Vopr Med Khim, 1976 May-Jun, 22(3), 343 - 6 {Enzyme splitting S-adenosylmethionine in the cells of Escherichia coli CK infected with phage CD}; Nikol'skaia II et al.; Extract of normal Escherichia coli CK cells methylated DNA of Cd phage in vitro in presence of 3H-S-adenosyl methionine, although in vivo methylation of viral DNA did not occur . Extract of cells, infected with Cd phage, also was unable to methylate the DNA in vitro due to de novo synthesis of the enzyme splitting S-adenosyl methionine to 5'-methyl thioadenosine and homoserine . The enzyme was not found in cells, infected with the phage in presence of chloramphenicol . The activity of the S-adenosyl methionine splitting enzyme was shown to be maximal between the fourth and fifth min of infection . This suggests that the enzyme is synthesized under control of the virus genome, it belongs to the early virus-specific proteins and inhibits the methylation of the phage DNA in vivo . In unpurified extracts of Escherichia coli CK the methylating activity was completely inhibited by 10(-5) M S-adenosyl homocysteine. Microsc Acta, 1976 May, 78(2), 131 - 48 Preparation and counts of particles in electron microscopy: application of negative stain in the agarfiltration method; Kellenberger E et al.; The agarfiltration method, published by Kellenberger and Arber in 1957, has been adapted to negative stain with sodium phosphotungstate and uranyl acetate . The technique is described in detail including all recent improvements . The precision obtainable in particle counts is discussed for agarfiltration and sprayed droplets. J Antibiot (Tokyo), 1976 May, 29(5), 579 - 83 The in vitro activity of ceftezol (demethylcefazolin) against dense populations of Escherichia coli; Greenwood D et al.; The activity of ceftezol was examined by continuous turbidimetric monitoring of dense populations of Escherichia coli exposed to the drug . Although ceftezol was found to be very active against strains of E . coli, its activity was consistently less than that of the closely related antibiotics, cefazolin . This difference was also found when strains of E . coli were examined in a dynamic system which simulates some of the conditions in which bacteria and drug interact in the treatment of bacterial cystitis . Evidence is presented that the difference in activity between the two cephalosporins resides in a differential ability to induce certain morphological changes in E . coli and in a differential rate of destruction by escherichial beta-lactamases. Cell, 1976 May, 8(1), 115 - 22 Effect of the RelA gene on the synthesis of individual proteins in vivo; Furano AV et al.; We have shown that the relA gene can affect the rates of synthesis of many nonribosomal proteins in E . coli . We used rel+ and relA strains that contain a temperature-sensitive valyl tRNA synthetase . Upon transfer from a permissive temperature (30degreesC) to a semi-resitrictive one (36.5degreesC), these strains continue to grow, although undergoing a partial deprivation of valyl tRNA, In the rel+ strain, the concentration of ppGpp increases immediately, and the accumulation of RNA ceases abruptly but temporarily . In contrast, in the relA strain, the concentration of ppGpp falls, whereas the rate of accumulation of RNA increases . As judged by gel electrophoresis, the rates at which individual polypeptides are synthesized by the strains after their transfer to 36.5degreesC depend to a large extent upon the allelic state of the relA gene . In both strains, the concentration of ppGpp changes soon enough to have altered the synthesis of some of the proteins by affecting the transcription of their genes. Zh Mikrobiol Epidemiol Immunobiol, 1976 May, (5), 68 - 71 {A new variant, for the USSR, of enteropathogenic escherichia 0151:K--, isolated from acute intestinal diseases under the conditional name "Crimea"}; Golubeva IV et al.; A study was made of enteropathogenic escherichia "Krym" isolated in the USSR from patients with acute enteric diseases; their antigenic structure was unknown . Also cultures of new serological groups officially recorded as standard test-strains were investigated . By its antigenic structure "Krym" escherichia corresponded to the standard strain of the international set 0151:K-- . Along with the H10 antigen described earlier "Krym" escherichia proved to possess H11 antigen; these two serological types of escherichia were revealed in the USSR, i.e . 0151:K--: H10 and 0151:K--: H11 . The statement on the presence of a new H50 antigen described in escherichia 880-67 with an antigenic formula of 0151:K--: H50 should be revised due to its complex identity with the 0151: K--: H10 strain . For proper serological identification the conditioned name "Krym" should be replaced by "escherichia of serological group 0151:K--". Nucleic Acids Res, 1976 May, 3(5), 1351 - 71 Cleavage of T4 species I ribonucleic acid by Escherichia coli ribonuclease III; Paddock GV et al.; T4 Species I RNA, a molecule 140 nucleotides in length with some structural features very much like a tRNA, is specifically cleaved by an enzymatic activity in Escherichia coli extracts to give three segments with 19, 48 and 73 nucleotides . We report the purification and characterization of the E . coli RNase which cleaves two 3' phosphodiester bonds of T4 Species I RNA . This reaction has many properties in common with those catalyzed by E . coli RNase III, although the optimal salt conditions for T4 Species I RNA cleavage differ significantly from those for other RNase III-catalyzed reactions . The reaction is not catalyzed by extracts from an E . coli strain lacking RNase III activity . Furthermore, T4 Species I RNA is cleaved by highly purified E . coli RNase III to yield the same three specific fragments . We conclude that this specific cleavage is due to the action of RNase III, and that the requirement for lower ionic strength may reveal further important properties about this RNA processing enzyme. Nucleic Acids Res, 1976 May, 3(5), 1307 - 22 Specific release of ribosomal proteins by nucleic acid-intercalating agents; Ballesta JP et al.; Increasing concentrations of ethidium bromide cause progressive inactivation of ribosomes, apparently by binding to double-stranded regions of the rRNA . At low drug concentrations (10(-4)M) the partial inhibition detected is due to specific release of proteins L7 and L12; activity can be restored by addition of an excess of these two proteins . At higher concentrations the inactivation is not reversed by supplementation with released proteins . The presence of ethanol affects the extent of ethidium binding and also the release of ribosomal proteins . In all tests the proteins most sensitive to the presence of the drug are L7 and L12, followed by L8/9, L11, L27, L28, L29 and L30 . Despite the fact that L7 and L12 are the first two proteins released by ethidium they are never totally missing from drug-treated ribosomes, though the other proteins can be displaced completely . About 50% of proteins L7 and L12 remain on the ribosomes at the highest drug concentrations tested, possibly indicating heterogeneity in the binding sites for the several copies present in the ribosome. Nucleic Acids Res, 1976 May, 3(5), 1215 - 24 Fluorescent 2'(3')-O-aminoacylnucleosides-acceptor substrates for ribosomal peptidyltransferase+; Chladek S et al.; 2'(3')-O-L-Phenylalanylderivatives of fluorescent 1,N6-ethenoadenosine and 3,N4-ethenocytidine were prepared by chemical synthesis . Both compounds are good acceptor substrates in ribosomal peptidyltransferase reactions . Since these compounds cannot form Watson-Crick base pairs, the results indicate that the terminal aminoacyladenosine unit of AA-tRNA is bound to ribosomal protein on the acceptor site of peptidyltransferase and not to rRNA. Nucleic Acids Res, 1976 May, 3(5), 1203 - 13 Stimulation of transcription on chromatin by polar organic compounds; Stratling WH; Polar organic compounds, including DMSO, increase RNA synthesis on isolated chromatin by E . coli RNA polymerase and RNA polymerase II from calf thymus . Transcription is stimulated on chromatin from Friend-virus-infected erythroleukemia cells and from various other sources . Using procedures which inhibit specifically the formation of a stable initiation complex, it is shown that the stimulation does not result from an increase in initiation of both E . coli and the eukaryotic RNA polymerase . After separation of chromatin into template active and inactive fractions, DMSO increases RNA synthesis by a factor of about 1.5 using the template inactive fraction, while stimulation of transcription on the template active portion is lower (factor of 1.2) . It is suggested that the effect on RNA synthesis is mediated by a weakening of the apolar interactions between histones in chromatin subunits, releasing transcription partially from the constraints imposed by histones. Biochem J, 1976 May 1, 155(2), 429 - 32 Spin-label study of the mobility of enzyme-bound lipoic acid in the pyruvate dehydrogenase multienzyme complex of Escherichia coli; Ambrose MC et al.; The lipoic acid residues covalently bound to the transacetylase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were selectively modified by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidino-oxyl . The electron-spin-resonance spectrum of the spin-labelled enzyme indicates that the bound nitroxide groups have high mobilities relative to the protein molecule . This physicochemical evidence is consistent with the view that the dithiolane ring of a lipoyl residue is capable of rapid migration between the active sites of the component enzymes in the catalytic mechanism. Biochem J, 1976 May 1, 155(2), 225 - 9 The concerted inactivation of Escherichia coli uridine diphosphate galactose 4-epimerase by sugar nucleotide together with a free sugar; Blackburn P et al.; 1 . The combined effect of the sugar nucleotides UDP-D-fucose or UDP-D-glucuronic acid together with the free sugars D-fucose or L-arabinose is the inactivation of the Escherichia coli enzyme UDP-galactose 4-epimerase (EC 5.1.3.2) . The sugar nucleotide or the free sugar alone or the sugar nucleotide plus 5'-Ump do not inactivate the enzyme . 2 . The inactivation of the enzyme by its substrate UDP-D-glucose was not affected by the presence of free sugar . 3 . In all cases the inactivation observed follows pseudo-first-order kinetics . 4 . A comparison of various sugar nucleotides indicates that the hydroxymethyl group at position 6 of the sugar moiety of the natural substrates is important for substrate binding. Biochem J, 1976 May 1, 155(2), 209 - 16 A cyanogen bromide fragment of beta-galactosidase from Escherichia coli with alpha-donor activity in complementation of the enzyme from mutant M15; Marinkovic DV et al.; Aminoethylated beta-galactosidase from Escherichia coli was cleaved by CNBr . The fragment C4a was purified by gel filtration and ion-exchange chromatography . The molecular weight of the fragment C4a was determined to be 9000 +/- 600 . The N-terminal amino acid was found to be isoleucine . Qualitative examination of homogeneity was carried out by disc-gel electrophoresis . The fragment C4a was shown to be active as an alpha donor in complementation of beta-galactosidase activity in vitro with E . coli mutant M15, which has a deletion in the alpha region of the z gene . The molecular weights of complementable fractions from mutant M15 were found to be 123 000 +/- 2500 and 507 000 +/- 11 000, and of the complemented enzyme 522 500 +/- 11 400. Res Vet Sci, 1976 May, 20(3), 233 - 6 Observations on the association of pathogenic Escherichia coli with small intestinal villi of pigs; Arbuckle JB; Pathogenic Escherichia coli were observed in association with microvilli of piglets experimentally infected with E coli O149:K91,K88a,c, but the bacteria were only rarely observed actually in contact with microvilli . The association of E coli with villi occurred most commonly in the ileum and least commonly in the duodenum: in some pigs, it was only observed in the ileum . The proliferation of pathogenic E coli in the small intestine and their association with villi commonly coexisted, although each phenomenon was also observed independently . It is suggested that the villous association of pathogenic E coli is not a prerequisite for their proliferation. J Gen Virol, 1976 May, 31(2), 277 - 80 Genes 46 and 47 of phage T4: possible compensation for loss of their function; Minner CA et al.; The gene 46 and 47 functions of phage T4 are required for normal DNA replication, recombination, u.v . repair and host DNA breakdown, and yet am mutants defective in these genes characteristically form tiny plaques on Escherichia coli strains lacking an am suppressor . Our results imply that this limited growth is not due to misreading of am codons or partial function of nearly complete poly-peptides terminated at the am mutation . Thus it appears that genes 46 and 47 are not entirely essential, perhaps because other phage or host products can partially compensate for their loss. J Gen Microbiol, 1976 May, 94(1), 75 - 89 Uptake of galactose into Escherichia coli by facilitated diffusion; Kornberg HL et al.; Strains of Escherichia coli devoid of systems for the active transport of galactose (galP mgl) still grow on galactose but at rates that are a function of the galactose concentration of the medium: half-maximal growth rates require more than 2 mM-galactose to be present . Evidence is presented that galactose is taken up by such strains by facilitated diffusion on a carrier specified by the umg gene (or by a gene highly co-transducible with it) which is thus a part of, or closely associated with, an enzyme II for glucose of the phosphoenolpyruvate-phosphotransferase system . However, the entry of galactose does not require phosphotransferase activity, and the sugar taken up appears in the cells as free galactose. Eur J Biochem, 1976 May 1, 64(2), 597 - 606 The subcellular distribution and state of the elongation factor Tu in extracts of Escherichia coli B; Furano AV; The concentration of elongation factor Tu (EF-Tu) is about 8 times that of ribosomes in extracts of Escherichia coli B grown in glucose-minimal medium . 90% of the EF-Tu is found in the ribosome-free fraction of the cell and 75% of this is free of other macromolecules as judged by chromatography on Sephadex G-100; about 10% is bound to the elongation factor EF-Ts and the remaining 10-15% is eluted from Sephadex G-100 earlier than expected for its molecular weight . Tryptic peptide maps of the three forms of EF-Tu are essentially indistinguishable. Eur J Biochem, 1976 May 1, 64(2), 511 - 8 The specific role of ribosomal protein S1 in the recognition of native phage RNA; van Dieijen G et al.; The previously reported requirement of ribosomal protein S1 for translation of phage RNA is now shown to be related to the involvement of the protein in initiation complex formation . The structure of the messenger RNA appears to be uniquely related to S1 function, since translation and initiation and midly unfolded phage RNA (by modification with formaldehyde) are independent of S1 . It is proposed that S1 functions in conjunction with initiation factor IF-3 by recognizing and unfolding elements of the tertiary structure of phage RNA . A model is suggested for S1 function in both initiation of protein synthesis and initiation of phage RNA replication. Clin Exp Immunol, 1976 May, 24(2), 336 - 45 Immunological enhancement in the pathogenesis of pyelonephritis; Miller TE et al.; The role of the immune response in pyelonephritis was investigated by manipulation of the host's immune capacity using the immunosuppressive drugs 6-mercaptopurine, cyclophosphamide and thiamphenicol . Treatment with 6-mercaptopurine depressed the humoral immune response but did not have an adverse effect on the course of renal infection . Thiamphenicol administration prevented the development of pathological lesions but this was due to the anti-bacterial activity of thiamphenicol and not to its immunosuppressive activity . Pyelonephritic animals treated with cyclophosphamide did not produce anti-bacterial antibody . Despite this, cyclophosphamide-treated animals were able to eliminate organisms more readily from the infected kidney than untreated animals with a normal humoral immune response . We believe that blocking of the phenomenon of immunological enhancement explains these unexpected results and that the immune response to renal infection may have an immunoenhancing role protecting the bacterial cell from otherwise effective host defence mechanisms. Can J Microbiol, 1976 May, 22(5), 745 - 51 Role of lipopolysaccharide in adsorption of coliphage T4D to Escherichia coli B; Watanabe T; Coliphage T4D was strongly adsorbed to intact lipopolysaccharides and alkaline and lipase-treated lipopolysaccharides from cells of Escherichia coli B, but was not so adsorbed to heat-treated cells . In contrast, coliphage T2h was not adsorbed to lipopolysaccharides and the heat-treated cells . Acid hydrolysate of lipopolysaccharides strongly inhibited the adsorption of phage T4D to acetone and ether-treated cells . The adsorption of phage T4D to the acetone and ether-treated cells was markedly inhibited by authentic D-glucosamine, N-acetyl-D-glucosamine, alpha-methyl-N-acetyl-D-glucosaminide, alpha-methyl-D-glucoside, and D-maltose . Authentic D-glucose and E,L-2,6-diaminopimelic acid also showed similar activity . These compounds did not affect the adsorption of phage T2h to the acetone- and ether-treated cells . Concanavalin A and wheat-germ agglutinin inhibited phage T4D adsorption to the acetone and ether-treated cells probably by blocking the phage-receptor sites on the cell wall . The blocking by concanavalin A and by wheat-germ agglutinin was reversed by alpha-methyl-D-glucoside and by alpha-methyl-N-acetyl-D-glucosaminide, respectively . Results suggested the possibility that coliphage T4D requires N-acetyl-D-glucosaminyl-glucose or glucosyl-D-glucosamine residues of the core of lipopolysaccharides for the initial attachment to the cell wall of Escherichia coli B. Can J Biochem, 1976 May, 54(5), 470 - 6 Protein binding and subunit association activity in particles reconstituted from Escherichia coli MRE600 50S ribosomal components; Chu FK et al.; Reconstituted 37S and 48S ribonucleoprotein particles were constructed by incubating Escherichia coli ribosomal RNA with total 50S ribosomal proteins by a sequential incubation method . By comparing the protein compositions of the two types of particles, the proteins that bind to 37S complexes to form 48S particles have been determined . Although only 48S particles could associate with 30S subunits, isolated 37S reconstituted particles could do so if incubated with exogenous 50S proteins . The proteins that bind under these conditions and confer upon particles the ability to associate are L2, L11, L15, L18, and L25 . The involvement of these proteins in 5S RNA binding is discussed. Am J Vet Res, 1976 May, 37(5), 531 - 4 Sensitivity of Escherichia coli after exposure to lincomycin in vitro and in vivo; DeGeeter MJ et al.; Exposure of 10 Escherichia coli isolates in vitro to a concentration of lincomycin found in the intestine of swine fed the maximun concentration recommended in feed did not significantly affect sensitivity to 8 antibiotics, 1 nitrofuran, and 1 sulfonamide when compared with sensitivity of E coli isolates not exposed to lincomycin . Changes in sensitivity, on the basis of Kirby-Bauer interpretation, did occasionally occur; however, these alterations were in zonal sizes, which were marginal for designation as sensitive, intermediate, or resistant . These same fluctuations were observed in E coli not exposed to lincomycin . Exposure of E coli to lincomycin in the intestinal tract of swine for 34 days did not alter sensitivity of E coli to tetracycline, dihydrostreptomycin, spectinomycin, lincomycin, or triple sulfa . The results indicated that addition of lincomycin to the feed did not appear to promote resistance transfer in E coli. Proc Natl Acad Sci U S A, 1976 May, 73(5), 1698 - 701 A mutant of Escherichia coli with an altered elongation factor Tu; Pedersen S et al.; A previously isolated mutant of E . coli K12 HAK 88 {Kuwano, M., Endo, h & yamamoto, M . (1972) J . Bacteriol, 112, 1150-1156}, contains a new protein that in two-dimensional gel electropherorgrams has the same molecular weight as normal elongation factor Tu, but whose isoelectric point is altered approximately 0.1 pH unit in the acidic direction . Peptide mapping, purification properties and the ratio of leucyl plus isoleucyl to methionyl plus cysteinyl residues of the normal elongation factor Tu protein and the new protein show a close similarity between the two . The mutation causing the altered electrophoretic mobility is located between argH and rif (79 min on the E . coli genetic map) . These biochemical and genetic data indicate that strain HAK 88 has a mutationally altered tufB gene. Proc Natl Acad Sci U S A, 1976 May, 73(5), 1518 - 22 Electron microscopy of gene regulation: the L-arabinose operon; Hirsh J et al.; Unlike normal cells, malignant rat and two simian virus 40-transformed human cell lines can neither grow nor survive in B12- and folate-supplemented media in which methionine is replaced by homocysteine . Yet three lines of evidence indicate that the malignant and transformed cells synthesize large amounts of methionine endogenously through the reaction catalyzed by 5-methyltetrahydropterolyl-L-glutamate: L-homocysteine S-methyltransferase (EC 2.1.1.13) . (1) The activities of this methyltransferase were comparable in extracts of malignant and normal cells . (2) The uptake of radioactive label from {5-14C}methyltetrahydropteroyl-L-glutamic acid (5-Me-H4PteGlu) was at least as great in the malignant cells as in the normals and was nearly totally dependent on the addition of homocysteine, the methyl acceptor; furthermore, 59-84% of the label incorporated by cells was recovered as methionine. Proc Natl Acad Sci U S A, 1976 May, 73(5), 1509 - 12 Synthesis of an R plasmid protein associated with tetracycline resistance is negatively regulated; Yang HL et al.; Synthesis of proteins encoded by the R222 plasmid was observed in a DNA-directed cell-free system and the products were compared to those plasmid proteins synthesized in Escherichia coli minicells . A greater number of plasmid-specified proteins was detected in the in vitro system than in the minicell, suggesting the presence of control factors for plasmid gene expression in the minicell . Synthesis of a newly detected plasmid protein (TET protein) is induced by tetracycline in minicells containing tetracycline-resistant plasmids, including R222, and this induced synthesis correlates with induced host resistance to the drug . This TET protein was synthesized in vitro from R222 DNA in the absence of tetracycline, indicating that no positive regulatory role for tetracycline is required for the protein's synthesis . TET proteon synthesis was inhibited in vitro when cell-free extracts prepared from cells containing the R222 plasmid were used. Klin Wochenschr, 1976 May 1, 54(9), 449 - 50 {Template activity of chromatin and DNA-dependent RNA polymerase III during postnatal development (author's transl)}; van der Meulen N et al.; The template-activity of chromatin isolated from liver nuclei of developing rats increases sharply at birth and decreases remarkable until the 10th day . Between the 10th and the 40th day a slow increase was shown in template activity of chromatin tested with purified E.-coli RNA-polymerase (EC 2.7.7.6) . We correlated these findings with changes of RNA-polymerase III-activity in rat liver during development. JACEP, 1976 May, 5(5), 332 - 3 Infection rate of simple suturing; Galvin JR et al.; Four hundred consecutive patients with superficial lacerations participated in a study of infection rate of lacerations sutured in an emergency department . Of these, 322 (85%) returned for suture removal . An infection rate of 5% was noted . Reported incidence of infection among the 68 (17%) non-returnees was 5.8%. Surgery, 1976 May, 79(5), 564 - 8 Direct effect of endotoxin on the gastric mucosal microcirculation and electrical gradient; Cheung LY et al.; The effects of intra-arterial infusion of E . coli endotoxin at 1.0 mg . per minute on the gastric total and mucosal blood flows, electrical potential difference, and ionic fluxes across the gastric mucosa were studied in an exteriorized, chambered preparation of canine fundic stomach . Gamma-labelled microsphere technique was used in addition to venous drainage and plasma aminopyrine clearance for the measurement of total and mucosal blood flow, respectively . In spite of normal systemic blood pressure throughout the experiment, E . coli endotoxin infusion caused a significant decrease in total gastric blood flow and in the fractional distribution of flow to the mucosae . There was no significant arteriovenous shunting of microspheres . Significant reduction in potential difference and hydrogen-ion back diffusion also was noted after endotoxin infusion, possibly as a consequence of reduced mucosal blood flow . The results indicate that significant gastric mucosal ischemia can occur and may represent a mechanism in the development of gastric erosions in endotoxemia, even in the absence of systemic hypotension. J Bacteriol, 1976 May, 126(2), 990 - 6 In vivo methylation by Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase protects against in vitro cleavage by the RII restriction endonuclease (R . Eco RII); Schlagman S et al.; We have analyzed the susceptibility of the deoxyribonucleic acid (DNA) of phage fd replicative form (RF) and of Escherichia coli to in vitro cleavage by purified RII restriction endonuclease (R . Eco RII) . The results are summarized as follows: (i) fd, mec- RFI, isolated from infected E . coli K-12 mec- bacteria (a mutant strain lacking DNA-cytosine methylase activity), is cleaved into at least two fragments, whereas fd . mec+ RFI, isolated from the parental mec+ strain, is not cleaved . (ii) E . coli mec- DNA is extensively degraded, whereas mec+ DNA-cytosine methylase acts as an RII modification enzyme. J Bacteriol, 1976 May, 126(2), 985 - 9 Pyrimidine dimer excision in surviving and nonsurviving cells of ultraviolet-irradiated cultures of Escherichia coli; Schenley RL et al.; We compared dimer excision in viable and nonviable cells fractions separated from Escherichia coli B/r cultures exposed to ultraviolet (UV) irradiation . For cells grown on minimal medium with glycerol as a carbon source, both fractions from the irradiated (20 J/m2, 5% survival) culture excised 60 to 70% of the thymine dimers from prelabeled DNA within 120 min . This percentage was, within experimental error, the same as that obtained from unseparated cultures . When isolated viable and nonviable populations were given a second UV exposure (20 J/m2) both types of cells were again able to excise dimers . The UV survival curve for the isolated viable population indicates that these cells are no more sensitive to radiation than exponentially growing cells not previously exposed to UV . The extent of dimer excision after UV irradiation was also the same in viable and nonviable cells separated from cultures grown on a glucose minimal medium in which both populations excised about 85% of the dimers within 120 min . These results show that the extent of removal of pyrimidine dimer from deoxyribonucleic acid is not precisely correlated with survival of repair-competent bacterial cells after exposure to UV light. J Bacteriol, 1976 May, 126(2), 977 - 84 Centrifugal separation of irradiated cultures of Escherichia coli cells into viable and nonviable populations; Schenley RL et al.; Incubation of ultraviolet-irradiated Escherichia coli B/r cultures with 0.7% Triton X-100 resulted in a large decrease in turbidity . Under phase-contrast optics, most of the irradiated detergent-treated cells were smaller than normal and of low phase density; only a small percentage were normal or larger than normal and of normal phase density . Irradiated cells not treated with detergent showed fewer pronounced morphological changes . Irradiated cells treated with detergent lost large amounts of proteins and ribonucleic acid, but not of deoxyribonucleic acid . Such cultures could be separated by centrifugation into populations of (i) slowly sedimenting cells consisting of small, phase-light cells of low viability and (ii) large cells of normal phase density and high viability (100%) . A similar separation was effected in gamma-irradiated cultures. J Bacteriol, 1976 May, 126(2), 951 - 8 Periplasmic protein related to the sn-glycerol-3-phosphate transport system of Escherichia coli; Silhavy TJ et al.; Two-dimensional gel electrophoresis of shock fluids of Escherichia coli K-12 revealed the presence of a periplasmic protein related to sn-glycerol-3-phosphate transport (GLPT) that is under the regulation of glpR, the regulatory gene of the glp regulon . Mutants selected for their resistance to phosphonomycin and found to be defective in sn-glycerol-3-phosphate transport either did not produce GLPT or produced it in reduced amounts . Other mutations exhibited no apparent effect of GLPT . Transductions of glpT+ nalA phage P1 into these mutants and selection for growth on sn-glycerol-3-phosphate revealed a 50% cotransduction frequency to nalA . Reversion of mutants taht did not produce GLPT to growth on sn-glycerol-3-phosphate resulted in strains that produce GLPT . This suggests a close relationship of GLPT to the glpT gene and to sn-glycerol-3-phosphate transport . Attempts to demonstrate binding activity of GLPT in crude shock fluid towards sn-glycerol-3-phosphate have failed so far . However, all shock fluids, independent of their GLPT content, exhibited an enzymatic activity that hydrolyzes under the conditions of the binding assay, 30 to 60% of the sn-glycerol-3-phosphate to glycerol and inorganic orthophosphate. J Bacteriol, 1976 May, 126(2), 861 - 8 Clo DF13 plasmid deoxyribonucleic acid-directed in vitro synthesis of biologically active cloacin DF13 and clo DF13 immunity protein; Konings RN et al.; Clo DF13 plasmid deoxyribonucleic acid (DNA) was used as a template to direct transcription and translation in a DNA-dependent cell-free system prepared from Escherichia coli . Analysis of the invitro products on sodium dodecylsulfate-polyacrylamide gels revealed that Clo DF13 DNA directs the synthesis of at least 10 polypeptides ranging in molecular weight from approximately 7,000 to 70,000 . Two of these polypeptides could be identified, with respect to their physiochemical and biological characteristics, as the products of the Clo DF13 genes coding for cloacin DF13 and Clo DF13 immunity protein . These results confirm previous findings, obtained which Clo DF13-harbouring minicells of E . coli, that the structural tenes for the latter proteins residue on the Clo DF13 genome. J Bacteriol, 1976 May, 126(2), 814 - 22 Thymidine uptake and utilization in Escherichia coli: a new gene controlling nucleoside transport; McKeown M et al.; A commonly used strain of Escherichia coli K-12 was shown to be deficient in the transport of a number of nucleosides, including thymidine . Thymidine incorporation was unaffected . Strain AB2497 exhibited a strikingly lower thymidine pulse-label incorporation at low (less than 1 mug/ml) thymidine concentrations than do many other strains . The deficiency appeared to be due to mutation in a single gene . This gene, which we designated nup (for nucleoside uptake), is located at 10 to 13 min on the E . coli linkage map . In nup+ strains, the transport of a given nucleoside was relatively insensitive to large excesses of other nucleosides but was competitively inhibited by the same nucleoside . Mutants deficient inthymidine kinase are deficient in thymidine uptake but normal in deoxyadenosine uptake . A two-step model for nucleoside transport is presented in which the first step, utilizing the nup gene product, is a nonspecific translocation of nucleoside to the interior of the cell . In the second step, the individual nucleosides are modified by cellular enzymes (e.g., nucleosides kinases) facilitate accumulation. J Bacteriol, 1976 May, 126(2), 593 - 600 Characterization of an Escherichia coli K-12 F-Con-mutant; Havekes LM et al.; An Escherichia coli K-12 F-mutant defective in conjugation was isolated by means of a zygotic induction enrichment procedure . The recipient ability of the mutant was reduced about 50 times owing to a block in one of the first steps of the conjugation process . In the mutant, cell envelope alterations could not be observed . Sensitivity toward detergents, antibiotics, and phages was unaltered . The mutation appeared to be co-transducible with pyrD . The linkage order in the region of the mutation is origin KL 99-con-pyrD-aroA. J Bacteriol, 1976 May, 126(2), 563 - 7 Extragenic suppression of two ribosomal protein cistrons lying near the rif locus in Escherichia coli; Molholt B; The experiments reported here involve temperature-sensitive mutations in two of five cistrons encoding 50S ribosomal proteins that lie near the rif locus in Escherichia coli . I selected spontaneous TS+ mutants able to grow at elevated temperatures in which the TS+ event takes place outside this tract of cistrons near rif . Six distinct classes of extragenic suppressors were found, five of which have been mapped . Two of these suppressors lie near 64 min, a region known to be rich in cistrons ribosomal proteins (Dennis and Nomura, 1975) . The remaining three extragenic suppressors were located near 16.5, 47, and 86 min. Biochem J, 1976 May 1, 155(2), 419 - 27 Selective inactivation of the transacylase components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli; Brown JP et al.; 1 . The reaction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli with maleimides was examined . In the absence of substrates, the complex showed little or no reaction with N-ethylmaleimide . However, in the presence of pyruvate and N-ethylmaleimide, inhibition of the pyruvate dehydrogenase complex was rapid . Modification of the enzyme was restricted to the transacetylase component and the inactivation was proportional to the extent of modification . The lipoamide dehydrogenase activity of the complex was unaffected by the treatment . The simplest explanation is that the lipoyl groups on the transacetylase are reductively acetylated by following the initial stages of the normal catalytic cycle, but are thereby made susceptible to modification . Attempts to characterize the reaction product strongly support this conclusion . 2 . Similarly, in the presence of N-ethylmaleimide and NADH, much of the pyruvate dehydrogenase activity was lost within seconds, whereas the lipoamide dehydrogenase activity of the complex disappeared more slowly: the initial site of the reaction with the complex was found to be in the lipoyl transacetylase component . The simplest interpretation of these experiments is that NADH reduces the covalently bound lipoyl groups on the transacetylase by means of the associated lipoamide dehydrogenase component, thereby rendering them susceptible to modification . However, the dependence of the rate and extent of inactivation on NADH concentration was complex and it proved impossible to inhibit the pyruvate dehydrogenase activity completely without unacceptable modification of the other component enzymes . 3 . The catalytic reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by NADH in the presence of the pyruvate dehydrogenase complex was demonstrated . A new mechanism for this reaction is proposed in which NADH causes reduction of the enzyme-bound lipoic acid by means of the associated lipoamide dehydrogenase component and the dihydrolipoamide is then oxidized back to the disulphide form by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) . 4 . A maleimide with a relatively bulky N-substituent, N-(4-diemthylamino-3,5-dinitrophenyl)maleimide, was an effective replacement for N-ethylmaleimide in these reactions with the pyruvate dehydrogenase complex . 5 . The 2-oxoglutarate dehydrogenase complex of E . coli behaved very similarly to the pyruvate dehydrogenase complex, in accord with the generally accepted mechanisms of the two enzymes . 6 . The treatment of the 2-oxo acid dehydrogenase complexes with maleimides in the presence of the appropriate 2-oxo acid substrate provides a simple method for selectively inhibiting the transacylase components and for introducing reporter groups on to the lipoyl groups covalently bound to those components. Can J Microbiol, 1976 May, 22(5), 712 - 8 Further studies on the relative antiviral efficacies of interferons induced by poly I:C and Mengo virus; Campbell JB; Mouse serum interferons induced by polyI:C, vesicular stomatitis virus (VSV), reovirus, and Mengo virus were assayed in monolayers of mouse L-929 cells by the plaque-reduction method using both VSV and Mengo as challenge viruses . Titers obtained with Mengo virus as challenge were all lower than with VSV . With the interferons induced by VSV, reovirus, and ployI:C, the reductions were of the order of two- to three-fold . With Mengo virus-induced interferon the reduction was much greater (about 17-fold) . This offers an explanation for the observation that, unit for unit (measured by the plaque reduction of VSV), Mengo virus-induced interferon is only about 1/10 as effective as polyI:C-induced interferon in protecting mice against lethal infection with Mengo virus . The data are consistent with the hypothesis that an interferon antagonist is produced in the serum of mice infected with Mengo virus . This antagonist, which is not produced in mice inoculated with polyI:C, or reovirus, effectively blocks the antiviral action of interferon during Mengo virus infections, both in vivo and in vitro. Bol Med Hosp Infant Mex, 1976 May-Jun, 33(3), 595 - 606 {New concepts on the etiopathogenesis of diarrhea}; Olarte J; The author discusses new knowledge concerning E . coli enterotoxins and Duovirus in the production of diarrheal disease. J Med Chem, 1976 May, 19(5), 643 - 7 Synthesis and biological studies of 3-(beta-D-ribofuranosyl)-2,3,-dihydro-6H-1,3-oxazine-2,6-dione, a new pyrimidine nucleoside analog related to uridine; Chwang TL et al.; Reaction of the trimethylsilyl derivative of 2,3-dihydro-6H-1,3-oxazine-2,6-dione (2, "uracil anhydride") with protected 1-O-acetylribofuranoses in the presence of stannic chloride gave the corresponding block nucleosides . 3-(2,3-5-Tri-O-2',2',2'-trichloroethoxycarbonyl-beta-d-ribofuranosyl)-2,3-dihydro-6H-1,3-oxazine-2,6-dione (4c) thus prepared from the protected sugar 3c, 1-O-acetyl-2,3,5-tri-O-(2,2,2-trichloroethoxycarbonyl)ribofuranose, gave, on removal of the protecting groups with zinc dust,3-(beta-d-ribofuranosyl)-2,3-dihydro-6H-1,3-oxazine-2,6-dione (1) . The structure of 1 was confirmed by uv, ir, NMR, and CD spectral data and was shown to be an N nucleoside . Uracil anhydride, 2, and, to a lesser extent, its ribonucleoside 1 exert a moderate growth inhibition of mouse leukemia L5178Y, HeLa, and Novikoff hepatoma cells i- culture . Both compounds produce weak inhibition of vaccinia viral replication in HeLa cells. Cancer Res, 1976 May, 36(5), 1761 - 70 Inhibition of rat liver RNA polymerases by action of the methylating agents dimethylnitrosamine in vivo and methyl methanesulfonate in vitro; Herzog J et al.; Dimethylnitrosamine maximally inhibits rat liver nuclear RNA synthesis by 50% at a dose of 40 mg/kg of body weight . The inhibition develops during the first 4 hr and persists through the 12th hr . All parenchymal cells of the lever lobule seem to be affected . The decreased RNA synthesis can be accounted for entirely by an inhibition of the RNA polymerase activities quantitatively solubilized and partially purified . A similar inhibition of the polymerase activities was demonstrated in the intact nuclei by inactivating the endogenous template with actinomycin D and assaying the polymerases with an added exogenous template, poly(deoxy-adenylate-deoxythymidylate) . Chromatin was prepared by two methods differing in the extent to which they remove the endogenous polymerase activity . Each preparation was transcribed with either added Escherichia coli or partially purified rat liver nucleoplasmic RNA polymerase . With either polymerase or chromatin preparation, no inhibition of the template activity of liver nuclear chromatin isolated from the DMN-treated animals was detected . A similar mechanism of inhibition of RNA synthesis was produced by the action of the methylating agent methyl methanesulfonate on whole nuclei in vitro . The dose-dependent inhibition of RNA synthesis could be accounted for by an inhibition of the RNA polymerase activities quantitatively solubilized and partially purified from the affected nuclei . Chromatin prepared from the methyl methanesulfonate-treated nuclei had a normal template capacity with either E . coli or rat liver nucleoplasmic RNA polymerase . No preferential methylation of the RNA polymerases by {14C}methyl methanesulfonate could be demonstrated . It is concluded that the action of the two methylating agents on RNA metabolism is similar and that the inhibition of liver nuclear RNA synthesis results from inactivation of the RNA polymerases . At the same time, dimethylnitrosamine and methyl methanesulfonate leave the chromatin template intact, at least quantitatively, for the synthesis of RNA . The implications of such an effect on RNA synthesis are discussed. J Bacteriol, 1976 May, 126(2), 999 - 1001 Altered deoxyribonucleotide pools in P2 eductants of Escherichia coli K-12 due to deletion of the dcd gene; Neuhard J et al.; Deletion of the Escherichia coli K-12 chromosome associated with P2 mediated education extend through the structural gene for uridine kinase, udk, and the dcd gene encoding 2'-deoxycytidine 5'-triphosphate deaminase . The lack of uridine kinase makes a positive selection possible for these strains . Due to the dcd mutation, P2 eductants show large alterations in their deoxyribonucleoside triphosphate pools. J Bacteriol, 1976 May, 126(2), 852 - 60 Lack of glucose phosphotransferase function in phosphofructokinase mutants of Escherichia coli; Roehl RA et al.; Phosphofructokinase (pfkA) mutants of Escherichia coli are impaired in growth on all carbon sources entering glycolysis at or above the level of fructose 6-phosphate (nonpermissive carbon sources), but growth is particularly slow on sugars, such as glucose, which are normally transported and phosphorylated by the phosphoenolpyruvate, (PEP)-dependent phosphotransferase system (PTS). J Bacteriol, 1976 May, 126(2), 785 - 93 Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12; Billen D et al.; Toluene-treated Escherichia coli mutants have been used to study the roles of deoxyribonucleic acid (DNA) polymerases I, II, and III, and of DNA ligase in repair synthesis and strand rejoining following X-irradiation . In cells possessing all three DNA polymerases, both a greater amount of repair synthesis ("exaggerated" repair synthesis) and failure of ligation are observed when DNA ligase activity is inhibited . In a mutant lacking the polymerizing activity of DNA polymerase I, exaggerated repair synthesis is not observed, and strand rejoining does not occur even if DNA ligase is fully activated . In a mutant possessing the polymerizing activity of DNA polymerase I but lacking its 5'leads to 3' exonuclease activity, exaggerated repair synthesis is minimal . After irradiation, DNA polymerases II and III are capable of carrying out an adenosine 5'-triphosphate-dependent repair synthesis,but rejoining of strand breaks does not occur and exaggerated synthesis is not seen whether DNA ligase is active or not . These results suggest that DNA polymerase I and DNA ligase act together to limit repair synthesis after X irradiation and that both are necessary in toluene-treated cells for strand rejoining . DNA polymerases II and III apparently cannot complete chain elongation and gap filling, and therefore repair carried out by these enzymes does not respond to ligase action. J Bacteriol, 1976 May, 126(2), 646 - 53 Methyl methane sulfonate-sensitive mutant of Escherichia coli deficient in an endonuclease specific for apurinic sites in deoxyribonucleic acid; Ljungquist S et al.; A methyl methane sulfonate (MMS)-sensitive mutant of Escherichia coli AB 1157 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment . The mutant strain, AB 3027, is defective both in endonuclease activity for apurinic sites in deoxyribonucleic acid (DNA) and in DNA polymerase I, as shown by direct enzyme assays . Derivative strains, which retained the deficiency in endonuclease activity for apurinic sties (approximately 10% of the wild-type enzyme level) but had normal DNA polymerase I activity, were obtained by P1-mediated transduction (strain NH5016) or by selection of revertants to decreased MMS sensitivity . These endonuclease-deficient strains are more MMS-sensitive than wild-type strains . Revertants of these deficients strains to normal MMS resistance were isolated . They had increased levels of the endonuclease activity but did not attain wild-type levels . The data suggest that endonuclease for apurinic sites is active in repair of lesions introduced in DNA as a consequence of MMS treatment . Two different endonucleases that specifically attack DNA containing apurinic sites arepresented in E coli K-12 . A heat-labile activity, sensitive to inhibition by ethylenediaminetetraacetate, accounts for 90% of the total endonuclease activity for apurinic sties in crude cell extracts . The residual 10% is due to a more heat-resistant activity, refractory to ethylenediaminetetraacetate inhibition . The AB3027 and NH5016 strains have normal amounts of the latter endonuclease but no or very little of the former activity. Z Naturforsch {C}, 1976 May-Jun, 31(5-6), 255 - 62 {Coalpha/Cobeta-isomerism of corrinoids . Partial synthesis and Escherichia coli activity of further isomer pairs of the (Co-methyl)-corrinoids (author's transl)}; Moskophidis M et al.; In 2-methyladenyl-(Cobeta-methyl)-cobamide and in adenyl-(cobeta-methyl)-cobamide the nucleotide base is coordinated to the cobalt atom in neutral and weak acidic aqueous solutions (in the corresponding adenosylcorrinoids the nucleotide base is not coordinated) . 2-Methylthioadenyl-(Cobeta-methyl)-cobamide resembles, with regard to the coordination of the nucleotide base, the benzimidazole-corrinoids . The partial synthesis via cobalt (I) corrinoids results in a variable proportion: (Coalpha-methyl) isomer/(Cobeta-methyl) isomer, e . g . 7/93 (cobalamin) and 50/50 (p-cresylcobamide) . This proportion is generally low, if in the corresponding cyanocorrinoid the nucleotide base is firmly coordinated; it is high, if the coordination in the corresponding cyanocorrinoid is weak or absent . These results are compatible with the assumption that in the cobalt (I) corrinoids the nucleotide base is to a certain extent coordinated . In Escherichia coli 113-3 the examined (Coalpha-methyl)-corrinoids show a weaker bioactivity than the corresponding (Cobeta-methyl)-corrinoids. J Biochem (Tokyo), 1976 May, 79(5), 927 - 37 A novel adenosine triphosphatase isolated from RNA polymerase preparations of Escherichia coli . II . Enzymatic properties and molecular structure; Ishihama A et al.; An adenosinetriphosphatase (ATPase) {EC 3.6.1.3} copurified with the DNA-dependent RNA polymerase {EC 2.7.7.6} from Escherichia coli was isolated to apparent homogeneity and some of its functional as well as structural properties were examined . Although the novel ATPase exhibited metal requirements similar to those of Mg2+, Ca2+-ATPase, its response to NaN3 and antisera appeared completely different from that of the Mg2+, Ca2+-ATPase . The purified ATPase was found to be a large protein with a molecular weight of 9.3X10(5) daltons, composed of identical subunits of 7X10(4) daltons . When viewed under an electron microscope, the ATPase appeared to be very similar to material previously misidentified as the RNA polymerase . The physiological role of the novel ATPase, however, remains unclear. J Biochem (Tokyo), 1976 May, 79(5), 917 - 25 A novel adenosine triphosphatase isolated from RNA polymerase preparations of Escherichia coli . I . Copurification and separation; Ishihama A et al.; Adenosinetriphosphatase (ATPase) {EC 3.6.1,3} activity has been found to exist in most preparations of DNA-dependent RNA polymerase {EC 2.7.7.6} obtained from Escherichia coli by a number of purification procedures so far established . Electrophoretic analysis on polyacrylamide gels demonstrated that ATP hydrolysis and RNA synthesis were catalyzed by two distinct enzyme proteins . It appears that the two enzymes are associated or have similar molecular properties . Separation of the two enzymes, the object of the present work, was achieved by three independent methods: ion exchange chromatography on a phosphocellulose column, electrophoresis in glycerol gradients, or high-salt glycerol gradient centrifugation. J Virol, 1976 May, 18(2), 709 - 18 Low-molecular- weight Rauscher leukemia virus protein with preferential binding for single-stranded RNA and DNA; Davis J et al.; A sensitive nitrocellulose filter assay that measures the retention of 125I single-stranded calf thymus DNA has been used to detect and purify DNA-binding proteins that retain a biological function from Rauscher murine leukemia virus . By consecutive purification on oligo (dT)- cellulose and DEAE-Bio-Gel columns and centrifugation in 10 to 30% glycerol gradients, RNA-dependent DNA polymerase has been separated from a second virion DNA-binding protein . The binding of this protein to DNA was strongly affected by NaCl concentration but showed little change in activity over a wide range of temperature or pH . After glycerol gradient purification, polyacrylamide gel electrophoresis of this protein showed one major band with a molecular weight of approximately 9,800 . This protein binds about as well as to single-stranded Escherichia coli or calf thymus DNA or 70S type C viral RNA . The binding to 125I single-stranded calf thymus DNA is very efficiently inhibited by unlabeled single-stranded DNA from either E . coli or calf thymus and by 70S murine or feline viral RNA . Much larger amounts of double-stranded DNA are required to produce an equivalent percentage of inhibition . This protein, therefore, shows preferential binding to single-stranded DNA or viral RNA. Lancet, 1976 May 1, 1(7966), 930 - 4 Enhanced antibody responses in active chronic hepatitis: relation to HLA-B8 and HLA-B12 and porto-systemic shunting; Galbraith RM et al.; Titres of antibodies to rubella, measles, smooth muscle, nuclei, and Escherichia coli were examined in relation to the presence of particular histocompatibility antigens in 57 patients with active chronic hepatitis, 8 of whom were HBsAg positive . With the exception of antibodies to E . Coli, the HBsAg-negative patients with HLA-B8 or HLA-B12 had higher titres than those with neither, and antibody titres were highest in the 7 cases with both these histocompatibility antigens . In contrast, E . coli antibody titres were not related to the presence of particular histocompatibility antigens but correlated closely with the degree of portosystemic shunting . None of the HBsAg-positive patients possessed HLA-B8, and titres of all the antibodies were significantly lower than in the HBsAg-negative cases . The increased antibody response in HBsAg-negative patients is likely to be due to a genetically determined increase in immunological responsiveness for which HLA-B8 and HLA-B12 are markers . The results obtained in healthy family members also suggest that this defect in immunoregulation is under polygenic control. Cell, 1976 May, 8(1), 123 - 8 The mechanism of action of ppGpp on rRNA synthesis in vitro; van Ooyen AJ et al.; We have studied the mechanism of the specific inhibition of ribosomal RNA synthesis by ppGpp in a purified system using as templates E . coli DNA and DNA from lambdad5ilv, which carries a rRNA cistron from E . coli . Ribosomal RNA synthesis, as well as its inhibition by ppGpp, are critically salt-dependent . Of a number of guanosine phosphates tested, only pppGpp (MS II) mimicked the action of ppGpp, establishing the specificity of ppGpp . The two templates gave similar results for rRNA synthesis in all experiments . By using the initiation inhibitor rifampicin, we could show that the specific inhibition of rRNA synthesis by ppGpp is due to its effect on rRNA initiation . The somewhat variable inhibition of RNA synthesis in general by ppGpp is mainly or wholly a consequence of premature chain termination . We propose that ppGpp specifically inhibits rRNA synthesis by acting on the formation of the so-called "closed-promoter complex". Can J Biochem, 1976 May, 54(5), 413 - 22 Modified 5'-nucleotides resistant to 5'-nucleotidase: isolation of 3-(3-amino-3-carboxypropyl) uridine 5'-phosphate and N2, N2-dimethylguanosine 5'-phosphate from snake venom hydrolysates of transfer RNA; Gray MW; A procedure for the quantitative measurement of the O2'-methylnucleoside constitutents of RNA has recently been developed in this laboratory (Gray, M.W . Can . J . Biochem . 53, 735-746 (1975)) . This assay method is based on the resistance of O2'-methylnucleoside 5'-phosphates (pNm) (generated by phosphodiesterase hydrolysis of RNA) to subsequent dephosphorylation by venom 5'-nucleotidase (EC 3.1.3.5) . In the present investigation, two base-modified 5'-nucleotides, each displaying an unusual resistance to 5'-nucleotidase, have been identified . These compounds have been characterized by a variety of techniques as N2, N2-dimethylguanosine 5'-phosphate (pm2/2G) and 3-(3-amino-3-carboxypropyl)uridine 5'-phosphate (p4abu3U) . Because of their resistance to 5'-nucleotidase, pm2/2G and p4abu3U are isolated along with the pNm in the mononucleotide fraction of venom hydrolysates of transfer RNA . Under hydrolysis conditions, the stability of p4abu3U is comparable to that of a pNm, allowing quantitative assay of the nucleotide . The proportion (mean +/- SD) of p4abu3U in venom hydrolysates of wheat embryo and Escherichia coli tRNA has been determined to be 0.35 +/- 0.03 (n=5) and 0.14 +/- 0.02 (n=4) mol%, respectively . The absence of p4abu3U in venom hydrolysates of yeast tRNA implies the absence of the corresponding nucleoside in yeast tRNA, in agreement with existing data . The variable recovery of pm2/2G from venom hydrolysates of wheat embryo and yeast tRNA indicates that under hydrolysis conditions, this base-modified nucleotide is only partially resistent to 5'-nucleotidase . The complete absence of pm2/2G in venom hydrolysates of E . coli tRNA is consistent with the known absence of N2, N2-dimethylguanosine in this RNA . These observations demonstrate that resistance to 5'-nucleotidase is a necessary but not sufficient criterion for concluding that a 5'-nucleotide is O2'-methylated . When applied to wheat embryo ribosomal RNA, the analytical methods described in this report failed to reveal any compound having the distinctive charge properties of p4abu3U . It therefore appears that 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, recently characterized as a constituent of the 18 S rRNA of Chinese hamster cells (Saponara, A.G . & Enger, M.D . Biochim . Biophys . Acta 349, 61-77 (1974)), may not be present in wheat embryo ribosomal RNA. J Bacteriol, 1976 May, 126(2), 601 - 8 Effect of colicin K on a membrane-associated, energy-linked function; Sabet SF; The purpose of this work was in investigate the capability of cell extracts of Escherichia coli and E . coli treated with colicin K to catalyze the following energy-dependent reverse transhydrogenase reaction: NADP + NADH + ATP in equilibrium NADPH + NAD +ADP + Pi . Under anaerobic conditions this reaction requires the presence of a specific portion of the electron transport chain, a functional energy coupling system, including an adenosine triphosphatase, enzyme, and ATP as energy source . The ATP-linked reaction was partially inhibited in French press extracts of E . coli K-12 C600 cells that had been pretreated with colicin K but not in extracts from similarly treated cells of a colicin-tolerant mutant . Ultracentrifugation of extracts yielded particulate fractions competent in catalyzing the reaction; this reaction is substantially inhibited in fractions from colicin-treated cells . The extent of inhibition increased with increasing concentration of colicin . Supernatants also supported ATP-linked formation of NADPH, but this reaction was insensitive to the colicin effect . A comparison between the requirement of the reaction in supernatant and particulate fractions suggests that the reaction in the supernatant is different from the one inhibited by colicin . The ATP-hydrolyzing ability of particulate fractions from the control or treated bacteria was identical . Likewise, the electron transport chain was not affected by colicin treatment, as evidenced from lack of effect on NADH oxidase, succinic dehydrogenase, and NADPH-NAD transhydrogenase . It is concluded that colicin K interferes with the coupling of ATP the utilization of the intermediate for the ATP-linked transdehydrogenase reaction. N Engl J Med, 1976 Apr 29, 294(18), 965 - 72 Human reovirus-like agent as the major pathogen associated with "winter" gastroenteritis in hospitalized infants and young children; Kapikian AZ et al.; We found a human reovirus-like agent in the stools of 42 per cent of 143 infants and young children hospitalized with acute gastroenteritis between January, 1974, and June, 1975 . Half the patients studied by electron microscopy and serologic technics had evidence of infection with the agent . The infection had a seasonal pattern: 59 per cent of those admitted during the cooler months (November to April) shed the agent, with a peak of 78 per cent in December, 1974, and January, 1975, combined . None of the patients admitted during the warmer months (May to October) shed the agent . None of 275 Escherichia coli isolates from 32 patients with diarrhea produced heat-labile enterotoxin, whereas 17 of the 32 had evidence of infection with the reovirus-like agent . In addition, 14 of 40 parents of 37 patients with diarrhea associated with the reovirus-like agent were also infected, but most infectious were inapparent . This agent appears to be the major cause of diarrheal illness in the young during the cooler months. Virchows Arch B Cell Pathol, 1976 Apr 29, 20(3), 229 - 38 Evaluation of a short term in vitro test for granulocyte chalone activity; Maurer HR et al.; A short term in vitro test for granulocyte chalone activity eas examined for its specificity and reliability . The test used the inhibition, by granulocyte extracts, of 3H-thymidine (3H-Tdr) uptake in to the acid-insoluble material by rat bone marrow cells in vitro to measure possible chalone activity . Among the many possible 3H-Tdr artifacts pool size dilution by Tdr contained in the extracts was excluded using an E . coli mutant requiring thymine . Several amino acids and biogenic amines do not affect the test . However, continuous and pulse labelling of bone marrow cells with 3H-Tdr, viability tests and micro flow fluorometric measurements of the cell cycle distribution following colcemid treatment strongly suggests that the cells do not proliferate in vitro during short term incubation, since practically no cells enter the S-phase, cells in the S-phase die and few if any cells proceed through G2 and mitosis . Moreover, the test cannot exclude cytotoxicity . Thus, the in vitro test may only sceem for an unspecific S-phase inhibitor and must hence be supplemented by another assay to prove the chalone nature of an extract or fraction . The test per se fails to meet most of the requirements of a valid granulocyte chalone assay. J Biol Chem, 1976 Apr 25, 251(8), 2493 - 8 Different mechanisms of energy coupling for transport of various amino acids in cells of Mycobacterium phlei; Prasad R et al.; Whole cells of Mycobacterium phlei were shown to actively accumulate proline, leucine, lysine, tryptophan, histidine, glutamine, and glutamic acid to different steady state levels . The transport of proline, in contrast to that of other amino acids, was found to be insensitive to various respiratory inhibitors, e.g . cyanide, arsenate, azide, and sulfhydryl reagents . However, oxygen was an obligatory requirement for the uptake of proline, as well as for the other amino acids . The results indicate that the energy requirements for proline uptake are different from those of other amino acids . In contrast to the system from Escherichia coli, the mode of energy transduction for the uptake of proline, glutamine, and glutamic acid is different even though these amino acids are shock resistant in the M . phlei system. J Biol Chem, 1976 Apr 25, 251(8), 2399 - 404 Terminal deoxynucleotidyltransferase . Serological studies and radioimmunoassay; Kung PC et al.; Mouse antisera against calf terminal deoxynucleotidyltransferase (terminal transferase) have been prepared . The sera have been used to characterize terminal transferase both by studying inhibition of enzyme activity and by developing a competition radioimmunoassay using highly purified 125I-labeled terminal transferase . By either assay, anti-terminal transferase serum did not cross-react significantly with calf DNA polymerases alpha and beta, Escherichia coli DNA polymerase I, or the reverse transcriptase of Moloney mouse leukemia virus . The calf terminal transferase did, however, share cross-reactive but not identical determinants with human and murine terminal transferase . The radioimmunoassay could detect as little as 2 ng of terminal transferase/mg of soluble protein in a tissue extract . Thymocytes were found to contain 280 ng of terminal transferase/mg of cell protein or about 1 X 10(5) molecules/cell; bone marrow had about 1% of the level of enzyme found in thymus . Extracts of spleen, peripheral white blood cells, lymph nodes, liver, muscle, and kidney all lacked detectable antigenicity of terminal transferase . These data indicate that terminal transferase is a tissue-specific enzyme and is not related to other DNA polymerases. J Biol Chem, 1976 Apr 25, 251(8), 2487 - 92 Factors affecting the acyl selectivities of acyltransferases in Escherichia coli; Okuyama H et al.; In Escherichia coli the synthesis of phosphatidic acid from glycerophosphate involves first the intermediate formation of 1-acyl glycerophosphate . This reaction is catalyzed by the membrane-bound acyl-CoA(acyl-ACP):glycerophosphate acyltransferase . The 1-acylglycerophosphate is then converted to phosphatidic acid by acyl-CoA(acyl-ACP):1-acylglycerophosphate acyltransferase (Okuyama, H., and Wakil, S.J . (1973) J . Biol . Chem . 248, 5197-5205) . In vitro both acyltransferases utilize various saturated and unsaturated acyl-CoAs at comparable rates, resulting in the incorporation of both saturated and unsaturated fatty acids into position 1 as well as position 2 of the glycerophosphate moiety . Thus, the specificities of acyltransferase systems as compared with regard to the maximal velocities for various acyl-AoAs, do not explain the positional distribution of the individual fatty acid in phospholipid molecules . The selectivities of the acyltransferases for acyl-CoAs are variable depending upon the acceptor concentration . In the presence of both palmitoyl-CoA and oleoyl-CoA and at low concentrations of the acceptors comparable to those found in vivo, the acylation at position 1 of glycerophosphate by acyl-CoA:glycerophosphate acyltransferase showed higher preference for palmitate, whereas the acylation at position 2 by acyl-CoA:1-acylglycerophosphate acyltransferase showed higher selectivity for oleate . In the presence of saturating amounts of the acceptors, the acylation at position 1 or position 2 was less selective for the acyl-CoAs . The ratios of saturated acyl-CoA to unsaturated acyl-CoA also affect the ratios of the fatty acids incorporated in vitro into positions 1 and 2 of phosphatidic acid; relatively more palmitate was incorporated when the proportion of palmitoyl-CoA among the acyl donors was higher and vice versa . Thus, highly selective positioning of various acyl-CoAs observed at lower concentrations of the acceptors in phosphatidic acid synthesis in vitro helps to explain the selective distribution of saturated and unsaturated fatty acids at positions 1 and 2 of glycerophospholipids in the membranes . Another factor, the availability of acyl donors, affects the proportions of different molecular species of phosphatidic acid, which at least partly explains the variability of molecular species of phospholipids found in vivo. J Biol Chem, 1976 Apr 25, 251(8), 2475 - 9 An antimutator deoxyribonucleic acid polymerase . I . Purification and properties of the enzyme; Lo KY et al.; The DNA polymerase induced by an antimutator T4 phage has been purified to apparent homogeneity and has been compared to the wild type polymerase . The mutant enzyme resembles the wild type in thermal stability, pH optimum, salt activation, divalent metal ion requirement, inhibition by a sulfhydryl reagent, and apparent affinity for DNA . However, the mutant enzyme differs from the wild type in its 8-fold higher 3'-exonuclease activity and in its decreased apparent affinity for deoxyribonucleoside triphosphates . Inhibition studies indicate that the exonuclease of the mutant enzyme is more vulnerable to physical and chemical modification than its wild type counterpart. J Biol Chem, 1976 Apr 25, 251(8), 2299 - 306 Mode of action of bottromycin A2 . Release of aminoacyl- or peptidyl-tRNA from ribosomes; Otaka T et al.; Bottromycin A2 inhibited MS2 phage RNA-dependent protein synthesis as well as polyuridylic acid-(poly(U))- or polyadenylic acid (poly(A))-dependent polypeptide synthesis . When the ribosomal complex with N-acetyl-{14C}phenylalanyl-tRNA (N-acetyl-{14C}Phe-tRNA) at the A site was subjected to bottromycin A2, the release of N-acetyl-{14C}Phe-tRNA was observed while no release of N-acetyl-{14C}Phe-tRNA from the donor site was observed, indicating that the action of bottromycin A2 is specific to the A site of ribosomes . Due to bottromycin's capacity to release {14C}Phe-tRNA or N-acetyl-{14C}Phe-tRNA from the ribosomal acceptor site (A site), bottromycin A2 inhibited the nonenzymatic binding of N-acetyl-{14C}Phe-tRNA and elongation factor T (EF-T)-dependent binding if the concentration of EF-Tu-GTP-{14C}Phe-tRNA ternary complex was low . Our data are consistent with the possibility that the inhibition of overall polypeptide synthesis by bottromycin A2 is at least partly due to bottromycin A2's activity to release aminoacyl- or oligopeptidyl-tRNA from ribosomes . Among 10 antibiotics tested, bottromycin A2 and lincomycin released aminoacyl-tRNA from ribosomes. J Biol Chem, 1976 Apr 25, 251(8), 2207 - 16 Association-dissociation behavior and subunit structure of heat-released nitrate reductase from Escherichia coli; Lund K et al.; Nitrate reductase, released from the membrane fraction of Escherichia coli by a neutral heat treatment, was purified to homogeneity by gel filtration chromatography . The purified enzyme behaved as an associating-dissociating system, exhibiting concentration-dependent sedimentation constants which ranged from 24 S at high concentrations in the ultracentrifuge down to 10 S at low concentrations in sucrose gradients . The molecular weight determined at high concentrations by sedimentation equilibrium was 880,000 +/- 30,000 . Large and small enzyme species were detected on polyacrylamide disc gels run with diluted samples of enzyme . The ratio of the two species was concentration-dependent and the dissociation was reversible . The purified enzyme appeared to be homogeneous and monodisperse in the ultracentrifuge, on sucrose gradients, during gel filtration on Bio-Gel and on polyacrylamide gels, but it had a heterogeneous subunit composition as determined by sodium dodecyl sulfate gel electrophoresis . Enzyme species with different subunit compositions were partially resolved by gel filtration . The fractions with the highest specific activity contained subunits of 150,000 and 55,000 daltons in a ratio of approximately 1:1 . Other fractions contained reduced amounts of the 55,000-dalton subunit and correspondingly increased amounts of 51,000-, 45,000-, and 10,000-dalton subunits, suggesting that the heterogeneity was the result of proteolytic degradation of the 55,000-dalton subunit . The enzyme contained approximately 12 non-heme irons, 12 acid-labile sulfides, 24 cysteine residues, and 1 molybdenum per 200,000 daltons. J Biol Chem, 1976 Apr 25, 251(8), 2462 - 8 Involvement of the glucose enzymes II of the sugar phosphotransferase system in the regulation of adenylate cyclase by glucose in Escherichia coli; Harwood JP et al.; The nature of the interaction of glucose with toluene-treated cells of Escherichia coli leading to inhibition of adenylate cyclase was examined by the use of analogues . Those analogues with variations of the substituents about carbon atoms 1 or 2 (e.g . alpha-methylglucoside or 2-deoxyglucose) are inhibitory, and they are also substrates of the phosphoenolpyruvate-dependent sugar phosphotransferase system . Analogues with changes in other parts of the molecule (e.g . 3-O-methylglucose or galactose), L-glucose and several disaccharides and pentoses, do not inhibit adenylate cyclase and are not substrates of the phosphotransferase system . This correlation suggests some functional relationship between the adenylate cyclase and phosphotransferase systems . Further studies were done with mutants defective in glucose enzymes II of the phosphotransferase system (designated GPT and MPT); these two activities are measured by phosphorylation of alpha-methyl-glucoside and 2-deoxyglucose, respectively . The wild-type parent phosphorylates both analogues, and both inhibit adenylate cyclase . In the GPT- mutant, alpha-methylglucoside does not inhibit adenylate cyclase and is not phosphorylated, while 2-deoxyglucose is inhibitory and phosphorylated . In the GPT- MPT- double mutant, adenylate cyclase activity is present, but neither alpha-methylglucoside nor 2-deoxyglucose inhibits adenylate cyclase, and neither sugar is phosphorylated . These studies demonstrate that glucose inhibition of adenylate cyclase in toluene-treated cells requires an interaction of this sugar with either the GPT or mpt enzyme II of the phosphotransferase system. J Biol Chem, 1976 Apr 25, 251(8), 2388 - 94 Increased intestinal chromatin template activity . Influence of 1alpha,25-dihydroxyvitamin D3 and hormone-receptor complexes; Zerwekh JE et al.; 1alpha,25-Dihydroxyvitamin D3 administration to rachitic chicks results in an increase in the chromatin template activity of intestinal target tissue assayed in vitro using Escherichia coli RNA polymerase . The maximum stimulation of template capacity was 12 to 20% over control values and occurred 2 hours after administration of the sterol . This rapid effect preceded the biologic response to 1alpha,25-dihydroxyvitamin D3 in the intestine and was not observed in other tissues such as liver or kidney . The in vivo enhancement of intestinal chromatin template activity was specific for the 1alpha,25-dihydroxyvitamin D3 hormone in that equivalent doses of 25-hydroxyvitamin D3 or vitamin D3 did not elicit a response in 2 to 3 hours . Only 1alpha-hydroxyvitamin D3, a synthetic sterol which is very rapidly metabolized to the 1alpha,25-dihydroxyvitamin D3 form, was able to minic the natural hormone in vivo . To further elucidate the nuclear mechanism of action of 1alpha,25-dihydroxyvitamin D3, the hormone was preincubated at 0 degrees with intestinal cytosol to form hormone-receptor complexes . After addition of the hormone-receptor complexes to purified intestinal mucosa nuclei and incubation for 1 hour at 25 degrees, chromatin isolated from this reconstituted system displayed a significant increase in template activity as compared to chromatin prepared from similar in vitro incubations not containing hormone . This stimulation was 12 to 24% over control values and exhibited an absolute requirement for intestinal cell cytosol . The response was specific for physiologic levels of 1alpha,25-dihydroxyvitamin D3, but occurred with pharmacologic doses of 25-hydroxyvitamin D3 . It is concluded that a stimulation of the chromatin template activity of intestinal target tissue by 1alpha,25-dihydroxyvitamin D3 may be an integral part of the ultimate physiologic response of enhanced calcium transport. J Biol Chem, 1976 Apr 25, 251(8), 2520 - 4 ATPase activity required for termination of transcription by the Escherichia coli protein factor rho; Howard BH et al.; The relationship between the RNA-dependent beta-gamma ATPase in purified rho preparations and rho-mediated termination of transcription has been investigated . In a purified in vitro system, transcription from lambdagal DNA has been carried out using either ribonucleoside triphosphates (NTPs) or four ribonucleoside 5'-(beta-gamma-imino)triphosphates (NMP-P(NH)Ps) as RNA precursors . In the presence of NTPs, rho termination activity results in (a) the synthesis of rho-dependent transcripts which are of discrete size by polyacrylamide gel analysis and (b) a marked reduction by hybridization assay in RNA transcribed distal to the rho-sensitive termination site tR . In the presence of four NMP-P(NH)Ps, which are not substrates for the beta-gamma ATPase, termination by rho is completely abolished, whereas rho-independent termination occurs normally . Addition of ATP to transcription reactions containing four NMP-P(NH)Ps restores termination, ruling out the possibility that the termination activity of rho is nonspecifically inhibited by the analog preparations . We interpret our data as strongly suggesting that the RNA-dependent beta-gamma ATPase activity of rho is required for rho-mediated termination of transcription. Mol Gen Genet, 1976 Apr 23, 145(1), 97 - 100 Stabilization and size of araC protein; Wilcox G et al.; AraC protein from Escherichia coli has been further stabilized and characterized . pH is a critical variable in conferring stability . araC protein has a sedimentation coefficient of 4.0 +/- 0.2s on standardized 5%-20% glycerol gradients . Its isoelectric point is at a pH of 7.1. Mol Gen Genet, 1976 Apr 23, 145(1), 65 - 71 Glyceraldehyde 3-p dehydrogenase, glycerate 3-P kinase and enolase mutants of Escherichia coli: genetic studies; Irani MH et al.; The genetic loci for two enzymes of glycolysis have been established by transductional crosses . The eno locus, likely to be the structural gene for enolase maps in the 52-minute region of the Escherichia coli chromosome . A structural determinant for glycerate 3-P kinase (pgk) is located near serA . The map order is speB-pgk-serA-lysA-argA-eno-cysC . In the 35-minute region maps a locus affecting the structure of glyceraldehyde 3-P dehydrogenase. Biochim Biophys Acta, 1976 Apr 22, 431(1), 54 - 61 Temperature-sensitive formation of the phospholipid molecular species in Escherichia coli membranes; Nishihara M et al.; Phospholipids in the membranes of Escherichia coli grown at 37 degrees C are composed of different proportions of molecular species than are those at 17 degrees C . The 1,2-disaturated and 1-saturated-2-unsaturated molecular species increased at 37 degrees C, but the 1,2-diunsaturated species increased at 17 degrees C . When the growth temperature was lowered from 37 to 17 degrees C during the middle exponential growth phase, phosphatidylethanolamine and phosphatidylglycerol composed of proportions of molecular species similar to those found at 17 degrees C were immediately synthesized . By using various membranes composed of different compositions of the phospholipid molecular species, the temperature-sensitive formation of the phospholipid molecular species was found to be independent of the composition of the membrane phospholipids and to be dependent on changes in the specificities of membrane-bound sn-glycerol 3-phosphate acyltransferase against the acyl-CoAs, due to temperature changes. Biochemistry, 1976 Apr 20, 15(8), 1610 - 4 Production of high levels of phosphorylated F1 histone by zinc chloride; Tanphaichitr N et al.; Methods have been sought to perturb the level of phosphohistones . ZnCl2 (10 mM) exhibits histone phosphate phosphatase in vivo in HTC cells and leads to hyperphysiological levels of F1 phosphohistone . Treatment of tissue culture cells with this concentration of ZnCl2 leads to a reduction in medium pH to 6.4 . Control experiments have indicated that HTC cells grow efficiently at this pH and that the reduction of pH does not produce the hyperphosphorylated state per se . The optimum conditions for the ZnCl2 effect are described . That the effect of ZnCl2 on the heterogeneity of F1 histone is due to an effect on phosphorylation was demonstrated by the observation that the entire effect is abolished by treatment with alkaline phosphatase . The site of phosphorylation is in the carboxy-terminal end of the F1 molecule . The inhibitory effect of ZnCl2 on F3 phosphorylation in metaphase cells is also described. Biochemistry, 1976 Apr 20, 15(8), 1755 - 60 Mechanistic studies of glutamine synthetase from Escherichia coli: kinetics of ADP and orthophosphate binding to the unadenylylated enzyme; Rhee SG et al.; The kinetics of protein fluorescence change exhibited by ADP or orthophosphate addition to the Mg2+-or Mn2+-activated unadenylylated glutamine synthetase from Escherichia coli were studied . The kinetic patterns of these reactions are incompatible with a simple bimolecular binding process and a mechanism which required protein isomerization prior to substrate binding . They are consistent with a mechanism in which direct substrate binding is followed by a substrate-induced conformational change step, ES in equilibrium ES . At pH 7.0 and 15 degrees C, the association constants for the direct binding (K1) of ADP to MnE1.0 and of Pi to MnE1.0ADP are 3.9 X 10(4) and 2.28 X 10(2) M(-1), respectively . The association constant for the direct binding of ADP to MnE1.0Pi is 2.3 X 10(4) M(-1) at pH 7.0 and 19 degrees C . The deltaG degrees for the substrate-induced conformational step are -3.5 and -1.3 kcal mol(-1) due to ADP binding to MnE1.0Pi and MnE1.0, respectively, and -1.4 kcal mol(-1) due to Pi binding to MnE1.0ADP . Rate constants, k2 and k(-2), for the isomerization step are: 90 and 9.5 s(-1) for ADP binding to MnE1.0, 440 and 0.36 s(-1) for ADP binding to MnE1.0Pi, and 216 and 1.8 s(-1) for Pi binding to MnE1.0ADP . Due to low substrate affinity, the association constant for direct Pi binding to MnE1.0 was roughly estimated to be 230 M(-1) and k2 = 750 s(-1), k(-2) = 250 s(-1) . At 9 degrees C and pH 7.0, the estimated association constants for the direct ADP binding to MgE1.0 and MgE1.0 Pi are 1.8 X 10(4) and 1.6 X 10(4) M(-1), respectively; and the rate constants for the isomerization step associated with the corresponding reaction are k2 = 550 s(-1), k(-2) = 500 s(-1), and k2 = 210 s(-1), k(-2) = 100 s(-1) . From the kinetic analysis it is evident that the inability of Mn2+ to support biosynthetic activity of the unadenylylated enzyme is due to the slow rate of ADP release from the MnE1.0PiADP complex . In contrast the large k(-2) obtained for ADP release from the MgE1.0ADP or MgE1.0PiADP complex indicates that this step is not rate limiting in the biosynthesis of glutamine since the k catalysis obtained under the same conditions is 7.2 s(-1). Biochim Biophys Acta, 1976 Apr 15, 432(1), 49 - 59 Purification and properties of DNA-dependent RNA polymerase from Mycobacterium tuberculosis H37RV; Harshey RM et al.; RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase DNA-dependent), EC 2.7.7.6) was purified approximately 200 fold from Mycobacterium tuberculosis H37RV cells . The purified enzyme has a molecular weight of about 330 000-350 000 and is composed of four subunits . The subunits beta', beta and sigma have molecular weights different from those of Escherichia coli polymerase; the fourth, alpha subunit has a similar weight . The purified enzyme is a thousand-fold more sensitive to rifampicin, a potent antitubercular drug than the E . coli RNA polymerase, probably because of the difference in the beta subunits . This, with other data presented in this paper, indicate that the RNA polymerase of M . tuberculosis differs in its properties from that of E . coli. Eur J Biochem, 1976 Apr 15, 64(1), 77 - 89 Studies on the environment of protein S7 within the 30-S subunit Escherichia coli ribosomes; Rinke J et al.; Analyses are described of three types of ribosomal fragment, all derived from the well-characterized ribonucleoprotein species consisting of proteins S7, S9, S10, S14 AND S19, together with RNA from sections O'-D-E'-K-P-P'-E-A of the 16-S sequence . 1 . When 30-S subunits were hydrolysed with ribonuclease T1 in presence of deoxycholate in addition to the components previously described, a fragment could be isolated which contained only proteins S7 and S19, together with minor amounts of S13 or S14 . Oligonucleotide analysis of this fragment showed that it contained RNA from sections O'-E' and P-A of the 16-S RNA, but that section K (from the middle of this area) was missing . 2 . It has previously been shown that when 30-S subunits are irradiated with ultraviolet light, protein S7 is the primary target of cross-linking of protein to RNA . By making use of this reaction, ribonucleoprotein fragments were isolated from irradiated 30-S subunits the unbound proteins were removed, and RNA fragments containing covalently linked protein S7 were identified . It was possible to demonstrate that the site of cross-linking lies within the O'-A region of the 16-S RNA, and more precise experiments showed that this site almost certainly is in the P-A region . 3 . When the parent five-protein ribonucleoprotein fragment (above) was deproteinized under very mild conditions, RNA complexes could be isolated which consisted of non-contiguous sequences, but which migrated as a single species into a polyacrylamide gel . Analysis of one of these complexes showed that it contained sequences from sections O'-E' and P-A, but that section K was missing (cf . first paragraph above) . This demonstrated that these two separate regions of RNA interact within the 30-S subunit, independently of the presence of protein. Eur J Biochem, 1976 Apr 15, 64(1), 313 - 20 The binding of indole to the alpha-subunit and beta2-subunit and to the alpha2beta2-complex of tryptophan synthase from Escherichia coli . Identification of a second indole-binding site on the alpha-subunit; Weischet WO et al.; The binding of indole and indolepropanol phosphate, an analogue of the substrate indoleglycerol phosphate, to the individual alpha and beta2-subunits and to the alpha2beta2-complex of tryptophan synthase was studied by equilibrium dialysis . The use of {14C}indole and indolepropanol {32P}phosphate permitted simultaneous binding studies to be carried out . Competition between indole and indolepropanol phosphate in binding to a particular site was taken as evidence for that site being part of the active site of the alpha-subunit . The binding of indole to the active site of the alpha-subunit is weak (Kd = 18mM) . A second distinct site binds indole more strongly (Kd = 1.5 mM) and interacts with the active site indirectly . It is therefore designated an effector site . Furthermore, the binding of indole and/or indolepropanol phosphate appears to stabilize different conformations of the alpha-subunit . The beta2-subunit binds indole only weakly (Kd = 12 mM) to many (n = 10) sites per polypeptide chain . The alpha2beta2-complex retains one or two sites per alphabeta-equivalent of relatively high affinity (Kd = 1.2 mM) . The active sites of the component alpha and beta-subunits probably belong to the second class of many (n = 40) sites of low (Kd = 30 mM) affinity for indole . These findings support conclusions from the literature that both bi-substrate reactions involving indole catalyzed by tryptophan synthase and its subunits must follow strictly ordered addition mechanisms with the respective other substrate adding first. Eur J Biochem, 1976 Apr 15, 64(1), 295 - 300 Small-angle x-ray and light-scattering study of native and trypsin-modified methionyl-tRNA synthetase from Escherichia coli; Gulik A et al.; Small-angle X-ray scattering experiments were performed on an absolute scale on solutions of methionyl-tRNA synthetase from Escherichia coli in its native and trypsin-modified forms . A light-scattering study was performed on the same solutions to verify monodispersity . The structural parameters for the trypsin-modified enzyme, radius of gyration (2.48 nm), volume (90 nm3), surface/volume (1.5 nm-1) and the distribution of chords can account for an equivalent prolate ellipsoid of revolution having an axial ratio 2.3 and a maximum length of 9 nm, with a creviced surface . The rsults obtained for the native enzyme {i.e . radius of gyration (4.3 nm), volume (244 nm3), distribution of the scattering intensity and distribution of chords} exclude the possibility of a very compact quaternary structure and suggest that the enzyme consists of at least two globular parts, probably the two protomers, linked together by interactions involving a limited region of the structure. Eur J Biochem, 1976 Apr 15, 64(1), 199 - 204 Purification of protease II from Escherichia coli by affinity chromatography and separation of two enzyme species from cells harvested at late log phase; Pacaud M; N-Acetyl-D-arginine linked to an agarose matrix has been used to purify protease II from Escherichia coli by affinity chromatography . The specific adsorption of protease II to this absorbent was achieved in 220 mM potassium phosphate buffer pH 7.6, and the enzyme was eluted with L-arginine . Enzyme preparations from cells harvested at late log phase have been resolved into two molecular species which differ in specific activity, kinetic constants and carbohydrate content . Both species appeared homogeneous by electrophoresis in conventional buffers and also in the presence of sodium dodecyl sulfate . Only one enzyme species was obtained by the same procedure using bacteria harvested at the middle of exponential growth. Eur J Biochem, 1976 Apr 15, 64(1), 177 - 88 Structural properties of Escherichia coli RNA polymerase Subunits; Lowe PA et al.; 1 . The surface of the RNA-polymerase-DNA complex possesses an exposed polypeptide loop . 2 . Proteinases with differing specificities (trypsin, chymotrypsin, subtilisin and clostripain) preferentially cleave the exposed region . 3 . The cleaved polypeptide is reassembled into RNA polymerase by renaturation from a solvent which promotes a random coil conformation . 4 . Isolated beta subunit has a proteolytically resistant nucleus of approximately 70000 molecular weight . This resistant polypeptide may be generated by trypsin, chymotrypsin, subiilisin or clostripain . 5 . Isolated alpha subunits are comparatively resistant to proteolysis . 6 . Although of similar molecular weights beta and beta' appear to have unrelated primary sequences and markedly different conformations in free solution . 7 . Digestion of the beta subunit may be blocked by formation of the alpha2beta subassembly . 8 . Evidence is presented suggesting that beta' in the intact enzyme (alpha2beta beta') possesses the exposed polypeptide loop. Eur J Biochem, 1976 Apr 15, 64(1), 115 - 21 Globin mRNA species containing poly(A) segments of different lengths . Their functional stability in Xenopus oocytes; Nudel U et al.; Rabbit globin mRNA species containing poly(A) segments of different lengths were prepared by partial phosphorolysis of mRNA with Escherichia coli polynucleotide phosphorylase . By varying the salt concentration and the time of incubation of the phosphorolysis mixture, as well as performing oligo(dT)-cellulose chromatography at 22 degrees C and at 4 degrees C, globin mRNA preparations containing poly(A) segments of approximately 122, 95, 68, 39, 32, 21, and 16 adenylate residues were obtained . It was found that the functional stability of the mRNA species containing 32 or more adenylate residues after injection into Xenopus laevis oocytes equaled that of the native globin mRNA . On the other hand, the functional stability of mRNA containing an average number of 21 adenylate residues was about 30% of the native mRNA, while that of mRNA containing 16 adenylate residues was as low as poly(A)-free globin MRNA. Experientia, 1976 Apr 15, 32(4), 520 - 1 Induction of immunological memory in mice by RNA extract; Ziska P et al.; RNA extract isolated from spleens of mice immunized with lipopolysaccharide from E . coli induced an immunological memory in normal mice . Application of small amounts of corresponding antigen provoked a specific secondary immune response in RNA primed mice. Biochim Biophys Acta, 1976 Apr 15, 432(1), 98 - 103 Release of certain ribosomal proteins from 70-S Escherichia coli ribosomes by mild ribonuclease digestion; Gast WH et al.; A method for the release of some proteins from Escherichia coli (MRE 600)ribosomes is described, avoiding extraction with denaturing reagents . High-salt-washed, 70-S ribosomes were treated with pancreatic ribonuclease which led to the release of 12 proteins of the larger ribosomal subunit . Separation of released protein was first attempted by gel filtration and by fractionation with (NH4)2SO4 . The number and type of the released proteins were identified by sodium dodecyl sulphate-polyacrylamide gels and two-dimensional gel electrophoresis . The method should prove of use in the large scale purification of proteins L1, L7, and L25. Eur J Biochem, 1976 Apr 15, 64(1), 27 - 34 Conformation of Escherichia coli glutamic acid tRNA II as studied by hydrogen-tritium exchange catalyzed by cysteine methyl ester; Effect of estrogen on gene expression in the chick oviduct; Hen oviduct RNA polymerase II and Escherichia coli RNA polymerase holoenzyme and core enzyme were used to study the initiation of RNA synthesis on chromatin . In either the presence or absence of estrogenic stimulation, changes in the level of oviduct chromatin initiation sites as measured in the presence of either homologous or heterologous polymerases followed a similar pattern . Comparison of the initiation sited utilized by these enzymes on chick oviduct chromatin indicated that these enzymes compete with each other for the same initiation regions . In contrast to chromatin, however, the majority of the initiation sites on DNA which are utilized by the oviduct RNA polymerase II are different from those utilized by E . coli holoenzyem . These results suggest that chromatin proteins are involved in the selection of initiation sites on chromatin for RNA polymerases . The in vitro transcripts of these RNA polymerases on stimulated chick oviduct chromatin were analyzed by hybridization to a cDNA probe transcribed from ovalbumin mRNA . The relative concentration of ovalbumin sequences transcribed by these three polymerases was 4:1.5:1 for oviduct RNA polymerase II, E . coli core enzyme, and holoenzyme respectively . Therefore, the efficiency of transcribing a specific gene appears to depend on the interaction between RNA polymerase and chromosomal elements in the initiation region. J Biol Chem, 1976 Apr 10, 251(7), 1896 - 901 Endonuclease II of Escherichia coli is exonuclease III; Weiss B; Exonuclease III, a phosphatase-exonuclease specific for bihelical DNA, wn the preparation was endonuclease II, an activity specific for DNA that has been partially depurinated by treatment with methyl methanesulfonate . The two activities, which could not be separated by electrophoresis, by sedimentation, or by gel filtration, were associated with a single monomeric protein of 28,000 daltons . To explain how a relatively small protein could have such diverse activities, it is proposed that one site on the enzyme can recognize interstrand spaces created either by depurination or by spontaneous terminal unwinding of a DNA duplex. J Biol Chem, 1976 Apr 10, 251(7), 2063 - 9 Acetyl coenzyme A carbosylase . Circular dichroism studies of Escherichia coli biotin carboxyl carrier protein; Fall RR et al.; The biotin carboxyl carrier protein (BCCP) component of Escherichia coli acetyl coenzyme A carboxylase and three peptides derived from BCCP by proteolytic digestion have been examined by circular dichroism spectroscopy . BCCP, which has a peptide molecular weight of 22,500, has a spectrum typical of globular proteins with negative extrema at 222 nm and 208 nm . The two smallest peptides, BCCP(SC) and BCCP(9,100), with molecular weights of 8,900 and 9,100, respectively, exhibit unusual positive CD bands centered at 237 nm and 220 nm . BCCP(10,400), with a molecular weight of 10,400, has a CD spectrum intermediate between BCCP and that of the smallest peptides . Since d-biotin exhibits a positive CD band at 233 nm, it was suspected that the biotin prosthetic group might be the chromophore responsible for the 237 nm CD band seen in BCCP(SC) and BCCP(9,100) . Enzymatic carboxylation of BCCP(SC) to form CO2-BCCP(SC) caused the CD spectrum to change with a shift of the 237 nm band to 232 nm . The positive CD band at 220 nm was unaffected by carboxylation of the biotin prosthetic group . These date suggest that the 237 nm signal may be due either to the biotin which acts as a chromophore directly or to a chromophore that is perturbed by the carboxylation of biotin . A spectropolarimetric titration was carried out to investigate the possible contribution of the single tyrosine residue of BCCP(SC) to the CD spectrum of this peptide . At pH values over 9 the CD spetrum changed with the disappearance of the 237 nm band, suggesting that tyrosine might contribute to this CD band . Denaturation of BCCP(SC) or BCCP(9,100) with 8 M urea of 6 M guanidine HCl abolished the positive CD bands and resulted in spectra typical of a random coil, whereas treatment of BCCP(SC) with 1% sodium dodecyl sulfate abolished the positive bands and left a spectrum exhibiting a shoulder at 222 nm and a negative band at 205 nm, suggestive of a high degree of ordered structure . It is concluded that the CD band at 237 nm in BCCP(SC) and BCCP(9,100) is prabably due to a noncovalent interaction of biotin with an amino acid residue(s) of the protein . It is suggested that the biotin prosthetic group is partially buried in the surface of the protein, rather than swinging free at the end of the lysine side chain through which it is covalently linked to the protein, to permit this interaction to occur. Biochim Biophys Acta, 1976 Apr 9, 430(1), 182 - 8 The accumulation of superoxide radical during the aerobic action of xanthine oxidase . A requiem for H2O4; Hodgson EK et al.; The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O2- during the first few minutes of the reaction . H2O2 decreases this accumulation of O2- presumably because of the Haber-Weiss reaction (H2O2+O2- leads to OH- +OH+O2) and very small amounts of superoxide dismutase eliminate it . This accumulation of O2- was demonstrated in terms of a burst of reduction of cytochrome c, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate . The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H2O4-, can now be satisfactorily explained entirely on the basis of known radical intermediates. Biochim Biophys Acta, 1976 Apr 8, 429(2), 352 - 8 Uridine phosphorylase from Escherichia coli . Kinetic properties and mechanism; Krenitsky TA; Type I and Type II uridine phosphorylases (uridine: orthophosphate ribosyltransferase EC 2.4.2.3) are distinguished by their pH optima (Krenitsky et al . (1965) J . Biol . Chem . 240, 1281-1286) . A Type I enzyme was partially purified from Escherichia coli . The crossing pattern of the initial velocity analysis indicated that the catalytic mechanism involved the sequential addition of substrates to the enzyme . Product inhibition by uracil or by ribose 1-phosphate was linear competitive with uridine or with concentrations of phosphate below 3 mM . This indicated that the sequence of substrate addition was random rather than ordered . At concentrations of phosphate above 3 mM, product inhibition by uracil was complex . The random mechanism of this Type I enzyme contrasts with the ordered mechanism of a Type II enzyme from rat liver (Kraut, A . and Yamada, E.W . (1971) J . Biol . Chem . 246, 2021-2030). Biochemistry, 1976 Apr 6, 15(7), 1510 - 6 Incorporation of noncomplementary nucleotides at high frequencies by ribodeoxyvirus DNA polymerases and Escherichia coli DNA polymerase I; Mizutani S et al.; The fidelity of DNA synthesis with synthetic homopolymer templates by two ribodeoxyvirus DNA polymerases and E . coli DNA polymerase I was examined by nearest neighbor frequency analyses . The experiments were designed to favor the incorporation of noncomplementary nucleotides, and the reactions were carried out only for short periods of time . All incorporations were template dependent . The frequency of incorporation of noncomplementary nucleotides next to complementary nucleotides by all three of these DNA polymerases significantly varied, depending on the conditions, from almost none (less than 0.2%) to 100% of the incorporation of the complementary nucleotide . In general, there was more incorporation of noncomplementary nucleotides in the presence of Mn2+ than in the presence of Mg2+ and with ribohomopolymer templates than with deoxyribohomopolymer templates . With a heteropolymer RNA template, the infidelity of DNA synthesis by one of the ribodeoxyvirus DNA polymerases was also increased by the presence of Mn2+ in the DNA synthesizing reaction . These findings indicate that, under appropriate conditions, any DNA polymerase has the potential to make a significatn number of errors in DNA synthesis and that altered conditions for DNA polymerase action might play a role in spontaneous mutation. Biochemistry, 1976 Apr 6, 15(7), 1569 - 80 Glutamine synthetase adenylyltransferase from Escherichia coli: purification and physical and chemical properties; Caban CE et al.; The glutamine synthetase adenylyltransferase (EC 2.7.7.42), WHIch catalyzes the adenylylation and deadenylylation of glutamine synthetase in E . coli, has been stabilized and purified 2200-fold to apparent homogeneity . Sedimentation and electrophoresis studies show that the native enzyme is a single polypeptide chain of 115,000 +/- 5000 molecular weight with an isoelectric pH (PL) OF 4.98, a sedimentation coefficient (S20.w0) of 5.6S, and a molar frictional coefficient (f/f0) of 1.52 . An alpha-helical content of approximately equal to 25% and approximately equal to 28% beta-pleated sheet and approximately equal to 47% random coil structures were estimated from circular dichroism measurements . The amino acid composition of the protein has been determined . The intrinsic tryptophanyl residue flourescence of adenylyltransferase is two fold greater than that of L-tryptophan; this property has been used to monitor ligand-induced conformational changes in the enzyme . Activators of the adenylylation reaction (ATP, L-glutamine, or the E . coli PII regulatory protein) produced an enhancement of fluorescence; alpha-ketoglutarate, an inhibitor of adenylylation and an activator of deadenulylation, caused a net decrease in fluorescence . The adenylytransferase has separate interaction sites for L-glutamine and the regulatory PII protein. Biochemistry, 1976 Apr 6, 15(7), 1547 - 61 31P nuclear magnetic resonance study of alkaline phosphatase: the role of inorganic phosphate in limiting the enzyme turnover rate at alkaline pH; Hull WE et al.; 31P nuclear magnetic resonance (NMR) was used to directly observe the binding of inorganic phosphate to alkaline phosphatase . Evidencq for the tight binding of 1.5-2.0 mol of inorganic phosphate per dimer of alkaline phosphatase is presented . Two distinct forms of bound phosphate are observed, one predominating above pH 7 and representing the non-covalent E-P1 complex and the other predominating below pH 5 and representing the covalent E-P1 complex . The 31P NMR line width of the E-P1 complex indicates that the dissociation of noncovalent phosphate is the rate-limiting step in the turnover of the enzyme at high pH. Biochemistry, 1976 Apr 6, 15(7), 1535 - 46 Fluorine-19 nuclear magnetic resonance study of fluorotyrosine alkaline phosphatase: the influence of zinc on protein structure and a conformational change induced by phosphate binding; Hull WE et al.; 19F nuclear magnetic resonance (NMR) spectroscopy has been used to study a fully active E . coli fluorotyrosine alkaline phosphatase . The fluorotyrosine resonances provide sensitive probes of the conformational states of the protein . They were used to follow the addition of zinc or cobalt to the apoprotein, and the titration of the protein with inorganic phosphate or the inhibitor 2-hydroxy-5-nitrobenzylphosphonate . The results indicate that 2 molecules of inorganic phosphate per dimer of alkaline phosphatase are required to complete a general conformational change in the protein involving perturbations to the environment of several tyrosines . Spectra of the cobalt enzyme indicate that on specific tyrosine per subunit may be near the metal site . The 19F NMR results, combined with the 31P NMR results in the accompanying paper, lead directly to the conclusion that dissociation of noncovalently bound inorganic phosphate from the enzyme is the rate-limiting process in enzyme catalysis at high pH . The local environment of the individual fluorotyrosines is also discussed. Biochemistry, 1976 Apr 6, 15(7), 1387 - 92 The state of energization of the membrane of Escherichia coli as affected by physiological conditions and colicin K; Brewer GJ; The bacterial protein colicin K, when added to sensitive Escherichia coli in the presence of 3,3'-dihexyloxacarbocyanine, cuases a doubling in fluorescence of the probe . Glucose and oxygen cause a decreased fluorescence while anoxia and cyanide cause a rise in fluorescence . These results in conjunction with the work of other laboratories suggest that colicin K causes a depolarization of the transmembrane electrical potential . Fluorescence in the absence of colicin K was relatively independent of KCl, NaCl, and MgCl2 concentrations below 0.1 M . Although colicin K caused rapid efflux of the K+ analogue 86Rb+, the fluorescence rise was only partially blocked by 0.13 M KCl . The level of fluorescence caused by the action of colcin K was inversely proportional to the logarithm of the concentration of MgCl2 over the range of 2 muM to 4 mM . This suggests that a Nernst electrochemical potential for an anion can counteract a membrane depolarization caused by colcin . After colcin K action, the fluorescence of the carbocyanine could be further increased by anoxia or cyanide . The distribution of the weak base dimethyloxazolidinedione indicated that the pH in the interior of aerobic E . coli supplied with lactate was alkaline by 0.1 unit and unaffected by colicin . These results suggest that colicin K does not completely depolarize the membrane potential and does not interfere with the component of membrane energization generated by electron transport . Colicin K does not act as a cationophore . The partial depolarization of the membrane may account for the inhibition of active solute transport caused by colicin K. Biochim Biophys Acta, 1976 Apr 5, 426(4), 711 - 22 Kinetic processes in Escherichia coli membranes and cells . A laser photolysis study using derivatives of pyrene; Wong M et al.; Pyrene and several derivatives of pyrene are used to investigate photo-induced kinetic processes in whole cells and membranes extracted from Escherichia coli . A mutant of E . coli was used which, under appropriate growth conditions, produced a complete or incomplete lipopolysaccharide in the outer membrane . The pyrene derivatives used were: pyrene sulfonic acid, pyrene butyric acid and the ester of pyrene butyric acid and 10-hydroxydecanoic acid . The pyrene chromophore was excited by the ultraviolet pulse from a Q switch, frequency-doubled, ruby laser . The lifetimes of the pyrene fluorescence in the presence of the quenchers O2, thallous ion (T1+), I-and CH3NO2 were measured and tabulated as second order rate constants . For the most part the quenching rate constants were much lower than the corresponding values observed in simple nonviscous solution, e.g . ethanol . This is interpreted as being due to the location of the probe within the membrane . The membrane inhibits the movement of the quenchers to the excited state . Cell membranes containing complete lipopolysaccharide showed significantly lower quenching rates for the probes pyrene and pyrene sulfonic acid than cell membranes with incomplete lipopolysaccharide . From an amalysis of the kinetic data it is suggested that pyrene and pyrene sulfonic acid are located near and under lipopolysaccharide and close to membrane proteins . On the other hand, no effect of lipopolysaccharide composition was observed for the probes pyrene butyric acid and pyrene butyroyl decanoic acid . This may suggest that these probes are located primarily in the lipid part of the membrane . A simple model for the outer membrane of E . coli is suggested that accounts for the observed laser-induced kinetic processes. Biochim Biophys Acta, 1976 Apr 2, 425(4), 396 - 400 Effects of irradiation on RNA synthesis primed by calf thymus deoxyribonucleoprotein treated with salt and salt-urea; Kobayashi T et al.; 1 . The effects of ionizing radiation on the activity of calf thymus templates were examined in a Escherichia coli RNA polymerase system . 2 . The template activity of native and 2 M NaCl-5M urea-treated deoxyribonucleoproteins was enhanced by relatively low doses of irradiation, while that of 2 M NaCl-treated deoxyribonucleoprotein was not enhanced by irradiation . 3 . The template activity of purified DNA was markedly decreased by irradiation, while that of native deoxyribonucleoprotein, 2 M NaCl-treated, and 2 M NaCl-5 M urea-treated ones were slightly decreased at a higher dose range . The doses for 50% inactivation of these templates were 1.3, 210, 140, and approximately 200 krad, respectively. Biochim Biophys Acta, 1976 Apr 2, 425(4), 492 - 501 Regulation of nitrate reductase at the transcriptional and translational levels in Escherichia coli; Ruiz-Herrera J et al.; Nitrate reductase from Escherichia coli is induced by nitrate and derepressed by oxygen removal after a lag phase . Elimination of inducer, shift to aerobic conditions and addition of actinomycin D causes the decline in the rate of its synthesis, which eventually may stop . Kinetic analysis of the sensitivity of the biosynthetic process to oxygen, chloramphenicol, actinomycin D and rifampicin gave results which we interprete as evidence that oxygen (and possibly nitrate) affect simultaneously both the transcriptional and translational processes. Biochim Biophys Acta, 1976 Apr 2, 425(4), 451 - 62 The effect of silver ion binding and pH on the buoyant density of DNA and its use in fractionating heterogeneous DNA; Rinehart FP et al.; 1 . The effect of pH on the buoyant density of the complexes of Ag+ with DNA has been studied using 3H-labeled human DNA and several bacterial DNAs to determine the conditions necessary for the maximum resolution of compositional heterogeneity . In neutral CS2SO4 density gradients, Ag+ complexes with (G - C)-rich components are always denser than those with (A - T)-rich components, since (G - C)rich DNAs have a larger affinity for Ag+ than (A - T)-rich DNAs and their complexes are denser than (A - T)-rich complexes . In alkaline (pH greater than 9) CS2SO4 gradients, the buoyant density of the Ag+ - DNA complex is not a simple function of base composition . The Ag+ affinity of (A - T)-rich DNA is larger than that of (G - C)-rich DNA but the density of a (G - C)-rich complex is larger . Thus the ordering of the buoyant density changes depends on the amount of added Ag+ . 2 . The problem of resolving the density heterogeneity within a tracer DNA, and minor components of DNA, is explored and useful fractionation techniques are developed. Br J Haematol, 1976 Apr, 32(4), 573 - 7 Fine structural studies of peripheral blood leucocytes from children with kwashiorkor: morphological and functional properties; Schopfer K et al.; This is the first report of electron microscopic observation on circulating leucocytes from children with kwashiorkor, Neutrophils showed evidence of immaturity, prominent Golgi zones, granule pleomorphism; strands of endoplasmic reticulum and Dohle bodies were frequent . Although an in vitro microbicidal defect occurs with S . aureus, E . coli and C . albicans, the postphagocytic morphological events, including vacuole formation and degranulation, were normal . In mononuclear cell pellets plasmacytoid cells were frequently observed, and rare lymphoblasts occurred . The findings suggest activation of the phagocytic system and stimulation of humoral immunity in children with kwashiorkor . These fine structural observations are comparable to those which occur in trauma or thermal injury, both conditions associated with tissue breakdown and extensive antigenic stimulation. Immunology, 1976 Apr, 30(4), 529 - 35 Changes in thymocyte reactivity to lectins induced by B-cell mitogens of the type of sulphated polyanions; Blitstein-Willinger E et al.; Dextran sulphate, polyvinyl sulphate and carrageenan (but not the other polyanions tested, such as poly I:poly C, poly-L-glutamic acid or E . coli lipopolysaccharide) act synergisticaly with PHA on mouse thymocyte stimulation, as measured by thymidine uptake and blast cell transformation . Furthermore the active compounds shift the dose response of thymocytes to Con A . The effect could be observed in four different strains of mice tested by using normal or cortisone-resistant thymocytes . Two possibilities are envisaged for explanation of these effects: (a) attachment of polyanions by their anionic groups to cell surface constituents and interaction of the sulphate groups of the polymers with plant lectins; and (b) modification of the cell membrane induced by sulphated polymers changing either the binding capacity or the effectiveness of the membrane-associated events leading to thymocyte stimulation by the lectins. Proc Natl Acad Sci U S A, 1976 Apr, 73(4), 1131 - 5 Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex; Brown S et al.; Escherichia coli phage Qbeta RNA replicase, an RNA-dependent RNA polymerase (RNA-dependent RNA nucleotidyltransferase), is a tetramer composed of one phage-coded polypeptide and three host-supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host . Two of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, can be covalently crosslinked with dimethyl suberimidate to form a complex which lacks the ability to catalyze the known host functions catalyzed by the individual elongation factors . Using a previously developed reconstitution system we have examined the effects of crosslinking the EF-Tu-Ts complex on reconstituted replicase