatment were not noted . 4 . The incidence of side effects can be reduced largely by rejecting form AMPC therapy the patients who are hypersensitive to drugs including penicillins. Infect Immun, 1976 Feb, 13(2), 533 - 42 Changes in lactoferrin, immunoglobulin G, bovine serum albumin, and alpha-lactalbumin during acute experimental and natural coliform mastitis in cows; Harmon RJ et al.; An experimentally induced Escherichia coli infection of a bovine mammary gland resulted in a 30-fold increase in lactoferrin (Lf) concentration in the mammary secretion by 90 h postinoculation and a 4-fold increase in total daily production of Lf by 264 h postinoculation in the infected quarter . A simultaneous rise and fall of bovine serum albumin (BSA) and immunoglobulin G (IgG) concentrations occurred during the acute phase of the infection . Peak BSA and IgG levels were reached 36 h before peak Lf levels . BSA concentrations declined rapidly after the acute phase, whereas IgG and Lf levels remained elevated and decreased slowly as the infection subsided . A decline in alpha-lactalbumin concentration by 48 h postinoculation indicated decreased synthetic capability . The increased Lf production may be a result of a specific response of secretory tissue to inflammatory agents and thus the infectious process . Analogous changes in Lf, IgG, and BSA were observed during a natural coliform infection . Sephadex G-200 chromatography of mastitis skim milk showed that Lf approximated the monomer (molecular weight 77,100) early in infections progressed and abated, the apparent molecular weight of Lf increased to approximately that of the trimer and subsequently decreased to about 1.5 times that of the monomer. Infect Immun, 1976 Feb, 13(2), 448 - 56 Effect of estradiol on immune competence: in vivo and in vitro studies; Kenny JF et al.; The administration of a single dose of 2.5 mug of microcrystalline estradiol-17 beta from 1 day before and up until 3.5 days after the administration of 3 X 10(5) heat-killed Escherichia coli significantly increased numbers of splenic anti-E . coli antibody-producing cells in male mice sacrificed 4 days after receiving anitgen . Administration early in the proliferative phase of antibody production, i.e., 1 day before or 1 day after the antigen, appeared to increase numbers of antibody-producing cells more than when it was administered at a later time . When given 2 days before the antigen or 2 h before sacrifice no effect was observed . Spleen cells harvested from male animals injected 3 days before with 5 X 10(6) heat-killed E . coli were incubated for 24 h in vitro with estradiol in concentrations ranging from 5 pg to 20 ng/ml . With concentrations of 500 pg to 5,000 pg/ml, significant increases in antibody-producing cells occurred, whereas at concentrations of 20 ng/ml some decrease was observed . The increase in antibody-producing cells was blocked by a mitotic inhibitor . Significant changes in numbers of antibody-producing cells were not observed after a 2-h incubation period . Uptake of titrated thymidine was increased in thymic and spleen cells incubated for 24 h with 500 pg of estradiol per ml; a concentration of 20 ng/ml slightly (but insignificantly) decreased uptake . Findings suggest that estradiol, in concentrations that approximate physiological serum levels in females, enhances mitosis of immunocompetent cells . This phenomenon may have bearing on the better immunological responsiveness of females than males. Can J Biochem, 1976 Feb, 54(2), 192 - 3 Ribosomal RNA: the message or matrix for ribosomal proteins; Miller DR et al.; The known nucleotide sequence of Escherichia coli 16S ribosomal RNA has been converted to amino acid sequences in all possible ways, and compared to known ribosomal protein sequences . The degree of similarity is precisely what one would expect by chance alone, providing additional evidence that ribosomal proteins cannot be coded for by ribosomal RNA. Am J Vet Res, 1976 Feb, 37(2), 159 - 63 Local immune responses in the bovine fetus vaccinated in utero with Escherichia coli antigen; Wamukoya JP et al.; Using specific immunofluorescent examinations, the local immune responses were studied in 14 calves prenatally vaccinated (10 to 50 days before birth) with Escherichia coli (O26:K60:NM) antigen or sterile saline solution . All calves were colostrum-deprived, were given oral doses of homologous organisms (killed or live), and were necropsied either at birth or within 12 days after birth . Immunofluorescent plasma cells were not seen in duodenum, jejunum, jejunal lymph nodes, ileum, ileal lymph node, or spleen of control calves prenatally vaccinated with sterile saline solution . All of these tissues, except ileal lymph node, from calves vaccinated in utero with E coli showed fluorescence . Jejunum, jejunal lymph node, and ileum had the greatest number of immunofluorescent plasma cells . There were more immunoglobulin G2-than immunoglobulin M-producing cells . The cells producing specific antibodies against E coli (1 calf studied) comprised approximately 33% of the total number of immunofluorescent cells. Proc Natl Acad Sci U S A, 1976 Feb, 73(2), 317 - 21 Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam; Hough PV et al.; Proteins, nucleic acids, and fluorescein-conjugated antibody are shown to be identifidable in situ via the fluorescence excited by the focused electron beam of a canning electron microscope . A molecular species is identified by its characteristic fluorescence spectrum and by a characteristic alteration of the spectrum with time under the electron beam . Primary protein fluorescence is relatively rapidly destroyed by the beam, but protein photoproduct fluorescence is more rugged and will in some cases permit detection of small numbers of protein molecules . Nucleic acid fluorescence is extremely long-lived and will permit detection of small numbers of nucleic acid residues . The theoretical resolution limit for localization of a particular molecular species -- about 20 A--is determined by the known maximum distance for molecular excitation by fast electrons . Drect extapolation from an observed resolution of 900 A in the localization of nucleic acid using a low-efficiency detector leads to an experimental resolution limit of less than 60 A . Fluorescence is strongly quenched by residual water in the specimen . Similar quenching is produced by some macromolecular associations and so may serve to localize such associations. Mutat Res, 1976 Feb, 38(1), 33 - 42 Use of a simplified fluctuation test to detect low levels of mutagens; Green MH et al.; As a mutagen screening procedure we have used a modification of the Luria and Delbruck fluctuation test in which the individual tubes are scored by eye for the presence or absence of a mutation . The test is simple and extremely sensitive, detecting concentrations of mutagens up to 100-fold lower than conventional tests . Measuring mutation to tryptophan independence in Escherichia coli strain WP2 we have found that methyl methanesulphonate (0.5 mug/ml), mitomycin C (0.0015 mug/ml), dichlorvos (5 mug/ml, and K2CrO4 (0.5 mug/ml) are all positively mutagenic in the test, whereas NiCl2 is negative . Chronic exposure to low levels of mutagens using this method appears to induce more mutations than might be predicted by extrapolation from short exposure experiments at higher doses . The procedure is applicable to any system which involves mutation to prototrophy from a non-leaky auxotrophic requirement and should prove valuable in detecting and investigating the effects of low doses and chronic exposures. Mutat Res, 1976 Feb, 38(1), 3 - 32 Mutagen testing using TRP+ reversion in Escherichia coli; Green MH et al.; Escherichia coli strain WP2 and its repair-deficient derivatives are suitable strains for mutagen screening . In these strains, agents which cause base substitution mutations can be shown to increase the frequency of Trp+ revertants . In addition, agents causing many types of DNA damage can be detected through increased killing of the repair deficient derivatives . Four ways of performing tests are described: (a) Spot tests in which a small amount of the agent under test is placed directly on a selective agar plate . Trp+ revertants are counted and increased sensitivity of repair-deficient strains determined from the size of the zone of inhibition of cell growth . (b) Treat and plate tests, where a strain is treated with the agent under test and subsequently plated to determine survival or frequency of Trp+ revertants . (c) A simplified fluctuation test which shows exceptional sensitivity in measuring mutation with low levels of mutagens . (d) Use of a liver microsomal fraction in conjunction with treat and plate tests to detect metabolically activated mutagens . The merits and defects of these systems are discussed . Common pitfalls in evaluating tests and procedures for avoiding them are described. Am J Surg, 1976 Feb, 131(2), 213 - 8 Gunshot wounds of the colon . A review of 100 consecutive patients, with emphasis on complications and their causes; Haygood FD et al.; The cases of one hundred civilian patients with gunshot wounds of the colon treated at the Louisville General Hospital have been reviewed . Most injuries were in the transverse colon (44%), followed by the ascending colon (27%), rectosigmoid (19%), and descending colon (10%) . Associated injuries occurred in 81 per cent of the patients; the small bowel was the most common structure injured . Primary closure was used in 52% of the patients, with a resultant 19% rate of wound infection and 14% rate of serious complication . When the extent of contamination or tissue destruction required resection, an attempted primary anastomosis was followed by a high rate of wound infection (57%) and serious complications (36%) as compared with end-colostomy and mucous fistula, which resulted in a 24% rate of wound infection and 24% rate of serious complication . The rate of wound infection between these groups is significant (p = 0.05) . Results end-colostomy and mucous fistula were better than with attempted primary anastomosis. Proc Soc Exp Biol Med, 1976 Feb, 151(2), 329 - 32 Endotoxin toxicity in rats is enhanced by tilorone; Levine S et al.; The relatively nontoxic drug, tilorone, greatly enhanced the susceptibility of rats to lethal effects of endotoxin . The magnitude of the synergy was similar to that produced by adrenalectomy, but the effect of tilorone was not mediated by the adrenal glands . The histopathologic effects of endotoxin plus tilorone resembled those produced by much larger doses of endotoxin alone, including instances of the generalized Shwartzman reaction. Proc Soc Exp Biol Med, 1976 Feb, 151(2), 271 - 4 Inhibition by nalidixic acid of post-uv survival of Escherichia coli; Nishida M et al.; Nalidixate inhibited the post-uv survival of E . coli B . TAU-bar and Ts-7, but not Bs-1 or B/r when included in the plating medium . Removal of the drug sensitivity by photoreactivation was consistent with pyrimidine dimers as the target for the effect . Nalidixate did not inhibit liquid-holding recovery from uv when included in the holding medium, but survival was inhibited if the drug was in the subsequent plating medium . In fact, there was an actual increase in the number of nonsurvivors due to the drug following a period of holding. J Immunol, 1976 Feb, 116(2), 379 - 86 The in vitro suppression of antigen- or mitogen-induced DNA synthesis by murine spleen cells after the addition of freshly prepared syngeneic cells; Ficho TW et al.; The suppression of antigen- and mitogen-induced DNA synthesis by murine spleen cells in vitro was investigated . It appears that cultures receiving two signals exhibit marked suppression of DNA synthesis . The addition of KLH, PPD, Con A, PHA, or LPS to 72-hr-lod cultures of KLH-stimulated murine spleen cells resulted in the suppression of DNA synthesis assayed at 144 hr . When small numbers of freshly prepared KLH-PPD SC or NSC were added to these cultures at 72 hr the suppression of DNA synthesis was abrogated . However, the addition of larger numbers of KLH-PPD SC or NSC resulted in increased suppression of DNA synthesis . Large numbers of KLH-PPD SC or NSC could substitute for the second stimulant (KLH, PPD, Con A, PHA, or LPS) in suppressing DNA synthesis . The addition of fresh cells obtained from KLH-PPD-immunized mice were more effective in eliciting the subsequent suppression than were cells obtained from non-immunized mice . Cells obtained 30 to 75 days post immunization were most effective in suppressing DNA synthesis. J Am Vet Med Assoc, 1976 Feb 1, 168(3), 231 - 2 Effect of supplemental dietary vitamin E on the serologic response of swine to an Escherichia coli bacterin; Ellis RP et al.; Three groups of 10 pigs (6 to 8 weeks old) were fed a nutritionally complete ration (control ration, CR), CR plus 20,000 IU of vitamin E/ton (CR + recommended E), and CR plus 100,000 IU of vitamin E/ton (CR + high E), respectively . Each pig was given an intramuscular injection of an Escherichia coli bacterin at experimental days 0 and 35 . Serums were collected 7 days prior to the first injection and at days 7, 14, 21, 35, 42, 49, and 56 . Pigs fed the CR + high E ration developed anti-E coli serum antibody titers two- to threefold higher than those of the controls . Pigs fed the CR + recommended E ration developed serum antibody titers intermediate between those of pigs in the other 2 groups. Chem Biol Interact, 1976 Feb, 12(2), 211 - 20 Removal of minor methylation products 7-methyladenine and 3-methylguanine from DNA of Escherichia coli treated with dimethyl sulphate; Lawley PD et al.; Persistence of methylpurines in DNA methylated in vitro and in vivo in Escherichia coli WP2 cells, by dimethyl sulphate (DMS) was studied, with particular reference to the minor products 7-methyladenine and 3-methylguanine, not previously investigated in this respect, but known to be removed from DNA in vitro by spontaneous hydrolysis at neutral pH . The half-life of 7-methyladenine in vivo was relatively short (2.6 +/- 0.2 h) but not significantly shorter than in vitro at pH 7.2, 37 degrees C . The half-life of 3-methylguanine was 3.6 +/- 0.3 h in vivo, markedly shorter than in vitro, where its stability was somewhat greater than that of 7-methylguanine . Enzymatic excision of 3-methylguanine was therefore indicated to occur in E . coli . Previous findings that 7-methylguanine is probably not enzymatically excised from DNA in vivo, whereas 3-methyladenine is rapidly removed, were confirmed, and additional support for the concept of enzymatic removal of 3-methyladenine was obtained by showing extensive inhibition of its removal from cells treated with iodoacetamide prior to methylation . It is suggested that methylations of adenine or guanine in DNA at N-3 constitute blocks to template activity of DNA and stimulate a "repair" response of enzymatic removal of 3-methylpurines . Possible valence bond structures for 3-methylpurine residues in DNA are discussed, leading to the suggestion that ionized forms with positively charged amino groups may be the most effective blocks to template activity. Biokhimiia, 1976 Feb, 41(2), 357 - 62 {Escherichia coli membrane-bound polyphosphatase}; Severin AI et al.; A complex of polyphosphatase with E . coli membranes has been isolated and studied . It is shown by gel-filtration through G-200 Sephadex and centrifugation in sucrose concentration gradient that about 5% of polyphosphatase total content in cells is bound with the heterogenous fraction containing smooth membranes and the ribosome-membrane complex . On the basis of the data obtained it is suggested that the formation of a complex of polyphosphatase with membranes is a stage of synthesis and secretion of this enzyme to protoplasm. Infect Immun, 1976 Feb, 13(2), 332 - 6 Interferon-producing capacity of germfree mice; Ito Y et al.; The general capacity of germfree mouse spleen cells to produce interferon in vitro in response to various stimuli was investigated . The interferon response of germfree mouse spleen cells in vitro, when compared with that of the conventionals, appears to be lower to some inducers . Interferon production in vitro stimulated by hemagglutinating virus of Japan (HVJ) or BHK-HVJ cells (BHK cells persistently infected with HVJ) was apparently suppressed in germfree mouse spleen cells as compared with the corresponding conventionals, whereas no difference of interferon production was observed between germfree and conventional mouse spleen cells in response to Newcastle disease virus, Escherichia coli endotoxin, poly(I:C), and phytohemagglutinin . Although monocontamination with HVJ had no enhancing effect on the interferon-producing ability of germfree mouse spleen cells in response to HVJ, conventionalization for 2 weeks greatly enhanced interferon-producing capacity. Scand J Haematol, 1976 Feb, 16(2), 144 - 53 Function of granulocytes with deficient myeloperoxidase-mediated iodination in a patient with generalized pustular psoriasis; Stendahl O et al.; The granulocytes of a patient with generalized pustular psoriasis (GPP) were found to have impaired ability to fix iodine after ingestion of yeast particles . Since hexose monophosphate shunt (HMS) activity was increased and the contents of 3 other lysosomal enzymes, beta-glucuronidase, N-acetyl-beta-glucosaminidase and lysozyme, were within normal range, the impaired iodination appeared to be due to a selective defect of myeloperoxidase (MPO) activity within the phagocytic cells . The deficient iodination was accompanied by a decreased intracellular killing of E . coli and C . albicans . Since hexose monophosphate shunt activity was enhanced and azide and cyanide inhibited the intracellular killing of E . coli only moderately, the patient's granulocytes may possess azide- and cyanide-resistant, MPO-independant microbicidal systems coupled to the oxidative metabolism . Assessment of granulocyte iodination and enzyme contents of the relatives of the patient revealed no hereditary transmission . Since GPP is characterized by the development of subcorneal pustules containing granulocytes, the MPO-deficiency may be the cause of or enhance the development of the disease. J Virol, 1976 Feb, 17(2), 614 - 21 Simian virus 40 DNA replication: characterization of gaps in the termination region; Chen MC et al.; A class of precursor DNA (pDNA) II molecules has been identified as the immediate precursor of simian virus 40 DNA I . A pDNA II molecule contains a strand of newly synthesized DNA with an interruption located in the region where DNA synthesis terminates (4) . These pDNA II molecules have been isolated and further characterized . They are converted to covalently closed structures (simian virus 40 DNA I) only when they are treated in vitro with both T4 DNA polymerase and Escherichia coli ligase . After in vitro repair of pDNA II with T4 DNA polymerase and nucleoside triphosphates, approximately 7 mol of alpha-{32P}dATP is incorporated per mol of DNA II . Alkaline sucrose analysis of these gap-filled molecules, after they have been cleaved with Eco RI restriction endonuclease, has demonstrated that gaps are specifically located in the termination region . alpha-{32P}dATP is incorporated equally into the two labeled products that are generated by RI cleavage of these molecules . This indicates the presence of gaps in both the newly synthesized plus the minus strands . Electrophoretic analysis of the gap-filled molecules, after they have been cleaved with endonuclease Hind, has shown that gaps are localized in Hind fragments G and B and to a minor degree in fragment J . pDNA II molecules have the following properties . There is a gap in the newly synthesized linear DNA strand contained in the pDNA II molecule . Nicked pDNA II molecules cannot be detected . The two molecules that arise by segregation contain gaps in both of the complementary strands . Based on the amount of alpha-{32P}dATP incorporated and the rate of exonuclease III digestion of gap-filled molecules, it is estimated that the size of the gaps is between 22 and 73 nucleotides . Models for termination of DNA synthesis are proposed based on these findings. Proc Natl Acad Sci U S A, 1976 Feb, 73(2), 366 - 70 "Early" simian-virus-40-specific RNA contains information for tumor antigen formation and chromatin replication; Graessmann M et al.; Simian virus 40 (SV40) induces tumor (T)-antigen formation, chromatin replication, and mitosis in primary mouse kidney cells arrested in G0 phase of the mitotic cycle . The temporal and quantitative relation between these early virus-specific reactions led to the hypothesis that the early SV40 mRNA contains information necessary for T-antigen formation and induction of cellular DNA synthesis . To get direct experimental evidence for this hypothesis, the early strand of SV40 DNA was transcribed in vitro by Escherichia coli DNA-dependent RNA polymerase and the SV40-specific cRNA was transferred by microinjection into epitheloid cells of confluent primary mouse kidney cultures . T-antigen formation and stimulation of DNA synthesis were investigated in the recipient cells . The experimental results obtained agree with the hypothesis that T-antigen is a virus-coded protein and that the early virus-specific mRNA contains information necessary for stimulation of cellular DNA replication in the arrested cells. J Bacteriol, 1976 Feb, 125(2), 575 - 80 Suppressor-induced structural changes in a missense L-ribulokinase of Escherichia coli; Cribbs RM et al.; A suppressor mutation specific for a missense codon in the L-ribulokinase structural gene of the L-arabinose operon of Escherichia coli B/r enhanced L-arabinose utilization by the strain containing the missense codon . Electrophoretic comparisons of the wild-type, missense, and suppressed missense L-ribulokinases indicated that the suppressor changed the structure of the missense kinase, thereby increasing its catalytic activity . Hyperinducibility imposed on an operator-distal gene by the missense codon was not affected by the suppressor mutation. J Immunol, 1976 Feb, 116(2), 454 - 61 Cellular mechanism of endotoxin unresponsiveness in C3H/HeJ mice; Glode LM et al.; B cells from C3H/HeJ mice fail to respond to an endotoxin (LPS K235) which is mitogenic for normal mice including the closely related C3H/HeN strain . The cellular basis for this unresponsive state has been investigated . The C3H/HeJ mice have normal numbers of B cells, which are capable of normal responses to other B cell mitogens, such as polyinosinic acid (Poly I) . Addition of normal macrophages or spleen cells fails to reconstitute the normal response . Furthermore, neither macrophages nor spleen cells from the C3H/HeJ strain suppress the normal C3H/HeN spleen cells . Finally, spleen cells enriched for B cells by the removal of macrophages or T cells demonstrate the same differences in responsiveness to LPS . These results indicate that LPS unresponsiveness is a defect of the B cell itself and not due to suppressor cells or the absence of helper cells . When LPS is added to Poly I-stimulated cultures, there is additional enhancement of the response of normal C3H/HeN spleen cells . However, LPS causes a dose-dependent suppression of the Poly I response of C3H/HeJ spleen cells . This suppression is dependent on the time of addition of LPS to the Poly I-stimulated cultures . These data are interpreted as indicating that the binding of LPS to the membrane of C3H/HeJ B cells results in their inactivation or suppression, and that this is the basis of LPS unresponsiveness in this mouse strain. J Bacteriol, 1976 Feb, 125(2), 524 - 30 Coordinated alterations in ribosomes and cytoplasmic membrane in sucrose-dependent, spectinomycin-resistant mutants of Escherichia coli; Mizuno T et al.; Alterations in cytoplasmic membrane and ribosomes from sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli, mutants that are resistant to spectinomycin in the presence of 20% sucrose but sensitive in the absence of sucrose, were studied . The protein composition of cytoplasmic membrane was analyzed by gel electrophoresis on polyacrylamide gel containing 8 M urea and 0.5% sodium dodecyl sulfate, which assured the reproducible separation of 28 protein bands . A major protein band, I-19, was missing in all cytoplasmic membrane preparations from 10 Sucd-Spcr mutants . Besides protein I-19, proteins I-13 and I-24 were missing in some mutants . On the other hand, the protein composition of cytoplasmic membrane from a sucrose-independent spectinomycin-resistant mutant was indistinguishable from that from the wild-type strain . The polypeptide synthetic activity of ribosomes from Sucd-Spcr mutants was resistant to spectinomycin . Studies on a revertant obtained from one of these mutants without any selection for sensitivity to spectinomycin revealed that a single mutation was responsible for both the ribosomal alteration, i.e., spectinomycin resistance, and the lack of protein I-19 in the cytoplasmic membrane . Studies on a transductant obtained with a Sucd-SPcr mutant as the donor also confirmed the single-mutation concept . It was concluded that in Sucd-SPcr mutants an alteration in the ribosomes caused the deletion of protein I-19 from cytoplasmic membrane. J Bacteriol, 1976 Feb, 125(2), 467 - 74 Mutant of Escherichia coli defective in response to colicin K and in active transport; Plate CA; A mutant of Escherichia coli has been isolated that grows poorly on succinate and exhibits a markedly reduced sensitivity to colicin K . This mutant is also deficient in the respiration-linked transport of proline and thiomethyl-beta-D-galactoside but appears normal for the adenosine triphosphate-dependent transport of glutamine and arginine . A temperature-conditional revertant of the mutant grows on succinate and is sensitive to colicin K at 27 C, but fails to grow on succinate and is insensitive to colicin K at 42 C . Proline transport in the temperature-conditional revertant is reduced at 42 C when either glucose or succinate is used as energy source . Glutamine transport, on the other hand, is normal at 42 C with glucose as energy source, but is reduced with succinate, although not to the same extent as is proline transport . The lack of growth on succinate and the deficiencies in transport at 42 C are not due to a temperature-dependent lesion in either the electron transport chain or in Ca2+, Mg2+-activated adenosine triphosphatase activity . Membrane vesicles prepared from the temperature-conditional revertant are impaired in proline transport at both 27 and 42 C . These findings suggest the existence in the cytoplasmic membrane of E . coli cells of a component, presumably protein, that is required for colicin K action and that functions in respiration-linked and, to a lesser degree, in adenosine triphosphate-dependent active transport systems . This protein may serve as the primary target of colicin K action. Cell, 1976 Feb, 7(2), 279 - 88 Enzymatic in vitro synthesis of globin genes; Efstratiadis A et al.; Full-length, single-stranded rabbit globin cDNA, synthesized by AMV reverse transcriptase, apparently contains a small double-stranded sequence (hairpin) at the 3' terminus . This cDNA can serve as template-primer for E . coli DNA polymerase I, which synthesizes a strand complementary to the cDNA and covalently bound to it . The loop connecting the two strands can be cut by S1 nuclease . Reassociation, hybridization, and restriction endonuclease studies, as well as electrophoretic analyses, indicate that the sequential actions of reverse transcriptase, DNA polymerase 1, and S1 nuclease generate full-length, double-stranded synthetic globin genes. Acta Virol, 1976 Feb, 20(1), 86 - 8 Consumption of complement in chelated and nonchelated guinea pig serum after challenge with some viral and nonviral interferon inducers; Rathova V et al.; The consumption of complement observed during the induction of interferon by various inducers may proceed via the alternate pathway . The alternate pathway requires the presence of Mg ions in the medium while Ca ions may be absent. Proc Natl Acad Sci U S A, 1976 Feb, 73(2), 476 - 80 Mechanistic studies of glutamine synthetase from Escherichia coli: kinetic evidence for two reaction intermediates in biosynthetic reaction; Rhee SG et al.; Fast reaction techniques were used to study the kinetics of protein fluorescence intensity changes that are associated with the reactions of unadenylylated Escherichia coli glutamine synthetase {L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2} with its substrates . It was established that the synthesis of glutamine occurs by a stepwise mechanism . During the catalytic process two fluorometrically distinct intermediates were observed . Both forward and reverse rate constants which lead to the formation and consumption of these intermediates were evaluated . The catalytic rate constant, kc, which was calculated from these rate constants agrees well with the values of kc which were determined by direct measurement of the overall biosynthetic activities by means of stopped-flow technique or the steady-state assay method. Nature, 1976 Jan 29, 259(5541), 285 - 90 Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons; Cabello F et al.; Although it carries two competent replication systems, a composite plasmid formed in vitro by linkage of the complete ColE1 and pSC101 plasmid replicons at their unique EcoRI endonuclease cleavage sites normally uses only the replication origin and functions of the ColE1 component . Restriction of ColE1 replication functions by DNA polymerase I deprivation results, however, in exclusive use of the pSC101 replication origin . When using the ColE1 replication system the composite plasmid is nevertheless incompatible with both the parent replicons . This suggests that a trans-dominant gene product is involved in plasmid incompatibility and supports negative control rather than positive control models for regulation of the initiation of DNA replication. Biochemistry, 1976 Jan 27, 15(2), 271 - 6 Replacement of metal in metalloenzymes . A lead-alkaline phosphatase; Sabbioni E et al.; Lead ions can interact with calf intestine alkaline phosphatase . Experiments using 203Pb-labeled Pb2+ ions showed that Pb2+ ions bind the native protein in a molar ratio of Pb/protein of 1:5 with moderate inhibition of its biochemical activity . The 4 g-atoms of Zn per mol present in the native enzyme may be removed by dialysis against EDTA . The inactive apoenzyme is capable of incorporating Pb2+ ions in a Pb/protein molar ratio of 2:1, giving a lead-protein complex still enzymatically active also when genetic material, such as nucleotides or DNA, has been used a a substrate . The reconstituted lead-protein is capable of binding Zn2+ ions without any release of the Pb2+ ions and with an increase in the catalytic activity of only 10-15% . The absence of Zn in the inactive apoenzyme as well as in the reconstituted lead-protein, the incorporation of Pb2+ ions in stoichiometric amounts in the apoenzyme, and the weak influence of the Zn2+ ions on the enzymatic assay of the lead-enzyme suggest that lead ions partially reactivate the calf intestine alkaline phosphatase apoenzyme. Biochemistry, 1976 Jan 27, 15(2), 422 - 5 Renaturation of a multisubunit multiactivity enzyme complex: recovery of phage Qbeta RNA replicase, EF-Tu, and EF-Ts activities after denaturation in urea; Blumenthal T et al.; Phage Qbeta RNA replicase consists of four nonidentical subunits three of which are required for poly(C)-directed synthesis of poly(G): a phage-coded polypeptide and the two host-supplied protein biosynthesis elongation factors EF-Tu and EF-Ts . After denaturation of the enzyme in 8 M urea, poly(G) polymerase activity can be renaturated by dilution of the denatured subunits into a high ionic strength buffer with glycerol . The renaturation reaction has a broad temperature optimum between 11 and 21 degrees . The extent of renaturation is dependent on enzyme concentration: at low enzyme concentrations and 21 degrees renaturation proceeds for more than 3 h with greater than 40% recovery of activity, whereas at high enzyme concentrations the reaction is complete by 1 h with less than 10% of the poly(G) polymerase activity regained . Activities catalyzed by the elongation factors can be measured while they are part of the replicase complex . Study of rates of renaturation of EF-Tu and EF-Ts dependent activities alone and in the replicase complex revealed that virtually 100% of the EF-Ts activity was recovered more rapidly than could be assayed at temperatures as low as 2 degrees, while the rate of recovery of EF-Tu activity was comparable to that of the poly(G) polymerase activity and was independent of either EF-Tu concentration or the presence of other enzyme subunits . The rate of recovery of the poly(G) polymerase activity was found to be limited by the renaturation of EF-Tu, since the rate was dramatically increased by the addition of undenatured EF-Tu. Biochemistry, 1976 Jan 27, 15(2), 328 - 34 Randomization of membrane lipids in relation to transport system assembly in Escherichia coli; Thilo L et al.; The distribution of newly synthesized lipid molecules in the pre-existing lipid phase of the membrane was studied in whole cells of the fatty acid requiring Escheria coli strain K1062 . The fluorescence probe N-phenyl-1-naphthylamine revealed reversible lipid phase transitions in cells supplemented with cis-delta9-octadecenoate (transition temperature Tt = 14 degrees C; width of the transition deltaT = 13 degrees C) or trans-delta9-hexadecenoate (Tt = 27 degrees C; deltaT = 7 degrees C) . Cells were first grown in the presence of cis-delta9-octadecenoate at 37 degrees C and subsequently for various periods in the presence of trans-delta9-hexadecenoate at 37 or 22 degrees C, i.e . above or below the transition of the newly formed lipids . Reproducible phase transitions with single, well-defined Tt values between 14 and 27 degrees C were observed under both conditions . Beta-Galactoside transport induced in a similar experiment before or during a change in the fatty acid composition showed a single change in activation energy at a temperature close to the lipid transition temperature, Tt . Starvation of cis-delta9-octadecenoate-supplemented cells for this fatty acid led to a gradual rise in the transition temperature, due to an increase in the percentage of saturated acyl chains in the membrane lipids . It is concluded that under all conditions investigated a mixed lipid phase composed of newly synthesized and pre-existing lipid molecules is formed in the membrane . Since conserved domains of newly synthesized lipids surrounding simultaneously formed transport proteins could not be demonstrated, the results do not support a membrane assembly mechanism proposed by N . Tsukagoshi and C . F . Fox {(1973), Biochemistry 12, 2822-2829} . It rather appears that newly formed lipid molecules are continuously released from their sites of synthesis into the lipid matrix by