Escherichia coli

J Bacteriol, 1980 Oct, 144(1), 451 - 3
Incorporation of acyl moieties of phospholipids into murein lipoprotein in intact cells of Escherichia coli by phospholipid vesicle fusion; Lai JS et al.; The biosynthesis of the acyl moieties in murein lipoprotein was studied by fusion of {3H}palmitate-labeled phospholipid vesicles with intact cells of an fadD mutant of Escherichia coli . A linear increase in the incorporation of {3H}palmitate radioactivity into both the ester- and amide-linked fatty acids in lipoprotein was observed during a 3-h chase after the fusion . Addition of chloramphenicol completely prevented the incorporation of {3H}palmitate from phospholipids to lipoprotein . These results strongly support our hypothesis that the acyl moieties in phospholipids are the precursors for the fatty acids in murein lipoprotein of E . coli . Among the major glycerophosphatides in E . coli, no specificity was observed regarding the efficacy of the donor.

J Bacteriol, 1980 Oct, 144(1), 45 - 52
Two different species of murein transglycosylase in Escherichia coli; Mett H et al.; We demonstrated that Escherichia coli murein transglycosylase exists in two forms . After mechanical disruption of the cells, one form was found in the soluble fraction and the other, in the cell envelope . The two enzymes differed with respect to molecular weight, isoelectric point, solubility in aqueous buffers, and to some extent in their requirements for maximal catalytic activity . The molecular weight of the membrane-bound transglycosylase (35,000) was half that of the soluble enzyme . Whether the high-molecular-weight soluble protein is a precursor of the membrane-bound enzyme species remains to be elucidated.

J Bacteriol, 1980 Oct, 144(1), 438 - 40
Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell envelope gene murG; Salmond GP et al.; We report the identification, cloning, and mapping of a new cell envelope gene, murG . This lies in a group of five genes of similar phenotype (in the order murE murF murG murC ddl) all concerned with peptidoglycan biosynthesis . This group is in a larger cluster of at least 10 genes, all of which are involved in some way with cell envelope growth.

J Bacteriol, 1980 Oct, 144(1), 435 - 7
Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell division gene ftsQ; Begg KJ et al.; We report the identification, cloning, and mapping of a new cell division gene, ftsQ . This gene formed part of a cluster of three division genes (in the order ftsQ ftsA ftsZ) which itself formed part of a larger cluster of at least 10 genes, all of which were involved in some step in cell division, cell envelope synthesis, or both . The ftsQAZ group was transcribed from at least two independent promoters.

J Bacteriol, 1980 Oct, 144(1), 425 - 7
Comparison of the polypeptide composition of Escherichia coli outer membranes prepared by two methods; Chopra I et al.; Escherichia coli outer membranes were prepared by centrifugation to equilibrium in sucrose gradients and then treated with Sarkosyl in the presence of ethylenediaminetetraacetate . The polypeptide profiles of the two outer membrane preparations were compared by two-dimensional polyacrylamide gel electrophoresis . The patterns obtained were not identical, and Sarkosyl removed several minor proteins from the outer membrane.

J Bacteriol, 1980 Oct, 144(1), 366 - 74
Effect of arsenate on inorganic phosphate transport in Escherichia coli; Willsky GR et al.; The effect of arsenate on strains dependent on the two major inorganic phosphate (Pi) transport systems in Escherichia coli was examined in cells grown in 1 mM phosphate medium . The development of arsenate-resistant Pi uptake in a strain dependent upon the Pst (phosphate specific transport) system was examined . The growth rate of Pst-dependent cells in arsenate-containing medium was a function of the arsenate-to-Pi ratio . Growth in arsenate-containing medium was not due to detoxification of the arsenate . Kinetic studies revealed that cells grown with a 10-fold excess of arsenate to Pi have almost a twofold increase in capacity (Vmax) for Pi, but maintained the same affinity (Km) . Pi accumulation in the Pst-dependent strain was still sensitive to changes in the arsenate-to-Pi ratio, and a Ki (arsenate) for Pi transport of 39 microM arsenate was determined . The Pst-dependent strain did not accumulate radioactive arsenate, and showed only a transient decrease in intracellular adenosine triphosphate levels after arsenate was added to the medium . The Pi transport-dependent strain ceased growth in arsenate-containing media . This strain accumulated 74As-arsenate, and intracellular adenosine triphosphate pools were almost completely depleted after the addition of arsenate to the medium . Arsenate accumulation required a metabolizable energy source and was inhibited by N-ethylmaleimide . Previously accumulated arsenate could exchange with arsenate or Pi in the medium.

J Bacteriol, 1980 Oct, 144(1), 36 - 44
Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli K-12: isolation and properties of mutants defective in leucine-repressible transport activities; Yamato I et al.; The characteristics of a mutant (hrbA) of Escherichia coli K-12 that is defective in a leucine-nonrepressible transport system, the LIV-3 system, for branched-chain amino acids were described previously (I . Yamato et al., J . Bacteriol 138:24-32, 1979) . New mutants requiring a high concentration of isoleucine for growth were isolated from strain B763 (hrbA ileA) after mutagenesis with ethyl methane sulfonate . These mutants had a defect of the leucine-repressible transport activities for branched-chain amino acids of the parental strain . One of these mutants, strain B7634, had defects of two independent genetic loci (hrbBC and hrbD) . The genes hrbBC were mapped at min 76 near malT, and the gene hrbD mapped at min 77 near xyl on the E . coli genetic map . The substrate specificity, kinetic properties, and source of coupling energy of the transport system coded for by each of these genes were studied using cytoplasmic membrane vesicles and intact cells . The results identified three transport systems with characteristic features other than the LIV-3 system . The hrbB and hrbC systems are responsible for the uptake activites of the LIV-2 system, with a high Km value, and the LIV-1 system, with a low Km value, respectively . Both activities are repressed by leucine and inhibited by threonine and the b(--) isomer of 2-aminobicycloheptyl-2-carboxylic acid . They both utilize adenosine 5'-triphosphate as coupling energy and are not detected in cytoplasmic membrane vesicles . The hrbD system is responsible for the LIV-4 system, with a high Km value . Its activity is repressed by leucine and partially inhibited by threonine . It is detected in cytoplasmic membrane vesicles with a proton motive force as the driving energy.

J Bacteriol, 1980 Oct, 144(1), 356 - 65
Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coli; Willsky GR et al.; Inorganic phosphate (Pi) transport by wild-type cells of Escherichia coli grown in excess phosphate-containing media involves two genetically separable transport systems . Cells dependent upon the high affinity-low velocity Pst (phosphate specific transport) system have a Km of 0.43 +/- 0.2 microM Pi and a Vmax of 15.9 +/- 0.3 nmol of Pi (mg {dry weight}-1min-1) and will grow in the presence of arsenate in the medium . However, cells dependent upon the low affinity-high velocity Pit (Pi transport) system have a Km of 38.2 +/- 0.4 microM and a Vmax of 55 +/- 1.9 nmol of Pi (mg {dry weight}-1min-1), and these cells cannot grow in the presence of an arsenate-to-Pi ratio of 10 in the medium . Pi transport by both systems was sensitive to the energy uncoupler 2,4-dinitrophenol and the sulfhydryl reagent N-ethylmaleimide, whereas only the Pst system was very sensitive to sodium cyanide . Evidence is presented that Pi is transported as Pi or a very labile intermediate and that accumulated Pi does not exit through the Pst or Pit systems from glucose-grown cells . Kinetic analysis of Pi transport in the wild-type strain containing both the Pst and Pit transport systems revealed that each system was not operating at full capacity . In addition, Pi transport in the wild-type strain was completely sensitive to sodium cyanide (a characteristic of the Pst system).

J Bacteriol, 1980 Oct, 144(1), 327 - 36
Pattern of meso-dl-2,6-diaminopimelic acid incorporation during the division cycle of Escherichia coli; Verwer RW et al.; The topography of meso-DL-2,6-diaminopimelic acid incorportion into the cell envelope of Escherichia coli W7 (doubling time, tau, = 70 min) has been studied by autoradiography . To follow the incorporation pattern during the division cycle, cells have been classified according to length and the silver grain distributions have been determined in the two cell halves . In particular, the question of equivalence (with respect to the grain distributions) of the two cell halves has been investigated statistically . The grain localizations have been determined separately for the left cell halves (highest number of grains) and right cell halves . The highest probability of finding grains was in the central area for cells of all length classes . In the longest cells (dividing or nondividing) incorporation occurred in the future septal regions of the prospective daughter cells . Autoradiography of tritiated thymidine-labeled cells indicated the presence of an atypical deoxyribonucleic acid replication cycle (at tau = 70 min) . Initiation of deoxyribonucleic acid replication occurred during the latter part of the division cycle, and its termination occurred in the next cycle.

J Bacteriol, 1980 Oct, 144(1), 312 - 21
Identification of a novel genetic element in Escherichia coli K-12; Greener A et al.; Induction of the SOS repair processes of Escherichia coli K-12 caused a 14.4-kilobase species of circular deoxyribonucleic acid, called element e14, to be excised from the chromosome . To aid further characterization of this species, an 11.6-kilobase segment of e14 was inserted into the HindIII site of plasmid pBR313 . To map e14 on the E . coli K-12 chromosome, the recombinant plasmid, pAG2, was used to transform a polA recipient, an event which required integration of pAG2 into the recipient chromosome . This recombinational event was dependent upon the region of homology between the incoming plasmid and the chromosome, as no transformants were scored when either a strain cured of the element was the recipient or pBR313 was the transforming deoxyribonucleic acid . Using these transformants, we have shown that e14 maps between the purB and pyrC loci near min 25 . Several strains of E . coli K-12 were found to contain e14; however, one strain, Ymel trpA36, did not . In addition, e14 was found to be absent in both E . coli B/5 and E . coli C . The approach to mapping developed for this work could be used to map other fragments of E . coli deoxyribonucleic acid which have no known phenotype.

J Bacteriol, 1980 Oct, 144(1), 28 - 35
Isolation and mapping of a mutation in Escherichia coli with altered levels of ribonuclease H; Carl PL et al.; A mutant of Escherichia coli with altered levels of ribonuclease (RNase) H was isolated after mutagenesis with ethyl methane sulfonate . A procedure for assaying RNase H in partially purified extracts was used to screen approximately 1,500 colonies for variations in RNase H activity . Confirmation of a lower level of RNase H in the mutant was accomplished by analysis of RNase H in sodium dodecyl sulfate-polyacrylamide gels . By Hfr, F', and P1 transduction mapping, the genetic locus responsible for the lower levels of RNase H was located at 5.1 min on the E . coli chromosome . This mutation (rnh) represents a new locus on the E . coli chromosome . The only phenotypic characteristic of this mutation which has been observed to date is the lower level of RNase H (30% of parental values).

J Bacteriol, 1980 Oct, 144(1), 274 - 8
Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli; Heller KB et al.; The specificity of the glycerol facilitator (glpF) of Escherichia coli was studied with an osmotic method . This transport system allowed the entry of polyols (glycerol and erythritol), pentitols, and hexitols . The analogous sugars were not transported . However, urea, glycine, and DL-glyceraldehyde could use this pathway to enter the cell . The glpF protein allowed the rapid efflux of preequilibrated xylitol . Glycerol surprisingly did not inhibit the uptake of xylitol, and xylitol only slightly reduced the uptake of glycerol . The observation and the insensitivity of the xylitol transport to low temperature suggest that the facilitator behaves as a membrane channel.

J Bacteriol, 1980 Oct, 144(1), 185 - 91
Further characterization of sfiA and sfiB mutations in Escherichia coli; Huisman O et al.; The sfiA and sfiB mutations, originally isolated in thermoresistant ultraviolet-resistant revertants of a tif lon strain, also suppressed filamentation in tsl strains (mutated at the lexA locus) . When deoxyribonucleic acid synthesis was arrested, however, sfi-independent filamentation occurred . Other SOS functions were not affected by sfiA and sfiB mutations; in particular, ultraviolet-induced repair and mutagenesis of bacterial deoxyribonucleic acid were normal, as was tsl-tif-induced synthesis of recA protein . Genetic studies (i) established the identity of map location of the sfiA and sulA loci, (ii) showed that the two sfiB mutations are recessive, and (iii) showed that of six independent sfiA mutations, three are recessive and three are dominant . One sfiB strain was shown to have a 6% growth disadvantage relative to a sfi+ or sfiA strain . It is proposed that the sfiA locus may define the structural gene of a hypothetical inducible SOS-associated division inhibitor.

J Bacteriol, 1980 Oct, 144(1), 179 - 84
Acetaldehyde coenzyme A dehydrogenase of Escherichia coli; Clark DP et al.; Mutants of Escherichia coli (adh) in which alcohol dehydrogenase is derepressed under aerobic conditions were also found to overproduce acetaldehyde coenzyme a dehydrogenase . However, acetaldehyde coenzyme A dehydrogenase was induced by ethanol or acetaldehyde and subject to strong catabolite repression, whereas alcohol dehydrogenase was little affected by these conditions . Mutants no longer able to use ethanol as carbon source were isolated from an adh strain . Some of these mutants were revertants at the adh locus and no longer produced either alcohol dehydrogenase or acetaldehyde coenzyme A dehydrogenase . Others, designated acd, were found to lack only acetaldehyde coenzyme A dehydrogenase . The acd mutation was located at min 62 of the E . coli genetic map, the gene order being thyA-lysA-acd-serA-fda . Isolation of Tn10 insertions cotransducible with acd greatly simplified the mapping procedure.

J Bacteriol, 1980 Oct, 144(1), 149 - 58
Biochemical and immunological characterization of an R plasmid-encoded protein with properties resembling those of major cellular outer membrane proteins; Ferrazza D et al.; MRB, a major R222 plasmid-encoded protein previously described by us, is synthesized in large amounts in host Escherichia coli cells, where it is located principally in the outer membrane . Most of this protein is also bound to the peptidoglycan layer in a form which is trypsin resistant . Its monomeric molecular weight is about 29,000, but it is isolated from cell membranes in aggregate molecular weights of more than 100,000 . These properties demonstrate a strong similarity between MRB and porins, major outer membrane proteins of host E . coli cells . They suggest that MRB may have an as-yet unidentified transport function, as do cellular outer membrane proteins with similar biochemical properties . By using antiserum specific for MRB, we demonstrated identity between MRB and the product of the traT gene, one of the surface exclusion proteins on the F plasmid . The synthesis of MRB was found to be constitutive, in contrast to other tra genes, which appear to be under more rigid regulation by the tra operon . These findings suggest that on R222 and other F-like R plasmids this protein has its own promoter.

J Bacteriol, 1980 Oct, 144(1), 114 - 23
Are growth rates of Escherichia coli in batch cultures limited by respiration?
Andersen KB, von Meyenburg K.
Batch cultures of Escherichia coli were grown in minimal media supplemented with various carbon sources which supported growth at specific growth rates from 0.2 to 1.3/h . The respiration rates of the cultures were measured continuously . With few exceptions, the specific rate of oxygen consumption was about 20 mmol of O2/h per g (dry weight), suggesting that the respiratory capacity was limited at this value . The adenosine triphosphate (ATP) required for the production of cell material from the different carbon sources was calculated on the basis of known ATP requirements in the biochemical pathways and routes of macromolecular synthesis . The calculated ATP requirements, together with the measured growth rates and growth yields on the different carbon sources, were used to calculate the rate of ATP synthesis by oxidative phosphorylation . This rate was closely related to the respiration rate . We suggest that aerobic growth of E . coli in batch cultures is limited by the rate of respiration and the concomitant rate of ATP generation through oxidative phosphorylation.

Surg Gynecol Obstet, 1980 Oct, 151(4), 477 - 80
Effects of cortisone on decrease of serum albumin secondary to experimental infections; Deysine M et al.; The observation that severe clinical bacterial infections are associated with decreases in total serum albumin levels was confirmed in experiments involving dogs and rats . In rodents, the injection of methylprednisolone sodium succinate after the onst of the infection significantly reduced the decrease in the serum albumin values . However, this protective or corrective effect did not occur if the steroid was given prior to the infection . The effect of cortisone may be due to its capacity to stimulate albumin synthesis by the parenchyma of the liver.

J Parasitol, 1980 Oct, 66(5), 730 - 4
Responses of B-cells to mitogens and antigen in mice receiving isogenic splenocytes from animals treated with Trichinella extract; Barriga OO; Splenocytes of C57BL/6J mice injected with a Trichinella spiralis larval extract for 7 consecutive days were transferred in two doses into isogenic, immunocompetent mice . On the 3rd day, some recipients were immunized with 10(9) sheep red blood cells and others were killed to investigate blastogenic response of their splenocytes to concanavalin A (Con A), Escherichia coli lipopolysaccharide (LPS), and Mycobacterium's purified protein derivative (PPD) . On the 8th day of immunization, the corresponding mice were killed to study rosette-forming cells (RFC) and direct and indirect plaque-forming cells (D- and I-PFC) in their spleens . Transfer of 10(6) cells depressed the Con A reactivity and the number of RFC and 1-PFC, but increased the PPD reactivity and the number of D-PFC in the recipients, as compared to control mice receiving splenocytes from donors injected with a saline solution . Ten million cells inhibited only the Con A reactivity, but enhanced the number of LPS- and PPD-responding cells and of D-PFC in the recipients over the controls . Inoculation of cells from mice injected with bovine serum albumin did not reproduce the same effects . Splenocytes of mice treated with T . spiralis extract simultaneously inhibit and enhance diverse functions of the immune system . Stimulation is exerted on IgG antibody production and appears to be mediated by suppressor T-cells . Stimulation is exerted mainly on IgM antibody formation . Depression seems to be antigen-specific; it is partially compensated by the concurrent suppression, and it is probably a result of macrophage activation.

Can J Biochem, 1980 Oct, 58(10), 885 - 97
Localization of the dicarboxylate binding protein in the cell envelope of Escherichia coli K12; Bewick MA et al.; Examination of the localization of the dicarboxylate binding protein (DBP) in the cell envelope of Escherichia coli K12 reveals that this protein is present on the cell surface, and also in the inner and outer regions of the periplasmic space . The cell surface DBP is release by treating the cells with EDTA . This protein can be surface labeled by lactoperoxidase radioiodination, and by diazo{125I}iodosulfanilic acid in whole cells . It also binds tightly, but not covalently, to lipopolysaccharide . The DBP located in the outer region of the periplasmic space is released when the outer membrane is dissociated by EDTA-osmotic shock treatment . The DBP located in the inner region of the periplasmic space is released only when the EDTA-osmotic shocked cells are subjected to lysozyme treatment . At the moment, it is not certain whether this protein is bound to or trapped by the peptidoglycan network . This protein cannot be surface labeled in whole cells or in EDTA-osmotic shock treated cells; and it is not associated with lipopolysaccharide . Analysis of transport mutants indicates that these DBP are coded by the same gene.

Can J Biochem, 1980 Oct, 58(10), 1172 - 8
The effect of amphipaths on the flavin-linked aerobic glycerol-3-phosphate dehydrogenase from Escherichia coli; Robinson JJ et al.; When assayed by the phenazine methosulphate coupled 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, the flavin-linked aerobic glycerol-3-phosphate dehydrogenase from Escherichia coli strain 7 showed a marked loss of activity if depleted of detergent, and this activity could be reconstituted with amphipaths . However, enzyme activity, as measured by the ferricyanide reduction assay, showed no loss of activity upon detergent depletion . Cross-linking studies indicated that amphipath was not inducing any significant quaternary structural change in the enzyme . Flavin fluorescence titration experiments indicated a single type of binding site for glycerol 3-phosphate whose affinity was unaffected by the presence of amphipath; apparent Kd values of 19.5 and 17.1 mM were measured in the presence and absence of amphipath, respectively . The Michaelis-Menten constants for DL-glycerol 3-phosphate, as measured by the phenazine methosulphate coupled MTT and ferricyanide reduction assays in the presence of amphipath, were found to be 1.9 and 10.2 mM, respectively . Cupric ions were found to specifically inhibit the phenazine methosulphate coupled MTT reducing activity while zinc ions inhibited the ferricyanide reducing activity . Circular dichroic studies provided initial evidence for a conformation change in the presence of the nonionic detergent Brij 58 and this was substantiated by quenching of tryptophan fluorescence at concentrations of Brij 58 equivalent to those needed to stimulate activity . We conclude from these studies that amphipaths may specificity induce a phenazine methosulphate binding site thus permitting reduction of this electron acceptor.

Infect Immun, 1980 Oct, 30(1), 231 - 43
Localization of electron-dense tracers during entry of Rickettsia tsutsugamushi into polymorphonuclear leukocytes; Rikihisa Y et al.; The invasion of Rickettsia tsutsugamushi, Gilliam strain, into guinea pig polymorphonuclear leukocytes (PMNs) and the localization and distribution of tracers were followed during the process by electron microscopy . The seven tracers used were: cationized ferritin, ferritin, thorium dioxide (ThO2), carbon particles, latex spheres, paraffin oil, and Escherichia coli . These markers were added to the incubation medium containing the PMNs before or simultaneously with R . tsutsugamushi-infected BHK-21 cells . Both morphologically intact and degenerating rickettsiae were present in the phagosomes in PMNs, but only the viable-appearing rickettsiae were free in the cytoplasm . The intact rickettsiae were singly and selectively phagocytized in tightly enclosed phagosomal membranes which usually excluded the tracers, except when ThO2 or ferritin was used . When ThO2, which labels the plasma membrane of PMNs, was used . ThO2-labeled phagosomal membranes enclosing rickettsiae were observed and short membrane fragments still labeled with this tracer were found in the vicinity of rickettsiae in the cytoplasmic matrix of PMNs . When ferritin or ThO2 was used as a tracer, some of the phagosomes contained rickettsiae still enclosed in an envelope of BHK-21 cytoplasm and cell membrane . Phagolysosomes preloaded with electron-dense markers fused with subsequently formed phagosomes containing degenerated rickettsiae but not with those containing intact rickettsiae . These results support our interpretation that viable rickettsial entry into PMNs is by selective phagocytosis and escape from these phagosomes.

Cancer Res, 1980 Oct, 40(10), 3508 - 11
Genetic factors in Escherichia coli that affect cell killing and mutagenesis induced by benzo(a)pyrene-7,8-dihydrodiol 9,10-oxide; Ivanovic V et al.; In the present study, the induction of base-pair reversion mutations from trp to Trp+ in the wild type and in uvrA, recA, and lexA mutants of Escherichia coli B/r was used to analyze the mechanism of benzo(a)pyrene-7,8-dihydrodiol 9,10-oxide (BPDE) mutagenesis . BPDE mutagenesis was compared to that of two well characterized mutagens: ethyl methanesulfonate and 4-nitroquinoline 1-oxide . We found that a uvrA mutant was about 20 times more sensitive to BPDE killing and BPDE-induced mutations than was wild-type E . coli . This suggests that BPDE-DNA lesions are removed by an excision repair mechanism similar to that which excises pyrimidine dimers . Although strains carrying either recA or lexA mutations were also more sensitive to BPDE killing, BPDE was not mutagenic in these strains . The recA+ and lexA+ gene products coordinately control the regulation of a set of genes that are expressed in response to DNA-damaging agents (SOS functions) . Therefore, our results indicate that in E . coli BPDE is an indirect mutagen that acts through host-mediated functions, i.e., the SOS pathway, in the process of mutation fixation . The possible significance of these findings to carcinogenesis are discussed.

J Natl Cancer Inst, 1980 Oct, 65(4), 759 - 68
Immunosuppressive factors from mastocytoma cells cultured in serum-free medium; Nakamura M et al.; Immunosuppressive factors were isolated from culture fluids of DBA/2 mouse mastocytoma cells grown in serum-free RPMI-1640 medium by measurement of inhibitory activity on tritiated thymidine uptake of DBA/2 spleen cells responding to Escherichia coli lipopolisaccharide (LPS) and concanavalin A (ConA) and by comparison of the number of hemolytic antibody-forming cells in vitro after simultaneous addition of the factors with sheep red blood cells (SRBC) . The culture fluids were separated into four fractions with immunosuppressive activities: F-I (mol wt, > 30,000), and F-II (mol . wt, 10,000-30,000), F-III (mol wt, 2,000-10,000), and F-IV (molwt, 700-2,000), F-IV specifically suppressed lymphocyte responses to E . coli LPS at concentrations of 50-200 microgram/ml . The other three fractions also showed immunosuppressive activities in both Con-A-induced and E . coli LPS-induced lymphocyte responses . The four fractions as well as crude culture fluids of mastocytoma cells expressed as immunosuppressive effect when injected into the peritoneal cavities of DBA/2 mice at doses of 24-180 microgram/day for 5 consecutive days . The fractions with a molecular weight equivalent to F-IV of mastocytoma culture supernatant were detected little, if any, in the supernatant from a nonmalignant cell culture . No suppressive effects of the fraction with a molecular weight greater than 2,000 (equivalent to F-I + F-II + F-III) from nonmalignant cell culture were found on lymphocyte responses to mitogens or SRBC at the concentrations used (100-200 microgram/ml).

J Bacteriol, 1980 Oct, 144(1), 291 - 9
In vitro formation of beta-galactosidase with a template containing the lac genes fused to gene ilvD; Noti JD et al.; An in vitro coupled transcription-translation system was used to synthesize transaminase B and beta-galactosidase in the presence of a deoxyribonucleic acid template containing lac deoxyribonucleic acid under normal lac-specific control and in the presence of several deoxyribonucleic acid templates containing lac deoxyribonucleic acid fused to the ilvD gene . Time course experiments revealed that transcription of the lacZ gene from the fusion template required a longer time than did that initiated at the lac promoter . With a phage template containing an intact ilvE gene but lacking the normal ilv-specific promoter, synthesis of ilvE message was completed before synthesis of lacZ message . A phage template that contained the normal ilv-specific promoter but from which part of ilvE had been deleted also allowed formation of beta-galactosidase . Three plasmids containing the ilv-lac fusion were also used as templates . Two plasmids that contained both an intact ilvE gene and the normal ilv-specific promoter required longer times for lacZ transcription but were more efficient templates than was a plasmid in which the ilv-lac fusion, the ilvE gene, and the contiguous non-specific ilvE promoter were inverted with respect to the normal ilv-specific promoter . beta-Galactosidase synthesis was stimulated by guanosine 3'-pyrophosphate-5'-pyrophosphate with all templates tested except that in which the ilv-lac fusion had been inverted . Presumptive evidence was obtained for the generation of a limiting isoleucine signal by incorporating inhibitors of isoleucyl transfer ribonucleic acid synthetase into the coupled transcription-translation system.

Biochim Biophys Acta, 1980 Oct 1, 632(2), 326 - 35
The interaction of the K88 antigen with porcine intestinal epithelial cell brush borders; Sellwood R; The interaction of 125I-labelled K88 antigen with brush borders of the epithelial cells of the pig small intestine has been studied . The iodinated antigen bound avidly to the brush borders prepared from adhesive (receptor-positive) pigs even after pretreatment of the brush borders with formaldehyde, whereas the brush borders from non-adhesive (receptor-negative) pigs failed to bind the antigen under these conditions . Treatment with glutaraldehyde rapidly destroyed the ability of both types of brush border to bind the K88 antigen . Studies on the binding of antigen to brush borders revealed the presence of high affinity receptors, but the non-linearity of the Scatchard plot could be explained by cooperative-like interactions, which view was supported by dissociation experiments . Rapid dissociation only in the presence of unlabelled K88 antigen suggested the existence of receptor site interactions of the negatively cooperative type . Attempts to inhibit the binding of 125I-labelled K88 with simple monosaccharides and oligosaccharides suggested that the binding of antigen to brush borders involves complex interactions and that galactosyl residues may be important.

Clin Exp Immunol, 1980 Oct, 42(1), 77 - 85
Immune complex glomerulonephritis in mice infected with Escherichia coli; Fournie GJ et al.; C57Bl/6 mice were injected intraperitoneally with 10(8) to 2 x 10(8) living K 38 Escherichia coli (E . coli) and serological changes and kidney involvement were studied . E . coli were found in the blood 45 min to 24 hr after injection . In serum, large amounts of deoxyribonucleic acid (DNA) were present 24 hr after E . coli injection, and thereafter disappeared . Seven days after infection, antibodies directed against E . coli, anti-DNA antibodies and C1q-binding substances were found in serum and the kinetics of the variations of these parameters were studied until day 35 . Kidney lesions were evaluated immunochemically and by optical and electron microscopy . In the glomeruli, heavy granular deposits of IgG and IgM were constantly found in mesangium and along capillary walls . In most kidneys slight granular deposits of IgG and IgM were also found in the tubules . Histological studies revealed in the glomeruli mild endocapillary cell proliferation, focal thickening of glomerular basement membrane and dense deposits in mesangial and subendothelial areas and inside the glomerular basement membrane; in the tubules dense deposits were focally observed inside the tubular basement membrane.

Gene, 1980 Oct, 11(1-2), 173 - 5
An easy method for the selection of restriction- and modification-deficient mutants of Escherichia coli K-12; Piechaczyk M et al.; An easy and rapid method for selecting restriction- and modification-defective mutants of Escherichia coli K-12 is described . This method employs selection of tetracycline resistant lysogens after infection with lambda::Tn10 phage and results in a high yield of spontaneous rk-mk- and rk-mk+ mutants.

Cell, 1980 Oct, 21(3), 709 - 15
Molecular cloning and selection of genes regulated in Aspergillus development; Zimmermann CR et al.; Over 350 clones homologous to poly(A)+ RNAs that are significantly more prevalent in conidiating cultures of Aspergillus nidulans than in somatic cells have been selected from a recombinant DNA library formed between nuclear DNA and lambda Charon 4A . The procedure used for this selection involved in situ hybridization to a cDNA probe which had been selectively depleted of sequences represented in somatic cells by complement hybridization . Five of these clones have been characterized further . All but one encoded poly(A)+ RNAs that were at least ten times more prevalent in conidiating cultures than in somatic cells . One clone hybridized to a single, developmentally regulated RNA . The three others were complementary to several RNAs having different molecular weights, each of which was more prevalent in condiating cultures than in vegetative cells . These results and quantitative aspects of the selection procedure suggest that developmentally controlled poly(A)+ RNA coding regions may not be distributed randomly in the Aspergillus genome.

J Clin Invest, 1980 Oct, 66(4), 621 - 8
Suppressor cell regulation of cell-mediated immune responses in renal infection in vitro modulation of suppressor cell activity; Miller T et al.; Infection-induced anergy is a frequent complication of bacterial, viral, and parsitic infection . A marked suppression of the thymus-derived (T) lymphocyte response to concanavalin A has been demonstrated in vitro during renal infection and the mechanisms by which suppression occurs have been investigated . In particular we have considered the possibility that suppression might result from the inhibitory effect of prostaglandins, secreted by activated macrophages with immunoregulatory potential . The experiments have shown that the T-lymphocyte effector status in experimentally-induced renal infection is determined by two suppressor cells, one infection-induced and the other naturally occurring . The inability to respond to mitogenic stimulation was reversible and restoration of immune responsiveness to splenic lymphocytes from infected animals could be achieved in two stepwise manipulations; differential centrifugation removed the infection-induced suppressor cells, and the suppressor activity of the naturally occurring suppressor cells could then be inhibited by indomethacin . Thus the two suppressor cells were distinguishable on the basis of their physical characteristics and their response to indomethacin . The dominant factor determining the immune responsiveness of splenic lymphocytes from the pyelonephritic animals was, however, the infection-induced suppressor cell . This cell has been characterized as a sedimentable cell (30 g) with suppressor activity demonstrable in co-culture experiments . Plastic-adherent cells from the sedimentable fraction of pyelonephritic animals' splenic cells were shown to have suppressor activity that was not inhibited by indomethacin . The infection-induced and naturally occurring suppressor cells can be viewed as prototypes for the equivalent cells in man and may be useful models for studying the role of these cells as determinants in the pathogenesis of infectious disease.

Biochim Biophys Acta, 1980 Oct, 615(2), 489 - 96
Murein transglycosylase from phage lambda lysate . Purification and properties; Bienkowska-Szewczyk K et al.; Lysates of induced E . coli (lambda) lysogens contain two enzymes acting on murein: endopeptidase and murein transglycosylase . The transglycosylase was separated from the endopeptidase and purified to homogeneity . Its bacteriolytic activity was 200-fold higher than of hen egg lysozyme . The bacteriolytic activity of the lysate depends on the presence of the enzyme . The endopeptidase alone not lyse the cells, but it enhances the extent of lysis . The properties of the transglycosylase (molecular weight 17 500, pH optimum at 6.6, inactivation by Zn2+), show that it is entirely different from the bacterial enzyme of the same specificity described by others . Data are presented, which suggest that this enzyme is the phage lambda R-gene product.

Biokhimiia, 1980 Oct, 45(10), 1909 - 12
{Localization and restriction mapping of the origin of replication of rat liver mitochondrial DNA}; Tomarev SI et al.; Based on plasmid pCV II, a recombinant comprising the origin of replication of rat liver mitochondrial DNA was obtained . A detailed restriction map of the region based on restrictases Hae III, Hpa II and Hind II was developed . The origin of mitochondrial DNA replication is localized in one of Hind II fragments.

J Gen Microbiol, 1980 Oct, 120(2), 475 - 83
Biochemical and genetic characterization of nirB mutants of Escherichia coli K 12 pleiotropically defective in nitrite and sulphite reduction; Cole JA et al.; Mutants of Escherichia coli K12 defective in the nirB gene lack NADH-dependent nitrite reductase activity and reduce nitrite slowly during anaerobic growth . With one exception these mutants require cysteine for growth . Cytochrome C552 synthesis and the assimilation of ammonia are unaffected by the nirB mutation . The defective gene is located between the crp and aroB genes at minute 73 on the E . coli chromosome . Mapping and reversion studies indicate the nirB is identical to the previously described cysG gene . It is suggested that the product of the cysG+ (nirB+) inverted question markgene is an enzyme required for the synthesis of sirohaem, a prosthetic group of enzymes which catalyse the six-electron reduction of nitrite to ammonia and sulphite to sulphide.

Eur J Biochem, 1980 Oct, 111(2), 419 - 23
Molecular cloning of bovine thyroglobulin complementary DNA . Characterization of 2500-base-pair and 1900-base-pair fragments; Christophe D et al.; Double-stranded thyroglobulin complementary DNA (cDNA) was synthesized from purified 33-S bovine thyroglobulin mRNA . This synthetic structural gene has previously been shown to contain three sites for the restriction endonuclease HindIII, yielding two internal fragments of 1900 and 2500 base pairs respectively . Recombinant molecules were prepared by ligating the HindIII-restricted cDNA to the plasmid pBR322 which had been linearized by the same enzyme . When Escherichia coli was transformed with this mixture, it yielded two kinds of colonies each harboring recombinant plasmids containing one of the two cDNA fragments . Both recombinant molecules hybridized specifically to translatable thyroglobulin mRNA . Sequence homology between the two cloned DNAs could not be detected by cross-hybridization experiments; this argues against the existence of internal structural repetition in thyroglobulin subunits . Together, the two cloned DNA fragments represent 55% of the 8000-base-pair double-stranded thyroglobulin DNA.

Proc Natl Acad Sci U S A, 1980 Oct, 77(10), 5799 - 801
Catabolite repression in Escherichia coli mutants lacking cyclic AMP receptor protein; Guidi-Rontani C et al.; Pleiotropic carbohydrate-positive pseudorevertants have been isolated from a specific class of rho-crp double mutants of Escherichia coli carrying both defective transcription termination protein, rho, and cyclic AMP receptor protein . The modulation of catabolite repression of beta-galactosidase, amylomaltase, and tryptophanase has been studied in the pseudorevertants . It has been found that these mutants exhibit catabolite repression . Because catabolite-sensitive operons can be expressed in the absence of functional cyclic AMP receptor protein, this would suggest on the one hand that the cyclic AMP-receptor protein complex is not the exclusive mediator of catabolite repression and on the other hand that rho might be involved in the regulation of catabolite-sensitive operons.

J Virol, 1980 Oct, 36(1), 125 - 32
Expression of the gene for the polyoma small T antigen in Escherichia coli; Horwich A et al.; A cloned segment of the polyoma virus genome encoding the small T antigen has been fused, in the correct phase for translation, to the 5' end of the beta-galactosidase gene . The hybrid gene, cloned in Escherichia coli, produces a protein resembling the small T antigen.

Gene, 1980 Oct, 11(1-2), 169 - 71
A cautionary note on the use of certain restriction endonucleases with methylated substrates; Backman K; Methylation of adenine and cytosine residues in DNA isolated from common strains of Escherichia coli K-12 can render that DNA resistant to cleavage by certain restriction endonucleases at those sites at which the recognition sequence for such an endonuclease overlaps (but does not include) a sequence recognized by methylases specified by the dam or dcm gene.

J Bacteriol, 1980 Oct, 144(1), 346 - 55
Identification of a second tetracycline-inducible polypeptide encoded by Tn10; Zupancic TJ et al.; Three Tn10 polypeptides were detected by analyzing the proteins synthesized in ultraviolet light-irradiated Escherichia coli cells after infection with lambda::Tn10 . One of these polypeptides was the previously identified 36,000-dalton TET polypeptide . The other two had approximate sizes of 25,000 and 13,000 daltons . The syntheses of both the TET polypeptide and the 25,000-dalton polypeptide were inducible by tetracycline in lambda-immune hosts . Similarly, the synthesis of the TET polypeptide was inducible in nonimmune hosts . However, the synthesis of the 25,000-dalton polypeptide was constitutive in nonimmune hosts . An amber mutation in a gene required for tetracycline resistance on lambda::Tn10 was isolated that eliminated the synthesis of the TET polypeptide in sup+ hosts but not the synthesis of the 25,000-dalton or the 13,000-dalton polypeptides . The expression of tetracycline resistance from wild-type Tn10 was found to be anomalous in E . coli strains carrying the amber suppressors supD, supE, and supF . In general, strains containing these nonsense suppressors were less resistant to tetracycline.

J Bacteriol, 1980 Oct, 144(1), 279 - 90
Regulation of ilvEDA expression occurs upstream of ilvG in Escherichia coli: additional evidence for an ilvGEDA operon; Subrahmanyam CS et al.; A low-copy-number plasmid was prepared that contained the entire ilv gene cluster of Escherichia coli . The introduction of an ilvO mutation allowed the ilvG gene of the plasmid to be expressed and imparted valine resistance to strains carrying it . Insertion of Tn10 into the ilvG gene of the plasmid resulted in a strong polar effect on ilv genes E, D, and A . Replacement of a region of ilv deoxyribonucleic acid between two KpnI sites on the high-copy-number plasmid carrying the entire ilv gene cluster with a KpnI fragment carrying an ilv-lac fusion but not extending into the ilv-specific control region resulted in a plasmid expressing the lacZ gene under ilv control when the fusion had been inserted in its normal orientation but not when it had been inserted in the opposite orientation . These experiments indicate that ilv-specific control over ilvE, ilvD, and ilvA expression is dependent on these genes being continguous with deoxyribonucleic acid that lies upstream of ilvG . The results also add further support to the concept of an ilvGEDA operon in E . coli.

J Bacteriol, 1980 Oct, 144(1), 222 - 31
Physical and functional mapping of RP4-TOL plasmid recombinants: analysis of insertion and deletion mutants; Nakazawa T et al.; Cleavage sites for the restriction endonucleases XhoI, BamHI, HindIII, and EcoRI were mapped on the pTN2 plasmid, a recombinant of TOL and RP4, which specifies the toluene-degrading enzymes in the same way as the wild-type TOL plasmid . The pTN2 plasmid, purified from a strain of Escherichia coli, contained the entire length of the RP4 plasmid (about 54 kilobase pairs {kb}) and the TOL segment (about 56 kb) . The TOL segment is inserted at about 12 and 5 kb away from the EcoRI and BamHI cleavage sites of RP4, respectively . Cleavage sites for XhoI, BamHI, HindIII, and EcoRI were also mapped on an insertion mutant, pTN1, and two deletion mutants, pTN81 and pTN9 . Analysis of pTN81 and pTN9 allowed estimation of the region of the gene cluster for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, as well as the region required for toluate oxygenase activity . Induction of TOL enzymes directed by pTN1 suggested the location and orientation of transcription of the gene cluster for catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase . Analysis of strains carrying both pTN9 and a xylR mutant of the TOL plasmid demonstrated that xylR+ is trans dominant over xylR.

J Bacteriol, 1980 Oct, 144(1), 205 - 9
Suppression of defects in cyclic adenosine 3',5'-monophosphate metabolism in Escherichia coli; Alexander JK; Strain MM6-13 (ptsI suc lacI sup) of Escherichia coli contains a suppressor of the succinate-negative phenotype . In MM6-13, sup caused enhanced growth in glycerol, maltose, melibiose, and succinate media and increased activity of beta-galactosidase and tryptophanase relative to an isogenic strain without sup . In strain A61 (cya sup), sup partially suppressed cya . Cyclic guanosine monophosphate increased beta-galactosidase activity sevenfold in A61 and enabled this strain to grow on maltose, galactose, succinate, and arabinose . Strain A61 responded to much lower concentrations of cyclic adenosine monophosphate than cyclic guanosine monophosphate . It appears that sup is located in the crp locus . These results suggest that sup mutants have an altered cyclic adenosine monophosphate receptor protein which is activated by cyclic guanosine monophosphate and has an increased affinity for cyclic adenosine monophosphate.

J Bacteriol, 1980 Oct, 144(1), 131 - 40
Insertion element IS102 resides in plasmid pSC101; Ohtsubo H et al.; In vivo recombination was found to occur between plasmid pHS1, a temperature-sensitive replication mutant of pSC101 carrying tetracycline resistance, and plasmid ColE1 after selection for tetracycline resistance at the restrictive temperature, 42 degrees C . Extensive analysis of the physical structures of three of these recombinant plasmids, using restriction endonucleases and the electron microscope heteroduplex method, revealed that the plasmid pHS1 was integrated into different sites on ColE1 . The recombinant plasmids contained a duplication of a unique 1-kilobase (kb) sequence of pHS1 in a direct orientation at the junctions between the two parental plasmid sequences . This was confirmed by comparing the nucleotide sequence of the recombinants and their parental plasmids . Nucleotide sequence analysis further revealed that nine nucleotides at the site of recombination of ColE1 were duplicated at the junction of each of the 1-kb sequences . The formation of recombinants was independent of RecA function . Based on our previous finding that a plasmid containing a deoxyribonucleic acid insertion (IS) element can recombine with a second plasmid to generate a duplication of the IS element, we conclude that the 1-kb sequence is an insertion sequence, which we named IS102 . For convenience, we have also denoted the IS102 sequence as eta theta to assign the orientation of the sequence . Eighteen nucleotides at one end (eta end) were found to be repeated in an inverted orientation at the other end (theta end) of IS102 . The nucleotide sequence of the eta end of the sequence was found to be identical to the sequence at the ends of the transposon Tn903, which is responsible for transposition of the kanamycin resistance gene.

J Bacteriol, 1980 Oct, 144(1), 468 - 72
Macromolecular syntheses in an Escherichia coli adk mutant; Suit JL et al.; Preferential inhibition by high temperatures of synthesis of newly induced enzymes in Escherichia coli K-12 CR341T28 adk is only apparent; syntheses of all macromolecules cease simultaneously.

J Membr Biol, 1980 Sep 30, 56(2), 169 - 75
Membrane transport of p-nitrophenyl-alpha-galactoside by the melibiose carrier of Escherichia coli; Ottina K et al.; p-Nitrophenyl-alpha-galactoside (alpha-pNPG) was found to be a substrate for the melibiose transport system of Escherichia coli . This sugar enters induced cells via the carrier and is split by alpha-galactosidase to galactose and p-nitrophenol . In mutant cells lacking the alpha-galactosidase {3H}-alpha-pNPG accumulated to concentrations 15 times higher than the external medium . The transport of alpha-pNPG is inhibited by both Na+ and Li+ . Na+ (10 mM) reduced the Km for alpha-pNPG from 0.45 to 0.18 mM and reduced the Vmax from 6.7 nmoles/min/mg dry wt to a value of 3.0.

Biochemistry, 1980 Sep 30, 19(20), 4627 - 32
Methylation of chemotaxis-specific proteins in Escherichia coli cells permeable to S-adenosylmethionine; Rollins CM et al.; Using a modification of the EGTA treatment of Oishi and Smith {Oishi, M., & Smith, C . L . (1978) Proc . Natl . Acad . Sci . U.S.A . 75, 3569}, Escherichia coli cells have been made permeable to S-adenosylmethionine and other related molecules in order to facilitate the study of methylation in chemotaxis . The permeable cells are nonmotile but respond to chemotactic stimuli by reversible methylation of their methyl-accepting chemotactic proteins (MCP I and MCP II) in a manner similar to that of untreated, motile cells . Addition of S-adenosyl-L-{methyl-3H}methionine to the permeable cells specifically labels two proteins, MCP I and MCP II . Methylation of these MCP's is dependent on the presence of wild-type gene products of flaI, flaA, cheB, cheX, tsr, and tar . The extent of methylation of the MCP's is affected by the presence of attractants or repellents: addition of attractant increases the steady-state level of methylation; addition of repellent causes rapid demethylation to a new steady-state level . Methylation is inhibited by the addition of the transmethylase inhibitors A9145C and Sinefungin, which are S-adenosylmethionine analogues, and by S-adenosylhomocysteine.

Biochemistry, 1980 Sep 30, 19(20), 4633 - 9
Chemotaxis in Escherichia coli: associations of protein components; Chelsky D et al.; Interactions between protein components of the chemotaxis mechanism in Escherichia coli were investigated by using the cleavable cross-linking reagent, dithiobis(succinimidyl propionate) . Two methods were used to allow detection of chemotaxis-specific proteins in intact cells . The first method was to program their synthesis in the presence of {35S}methionine using lambda E . coli hybrid phages which carry the chemotaxis genes . The second method was to label endogenous methyl-accepting chemotaxis proteins (MCP's), with the methyl donor S-adenosyl-L-{methyl-3H}methionine, after permeabilizing the cells with EGTA . Physical associations between proteins were analyzed, after cross-linking, by two dimensional NaDodSO4-polyacrylamide gel electrophoresis . Both labeling methods demonstrate that MCP I and MCP II exist as functional tetramers . Other proteins involved with chemotaxis were found to form dimers and higher polymers . Phage-directed products of cheW, cheX, motA, and cheA formed dimers . CheB and hag products formed multimers . A number of apparent interactions between different gene products were detected as well . Products of cheB, cheW, cheZ, motA, and motB were found to form complexes with other gene products . Included are results consistent with interactions between the products of cheB and cheZ.

Proc R Soc Lond B Biol Sci, 1980 Sep 26, 209(1176), 431 - 9
The early pathogenesis of bovine mastitis due to Escherichia coli; Frost AJ et al.; The pathogenesis of coliform mastitis was studied after infusing each of ten lactating quarters of three dairy cows with a large dose (ca . 1 x 10(9) colony-forming units) of virulent Escherichia coli strain B117 . This approach was adopted first to maximize the chance of observing microscopic lesions in the tissues of a gland and secondly to overwhelm the differences that might be shown between animals in their response to the infection . The infected glands were examined at intervals of up to 4 h after infection by scanning and transmission electron microscopy and by light microscopy . The earliest lesions were seen after 1 h and consisted of necrosis and sloughing of the epithelial cells of the teat and lactiferous sinuses . After 2 h this was more severe, and was followed by an intense neutrophil response . Neutrophils migrated through the epithelial lesions and at first remained attached to the epithelial surface, forming large mounds . This resulted in gross underestimation of the number of cells in the lumen of the gland when neutrophils in the secretion were counted . At no stage was there evidence of attachment of organisms to the epithelial cells . Tissue damage did not extent beyond the basement membrane, which helps to explain the rapid clinical resolution seen in most field cases of the disease . There was considerable variation in the degree of response shown by the three cows, and also within the infected glands, where the damage was most severe in the lactiferous and teat sinuses . It seems unlikely that all aspects of the disease could be attributed to endotoxin.

Nucleic Acids Res, 1980 Sep 25, 8(18), 4201 - 19
In vitro transcription of chromatin containing histones hyperacetylated in vivo; Dobson ME et al.; The culture of cells in the presence of sodium n-butyrate causes an accumulation of histones that are highly acetylated . When chromatin containing these histones was transcribed with E . coli RNA polymerase, an increase in the template activity compared to control chromatin was observed . Titration of chromatin with polymerase under both reinitiating and non-reinitiating conditions showed there was no increase in the number of regions available for transcription . Comparison of the kinetics for single and multiple rounds of transcription indicated that the rate of elongation was increased and probably the rate of reinitiation as well . Comparison of the size of transcripts from control and acetylated chromatin showed a small increase in the average size of transcripts from acetylated chromatin . When transcription was compared using partially purified HeLa polymerase, an increase was also seen . Studies under various ionic conditions showed that control chromatin required a higher salt concentration for optimum activity than did acetylated chromatin . In addition, at the optimum salt concentration for each chromatin, there was very little difference in the transcriptional activity using exogenous HeLa RNA polymerase.

J Biol Chem, 1980 Sep 25, 255(18), 8431 - 6
Transient kinetic analysis of the catalytic cycle of alkaline phosphatase; Bale JR et al.; Transient kinetic studies were carried out to elucidate the catalytic cycle of Escherichia coli alkaline phosphatase at ph 8.3, 10 degrees C, and to evaluate the rate constants for the individual steps . Using a rapid mixing cell, we were able to detect the burst phase of the reaction, which could not be obtained at alkaline ph values with conventional mixing devices . Analysis of the burst phase revealed that the equilibrium of the initial binding of the substrate, 4-methylumbelliferyl phosphate, to the enzyme is attained rapidly, and that the production of the alcohol, 4-methylumbelliferone, is fast . Analysis of the steady state phase of the reaction yielded a phosphate release rate constant which agrees very well with the kcat determined by initial rate studies . Dephosphorylation of the phosphoryl enzyme prepared at pH 5.7 was studied by the pH-jump technique, using a three-syringe stopped flow apparatus . The results showed that the dephosphorylation step is not rate-limiting in the catalytic cycle and that the presence of substrates or inhibitor has no effect on this step . The lack of effect of substrates on the rate of dephosphorylation and on the rate of phosphate dissociation indicates that the flip-flop mechanism, in which the product release is supposedly facilitated by the binding of a 2nd molecule of substrate, is not valid for alkaline phosphatase . Kinetic constants for various steps in the catalytic cycle of alkaline phosphatase at pH 8.3, 10 degrees C, are reported and a reaction scheme which is in harmony with previous observations from other laboratories is described.

J Biol Chem, 1980 Sep 25, 255(18), 8366 - 9
Identification of a cytoplasmic membrane-associated component of the maltose transport system of Escherichia coli; Bavoil P et al.; The maltose transport system of Escherichia coli contains at least five components, three of which, i.e . the products of lamB, malE, and malF genes, have so far been identified as constituents of the outer membrane, periplasmic space, and cytoplasmic membrane, respectively . We identified another component, a cytoplasmic membrane protein of an apparent molecular weight of 43,000, as the product of the malK gene on the basis of polyacrylamide gel electrophoretic analysis of various mutants and suppressed strains and by the incorporation of extra tyrosine residue into this proten in malK amber mutants containing the suppressor Su3+ allele . The transport of maltose thus appears to require at least two proteins associated with the cytoplasmic membrane.

J Biol Chem, 1980 Sep 25, 255(18), 8706 - 10
Methylation of newly synthesized ribosomal protein L11 in a DNA-directed in vitro system; Jerez C et al.; The methylation of newly synthesized ribosomal protein L11 has been obtained in an in vitro system using lambda rifd 18 DNA as template and S-adenosyl{3H}methionine as methyl donor . About four methyl groups are incorporated per mol of L11 synthesized and the bulk of the methyl groups are present in the protein as trimethyl-lysine . lambda rifd 18 DNA also contains the genes for rRNA and newly synthesized 16 S and 23 S RNA are also methylated in this in vitro system.

Nucleic Acids Res, 1980 Sep 25, 8(18), 4235 - 46
Involvement of DNA gyrase in the transcription of ribosomal RNA; Oostra BA et al.; The DNA gyrase inhibitor novobiocin specifically inhibits the transcription of ribosomal RNA in vivo while protein synthesis and the mRNA transcription are only partly affected . In vitro the novobiocin inhibition is only observed when protein fraction, which stimulates ribosomal RNA synthesis, is present . These results indicate that DNA gyrase is involved in the transcription of ribosomal RNA, probably at an initiation step.

Nucleic Acids Res, 1980 Sep 25, 8(18), 4165 - 84
Equilibrium melting of plasmid ColE1 DNA: electron-microscopic visualization; Borovik AS et al.; The fine structure of the melting curve for the linear colE1 DNA has been obtained . To find the ColE1 DNA regions corresponding to peaks in the melting curve's fine structure, we fixed the melted DNA regions with glyoxal /12/ . Electron-microscopic denaturation maps were obtained for nine temperature points within the melting range . Thereby the whole process of colE1 DNA melting was reconstructed in detail . Spectrophotometric and electron microscopic data were used for mapping the distribution of Gc-pairs over the DNA molecule . The most AT-rich DNA regions (28 and 37% of GC-pairs), 380 and 660 bp long resp., are located on both sides of the site of ColE1 DNA's cleavage by EcoR1 endonuclease . The equilibrium denaturation maps are compared with maps obtained by the method of Inman /20/ for eight points of the kinetic curve of ColE1 DNA unwinding by formaldehyde.

J Biol Chem, 1980 Sep 25, 255(18), 8928 - 35
Cloning of fragments of lambda dapB2 DNA and identification of the dapB gene product; Mackie GA; DNA of the specialized transducing phage lambda dapB2 has been digested with the restriction endonucleases Bam I, HindIII, or both together to generate fragments originating from the bacterial substitution on the phage . Seven such fragments ranging in size from 0.8 to 7.1 kilobases and encompassing the entire bacterial substitution of 18 kilobases of DNA have been covalently ligated to the vector pBR322 . The recombinant plasmids so constructed have been tested for their ability to complement the dapB17 allele in Escherichia coli strain AT999 . Only pGM4, which contains a 7.1 kilboase fragment generated by Bam I cleavage of lambdadapB2 inserted into pBR322, relieves this strain's requirement for DL-diaminopimelic acid and restores dihyrodipicolinic acid reductase activity to wild type levels . Deletions were obtained in pGM4 by two methods . None of the resultant shortened plasmids were proficient in complementation of the dapB17 allele . The proteins encoded by the parental plasmid and by those of seven deletions derived from it have been identified by coupled transcription and translation of plasmid DNA templates in vitro or by the stimulation of protein synthesis promoted by these plasmids in an ultraviolet irradiated host . The parent encodes four proteins unique to the 7.1 kilobase insert whose apparent molecular weights are 48,000, 36,000, 32,000, and 17,000 . Of these, the protein of 32,000 is consistently missing when noncomplementing pasmids harboring deletions are used as templates . This protein is tentatively identified as the product of the dapB gene . The role of the other three proteins whose genes are closey linked to the dapB gene is unknown . There appear to be at least two transcriptional units within this cluster of genes, however, suggesting independent regulation and, possibly, function.

J Biol Chem, 1980 Sep 25, 255(18), 8655 - 62
Structure of the rat prolactin gene; Gubbins EJ et al.; The organization and sequence of the rat preprolactin gene has been investigated . Analysis of two different plasmids containing pituitary cDNA inserts has provided the complete 681-nucleotide coding sequence of preprolactin as well as 17 nucleotides preceding the initiation codon and 90 nucleotides following the termination codon . Digestion of rat chromosomal DNA with the restriction endonuclease Eco RI followed by size fractionation and hybridization to a labeled prolactin cDNA probe has demonstrated that prolactin genomic sequences are located on 6.0-, 3.9-, and 2.9-kilobase fragments . The 6.0- and 3.9-kilobase fragments were isolated from a library of cloned rat DNA fragments . The sequence of more than 1800 nucleotides of the cloned DNA has been determined . The sequenced region contains coding regions of 180 and 189 nucleotides which specify the COOH-terminal 123 amino acids of the 227-amino-acid sequence of rat preprolactin . These coding regions are separated by an intervening sequence of 597 nucleotides . At least one other large intervening sequence separates this region from the region coding for the NH2-terminal portion of preprolactin . Hybridization experiments suggested that the intervening sequences of the rat prolactin gene contain DNA sequences which are repeated elsewhere in the rat genome.

J Biol Chem, 1980 Sep 25, 255(18), 8623 - 8
Characterization of yeast iso-1-cytochrome c mRNA; Boss JM et al.; The iso-1-cytochrome c mRNA has been identified by hybridization of a 32P probe prepared from a plasmid containing the iso-1-cytochrome c gene to RNA size-fractionated on agarose gels and transferred to paper . A hybridization band was visible with RNA prepared from wild type cells, but not with RNA prepared from an iso-1-cytochrome c deletion mutant . RNA prepared from cells containing a nonsense mutation in the iso-1-cytochrome c gene showed reduced levels of hybridization . The RNA that hybridized to the probe was 700 +/- 50 nucleotides in length and was polyadenylated . The cellular levels of this RNA were repressed by glucose, and this repression was achieved within 5 min after glucose addition to a derepressed culture . No precursors of this RNA were detected in wild type cells or in an RNA1 mutant, temperature-sensitive for RNA metabolism . The length of the 3' noncoding region of this RNA was determined to be 200 +/- 25 nucleotides (excluding the poly(A) tail) and the 5' noncoding region was estimated to be about 120 nucleotides in length.

Nucleic Acids Res, 1980 Sep 25, 8(18), 4131 - 42
The role of the basic N-terminal region of protein L18 in 5S RNA-23S RNA complex formation; Newberry V et al.; Of the three proteins, L5, L18 and L25, which bind to 5S RNA, the former two effect the interaction of 5S RNA with 23S RNA . We have used trypsin as a probe to investigate the roles of the proteins in this RNA-RNA assembly, with the following results: (1) In complexes with 5S RNA, the highly basic N-terminal region of L18 is accessible to trypsin . This accessibility is unaffected by L25 . However, its presence is essential for stimulating L5 binding . (2) In 5S RNA-protein-23S RNA complexes proteins L5 and L18 are both strongly resistant to proteolysis . (3) No 5S RNA-23S RNA complex formation occurs in the presence of L5 and the C-terminal L18 fragment . Two possible models for the mechanism of RNA-RNA assembly are proposed.

Biochim Biophys Acta, 1980 Sep 19, 609(2), 313 - 20
A circular dichroism study of Escherichia coli Initiation Factor-1 binding to polynucleotides; Schleich T et al.; Binding of Escherichia coli Initiation Factor-1 protein to the nucleic acid lattice induces alterations in the secondary structures of a variety of purine and pyrimidine containing polynucleotides in both the single and double stranded conformations, as assessed by circular dichroism spectroscopy . The helical hairpin form of poly(U), the single-stranded stacked form of poly(C), and the duplex poly(A) x poly(U) (in the presence of Mg2+) are stoichiometrically converted by Initiation Factor-1 (IF-1) to structures spectrally indistinguishable from their partially or completely thermally denatured forms . By contrast, the binding of IF-1 to double stranded poly(C), single- and double-stranded poly(A) elicited spectral responses which were interpreted in terms of diminished base-base interaction, not equivalent to that induced by thermal means . Stoichiometric endpoints of 3-5 nucleotide residues/IF-1 were determined for polynucleotide structures in those cases where light scattering artifacts at low nucleotide residue to protein ratios were absent . In the absence of Mg2+ IF-1 was unable to elicit a conformation alteration effect in poly(A) x poly(U), while for poly(U) much less of an effect was observed than in the presence of this divalent ion . The functional significance of these results is briefly considered.

Biochim Biophys Acta, 1980 Sep 19, 609(2), 264 - 71
Specificity of the spermidine requirement for the replication of phi X174 DNA be cell-free extracts of Escherichia coli; Geiger LE et al.; A new experimental approach for assessing the biological significance of spermidine interactions in isolated systems is applied to the stimulation by spermidine of the conversion of phi X174 virion DNA to its replicative form by cell-free extracts of Escherichia coli . At 2.5 mM Mg2+, spermidine activated the reaction 20-fold . Varying the spermidine concentration affected both the rate and extent of this DNA synthetic reaction without altering the nature of the reaction products . We evaluated the biological significance of the spermidine requirement by measuring reaction rates in the presence of a homologous series of spermidine analogs of known activity in vivo . There was a lack of specificity, in that all of these analogs were capable of efficiently substituting for spermidine in stimulating the reaction rate . The relevance of this in vitro spermidine stimulation to Escherichia coli chromosome replication in vivo is discussed in light of the results obtained with the spermidine analogs.

Science, 1980 Sep 19, 209(4463), 1343 - 7
At least three human type alpha interferons: structure of alpha 2; Streuli M et al.; The sequence of a human leukocyte-derived complementary DNA (cDNA), Hif-2h, which directs the formation in Escherichia coli of a polypeptide, IFN-alpha 1, with interferon (IFN) activity has been described . A second IFN cDNA, Hif-SN206, which also elicits synthesis of a biologically active IFN, IFN-alpha 2, is described in this article . Whereas IFN-alpha 2 is twice as active on human as on bovine cells, IFN-alpha 1 is 10 to 20 times more active on bovine than on human cells . As deduced from the cDNA's, the messenger RNA's for the two IFN's differ in length and in 20 percent of the nucleotides; the mature IFN polypeptides differ in 17 percent of the amino acids . Both IFN-alpha 1 and IFN-alpha 2 differ from the lymphoblastoid IFN described by others . Therefore, at least three different IFN-alpha genes are expressed in man; studies on genomic DNA reveal the presence of at least eight IFN-related genes.

Nature, 1980 Sep 18, 287(5779), 203 - 8
DNA N-glycosylases and UV repair; Demple B et al.; Repair of some DNA photoproducts can be mediated by glycosylic bond hydrolysis . Thus, Escherichia coli endonuclease III releases 5,6-hydrated thymines as free bases, while T4 UV endonuclease releases one of two glycosylic bonds holding pyrimidine dimers in DNA . In contrast, uninfected E . coli apparently does not excise pyrimidine dimers via a DNA glycosylase.

Biochim Biophys Acta, 1980 Sep 17, 632(1), 35 - 46
Mode of action of heat-stable Escherichia coli enterotoxin . Tissue and subcellular specificities and role of cyclic GMP; Rao MC et al.; Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E . coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) . We have examined the tissue spcificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP . Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP concentration throughout the small and large intestine . It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon . This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP . However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments . The enterotoxin failed to stimulate guanylate cyclase inliver, lung, pancreas or gastric antral mucosa . In the intestines, it stimulated only the particulate and not the soluble form of the enzyme . Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase . Cytosol factors did not enhance the toxin's stimulation of intestinal guanylate cyclase . This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines.

Biochemistry, 1980 Sep 16, 19(19), 4527 - 33
Mechanism of reconsitution of the apo beta 2 subunit and the alpha 2 apo beta 2 complex of tryptophan synthase with pyridoxal 5'-Phosphate: kinetic studies; Bartholmes P et al.; The mechanism of pryidoxal 5'-phosphate (PLP) binding to both the alpha apo beta 2 complex and the apo beta 2 subunit of tryptophan synthase was investigated by rapid mixing experiments . Absorption and fluorescence changes were used to monitor the binding reaction directly . Reduction with sodium borohydride provided the rate of formation of the internal aldimine with the lysine amino group of the enzyme, and substrate turnover monitored the rate of formation of acive enzyme . The alpha 2 apo beta 2 complex binds PLP in a sequence of three steps of decreasing rate: formation of a noncovalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by formation of an active alpha 2 holo beta 2 complex . The two binding sites appear to bind PLP independently . The apo beta 2 subunit binds PLP cooperatively in a sequence of three steps of decreasing rate: formation of a nonconvalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by the formation of the enzymically active holo beta 2 subunit . Taken together with kinetic studies of pyridoxine phosphate binding {Tschopp, J., & Kirschner, K . (1980) Biochemistry (second paper of three in this issue)}, the rate data of the apo beta 2 subunit are shown to be consistent with the concerted mechanism . The difference between the values of the isomerization rate constants of bound PLP and bound PNP appear to result from the convalent internal aldimine, which is formed with PLP but not with PNP.

Biochemistry, 1980 Sep 16, 19(19), 4514 - 21
Subunit interactions of tryptophan synthase from Escherichia coli as revealed by binding studies with pyridoxal phosphate analogues; Tschopp J et al.; An improved purification procedure for the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been developed . It consists of DEAE-Sephacel chromatography, followed by hydrophobic chromatography on Sepharose CL 4B, and leads to material with a higher specific activity than reported previously . Inhibition studies, equilibrium dialysis, and spectrophotometric titration were used to study the binding both of pyridoxal phosphate analogues and of bisubstrate analogues . Pyridoxine 5'-phospate and N-phosphopyridoxyl-L-serine bind to the enzyme, but pyridoxamine 5'-phoshate and N-phosphopridoxyl-L-alanine do not . N-Phosphopyridoxyl-L-tryptophan is bound only weakly, although L-tyrptophan binds strongly to the alpha 2 holo beta 2 complex . It is likely that either differences is protonation or in geometry are responsible for the low affinity of the bisubstrate analogues in comparison to that of the external aldimines of either L-serine or L-tyrptophan with pyridoxal 5'-phosphate . As previously found with pyridoxal 5'-phosphate, pyridoxine 5'-phosphate, and N-phosphopryidoxyl-L-serine bind noncooperatively to two identical binding sites in the alpha 2 apo beta 2 complex . The same ligands bind with positive cooperatively to two binding sites in the apo beta 2 subunit . Because the analogues mimic the binding behavior of pyridoxal 5'-phosphate to both proteins, the internal aldimine of pyridoxal 5'-phosphate to the lysine amino group contributes only to the strength of that binding . The nickel apo beta 2 subunit, which is produced by limited proteolysis with trypsin, binds pyroxine 5'-phosphate noncooperatively to two identical sites . Therefore, the loop of polypeptide chain connecting the two autonomous domains of folding must be intact for enzyme activity, for the binding of the alpha subunit, and for cooperative binding of pyridoxine 5'-phosphate.

Biochemistry, 1980 Sep 16, 19(19), 4486 - 92
Circular dichroism study of Escherichia coli initiation factor 3 binding to nucleic acids; Schleich T et al.; The circular dichroic spectral features of (A)10-20, (C)10-20, A8UGU6, poly(A), and poly(C), at both neutral and acidic pH values and in the presence and absence of Mg2+, are significantly altered by Escherichia coli initiation factor 3 (IF3), implying the occurrence of protein-induced changes in nucleic acid secondary structure . Similarly, the circular dichroic spectral characteristics of helical poly(U), poly-(A)-poly(U), and poly(I)-poly(C) are modified by IF3 . However, no structural perturbation of poly(A)-poly(U) occurs in the absence of Mg2+ by IF3 . The oligonucleotides (A)10-20 and (C)10-20 at both pH 7.5 and 5.5 titrate to end point of 26 +/- 4 nucleotide residues per IF3 {except (C) 10-20 at pH 5.5 which titrates to 17 +/- 1 nucleotide residues per IF3}, whereas the hairpin A8UGU6 under similar conditions at neutral pH and in the presence of Mg2+ titrates to an end point of 56 +/- 3 nucleotide residues per IF3, thereby suggesting the presence of multiple binding sites on the protein . By contrast, poly(A) and poly(C) at neutral pH and in the absence of Mg2+ titrate to an end point of 13 +/- 1 nucleotide residues per IF3 . The occurrence of significant light-scattering artifacts precluded a determination of the end point stoichiometry in most other cases . The circular dichroic spectra of E . coli tRNA, MS2 RNA, phiX174 DNA, and sonicated calf thymus DNA were unaffected by IF3 at physiological concentrations . Addition of an equimolar mixture of IF3 and ribosomal protein S1 titrates the circular dichroism of poly(C) at acid pH as did S1 alone . However, addition of IF3 to mixture of poly(A) and S1 at neutral pH did not result in significant titration of the optical activity until IF3 was in excess over S1, even though filter binding assays indicate normal IF3 binding to the polynucleotide . The possible relation of these observations to the biological function of IF3 is briefly considered.

Biochemistry, 1980 Sep 16, 19(19), 4521 - 7
Kinetics of cooperative ligand binding to the apo beta 2 subunit of tryptophan synthase and its modulation by the alp ha subunit; Tschopp J et al.; The different binding mechanisms of pyridoxine 5'-phosphate and N-phophopridoxyl-L-serine have been investigated by kinetic studies with rapid reaction techniques . Pyridoxine 5'-phosphate binds in a single rapid step to the alpha 2 apo beta 2 complex and in a single slow step to the nicked apo beta 2 subunit that is obtained by limited proteolysis with trypsin . Both pyridoxine 5'-phosphate and N-phosphopyridoxyl-L-serine bind to the apo beta 2 subunit with a comparatively slow binding step, followed by an event slower isomerization reaction . These findings are consistent with nonexclusive concerted mechanism of cooperative binding but cannot be explained by the simple sequential mechanism . A quantitative fit of the rate and equilibrium data to the concerted mechanism generally yielded the pertinent rate and equilibrium constants . In particular, the same value of L0 = {T0}/{R0} = 200 +/- 50 simultaneously satisfies the data obtained with three different ligands . The comparison of the mechanisms of ligand binding to the three states of the apo beta 2 subunit suggests that the alpha 2 apo beta 2 complex is similar to the high-affinity R state and the nicked apo beta 2 subunit is similar to the low-affinity T state of the apo beta 2 subunit . The slow isonerization involved in the cooperative binding of the ligands to the intact apo beta 2 subunit is discussed in terms of local and concerted conformational changes involving the two autonomously folding domains of the beta protomer.

C R Seances Acad Sci D, 1980 Sep 15, 291(2), 203 - 6
{Inhibition of the 3' to 5' exonuclease activity of the DNA polymerase I of Escherichia coli by deoxyribonucleotides}; Granger M et al.; The 3' a 5' exonuclease activity of E . coli DNA-polymerase I is inhibited by nucleotides and deoxynucleotides at concentrations (< 1 mM) where polymerase activity is not affected . This inhibitory effect depends on the nature of the excised deoxynucleotide, excision of purines being much less inhibited than that of pyrimidines . It does not depend on the purine or pyrimidine nature of the inhibitor.

Nucleic Acids Res, 1980 Sep 11, 8(17), 3851 - 64
Use of RNA polymerase as an enzymatic probe of nucleosomal structure; Hodo HG 3rd et al.; Nucleosomes prepared from human placental nuclei and Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyl transferase EC.2.7.7.6) form stable initiation complexes . This property is utilized as a probe of nucleosome structure . RNA polymerase initiation has been studied on purified nucleosomes, nucleosome cores, and nucleosomal DNA . The affinity of E . coli RNA polymerase for both nucleosome cores and monomers was 5-6 fold less than found for nucleosomal DNA . No difference in apparent initiation Km was found between cores and mononucleosomes . This suggests that initiation does not preferentially occur on the DNA tails of nucleosomes . Once initiated and allowed to form nascent RNA, these complexes are very stable to ionic strength changes . Under conditions in which free enzyme is inactivated with rifampicin, the enzyme in the complex retains activity as demonstrated by its ability to transcribe and reinitiate on both nucleosomes and free DNA . These complexes can be well resolved from free nucleosomes on preparative polyacrylamide gels and both can be eluted from gels for analysis of proteins and DNA sequence complexity . Studies using (125I) labelled nucleosomes show that histones are retained in the initiation complex, and are not dissociated by the enzyme during initiation.

Nucleic Acids Res, 1980 Sep 11, 8(17), 3865 - 74
DNA gyrase subunit stoichiometry and the covalent attachment of subunit A to DNA during DNA cleavage; Sugino A et al.; Escherichia coli DNA gyrase contains a 1:1 ratio of protomers coded by the genes gyrA and gyrB . This along with previous results shows that the enzyme has two copies of each protomer and thus a molecular weight of 400,000 . Abortion of the gyrase reaction results in double-strand breakage of the DNA and covalent attachment of both gyrA protomers to the 5'-cut ends . We conclude that the gyrA protomer contains a critical part of the active site for the concerted breakage and reunion reaction of gyrase, the topoisomerase activity of the enzyme.

Nucleic Acids Res, 1980 Sep 11, 8(17), 3809 - 27
Sequence of the distal tRNA1Asp gene and the transcription termination signal in the Escherichia coli ribosomal RNA operon rrnF(or G); Sekiya T et al.; Several DNA fragments carrying tRNA genes have been cloned from EcoRI endonuclease digests of Escherichia coli DNA . Using cloned DNA, the sequence of the region around the distal gene for tRNA1Asp (F(or G)) in the E . coli ribosomal RNA operon {rrnF(or G)} has been determined . In the distal portion of rrnF(or G), the genes for 23S, 5S rRNA and tRNA1Asp (F(or G)) are located in that order and separated by intergenic spacers of 93 and 52 base pairs, respectively . A possible hairpin structure, with its center between the 22nd and 23rd base pair downstream from the 3'-end of the tRNA1Asp(F(or G)) gene, followed by a sequence of eight thymidine residues was identified as the transcription termination signal for rrnF(or G) . The termination is rho-independent, at least in vitro, and occurs within the region of the contiguous thymidine residues . A possible promoter for a protein gene is present about 50 base pairs downstream from the rrnF(or G) terminator.

J Biol Chem, 1980 Sep 10, 255(17), 8109 - 15
Subunit interaction during catalysis . ATP modulation of catalytic steps in the succinyl-CoA synthetase reaction; Bild GS et al.; A new approach for assessing of catalytic cooperativity may occur between subunits has been applied to succinyl-CoA synthetase . This is based on the extent of oxygen exchange between medium {18O}Pi and succinate per molecule of ATP cleaved during steady state succinyl-CoA synthesis . Suitable traps are used to remove succinyl-CoA and ADP as soon as they are released to the medium . With the Escherichia coli enzyme, which has an alpha 2 beta 2 structure, a pronounced increase in oxygen exchange per ATP cleaved occurs as ATP concentration is lowered . In contrast, when the CoA concentration is varied, the oxygen exchange per molecule of product formed remains constant . Also, with the pig heart enzyme, which is shown to retain its alpha beta structure during catalysis and thus has only one catalytic site, no modulation of oxygen exchange by ATP concentration is observed . These experimental findings show that the binding of an ATP either promotes the dissociation of bound succinyl-CoA or decreases its participation in exchange . Measurement of the distribution of {18O}Pi species found as exchange occurs shows that only one catalytic sequence is involved in exchange at various ATP concentrations . These observations along with other controls and results eliminate most other explanations of the ATP modulation of the exchange and suggest that binding of ATP at one catalytic site promotes catalytic site promotes catalytic events at an alternate catalytic site.

J Biol Chem, 1980 Sep 10, 255(17), 8027 - 30
On the mechanism of ribonucleoside diphosphate reductase from Escherichia coli . Evidence for 3'-C--H bond cleavage; Stubbe J et al.; The 3'-carbon--hydrogen bond of {3'-3H}uridine 5'-diphosphate is cleaved during its conversion to 2'-deoxyuridine 5'-diphosphate catalyzed by Esherichia coli ribonucleoside diphosphate reductase . A selection against 3H of approximately 3.3 is observed on this reduction reaction . During the course of this reaction, a small but significant amount of 3H is released to the solvent.

J Biol Chem, 1980 Sep 10, 255(17), 8116 - 20
Interaction of 70 S ribosomes from Escherichia coli with spin-labeled N-Cbz-Phe-tRNAPhe . An electron paramagnetic resonance study; Rodriguez A et al.; Two selectively spin-labeled Cbz-Phe-tRNAsPhe, one at position s4U8 and the other at position U33, have been used to study the dynamics of tRNA-ribosome interaction in the presence of poly(U) and factors washable from ribosomes . Upon binding to the ribosome, the correlation time of the spin label at position s4U8 decreases markedly while the same parameter for the label in the anticodon increases . The presence of poly(U) is not a prerequisite condition for the EPR spectral changes observed but larger variation occurs in the presence of factors washable from ribosomes . No variation in the correlation time is observed if uncharged spin-labeled tRNAPhe (on the s4U8 residue) is used in these experiments . Most of the ribosome-bound spin-labeled Cbz-Phe-tRNAPhe are puromycin-reactive, and consequently, the observed effect is manifested mainly at the ribosomal P site . These observations seem to suggest that the interaction between the N-blocked aminoacyl residue on the tRNA and the ribosome results in a conformational change on the tRNA, possibly involving tertiary interactions in a region close to s4U8 . The role that the amino acid at the 3'-end can possibly play on this structural change is discussed.

J Biol Chem, 1980 Sep 10, 255(17), 8069 - 73
Enzyme-catalyzed DNA unwinding . The role of ATP in helicase III activity; Das RH et al.; The enzyme helicase III catalyzes ATP-dependent unwinding of double-stranded DNA (Yarranto, G . T., Das, R . H., and Gefter, M . L . (1979) J . Biol . Chem . 254, 11997-12001) . The free enzyme is able to bind to double- and single-stranded DNA . In the presence of ATP the enzyme can bind single- but not double-stranded DNA . The enzyme catalyzes an ADP-ATP exchange reaction in the absence of DNA . It is suggested that there is an enzyme.phosphate complex that discriminates between the two forms of DNA . These results are discussed in relation to a model that accounts for catalytic unwinding of DNA coupled to ATP hydrolysis.

Biochim Biophys Acta, 1980 Sep 9, 615(1), 59 - 69
Incorporation of amino acid analogs during the biosynthesis of Escherichia coli aspartate transcarbamylase; Gueguen P et al.; Amino acid-requiring mutants capable of producing derepressed levels of aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) were obtained and used for the incorporation in this enzyme of eight different amino acid analogs . These amino acid replacements enabled the biosynthesis of a series of modified aspartate transcarbamylases altered in their catalytic or regulatory properties . The enzyme in which phenylalanine was rereplaced by 2-fluorophenylalanine was purified to homogeneity and appeared to have the same specific activity as normal asparate transcarbamylase but lacking both homotropic and heterotropic interactions.

Biochim Biophys Acta, 1980 Sep 9, 615(1), 10 - 8
The binding of a fluorescent activator 2-(N-decyl)aminonaphthalene-6-sulfonic acid to pyruvate oxidase; Schrock HL et al.; E . coli pyruvate oxidase (pyruvate:ferricytochrome b1 oxidoreductase, EC 1.2.2.2) is a peripheral membrane flavoenzyme which has been purified to homogeneity . In vivo the oxidase resides on the inner surface of the cytoplasmic membrane and is coupled to the bacterial electron transport chain . In vitro, the purified oxidase requires lipids for full enzymatic activity . Previous studies have characterized the conformational and energetic coupling between the lipid-binding site(s) and the catalytic active site . The affinity of the enzyme for phospholipids and detergents is significantly enhanced when the flavoprotein is in the reduced form, i.e., in the presence of pyruvate and the required cofactor, thiamin pyrophosphate . The lipid-binding studies were hindered due to the complicating factor of the self-association of the substrate-reduced flavoprotein . In this paper, fluorescence techniques are employed to measure the binding of a detergent-like activator to the oxidase . The experiments are performed at much lower protein concentrations than previously employed, so that protein aggregation is not a problem . The chromophore on the activator, 2-(N-decyl)aminonaphthalene-6-sulfonic acid is effective at quenching the pyruvate oxidase intrinsic tryptophan fluorescence . Quenching titrations are used to obtain the binding isotherm . AT DNS concentrations less than 10(-5) M, the results show a larger amount of DNS binding to the reduced flavoprotein than to the oxidized form of the enzyme . This is the concentration range where DNS is an effective activator of the enzyme . This represents a class of binding sites specifically found on pyruvate oxidase and not apparent in other proteins such as lysozyme or aldolase . At the DNS concentration which is optimum for activation approx . 20 molecules of DNS are bound per enzyme tetramer in the absence of the substrate . The pyruvate-reduced form of the enzyme binds about 40--50 molecules of DNS per tetramer . Qualitatively, the results are similar to what was previously found for both sodium dodecyl sulfate and cetyl trimethylammonium bromide . However, in both these cases, the amount of bound detergent was nearly an order of magnitude less than the values obtained using DNS.

Biochim Biophys Acta, 1980 Sep 9, 615(1), 103 - 12
Further kinetic characterization of the non-allosteric phosphofructokinase from Escherichia coli K-12; Ewings KN et al.; The labile non-allosteric form of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified to a specific activity of 107 U/mg (2078-fold) from aerobic cultures of Escherichia coli K-12 . The enzyme has an isoelectric point (pI) of 5.1, a native molecular weight of 67 000 +/- 3000 and a subunit weight of 34 000 +/- 400 . A number of divalent metal ions can substitute for Mg2+ in the enzyme reaction in decreasing order Mn2+ > Mg2+ > Co2+ > Ca2+ . In the presence of excess Mg2+, nucleotides do not affect the Km for fructose 6-phosphate with a value of 0.042 mM . The order of efficiency for nucleotides to act as phosphoryl donors is ATP > ITP > GTP > UTP > CTP . This remains unchanged in the presence of excess Mn2+, but V is increased 2.4-fold with ATP . A 2 : 1 ratio of Mn2+/nucleotide 5'-triphosphate produced an equivalent dissociation constant of 1.1 mM for all nucleotides, which was markedly decreased at a high Mn2+ level . The rate of enzyme catalysis was found to be dependent on the concentration of MnATP2- . Mn2+ at non-limiting values does affect the binding of fructose 6-phosphate to the enzyme.

Biochim Biophys Acta, 1980 Sep 9, 615(1), 121 - 31
Inactivation of Escherichia coli acetate kinase by N-ethylmaleimide . Protection by substrates and products; Wong SS et al.; Acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) from Escherichia coli exhibited a time-dependent loss of activity when incubated with N-ethylmaleimide at micromolar concentrations . However, prolonged incubation did not eliminate all catalytic activity and generally about 15% of its initial activity remained . When incubated with 7.2 microM N-ethylmaleimide, acetate kinase was inactivated with a rate constant of 0.063 min-1 . Adenine nucleotides, ATP, ADP and AMP, protected the enzyme against such inactivation, but acetate up to 3.0 M and in the presence of 0.2 M MgCl2 and acetyl phosphate at 24 mM did not interfere with the rate of inactivation . While both acetate and acetyl phosphate did not affect the protection rendered by AMP, the presence of acetyl phosphate altered ADP protection . However, both substrates prevented ATP from protecting the enzyme . These data suggest that the binding sites for acetate and acetyl phosphate are different from that of the adenosine binding domain, but are in close vicinity to the phosphoryl binding regions of the nucleotides.

Biochemistry, 1980 Sep 2, 19(18), 4237 - 43
Mechanism of the melibiose porter in membrane vesicles of Escherichia coli; Cohn DE et al.; The melibiose transport system of Escherichia coli catalyzes sodium--methyl 1-thio-beta-D-galactopyranoside (TMG) symport, and the cation is required not only for respiration-driven active transport but also for binding of substrate to the carrier in the absence of energy and for carrier-mediated TMG efflux . As opposed to the proton--beta-galactoside symport system {Kaczorowski, G . J., & Kaback, H . R . (1979) Biochemistry 18, 3691}, efflux and exchange of TMG occur at the same rate, implying that the rates of the two processes are limited by a common step, most likely the translocation of substrate across the membrane . Furthermore, the rate of exchange, as well as efflux, is influenced by imposition of a membrane potential (delta psi; interior negative), suggesting that the ternary complex between sodium, TMG, and the porter may bear a net positive charge . Consistently, energization of the vesicles leads to a large increase in the Vmax for TMG influx, with little or no change in the apparent Km of the process . It is proposed that the sodium gradient (Na+out < Na+in) and the delta psi (interior negative) may affect different steps in the overall mechanism of active TMG accumulation in the following manner: the sodium gradient causes an increased affinity for TMG on the outer surface of the membrane relative to the inside and the delta psi facilitates a reaction involved with the translocation of the positively charged ternary complex to the inner surface of the membrane.

Biochemistry, 1980 Sep 2, 19(18), 4208 - 13
Elementary steps in the reaction mechanism of the pyruvate dehydrogenase multienzyme complex from Escherichia coli: kinetics of acetylation and deacetylation; Akiyama SK et al.; The kinetics of the acetylation of the pyruvate dehydrogenase complex from Escherichia coli by {3-14C}pyruvate and of the deacetylation of the complex by coenzyme A have been studied by using rapid mixing-quench techniques . The time course for acetylation in 4 mM thiamin pyrophosphate, 2 mM MgSO4, and 0.02 M potassium phosphate (pH 7.0) at 4 degrees C can be analyzed in terms of two kinetic processes . At long times 10 nmol of acetyl groups is incorporated per mg of enzyme complex (48 sites per complex of molecular weight 4.8 X 10(6)) . The slower process is much too slow to be of catalytic significance . The rate constant for the faster process is not dependent on enzyme concentration and reaches a limiting value of 40--65 s-1 at high pyruvate concentrations; the exact value is dependent on the detailed acetylation mechanism assumed . The minimum molar turnover number of the enzyme complex is 420 s-1 (17.5 s-1 per pyruvate decarboxylase) . The acetylated lipoic acids are deacetylated by coenzyme A at a rate much faster than that of acetylation . Complete deacetylation is obtained only if the deacetylation is carried out within seconds of the acetylation, apparently because dead-end intramolecular transfers of acetyl groups from the lipoic acids to other functional groups on the enzyme not essential for catalytic activity can occur . The results obtained suggest only about half of the acetylation reactions are on the main catalytic pathway.

Biochemistry, 1980 Sep 2, 19(18), 4179 - 88
Precise localization of the site of cross-linking between protein L4 and 23S ribonucleic acid induced by mild ultraviolet irradiation of Escherichia coli 50S ribosomal subunits; Maly P et al.; Mild ultraviolet irradiation of Escherichia coli 50S ribosomal subunits causes a cross-linking reaction between protein and RNA, whose primary target is protein L4 {Moller, K., & Brimacombe, R . (1975) Mol . Gen . Genet . 141, 343} . Here we have determined the site of this cross-link both on L4 and on 23S RNA . For the site on the protein, a cross-linked protein-oligonucleotide complex was isolated and subjected to successive digestions with various proteases . In each case the peptide-oligonucleotide complexes formed were analyzed . It could clearly be shown that the cross-link site was contained within a characteristic peptide 16--20 amino acids long and that the amino acid concerned was the tyrosine residue at position 35 in the recently completed L4 sequence (M . Kimura and B . Wittmann-Liebold, personal communication) . For the site on the RNA, a cross-linked L4--23S RNA complex was subjected to mild nuclease digestion, producing a range of L4--RNA fragments which were isolated with the help of a new two-dimensional gel electrophoresis system . Oligonucleotide analyses of these fragments, combined with successive nuclease digestions of the residual oligonucleotide attached to protein L4, established that the site of cross-linking was homogeneous, involving a uridine residue at position 615 in the recently determined 23S RNA sequence {Brosius, J., Dull, T . J., & Noller, H . F . (1980) Proc . Natl . Acad . Sci . U.S.A . 77, 201}.

Biochemistry, 1980 Sep 2, 19(18), 4201 - 8
Escherichia coli photoreactivating enzyme: purification and properties; Snapka RM et al.; We have purified large quantities of Escherichia coli photoreactivating enzyme (EC 4.1.99.3) to apparent homogeneity and have studied its physical and chemical properties . The enzyme has a molecular weight of 36 800 and a S020,W of 3.72 S . Amino acid analysis revealed an apparent absence of tryptophan, a low content of aromatic residues, and the presence of no unusual amino acids . The N terminus is arginine . The purified enzyme contained up to 13% carbohydrate by weight . The carbohydrate was composed of mannose, galactose, glucose, and N-acetylglucosamine . The enzyme is also associated with RNA (approximately 10 nucleotides/enzyme molecule) containing uracil, adenine, guanine, and cytosine with no unusual bases detected.

Kidney Int, 1980 Sep, 18(3), 366 - 74
Immunologic studies in IgA nephropathy; Woodroffe AJ et al.; Circulating immune complexes (CIC) were detected in 43.6% of 78 patients with primary IgA nephropathy by the solid-phase Clq radioimmunoassay . The IC were intermediate (9 to 17S) in size and contained IgA, IgG, and less commonly IgM . CIC were often present intermittently, correlating with episodes of macroscopic hematuria . Elevated serum IgA concentrations (38.7%) did not correlate with the detection of CIC . Similar findings were observed in sera samples from patients with Henoch Schonlein purpura and in IgA glomerulonephritis associated with alcoholic cirrhosis and/or portal systemic shunts . The factors responsible for the mesangial localization of the IC are not clear, but elevations in serum antibody titers to respiratory pathogens (mycoplasma pneumoniae, herpes virus, influenza), gut flora (E . coli 07), and bovine serum albumin suggest that common exogenous antigens may be involved in the pathogenesis . Primary defects in either mucosal antigen exclusion or reticuloendothelial IC sequestration are proposed to account for these findings.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1980 Sep, 171(4-5), 448 - 54
{The in vivo influence of gibberellic acid (GA3) on the phagocytic properties of peritoneal macrophages (author's transl)}; Schwartz E et al.; Different concentrations of gibberellin acid GA3 - 10.0 mg, 1.0 mg and 0.1 mg - were applied intraperitoneally to white mice of the SPF strain on four consecutive days . Following the last application we injected E . coli, P . aerug . and Pr . vulg . intraperitoneally into the peritoneum of these animals, in a quantity of 1.5-2.0 X 10(6)/0.5 ml of sterile phosphate buffer . The peritoneal macrophages obtained by puncturing the peritoneum of the animals killed under chloroform anaesthesia showed, as compared with those of the control animals statistically most significantly higher values (P < 0.001) of the phagocytic factor and phagocytic index as well as of the activity of the acid phosphatase . In the test animals which received gibberellin acid, the inhibition of the lysosomal activity of peritoneal macrophages as a result of the application of trypan blue did not reach the level which was found to exist in the control animals and those infected with B CG - Praha.

Am J Trop Med Hyg, 1980 Sep, 29(5 Suppl), 1033 - 6
The genes for variant antigens in trypanosomes; Borst P et al.; We have studied the mechanism of antigenic variation by using DNA complementary to the messenger RNAs for four variant surface glycoproteins of Trypanosoma brucei . Pure complementary DNAs were obtained by cloning as recombinant DNA in Escherichia coli . Using these complementary DNAs as hybridization probes, we have analyzed the genes for these variant surface glycoproteins . The results provide new information on the origin and evolution of antigenic variation, and on the mechanism involved in switching from one antigenic type to another.

Nippon Yakurigaku Zasshi, 1980 Sep, 76(5), 363 - 72
{Effect of prednisolone 17-valerate 21-acetate on immunological responses in mice (author's transl)}; Kobayashi F et al.; The in vivo effects of prednisolone 17-valerate 21-acetate (PVA), an anti-inflammatory glucocorticoid on several immunological responses in mice were investigated in comparison with hydrocortisone 17-butyrate (HB) and betamethasone 17-valerate (BV), when given subcutaneously . PVA reduced the spleen weight, the number of splenic nucleated cells, the formation of hemolytic plaque forming cells (PFC), delayed type footpad reaction and the responsiveness of splenic lymphocytes to concanavalin A . These suppressive effects were almost the same as those seen with HB and weaker than those of BV . However, the responsiveness of splenic cells to lipopolysaccharide and circulating IgM antibody response to sheep red blood cells were suppressed by a smaller dose of PVA than that of HB . PVA had no effect on the responsiveness to phytohemagglutinin-P, whereas HB and BV enhanced the phytohemagglutinin-P responsiveness . The suppressive effect of PVA on the host defense to experimental infection with Escherichia coli was weaker than those of HB and BV . From these results, PVA appears to be similar to other glucocorticoids in that it exerts complicated effects on several immunological responses in mice.

Eur J Biochem, 1980 Sep, 110(2), 555 - 63
The role of eucaryotic factor Tu in protein synthesis . The measurement of the elongation factor Tu content of rabbit reticulocytes and other mammalian cells by a sensitive radioimmunoassay; Slobin LI; A sensitive radioimmunoassay for eucaryotic elongation factor Tu (eEF-TU) was developed using radioiodinated elongation factor T (eEF-T) and goat anti-(rabbit eEF-T) immunoglobulins coupled to a solid support . eEF-T was iodinated with 125I to a specific activity of 7 x 10(3) counts min-1 ng-1 using a system employing lactoperoxidase and glucose oxidase coupled to a solid support . The assay exhibits a limit of detection of about 1 ng eEF-TU and an intraassay variability of < 10% . By using the radioimmunoassay, it was found that eEF-Tu is a major non-hemoglobin protein of rabbit reticulocyte postribosomal supernatant proteins, comprising about 3% of the total hemoglobin and 10--13% of the non-hemoglobin proteins . Similar results were found for a number of different tissues and cells, including rabbit heart, brain, liver and kidneys, as well as both induced and non induced Friend erythroleukemia cells . Values of eEF-Tu ranged from 1% of supernatant proteins in heart to about 11% in noninduced erythroleukemic cells . The levels of eEF-Tu in these mammalian tissues were comparable to the level of the homologous factor EF-Tu in Escherichia coli . It has previously been found that EF-Tu constitutes about 6--8% of the supernatant proteins of E . coli {Furano, A . V . (1975) Proc . Natl Acad . Sci . USA, 72, 4780--4784} . The level of eEF-Tu in reticulocytes was compared to the abundance of other components of protein synthesis in reticulocytes, such as translocase (eEF-G), tRNA, ribosomes and eIF-2 . In all cases eEF-Tu was present in large excess over these other components.

Pflugers Arch, 1980 Sep, 387(2), 175 - 81
Effects of potential blood substitutes (perfluorochemicals) on rat liver and spleen; Lutz J et al.; The effect of an emulsion of perfluorochemicals (PFC) (7 parts perfluorodecalin and 3 parts perfluorotripropylamine, 4.4 g PFC/kg body weight) on organ function was determined . Whereas maximal storage of PFC was reached in the spleen as early as 12 h after PFC administration, the liver attained a maximal PFC content only after 2 days . The increase in weight also differed: a maximum occurred in the spleen on the 4th day, in the liver on the 8th day . Indocyanine green (ICG) clearance showed a small decrease, statistically significant after 12 and 24 h . Colloidal carbon clearance, used as a measure of the function of the reticuloendothelial system (RES) decreased instantly after PFC to less than half the control value; after full recovery a second decrease was seen which lasted till the 4th day after PFC . Pretreatment with C 48/80 or with increasing doses of E . coli endotoxin could largely obviate the depressive effect of PFC-loading on carbon clearance . Serum transaminases increased to about twice the control levels but were normal by the 2nd day, and thereafter . Alkaline phosphatase showed a 2.5 fold increase but returned to control level after the 2nd day . It is concluded that while a severe disturbance of liver function did not occur, the reduction in the capacity of the RES can become a serious factor in the defence against a simultaneously appearing infection if not compensated by activating the RES.

Biophys J, 1980 Sep, 31(3), 381 - 92
Sensitivity to ultraviolet radiation as a function of DNA content in Escherichia coli B/r; Bronk BV et al.; Populations of Escherichia coli B/r A were grown to log phase at various growth rates determined by the richness of the medium . The genome content, G, was calculated from log phase doubling times by means of the Cooper-Helmstetter formula . Cell volumes were measured and found to vary linearly with this genome content . Cells with various DNA contents were prepared for ultraviolet irradiation and plated for dark repair under similar conditions . The resulting logarithmic survival curves were all similar in shape: convex up, with straight line portions having approximately the same slope (D0 = 11.4 +/- 0.2 J/m2) . The shoulders however increase in width with calculated DNA content giving an extrapolation number which varies roughly as exp(G) or exp (0.6 Gmax).

Int J Radiat Biol Relat Stud Phys Chem Med, 1980 Sep, 38(3), 323 - 34
Cerenkov ultraviolet radiation (137Cs gamma-rays) and direct excitation (137Cs gamma-rays and 50 kVp X-rays) produce photoreactivable damage in Escherichia coli; Moss SH et al.; The mechanism of formation of photoreactivable damage in deoxyribonucleic acid (DNA) by ionizing radiation in a dark repair deficient strain of Escherichia coli (uvrA recA) has been investigated . By altering the ratio of damage produced directly (by ionization) to that formed indirectly (by Cerenkov ultraviolet (U.V.) radiation by 137Cs gamma-rays, it has been demonstrated that the major portion of the photoreactivable damage is produced by Cerenkov U.V . radiation . The amount of photoreactivable damage produced by 50 kVp X-rays, which cannot generate Cerenkov radiation, is similar to that component of photoreactivable damage produced by 137Cs gamma-rays that is not attributed to Cerenkov radiation . It is suggested that the second mechanism of formation of photoreactivable damage in DNA by ionizing radiation is the direct excitation of DNA . The possible role of Cerenkov U.V . radiation in ionizing radiation mutagenesis is discussed.

Biochem J, 1980 Sep 1, 189(3), 421 - 33
Analysis of progress curves . Rate law of pyruvate kinase type I from Escherichia coli; Markus M et al.; Progress curves of the reaction catalysed by pyruvate kinase from Escherichia coli K12, designed to cover the four-dimensional concentration space of phosphoenolpyruvate, ADP, Mg2+ and ATP in the regulatory region, were recorded with the pH-stat method (pH 7.0 and 25 degrees C) . Additional initial-rate measurement were performed to assess specific points . Two methods for the evaluation of progress curves were used: fitting the rate law to the rates obtained from the tangents of the progress curves and fitting the integrated rate law directly to the curves . Two models, both extensions of the concerted model given by Monod, Wyman & Changeux {(1965) J . Mol . Biol . 12, 88--118} with four protomers, could be fitted to the data within the experimental error . Model discrimination in favour of one of these models was possible by proper experimental design . In the selected model one conformational state of the enzyme forms the active complex . The active site of a second conformational state forms abortive complexes with Mg2+, causing strong inhibition at high Mg2+ concentrations . In the absence of ligands, most of the enzyme is in a third state that binds ATP at an allosteric site.

J Appl Physiol, 1980 Sep, 49(3), 516 - 20
Diphenhydramine reduces endotoxin effects on lung vascular permeability in sheep; Brigham KL et al.; Because, in sheep, histamine-induced increased lung vascular permeability is prevented by diphenhydramine, we tested the effects of diphenhydramine on the sheep lung vascular response to endotoxin . We infused E . coli endotoxin (0.40-1.00 micrograms/kg) with and without diphenhydramine (3.0 mg/kg bolus + 1.5 mg . kg-1 . h-1) in the same unanesthetized sheep while measuring pulmonary arterial (Ppa) and left atrial (Pla) pressures, lung lymph flow (Qlym) and lymph (L) and plasma (P) protein concentrations . Endotoxin caused pulmonary hypertension soon after infusion (base-line Ppa = 22 +/- 3 (SE) cmH2O; after endotoxin Ppa = 40 +/- 2; P less than 0.05, n = 6) and after several hours an increase in permeability reflected in high flow of protein-rich lymph (base-line; Qlym = 7.5 +/- 1.4 (SE) ml/h, L/P protein concentration = 0.60 +/- 0.02: after endotoxin; Qlym = 21.4 +/- 3.1, P less than 0.05; L/P = 0.66 +/- 0.03, P less than 0.05) . In the presence of diphenhydramine, endotoxin caused identical pressure changes but Qlym was lower during the period of increased permeability (16.7 +/- 3.0 (SE) ml/h, P less than 0.05 compared to endotoxin alone) and L/P protein concentration was similar (0.68 +/- 0.04, P = NS) . We conclude that endogenous histamine may be partly responsible for the increase in lung vascular permeability after endotoxemia, but that histamine probably is not the sole mediator of the permeability change.

Genetics, 1980 Sep, 96(1), 59 - 77
A new map location for the ilvB locus of Escherichia coli; Newman TC et al.; We isolated, in E . coli K12, new alleles of the ilvB locus, the structural gene for acetolactate synthase isoenzyme I, and showed them to map at or near the ilvB619 site . The map position of the ilvB locus was redetermined because plasmids containing the ilvC-cya portion of the chromosome did not complement mutations at the ilvB locus . Furthermore, diploids for the ilvEDAC genes formed with these plasmids in an ilvHI background facilitated the isolation of the new ilvB alleles . The ilvB locus was remapped and found to be located at 81.5 minutes, between the uhp and dnaA loci . This location was determined by two- and three-point transductional crosses, deletion mapping and complementation with newly isolated plasmids . One of the new alleles of the ilvB gene is a mu-1 insertion . When present in the donor strain, this allele interferes with the linkage of genes flanking the mu-1 insertion, as well as the linkage of genes to either side of the mu-1 insertion.

Am J Vet Res, 1980 Sep, 41(9), 1419 - 22
Immunoglobulin A response of the bovine fetus and neonate to Escherichia coli; Yamini B et al.; The chronologic appearance of immunoglobulin (Ig) A-containing plasma cells and their distribution and numbers in the intestinal tract, spleen, and mesenteric lymph nodes were determined in beef calves inoculated in utero with Escherichia coli O26-K60:NM bacterin or with saline solution . Secondary responses were assessed by oral revaccination or by challenge exposure to live E coli . Specific immunofluorescent procedures were used to count IgA-containing plasma cells . Appreciable numbers of IgA-containing plasma cells were seen in in utero-vaccinated calves at birth . Oral vaccination or challenge exposure with E coli increased the number of plasma cells . The caudal part of the jejunum and the ileum and related lymph nodes had more IgA-containing cells than any of the other tissues examined . In revaccinated and challenge exposed calves, the spleen was especially active in the formation of IgA-containing plasma cells . The results indicate that the entire small intestine, the draining lymph nodes, and the spleen were involved in IgA formation in these young calves . Age as a factor in IgA production was seen in the control calves which had no indication of IgA-containing cells before 9 days of age . None of the in utero-vaccinated calves at birth or at necropsy had evidence of IgA in serum.

Am J Vet Res, 1980 Sep, 41(9), 1416 - 8
Antibody-producing cells in bovine lacteal secretions after local immunization; Chang CC et al.; The indirect Jerne plaque assay was used to determine the presence of antibody-forming cells in lacteal secretions of 2 cows inoculated in the mammary gland with T4 phage . Two adjacent mammary glands of 2 nonlactating 7-months pregnant cows were inoculated by intramammary injection 4 times at 3- to 5-day intervals . The presence of plaque-forming cells (PFC) was assessed in each quarter beginning on postinoculation day 8 and at 4- to 14-day intervals thereafter . Amounts of antibodies in serum and secretions were measured by an indirect hemagglutination test . The PFC were detected in secretions from all quarters of both cows between postinoculation days 8 and 32 . Concentrations of PFC fluctuated within quarters during the course of the experiment but no relationship was evident between numbers of PFC in a quarter and its inoculation status . The use of monospecific antiglobulin sera at 1 sample-collection period revealed that cells synthesizing immunoglobulin (Ig)G and IgA antibodies were predominant in lacteal secretions . Antibody amounts in serum and secretions rose after inoculation, and titers in secretions were markedly higher in most instances . Antibody-forming cells were thus demonstrated to accumulate in the mammary gland after intramammary inoculation . The presence of antibody-forming cells in non-inoculated glands may have been the result of antigen transfer among quarters, but was considered more likely to have resulted from systemic migration of antigen-stimulated cells with migration into all quarters, regardless of inoculation status . Antibodies in lacteal secretions may have accumulated through a combination of local synthesis and selective transport from the bloodstream.

Mutat Res, 1980 Sep, 72(2), 311 - 22
Differential antimutagenic effects of caffeine and the protease inhibitor antipain on mutagenesis by various mutagens in Escherichia coli; Ichikawa-Ryo H et al.; The effects of caffeine (2 mg/ml) and the protease inhibitor antipain (1.75 mg/ml) in the plating agar medium on the yields of prototrophic revertants induced by 10 mutagens in E . coli uvrA- strains were tested . Mutagenesis by 4-nitroquinoline 1-oxide was greatly diminished by both caffeine and antipain . UV mutagenesis was decreased moderately by caffeine, and greatly by antipain . X-Ray mutagenesis was decreased very slightly by both caffeine and antipain . Mutagenesis by N-hydroxyurethan was inhibited moderately by caffeine, and greatly by antipain; that by methyl methanesulfonate was inhibited moderately by both caffeine and antipain, and that by N-methyl-N'-nitro-N-nitrosoguanidine was not suppressed by caffeine but was inhibited moderately by antipain . Mutagenesis by ethyl methanesulfonate was inhibited greatly by caffeine, but only slightly by antipain . The antimutagenic effect of caffeine was strong on furylfuramide (AF-2) mutagenesis, moderate on that of mitomycin C (tested with B2r type strain) and negligible on that of N-methyl-N-nitrosourea . These diverse antimutagenesis patterns are briefly discussed in relation to the current idea that antipain antimutagenesis is due to inhibition of inducible error-prone repair.

Mikrobiologiia, 1980 Sep-Oct, 49(5), 776 - 82
{Adaptation of certain strains of Escherichia coli to different sea water temperatures}; Alton LV; The purpose of this work was to study the adaptation of Escherichia coli strains to different temperatures (from 0 to 20 degrees C) of sea water . Three out of the four studied strains were capable of growing in sea water . Two of the strains could grow at 20, 10 and 5 degrees C while the third one did not grow below 20 degrees C . After the strains had been kept for a long time in sea water, some cells lost the ability to grow at 37 degrees C; a temperature considerably below 37 degrees C was optimal for their growth . The absence of growth when cells kept for a long time in sea water at 20, 10 and 5 degrees C were inoculated in the Endo medium does not signify that they died, since some of the cells were found to be capable of growth upon the subsequent incubation at 37 degrees C.

Eur J Biochem, 1980 Sep, 110(2), 599 - 604
Role of 16-S RNA in ribosome messenger recognition; Backendorf C et al.; The deoxyoctanuclotide (5'-3')d(A-A-G-G-A-G-G-T), which is complementary to the 3' end of 16-S RNA, inhibits the formation of the complex between the 30-S subunit and MS2 RNA described in the preceding paper . If the complex is preformed, the octanucleotide cannot prevent entry of the complex into the ribosome cycle upon supplementation with the components for protein synthesis . The subunit . MS2-RNA complex is unable to bind the octanucleotide . It is concluded that in the subunit . phage-RNA initiation precursor the 16-S terminus is base-paired with a complementary MS2 RNA sequence . Edeine, aurintricarboxylic acid and antibodies against ribosomal protein S1 prevent the association of phage RNA with 30-S subunits . These compounds do not, however, inhibit the binding of (5'-3')d(A-A-G-G-A-G-G-T) to 3-S subunits . It is concluded that formation of the complex between MS2 RNA and 30-S subunits does not depend solely on the Shine and Dalgarno base-paring reaction.

Eur J Biochem, 1980 Sep, 110(2), 593 - 7
Functional recognition of phage RNA by 30-S ribosomal subunits in the absence of initiator tRNA; Van Duin J et al.; 30-S ribosomal subunits of Escherichia coli form a stable complex with MS2 RNA or Q beta RNA at 37 degrees C in the absence of initiator tRNA . The complex functions as a precursor of initiation since it can enter the ribosome cycle in the presence of inhibitors of de novo initiation.

Eur J Biochem, 1980 Sep, 110(2), 493 - 8
On the renaturation of ribosomal protein L11; Kime MJ et al.; When urea-denatured preparations of protein L11 from the ribosome of Escherichia coli are introduced into physiological buffers, two completely different configurations can be obtained . One form, by NMR criteria, shows little evidence of stable tertiary interactions; the other shows strong indications of a distinctive folding pattern . The configuration obtained depends on minor details of the method used for returning samples to non-denaturing conditions.

Eur J Biochem, 1980 Sep, 110(2), 485 - 92
Synthesis of a new reagent, ethyl 4-azidobenzoylaminoacetimidate, and its use for RNA-protein cross-linking within Escherichia coli ribosomal 30-S subunits; Millon R et al.; A new reagent, ethyl 4-azidobenzoylaminoacetimidate, was prepared in a four-step synthesis starting from 4-aminobenzoic acid . This compound was used to cross-link RNA with proteins within the Escherichia coli 30-S ribosomal subunits . Following the reaction of the imidoester function with protein NH2 groups, photoactivation of the azide binds the other end of the reagent to RNA . The cross-linked proteins were labelled with 125I and identified by bidimensional gel electrophoresis . Proteins S3, S4, S5, S7, S9, S17, S18, and in a lower and more variable yield, S12, S13, S14 and S16 were bound to 16-S RNA . These results were confirmed by isolating cross-linked protein-oligonucleotide complexes from 30-S subunits containing 32P-labelled RNA.

Clin Exp Immunol, 1980 Sep, 41(3), 453 - 8
Specific IgM and IgG antibodies in IgA deficiency; Heddle RJ et al.; Evidence of an abnormality of IgM- and IgG-specific antibody responses was sought in subjects with selective IgA deficiency . Twenty-four patients having a serum IgA level less than 0 . 37 g/l were detected by local-population screening . Total IgM and IgG levels were measured as well as specific antibody to the lipopolysaccharides of six 0 serotypes of E . coli which are frequently isolated from human faeces . Serum IgM-class E . coli antibodies were significantly lower in IgA-deficient patients than in age- and sex-matched controls . The respective IgG-class antibody was generally elevated although more variation existed in this class . There was no correlation of specific antibody in either class with the degree of IgA deficiency . Possible explanations for these patterns of antibody response are discussed . It is concluded that some subjects with 'selective IgA deficiency' may be deficient in specific IgM antibody responses to certain antigens.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5135 - 9
Comparison of the structures of free and ribosome-bound tRNAPhe by using slow tritium exchange; Farber N et al.; The rate of incorporation of tritium from the solvent into the C-8 position of purines in RNA is markedly sensitive to the microenvironment . This slow tritium exchange reaction has been used to study the structure and interactions of yeast tRNAPhe bound to poly(U)-programed tight-couple 70S ribosomes of Escherichia coli . The tritium incorporation into specific sites of the tRNA was determined by enzymatic digestion and measurement of the specific activity of each of the isolated radioactive fragments . Ribosome binding leads to marked suppression in the exchange rate of a number of fragments . This delineates extensive regions of tRNA-ribosome contact . No change in exchange rates is seen for fragments from the corner of the molecule, indicating that this region of bound tRNA is readily accessible to the solvent . Ribosome binding results in an enhanced exchange rate at the T loop . This appears to be the result of a conformational change that is most likely an unfolding of the T and D loops . Additional tritium exchange reactions suggest this conformational change is induced by ribosomes and not by messenger.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5064 - 8
Differential amplification of specific areas of phage T4 genome as revealed by hybridization to cloned genetic segments; Kozinski AW et al.; Under various conditions, specific genetic areas of the phage T4 DNA molecule are preferentially and repeatedly replicated, resulting in the amplification of these areas . These areas are found to lie in the vicinity of the known origins of DNA replication.

Immunology, 1980 Sep, 41(1), 115 - 21
Immunological cross-reactivity of heat-labile enterotoxins produced by enterotoxigenic and enteropathogenic strains of Escherichia coli; Klipstein FA et al.; Weanling rats were immunized with a heat-labile enterotoxin contained in whole cell lysate (WCL) ultrafiltrate preparations of enteropathogenic (EPEC) and enterotoxigenic (ETEC) strains or with a purified preparation of heat-labile toxin (LT) from the ETEC strain and then challenged either with viable bacteria of each strain or the purified ETEC LT by means of the ileal ligated loop technique . Immunization with the WCL toxin preparations of either the EPEC or ETEC strain conferred protection against challenge with viable organisms of both strains; immunization with a similar preparation from a nontoxigenic strain did not yield protection . Immunization with either the WCL or purified LT toxin from ETEC strain afforded protection against challenge with the ETEC LT toxin, but immunization with the EPEC WCL preparation did not . The antigenicity of all of the toxin preparations was destroyed by heat-treatment . Possible contributory protective effects of somatic or colonization factor (CFA) antigens present in the WCL were excluded by the findings that protection was afforded against a heterologous somatic serotype, ileal bacterial counts were not reduced in protected animals, and WCL preparations of strains containing or lacking CFA yielded equal protection . These observations indicate that the heat-labile enterotoxin of EPEC strains is antigenic and is immunologically related to a heat-labile toxin present in similarly prepared material from an ETEC strain but not to the conventional LT toxin of ETEC strains . They suggest that the WCL preparation of the ETEC strain contains two heat-labile enterotoxins, one of which is conventional LT and the other of which resembles the EPEC toxin.

Cancer Lett, 1980 Sep, 10(3), 199 - 205
Cytotoxic effects of protease inhibitors on human cells . 1 . High sensitivity of xeroderma pigmentosum cells to antipain; Ishizaki K et al.; Antipain had little effect on UV-survival and UV-induced sister chromatid exchanges in normal and xeroderma pigmentosum (XP) cells, suggesting that it may not affect DNA repair . Antipain itself produced a small, but significant, amount of sister chromatid exchanges . XP cells showed very high sensitivity to the cytotoxic effect of antipain, but an SV40-transformed XP cell strain did not demonstrate high sensitivity.

Arch Microbiol, 1980 Sep, 127(2), 81 - 6
Molecular composition of the outer membrane of Escherichia coli and the importance of protein-lipopolysaccharide interactions; Gmeiner J et al.; Whole cells of Escherichia coli strains 0111, K12 and B as well as the ampicilln-resistant mutant K12 D21 and several lipopolysaccharide (LPS) mutants derived from this strain were analyzed for their molar LPS content per mg dry weight . An increase of the LPS concentration in some LPS mutants was substantiated by analyzing isolated cell walls and relating the molar LPS content to the murein subunit as measure of cell surface area . The increase of LPS was paralleled by increasing amounts of phospholipid while the overall protein content in the outer membrane decreased . According to the pattern of major outer membrane proteins in the various strains and the respective LPS structures, protein-LPS interactions are discussed as important requirements for outer membrane assembly and stability.

Mol Biol (Mosk), 1980 Sep-Oct, 14(5), 1080 - 7
{Affinity modification of Escherichia coli Dna-polymerase I by 4-(N-2-chloroethyl-N-methylamino)-benzyl-gamma-amides of dTTP and dATP}; Buneva VN et al.; The interaction of dTTP and dATP gamma-4-(N-2-chloroethyl-N-methylamino) benzylamidates with E . coli DNA-polymerase I were studied . dTTP and dATP gamma-4-(N-2-hydroxyethyl-N-methylamino) benzylamidates act as competitive inhibitors of DNA-polymerase I . Ki values for gamma-analogues of dTTP and dATP have been determined . Reactive dTTP and dATP derivatives are shown to be affinity reagent for this enzyme.

Mol Biol (Mosk), 1980 Sep-Oct, 14(5), 1023 - 38
{Study of the kinetics of the reaction reuniting DNA fragments catalyzed by DNA-ligase . Case of homogeneous molecules}; Mashko SV et al.; The kinetics of different DNA forms formation were studied during the ligation reaction . Reaction was carried out using as a model BamH1 DNA fragments of plasmid pBR322 and DNA-ligase of bacterophage T4 . The products of the reaction were separated with 1% agarose gel, followed by DNA quantitation in the corresponding bands by scanning fluorimeter . The kinetics of the ligation reaction were measured in the range of DNA concentrations from 1 to 100 micrograms/ml . The system of differential equations, that described the kinetics of the ligation reaction, was obtained . This has no analytical solution, but may be approximately calculated with a computer . The analysis of kinetic equations was made in order to optimize the ligation reaction . The optimal DNA concentrations were found allowing the maximal yield of multimer circular DNA forms . The simple formula for calculatng these concentrations were developed . The yield of circular DNA multimers might be increased if the reaction is carried out at two stages: initially at high DNA concentration for linear multimers formation followed then a lower concentration after appropriated dilution . The formula for determiantion of the dilution time is presented . The data obtained allow to optimize the reaction conditions providing the recombinant DNA molecules formation.

Am J Dis Child, 1980 Sep, 134(9), 845 - 7
Mitogenic response to Toxocara antigen and chemotactic defect in visceral larva migrans; Caldwell K et al.; A 3-year-old girl had the typical features of visceral larva migrans syndrome, including hypereosinophilia, recurrent pulmonary infiltrates, and elevated serum IgG, IgE, and isohemagglutinin titers . A marked mitogenic response of her cultured lymphocytes to Toxocara antigen but not to Ascaris antigen was found . A neutrophil chemotactic abnormality ot Escherichia coli culture filtrates and zymosan-activated sera was also demonstrated, which partially corrected after she was treated with diethylcarbamazine citrate.

Ultrasonics, 1980 Sep, 18(5), 224 - 8
Cavitational bio-effects of 1.5 MHz; Graham E et al.; The effects of continuous wave ultrasound on three different classes of biosystems have been investigated at a frequency of 1.5 MHz . The criteria for cavitation are given, and these are applied to experimentally observed growth retardation of plant roots, cell death and DNA degradation in bacteria and pyknosis of human lymphocytes . An attempt is being made to find common physical mechanisms for all these biological responses, and cavitation processes in particular are examined here . A description is given of the techniques used to monitor the presence of cavitation, and indirect evidence, drawn from pulsed field and elevated pressure experiments, is presented to show that other-non-linear processes are also operative.

J Bacteriol, 1980 Sep, 143(3), 1538 - 41
Intermediate location in the assembly of the matrix protein or porin into the outer membrane of Escherichia coli; Boyd A et al.; Evidence from pulse-chase experiments indicates that the outer membrane matrix protein or porin of Escherichia coli B/r passes through a Sarkosyl-soluble membrane pool on the way to its eventual Sarkosyl-insoluble state in the outer membrane.

J Bacteriol, 1980 Sep, 143(3), 1513 - 8
tonB-independent ferrichrome-mediated iron transport in Escherichia coli spheroplasts; Weaver CA et al.; Although a functional tonB gene product was required for ferrichrome-mediated iron transport in whole cells of Escherichia coli K-12, such transport did not require the tonB+ function in spheroplasts . We suggest that in spheroplasts ferrichrome has direct access to the cytoplasmic membrane and that this is reflected in tonB-independent accumulation of ferrichrome iron . Therefore, the tonB gene product does not function in the translocation of ferrichrome iron across the inner membrane.

J Bacteriol, 1980 Sep, 143(3), 1506 - 8
recA gene product is responsible for inhibition of deoxyribonucleic acid synthesis after ultraviolet irradiation; Trgovcevic Z et al.; Deoxyribonucleic acid synthesis after ultraviolet irradiation was studied in wild-type, uvrA, recB, recA recB, and recA Escherichia coli strains . Inhibition of deoxyribonucleic acid synthesis, which occurs almost immediately after exposing the cells to ultraviolet radiation, depends on the functional gene recA.

J Bacteriol, 1980 Sep, 143(3), 1504 - 5
Map location of the ssd mutation in Escherichia coli K-12; Morris JF et al.; A pleiotropic mutation at the ssd locus was mapped at 86 min near rha . A mutation at the ssd locus resulted in elevated L-serine deaminase activity, inability to grow with succinate as the carbon source, and inability to grow anaerobic conditions.

J Bacteriol, 1980 Sep, 143(3), 1289 - 94
Genetic characterization of a filament-forming, lipoprotein-deficient mutant of Escherichia coli; Torti S et al.; The fam-715 allele of Escherichia coli ST715, previously described as a temperature-sensitive filament former with reduced levels of lipoprotein at the nonpermissive temperature (S . V . Torti and J . T . Park, Nature {London} 263: 323--326, 1976), was mapped at 74 min . This mutation appears to be amber . It is recessive and can be complemented by F' plasmids carrying the wild-type allele or by an F' plasmid carrying an amber suppressor . Isotopic labeling experiments as well as map position differentiate the fam-715 allele from lipoprotein structural gene mutations.

J Bacteriol, 1980 Sep, 143(3), 1241 - 52
Correlation between size and age at different events in the cell division cycle of Escherichia coli; Koppes LJ et al.; The variability of (i) the B period between birth and initiation of chromosome replication, (ii) the U period between initiation of chromosome replication and initiation of cell constriction, and (iii) the interdivision period (tau) have been estimated for slowly growing Escherichia coli B/r F . Cultures synchronized by the membrane elution technique were pulse-labeled with {3H}thymidine or continuously labeled with {3H}thymine . After fixation, the pattern of deoxyribonucleic acid replication was analyzed by electron microscopic radioautography . Cell length was found to increase exponentially with age at two different slow growth rates . The coefficient of variation of the B period was estimated to be 60%, that of the U period was 29%, and that of the interdivision period was 12% . From these values and the coefficient of variation of length at different cell cycle events were calculated a negative correlation between the B and U period (r = -0.9) and a positive correlation between length at birth and cell separation (r = 0.6) . Initiation of chromosome replication and cell constriction were strictly correlated both with respect to age (r = 0.7) and length (r = 0.8) . On the other hand, length at initiation of chromosome replication was distantly correlated with age (r = 0.1) or length at birth (r = 0.3) . This low correlation excludes a model in which chromosome initiation is controlled by a random event in the B period . It favors a model in which chromosome initiation occurs at a particular distributed size independent of cell division.

J Bacteriol, 1980 Sep, 143(3), 1135 - 41
Genetic analysis of mutations causing borrelidin resistance by overproduction of threonyl-transfer ribonucleic acid synthetase; Frohler J et al.; Mutations leading to borrelidin resistance in Escherichia coli by overproduction of threonyl-transfer ribonucleic acid synthetase were anaylzed genetically . The regulatory mutations were closely linked to the treonyl-transfer ribonucleic acid synthetase structural gene (thrS), located clockwise to it . The mutation that causes the threefold-increased enzyme level was more distant from thrS than the mutation responsible for the ninefold overproduction . Both mutations were cis dominant in merodiploid strains, indicating that they affected promoter-operator-like control elements . Overproduction was restricted to threonyl-transfer ribonucleic acid synthetase and was not observed for the products of genes neighboring thrS (e.g., infC, pheS, pheT, and argS), providing evidence that thrS is transcribed singly and that gene amplificationis not a likely basis for increased thrS experession.

J Bacteriol, 1980 Sep, 143(3), 1116 - 26
Construction and expression of hybrid plasmids containing Escherichia coli K-12 uxu genes; Ritzenthaler P et al.; The three genes of the Escherichia coli K-12 uxu region (uxu operon and regulatory gene) were isolated on a ColE1-uxu hybrid plasmid from the bank of Clarke and Carbon, and a restriction map of this region was established . In vitro recombination techniques were used to subclone the uxu restriction fragments into the plasmid vector pBR322 or pBR325 . The various chimeric plasmids obtained were analyzed by restriction mapping and characterized genetically by introducing them in uxu mutant or wild-type strains . Differential rates of synthesis of the enzymes coded for by the uxu region were measured in the plasmid-containing strains; amplification of the products of the cloned genes was up to 40-fold the level found in haploid strains . The enzymes coded for by uxuA and uxuB were synthesized in vitro in a coupled transcription-translation system, confirming the results of the cloning experiments . The restriction analysis also suggests that the transcriptional direction of the uxu operon is from uxuA to uxuB and that the order of the loci in this region is: uxuR (regulatory gene), uxuB, uxuA, uxuAp (promoter), uxuAo (operator).

J Bacteriol, 1980 Sep, 143(3), 1095 - 107
Regulation of Escherichia coli K-12 hexuronate system genes: exu regulon; Portalier R et al.; Two types of Escherichia coli K-12 regulatory mutants, partially or totally negative for the induction of the five catabolic enzymes (uronic isomerase, uxaC; altronate oxidized nicotinamide adenine dinucleotide: uxaB; mannonate hydrolyase, uxuA) and the transport system (exuT) of the hexuronate-inducible pathway, were isolated and analyzed enzymatically . Hexuronate-catabolizing revertants of the negative mutants showed a constitutive synthesis for some or all of these enzymes . Negative and constitutive mutations were localized in the same genetic locus, called exuR, and the following order for the markers situated between the min 65 and 68 was determined: argG--exuR--exuT--uxaC--uxaA--tolC . The enzymatic characterization of the pleiotropic negative and constitutive mutants of the exuR gene suggests that the exuR regulatory gene product exerts a specific and total control on the three exuT, uszB, and uxaC-uxaA operons of the galacturonate pathway and a partial control on the uxuA-uxuB operon of the glucuronate pathway . The analysis of diploid strains conatining both the wild type and a negative or constitutive allele of the exuR gene, as well as the analysis of thermosensitive mutants of the exuR gene, was in agreement with a negative regulatory mechanism for the control of the hexuronate system.

Clin Chem, 1980 Sep, 26(10), 1403 - 12
Data-base techniques for multiple two-dimensional polyacrylamide gel electrophoresis analyses; Lipkin LE et al.; Two-dimensional protein electrophoresis can benefit from a powerful set of computer-supported image processing and data structure/management procedures . Detection of quantitative differences is complicated by local inhomogeneities in the polyacrylamide base; biochemical changes and variations in temperature and preparative technique also make the between-gel density and x-y coordinate correspondences quite imprecise . The program presented here provides local alignment and computer-controlled variable "flicker" rates for multiple gels, with use of an interactive display system . Manual spot densitometry, referred to a National Bureau of Standards density wedge, can be complemented by a set of automatic densitometry routines for previously established lists of spots . The ability to establish a set of local landmarks, either by included standards or user identification, provides a basis for automatic n-way gel comparison for subsets or for the entire set of spots . Automatic segmentatin algorithms allow isolatin of spots and separation of touching and partially overlapping regions . Various analytical and statistical facilities are part of the user's access to the interactively developed data base . The data-structure and image-manipulation techniques developed here allow for user-directed and heuristic comparisons with online presentation of intermediate and final results.

Endocrinology, 1980 Sep, 107(3), 851 - 3
Cloning of DNA complementary to bovine prolactin mRNA; Miller WL et al.; We have cloned DNA complementary to mRNA coding for bovine prolactin (bPrl) . Double-stranded cDNA prepared from bovine pituitary mRNA was inserted into the Pst I site of plasmid bPR322 by the dC x dG tailing technique and amplified in E . coli chi 1776 . A recombinant plasmid containing bPrl cDNa was identified by hybridization to cloned rat Prl cDNA . It contains cDNA corresponding to the region of the mRNA coding for the carboxy terminal 101 amino acids of bPrl, as well as 42 nucleotides in the 3' untranslated region of the mRNA . Nucleotide sequencing confirmed the amino acid sequencing of this region of bPrl, and permitted the assignment of asparagine or glutamic acid at seven previously equivocal loci . Codon use in bPrl mRNA is comparable to that found in rat and human Prl mRNA's and differs from that in bovine, rat, and human growth hormone mRNA's.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5187 - 91
Communication between catalytic subunits in hybrid aspartate transcarbamoylase molecules: effect of ligand binding to active chains on the conformation of unliganded, inactive chains; Yang YR et al.; In the regulatory enzyme aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) of Escherichia coli, the six catalytic polypeptide chains are arranged as two distinct catalytic trimers "crosslinked" by three regulatory dimers . Because in allosteric proteins it is assumed that the binding of a ligand to one site promotes a conformational change that affects the subsequent binding to other sites in the oligomeric protein, it was of interest to determine directly whether the effects of ligand binding to chains in one catalytic subunit are "communicated" to unliganded chains in the other subunit . Accordingly, hybrid enzyme molecules were constructed containing sensitive chromophores on the three inactive catalytic chains in one subunit along with an active catalytic subunit and three native regulatory subunits . The derivative exhibited the allosteric properties characteristic of the native enzyme . Communication between the two catalytic subunits was demonstrated by spectral measurements showing that the effects of ligand binding to the three active chains are propagated to the chromophores on the unliganded, inactive chains in the other subunit . Moreover, the change in the tertiary structure of the unliganded catalytic chains is tightly linked to the alteration in the quaternary structure.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5149 - 52
Enzymatic reduction of alloxan by thioredoxin and NADPH-thioredoxin reductase; Holmgren A et al.; Alloxan reacts with certain sulfhydryl groups either by chemical modification or reduction to dialuric acid . The effects of the drug on NADPH-thioredoxin oxidoreductase, EC 1.6.4.5} and thioredoxin-(SH)2, a ubiquitous thiol-dependent disulfide reductase system, are described . Alloxan was a direct substrate for a nearly homogenous preparation of calf thymus NADPH-thioredoxin reductase with an apparent Km of 330 microM and a Kcat of 1000 min-1 at pH 7.0 and 25 degrees C . Alloxan was not a substrate for the corresponding Escherichia coli NADPH-thioredoxin reductase . However, E . coli and calf thymus thioredoxin-(SH)2 both efficiently reduced alloxan . Thus, alloxan showed an apparent Km of 70 microM in the presence of 3.4 microM E . coli thioredoxin, 0.2 microM thioredoxin reductase, and 0.4 mM NADPH . The insulin disulfide reductase activity of the complete calf thymus thioredoxin system was inhibited by alloxan, as predicted from the reaction of the drug with both thioredoxin-(SH)2 and thioredoxin reductase . The toxic action of alloxan on animal cells, particularly the beta cells of pancreas, may be caused by rapid oxidation of cellular NADPH and generation of cytotoxic dialuric acid catalyzed by the thioredoxin system.

Ukr Biokhim Zh, 1980 Sep-Oct, 52(5), 652 - 67
{Proteins specifically binding thiamine and their biological role}; Rybina AA et al.; The paper deals with generalization and a critical review of modern ideas of thiamine-binding proteins and their biological role in the vitamin transport in micro and macroorganisms . The significance of the given problem as applied to physiology and medicine is shown.

Ukr Biokhim Zh, 1980 Sep-Oct, 52(5), 547 - 50
{Nucleotide sequence in tRNA Leu 2 from cow mammary gland}; Tukalo MA et al.; The primary structure of the tRNA2Leu from a lactating cow mammary gland was established T1 and pancreatic ribonuclease digestion were determined by an ultramicrospectrophotometric method using the equipment for microcolumn chromatography . tRNA2Leu is composed of 85 nucleotide residues, including 15 modified nucleotides . The anticodon region of tRNA2Leu is respresented by the sequence CAG . The primary structures of tRNA2Leu from the cow mammary gland and leucine tRNAs from E . coli and yeast were compared.

Pathol Biol (Paris), 1980 Sep, 28(7), 435 - 9
{An electrochemical evidence that some hormones influence the transmembrane transport of lipoic acid by Escherichio coli K 12 (author's transl)}; Junter GA et al.; The influence of diverse substances on the transmembrane transport of lipoic acid can be followed by a recently developed method based on electrochemical measurements of the oxydo-reduction of lipolic acid . This technique was applied to study some hormonal effects on E . coli . The results show that the active transport of lipoic acid (at low lipoic acid concentration) through the E . coli K 12 membrane is increased by hydrocortisone and insulin, but remains unaffected by adrenalin . At high lipoic acid concentration, its passive diffusion through the E . coli membrane is enhanced by adrenalin, but decreased by insulin and hydrocortisone . These results are similar to those reported for rat liver mitochondria and erythrocites.

J Bacteriol, 1980 Sep, 143(3), 1436 - 43
Use of Escherichia coli operon-fusion strains for the study of glycerol 3-phosphate transport activity; Miki K et al.; Strains of Escherichia coli K-12 deleted in the native lac operon and bearing both a wild-type glpT operon encoding for sn-glycerol 3-phosphate (G3P) transport and a hybrid operon in which glpT operator and promoter regions are fused to the lacZ gene were constructed . In strains with such a hybrid operon, beta-galactosidase and beta-galactoside permease become inducible by G3P . In these mutants the function and maturation of the glpT-coded proteins should be distinguishable from the level of gene expression, since the beta-galactosidase activity can serve as an index of the latter . With the aid of such mutants, it was shown that: (i) the expressions of the two neighboring operons, glpT and glpA (encoding anaerobic G3P dehydrogenase), are not coordinate; (ii) upon induction, the appearance of the cytoplasmic beta-galactosidase activity preceded that of methyl-beta-D-thiogalactoside transport activity (requiring only a cytoplasmic membrane protein) by about 4 min and that of G3P transport activity (requiring both a cytoplasmic membrane protein and a periplasmic protein) by about 9 min; and (iii) when cells grown at several temperatures from 24 to 42 degrees C were measured for G3P transport activity at 30 degrees C, the activity increased with the growth temperature, indicating that, within the range studied, the rate of transport increases with the fluidity of membrane phospholipids.

Plasmid, 1980 Sep, 4(2), 215 - 27
Partitioning of plasmid R1 in Escherichia coli . I . Kinetics of loss of plasmid derivatives deleted of the par region; Nordstrom K et al.; The stability of inheritance of plasmid R1drd-19 was tested . The copy number of the plasmid was determined in two different ways: As the ratio between covalently closed circular DNA and chromosomal DNA, and by quantitative determination of single-cell resistance to ampicillin . In the latter case, strains carrying the R1 ampicillin transposon Tn3 on prophage lambda was used as standard . The values were transformed to copy number per cell by using the Cooper-Helmstetter model for chromosome replication as well as by determination of chromosomal DNA per cell by the diphenylamine method . The copy number was found to be five to six per cell (or about four per newborn cell) . Nevertheless, plasmid R1drd-19 was found to be completely stably inherited . This stability was shown not to be due to retransfer of the plasmid by the R1 conjugation system, since transfer-negative derivatives of the plasmid were also completely stably inherited . Smaller derivatives of plasmid R1drd-19 were found to be lost at a frequency of about 1.5% per cell generation . The copy-number control was not affected in these miniplasmids, since their copy numbers were the same as that of the full size plasmid . Quantitatively, the instability of the miniplasmids was in accord with random partitioning . It is, therefore, suggested that the plasmid R1drd-19 carries genetic information for partitioning (par) of plasmid copies at cell division, and that the par mechanism is distinct from the copy number control (cop) system . Finally, the par gene maps on the resistance transfer part of the plasmid, but far away from the origin of replication and the so-called basic replicon; this is in accord with the approximate location of the repB gene (Yoshikawa, 1974, J . Bacteriol., 118, 1123-1131).

Tsitol Genet, 1980 Sep-Oct, 14(5), 73 - 6
{Genetic properties of phage lambda recombinant molecules . The effect of an inserted fragment on the hybrid yield and recombination with respect to the h- and vir-markers}; Maliuta SS et al.; The yield of mature phages in lambda gt-lambda Dm34, lambda gt-lambda DM225 and lambda gt-lambda Dm234 is 2, 8 and 8 times lower (respectively) as compared to the wild type . The output of hvir-recombinants in lambda gt-lambda Dm225 is more than an order lower as compared to the other phages . As distinct from two other hybrids, the decrease in lambda gt-lamba DM225 yield cannot be explained by red-genotype and a shortening of the phage genome as well as the decrease in the output hvir-recombinants cannot be attributed to a decrease in the yield of mature particles and to a shortening of the distance between markers . The data obtained show a contribution of Drosophila DNA to the observed effects.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5370 - 4
Overlapping genes at the cheA locus of Escherichia coli; Smith RA et al.; The cheA locus of Escherichia coli, which is required for chemotactic behavior, encodes two polypeptide products designated p{cheA}L and p{cheA}S . The mode of synthesis of these two proteins was investigated by transferring various missense and nonsense mutations to a lambda transducing phage and observing the mutant cheA products made after infection of ultraviolet-irradiated host cells . Missense mutations had no effect on either the size or the relative amounts of the two cheA polypeptides . Most nonsense mutations caused premature translational termination of both cheA products, indicating that p{cheA}L and p{cheA}S must be translated from the same coding sequence in the same reading frame . Two exceptional nonsense alleles at the promoter-proximal end of cheA made an intact p{cheA}s but no detectable p{cheA}L . These findings show that the cheA locus may contain two different sites for initiation of translation . The synthesis of both proteins can be effected by the same promoter, but it is not yet clear whether both are translated from identical mRNA molecules . Complementation studies of cheA mutants provided evidence for two functional activities, one associated with the amino terminus of p{cheA}L and the other with the common portions of p{cheA}L and p{cheA}S . It is possible that each cheA product has a different function required for chemotaxis . The possible roles of these two products and the functional significance of bacterial genes with overlapping coding sequences are discussed.

Appl Environ Microbiol, 1980 Sep, 40(3), 533 - 8
Limulus amoebocyte lysate assay for detection and quantitation of endotoxin in a small-volume parenteral product; Tsuji K et al.; A Limulus amoebocyte lysate gel-clotting method for the determination of endotoxin in a small-volume parenteral product has been described . Sample dilution with 0.1 M potassium phosphate monobasic buffer (pH 8.0) effectively eliminated assay interference, whereas dilution with water did not . The threshold pyrogenic dose for Escherichia coli EC-2 and O127:B8 endotoxins was determined to be 1.0 ng of endotoxin per kg of body weight . Not more than 1.0 ng of endotoxin (the threshold pyrogenic dose) per the highest recommended human dose or the USP pyrogen test dose per kg of body weight, whichever dose is more stringent, is a logical limit for the quantity of bacterial endotoxin in small-volume parenteral products . Excellent correlation was attained when this criterion was used to compare the Limulus amoebocyte lysate assay with the USP pyrogen test.

Tijdschr Diergeneeskd, 1980 Sep 1, 105(17), 706 - 10
{Veterinary immunology: some recent developments (author's transl)}; Bokhout BA; Veterinary Immunology has become a valuable link in the field of veterinary research . Recent developments in veterinary immunology in the Department of Immunochemistry of the Central Veterinary Institute are reviewed . In 1978, Counter Immuno-Electrophoresis was introduced for the diagnosis of Aleutian Disease (AD) in mink . Since then, more than 100,000 sera were tested (1) . It is not clear whether the enormous amounts of specific anti-AD antibodies found in the serum of diseased mink have a biological significance . The present author recently developed an indirect haemagglutination test for the diagnosis of Dictyocaulus viviparus infection in cattle . The test compares favourably with the parasitological diagnostic methods so far used (2) . The interpretation of levels of specific and anti-D . viviparus antibodies continues to be open to discussion, and the question whether antibodies are induced by larvae and/or other developmental stages of D . viviparus is still unanswered . With regard to E . coli infections, the question is asked whether research on 'minimum' vaccination against E . coli during the first day of life may help to reduce diarrhoea in weaner pigs . As intradermal injections resulted in significantly higher antibody titre in an anti-bovine albumn-immunisation model in pigs, alternative modes of immunisation are worth investigating . Recent developments in the production and use of class-specific antibodies are reviewed (3, 4).

J Biochem (Tokyo), 1980 Sep, 88(3), 695 - 703
Coupling factor F1 ATPase with defective beta subunit from a mutant of Escherichia coli; Kanazawa H et al.; The defective coupling factor F1 ATPase from a mutant strain (KF11) of Escherichia coli was purified to a practically homogeneous form . The final specific activity of Mg2+-ATPase was 6-9 units/mg protein, which is about 10-15 times lower than that of F1 ATPase from the wild-type strain . The mutant F1 had a ratio of Ca2+-ATPase to Mg2+-ATPase of about 3.5, whereas the wild-type F1 had ratio of about 0.8 . The mutant F1 was more unstable than wild-type F1: on storage at -80 degrees C for 2 weeks, about 80% of its activity (dependent on Ca2+ or Mg2+) was lost, whereas none of the activity of the wild-type F1 was lost . The following results indicate that the mutation is in the beta subunit . (i) High Mg2+-ATPase activity (about 20 units/mg protein) was reconstituted when the beta subunit from wild type F1 was added to dissociated mutant F1 and the mixture was dialyzed against buffer containing ATP and Mg2+ . (ii) Low ATPase activity having the same ratio of Ca2+-ATPase to Mg2+-ATPase as the mutant F1 was reconstituted when a mixture of the beta subunit from the mutant F1 and the alpha and gamma subunits from wild-type F1 was dialyzed against the same buffer . (iii) Tryptic peptide analysis of the beta subunit of the mutant showed a difference in a single peptide compared with the wild-type strain.

J Bacteriol, 1980 Sep, 143(3), 1281 - 8
Determination of transcriptional units and gene products from the ftsA region of Escherichia coli; Lutkenhaus JF et al.; Lambda transducing phage gamma 16-2 carries the genes envA, ftsZ, ftsA, ddl, and murC and directs the synthesis of six unique proteins in ultraviolet-irradiated cells . Various derivatives of gamma 16-2 carrying smaller segments of the bacterial deoxyribonucleic acid have also been analyzed for their capacity to direct protein synthesis in ultraviolet-irradiated cells . These results, in combination with genetic results, have allowed the gene product of each of these genes to be assigned . In addition, an unidentified gene was located counterclockwise to murC between murC and murF . Analysis of the direction of transcription indicates that murC, ddl, ftsA, and ftsZ are transcribed clockwise on the Escherichia coli genetic map, and envA is transcribed counterclockwise . In addition, it is shown that each of the genes envA, ftsZ, and ftsA can be expressed independently.

J Bacteriol, 1980 Sep, 143(3), 1156 - 64
purF-lac fusion and direction of purF transcription in Escherichia coli; Smith JM et al.; The purF locus codes for the first enzyme, glutamine phosphoribosylpyrophosphate amidotransferase, of the purine biosynthetic pathway . A strain of Escherichia coli K-12 was isolated in which the lac structural genes were fused to the control region of the purF locus . This purF-lac fusion was shown to respond to purine-specific regulatory signals . A plaque-forming lambda transducing phage bearing this purF-lac fusion was isolated . This phage was used to genetically determine the direction of transcription for the pufF locus by two independent means . Results from both methods agreed that the direction of transcription of the purF locus was clockwise on the standard Escherichia coli K-12 genetic map.

Genetics, 1980 Sep, 96(1), 43 - 57
Chi mutation in a transposon and the orientation-dependence of Chi phenotype; Yagil E et al.; Chi, an element that stimulates recombination via the E . coli RecBC pathway, can arise by spontaneous mutation in the transposon Tn5 . When in phage lambda in one orientation, the mutant transposon confers Chi+ phenotype (large plaque and a high rate of exchange near the transposon) . In the other orientation, however, the transposon does not confer Chi+ phenotype . The mobility of the transposon allows us to show that the Chi+ orientation of the mutant Tn5 is the same at different locations in lambda . These include a site near gene J, one in gam at 69, one to the right of gam at 73 and several to the right of R between 95.7 and 99.5 . To the right of R, the mutant transposon could be found in only one orientation, that which confers Chi+ phenotype . We speculate that the other orientation of Tn5 in that locale is lethal to lambda . The orientation-dependence of Chi+ phenotype also revealed that Tn5 flip-flops in lambda.

Eur J Biochem, 1980 Sep, 110(1), 161 - 6
The modification of the peptidyltransferase activity of 50-S ribosomal subunits, LiCl-split proteins and L16 ribosomal protein by pyridoxal phosphate; Baxter RM et al.; Pyridoxal phosphate photoinactivates the peptidyltransferase activity of 50-S ribosomal subunits, LiCl split proteins and protein L16 . Ethyromycin exhibits significant protection . These results, taken together with earlier reports, indicate the involvement of the single histidine of L16 in peptidyltransferase activity . The adjacent association in L16 of histidine and lysine indicates that pyridoxal phosphate should represent a selective inhibitor of peptidyltransferase activity.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5399 - 403
Replication and expression of thymidine kinase and human globin genes microinjected into mouse fibroblasts; Anderson WF et al.; A mixture of two recombinant plasmids was microinjected into mouse thymidine kinase-negative fibroblasts (L cells) . One plasmid contained the thymidine kinase gene of herpes simplex virus type I and the other contained the human beta globin gene . Seven fibroblast colonies arising from injected cells incubated in hypoxanthine/aminopterin/thymidine medium were analyzed . These microinjected cells were shown to: (i) produce functionally active herpes simplex type I thymidine kinase enzyme, (ii) replicate the human beta globin gene, and (iii) produce human beta globin mRNA sequences at low levels . Thus, the genetic defect (lack of thymidine kinase activity) was corrected by the microinjected thymidine kinase gene, and a coinjected human beta globin gene was replicated and weakly expressed.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5390 - 3
Four heat shock proteins of Drosophila melanogaster coded within a 12-kilobase region in chromosome subdivision 67B; Corces V et al.; Unique coding sequences for four heat shock proteins of Drosophila melanogaster, hsp 28, hsp 26, hsp 23, and hsp 22, are clustered in a 12-kilobase interval at chromosome subdivision 67B . The four genes are not transcribed in the same direction and each gives rise to a separate messenger RNA, with no indication of intervening sequences . Including the present results, the genes for all seven major heat shock proteins of D . melanogaster are now cloned are found to exhibit a variety of patterns of organization at the five loci they occupy.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5336 - 40
Putative actin genes in the macronucleus of Oxytricha fallax; Kaine BP et al.; Previous work has shown that the macronuclear DNA of the hypotrichous ciliate Oxytricha fallax is arranged as short achromosomal pieces, 22 to 0.5 kilobase pairs (kb) in length . Micronuclear DNA has a typical chromosomal organization . Macronuclear DNA is derived from micronuclear DNA through a process of polytene chromosome fragmentation with a resultant decrease in DNA sequence complexity . Three putative actin genes have been identified in macronuclear DNA by using a cloned yeast actin gene as a hybridization probe . A restriction fragment of the yeast gene containing both actin coding and noncoding DNA hybridizes strongly to two macronuclear DNA pieces, 1.6 and 1.4 kb in length, and weakly to a 1.2-kb piece . The entire 1.6-kb piece has been cloned in plasmid pBR322 and the resulting recombinant plasmid has been designated pOfACT(1.6) . The 1.6-kb pOfACT(1.6) insert hybridizes only to those restriction fragments of the yeast actin gene containing actin coding sequences . When hybridized to macronuclear DNA under conditions that allow the yeast probe to hybridize to all three macronuclear pieces, the pOfACT(1.6) insert hybridizes only to the 1.6-kb piece . Under less stringent conditions the insert also hybridizes to the 1.4-kb piece, but it shows no hybridization to the 1.2-kb DNA . The three macronuclear pieces homologous to the yeast actin gene thus differ in sequence and are interpreted as a related family of actin genes . Each of these pieces could accommodate an actin coding sequence, which in yeast, Dictyostelium discoideum, and Drosophila melanogaster is 1.1 kb, and an additional 0.1-0.5 kb of noncoding DNA.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5317 - 21
Messenger RNA prevalence in sea urchin embryos measured with cloned cDNAs; Lasky LA et al.; mRNA prevalence during sea urchin development was measured by treating cDNA clone colonies with labeled cDNAs transcribed from unfertilized egg and embryo poly(A)-RNAs . The number of cytoplasmic transcripts per embryo complementary to several clones was determined independently by titration with poly(A)-RNA in solution, and the amount of cDNA bound to these clones in colony hybridizations was shown to be proportional to the concentration of the respective poly(A)-RNAs in the embryo cytoplasm . At the gastrula stage, the most prevalent mRNA species occur in about 10(6) molecules per embryo . If all cells were equivalent, this would be a few hundred molecules per cell . By pluteus stage, the prevalence of some sequences has increased more than 10-fold . Most, though not all, sequences prevalent in later embryos are also present in the maternal RNA of the unfertilized egg . For most poly(A)-RNA sequences, the prevalence levels determined during oogenesis are maintained through the pluteus stage, whereas a minority of sequences display sharp stage-specific changes in representation during development.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5292 - 6
Cloning of an origin of DNA replication of Xenopus laevis; Watanabe S et al.; DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Escherichia coli plasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X . laevis . After measurement of the {3H}-thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (550 base pairs) and several kilobases of frog DNA, were selected for more extensive analysis . In spite of the small size of the segment in pSW14, it incorporates in 2 hr at least 3 times as much labeled thymidine as either pSW9 or the vector alone . The DNA synthesis in pSW14 was shown to be replication rather than repair synthesis, based on a buoyant density shift of the product when iododeoxyuridine was used for labeling . To determine the number of replications of pSW14, a novel method was employed . Because pSW14 is a head-to-head dimer of the vector with the Xenopus fragment inserted at an EcoRI site, the plasmid has three methylatable sites--two bracketing the Xenopus fragment and one opposite the fragment . By cotransformation of E . coli with pSW14 and pBR322 containing the EcoRI methylase gene, supercoiled pSW14 was methylated and injected into eggs with {3H}thymidine . Disappearance of modified EcoRI sites by semiconservative replication was followed by measuring the sensitivity to EcoRI endonuclease over time . The results showed that about 50% of the labeled, supercoiled DNA recovered from eggs after 4 hr was sensitive to EcoRI digestion, which indicates that most of the DNA that incorporated {3H}thymidine had replicated twice during the 4 hr in the unfertilized eggs of X . laevis . We conclude that pSW14 has a functional origin in the Xenopus DNA segment.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5182 - 6
In vitro DNA replication of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage phi X174; Zipursky SL et al.; The origin of phage phi X174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned . In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for phi X174 RFI DNA as template in phi X174-specific reactions in vitro . We have shown that the recombinant plasmids: (i) are cleaved by the phi X174 A protein; (ii) support net synthesis of unit-length single-stranded circular DNA in the presence of the phi X174 A protein and Escherichia coli rep protein, DNA-binding protein, and DNA polymerase III elongation system; (iii) support replication of duplexes catalyzed by the phi X174 A protein and extracts of E . coli.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5177 - 81
Cellular sequences are present in the presumptive avian myeloblastosis virus genome; Souza LM et al.; EcoRI restriction endonuclease fragments from a lambda proviral DNA hybrid containing the entire presumptive avian myeloblastosis virus (AMV) provirus, and from a lambda proviral hybrid containing a partial myeloblastosis-associated virus type 1 (MAV-1)-like provirus were compared by heteroduplex analysis . The cloned presumptive AMV provirus was also analyzed by electron microscopy, using R-loop formation with purified 35S RNA isolated from virions of the standard AMV complex . The results indicate that the putative AMV genome contains a segment absent in its MAV-1-like helper virus . This segment represents a substitution in the region of the genome that in MAV-1 virus is occupied by the envelope gene and is approximately 900 +/- 160 nucleotide pairs in length . Hybridization of specific probes from the presumptive AMV genome to Southern blots of EcoRI-digested cellular DNA has revealed that these substituted sequences are homologous to chicken and duck DNA that is not related to chicken endogenous proviral sequences.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5163 - 6
Acyclovir inhibition of Epstein-Barr virus replication; Datta AK et al.; Acyclovir {9-(2-hydroxyethoxymethyl)guanine} triphosphate inhibits Epstein-Barr virus (EBV)-associated DNA polymerase (DNA nucleotidyltransferase; EC 2.7.7.7) to a greater extent than it inhibits host alpha and beta DNA polymerases . The affinity of the compound for viral polymerase is 100-fold higher than for alpha-polymerase . The extent of inhibition is dependent upon the base composition of the template-primer . The inhibition is prevented by increasing concentrations of deoxyguanosine triphosphate . The EBV-associated DNA polymerase reaction in the presence of the inhibitor, although depressed, proceeds at a linear rate over a long period of time . In contrast, the reaction of Escherichia coli DNA polymerase I in the presence of 2',3'-dideoxythymidine 5'-triphosphate, a DNA chain terminator, levels off after initial linearity . Preincubation of acyclovir triphosphate with DNA and enzyme does not increase its inhibitory activity . The virus-producing cell line P3HRF-1 consistently shows reduced viral genome numbers and viral capsid antigen on prolonged exposure to acyclovir . The number of EBV genomes returns to the control level when the cells are grown in drug-free medium . The results suggest that a competitive mechanism is the major mode of acyclovir inhibition of EBV replication.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5153 - 7
Cloning and analysis of a cDNA coding for bovine prothrombin; MacGillivray RT et al.; Poly(A)-RNA enriched for prothrombin was isolated by specific immunoprecipitation of bovine liver polysomes . Prothrombin consisted of about 8% of the cell-free translation products of this RNA . A double-stranded cDNA was synthesized by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and made blunt-ended with nuclease S1 . After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved previously at the single Pst I site and similarly tailed with dGTP . The resulting plasmids were used to transform Escherichia coli strain RR1 under P3-EK1 conditions . Sixty-three tetracycline-resistant clones were obtained that hybridized to 32P-labeled cDNA synthesized from prothrombin-enriched mRNA . Recombinants containing cDNA to prothrombin mRNA sequences were screened by a solution hybridization assay with a {3H}cDNA synthesized from mRNA . This enriched mRNA was 50% prothrombin mRNA, as determined by a reticulocyte lysate translation assay . Three positive clones were identified by this assay; they contained bovine DNA inserts of 700, 500, and 400 base pairs . The DNA sequence of the 700-base-pair insert was then determined . This recombinant plasmid contained DNA coding for the carboxyl-terminal 160 residues of bovine prothrombin followed by a noncoding region of 119 base pairs and a poly(A) tail of 60 base pairs.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5092 - 6
Construction of a composite tRNA gene by anticodon loop transplant; Yarus M et al.; By using sites for the restriction nuclease Hpa II, the information for the anticodon stem and loop of an altered Su+2 amber suppressor tRNA (a mutant of tRNAGln) has been transplanted to a specially prepared tRNATrp gene, which lacks it homologous anticodon stem and loop sequence . The resulting tRNA gene was cloned under lac operator-promoter control . The result is a functional, hybrid, amber-suppressor tRNA that can exhibit a moderately high efficiency in translation . It appears less efficient, however, than Su+7 tRNA, the amber suppressor that results from a direct anticodon mutation in tRNATrp . As judged by its suppressor spectrum, which is almost identical to the spectra of Su+2, and Su+7, the recomposed tRNA inserts glutamine at amber sites . This experiment is the prototype of a series of construction that examine the role of the nucleotides in the anticodon region.

Gene, 1980 Sep, 10(4), 307 - 18
Restriction endonuclease mapping of ColE2-P9 and ColE3-CA38 plasmids; Watson R et al.; Using single and double restriction-endonuclease digestions, 16 and 17 cleavage sites have been mapped for the ColE2-P9 and ColE3-CA38 plasmids, respectively . One or more sites for AvaI, BglI, EcoRI, HincII, PvuI, PvuII, SmaI and XhoI endonucleases were found in both plasmids, two BglII sites were found only in ColE2-P9, and one KpnI site was unique to ColE3-CA38 . ColE2-P9 was found to be slightly smaller than ColE3-CA38, 4.4 Md compared to 4.6 Md . Eleven restriction sites are common to both plasmids in that they are identically placed relative to each other . These sites define a continuous DNA segment equal to over 60% of each plasmid . The remaining portions of the plasmids, which contain the non-homologous regions identified by Inselburg and Johns (1975) have no restriction sites in common, and differ in size by about 0.2 Md.

Mol Biol (Mosk), 1980 Sep-Oct, 14(5), 1098 - 1109
{Simple method of cloning eukaryote DNA: production of certain new ribosomal genes of Drosophila}; Kolchinskii AM et al.; We propose a simple method which allows to receive a collection of clones containing recombinant plasmids . It is based on the ligation of the longer fragment of pBR332 formed by EcoRI and BamH1 with eukaryotic DNA (from Drosophila melanogaster embryo in this case) partially cleaved with EcoRI and BamHI . This approach gave us 10(4) colonies from 1 microgram of Drosophila DNA and 0.1 microgram of the BamHI--EcoRI "vector" . About 0.5% of all clones carried the fragments of ribosomal genes with insertions in the 26S gene . Ribosomal genes lacking insertions did not enter the collection due to some peculiarities in their restriction map . The sites of cleavage are mapped in eight recombinant plasmide for HindIII, BamHI and EcoRI . These maps show that some insertions within 26S gene have not been cloned earlier . The mean length of cloned fragments is 11.8 kilobases, the mean number of EcoRI and BamHI restriction sites are 1.2 and 1.0, respectively . The electrophoretical screening of plasmids using cetyl trimethyl ammonium bromide was developed.

J Bacteriol, 1980 Sep, 143(3), 1362 - 73
Genetic organization of the broad-host-range IncP-1 plasmid R751; Meyer RJ et al.; We have identified regions encoding conjugal transfer, plasmid maintenance, and trimethoprim resistance on the IncP-1 plasmid R751 by complementation tests with cloned deoxyribonucleic acid fragments and self-replicating derivatives constructed in vitro . The genes for replication and transfer show a scattered organization similar to that previously determined for RK2, another IncP-1 plasmid . Derivatives of RK2 are able to complement R751 derivatives defective in these functions . Restriction enzyme cleavage sites in R751 deoxyribonucleic acid are clustered in regions of the plasmid physical map . Neither region is required for plasmid maintenance or transfer, although one determines resistance to trimethoprim . A similar clustering of cleavage sites is seen with RK2, which nevertheless has a very different restriction map.

J Bacteriol, 1980 Sep, 143(3), 1171 - 8
Tn2301, a transposon construct carrying the entire transfer region of the F plasmid; Johnson DA et al.; The largest R . BamHI fragment of the plasmid F, which carries the entire F conjugation system, has been cloned into the single R . BamHI site of the ampicillin (Ap) resistance transposon TN1 . pDS1106 (ColE1 mob::Tn1) was the vector plasmid, and the resultant conjugative plasmid, pED830, was characterized both genetically and by restriction enzyme analysis . The transposon construct, denoted Tn2301, was transposable at frequencies similar to Tn1 to small nonconjugative plasmids or to the Escherichia coli host chromosome . In the former case, Apr conjugative plasmids were obtained, whereas in the latter case, Hfr strains resulted . Representative Hfr strains were characterized by quantitative and interrupted mating experiments . Extension of this technique for Hfvr formation should aid chromosome mapping both in E . coli and in other bacterial genera.

J Gen Microbiol, 1980 Sep, 120(1), 183 - 98
Adenosine triphosphatase activity and its sensitivity to ruthenium red oscillate during the cell cycle of Escherichia coli K12; Scott RI et al.; A procedure has been developed for the large-scale fractionation into size and age classes of bacteria from exponentially growing cultures of Escherichia coli K12 by centrifugation through an equivolumetric gradient of sucrose in a zonal rotor . The resolution attained is superior to that in methods of this type that have been described previously . The activity of adenosine triphosphatase (ATPase) was assayed in extracts from bacteria separated into size classes by this method and from synchronous cultures prepared by size selection . Activity approximately doubled during a cell cycle, but the experimental data did not fit models of either continuously or exponentially increasing activity during the cycle . It is suggested that ATPase activity oscillates during the cell cycle with maxima at about 0.37 and 0.80 of a cycle . The fluctuations in activity greatly exceed the variations due to experimental error and, in the case of synchronous cultures, do not arise from perturbations in growth behaviour following zonal gradient selection . Sensitivity of ATPase activity to 75 micrometer-Ruthenium Red also fluctuates during the cell cycle, with maximum inhibition (60 to 80%) occurring near the middle of the cycle, a time that does not coincide with maximum enzyme activity.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5230 - 3
Expression of the human fibroblast interferon gene in Escherichia coli; Taniguchi T et al.; We applied the method of Guarente et al . {Guarente, L., Lauer, G., Roberts, T.M . & Ptashne, M . (1980) Cell 20, 543-553} to construct plasmids that direct expression in Escherichia coli of the human fibroblast interferon (F-IF) gene . Two plasmids were recovered . One directs efficient synthesis of a protein whose primary sequence is that of pre-F-IF and the other, that of mature F-IF . Extracts of bacteria synthesizing mature F-IF display antiviral activity characteristic of human F-IF . This activity is lower than that expected from the differential rate of synthesis of the protein . We have detected no such activity in extracts of bacteria synthesizing pre-F-IF.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5110 - 4
Mechanism of translocation: relative arrangement of tRNA and mRNA on the ribosome; Matzke AJ et al.; AcPhe-tRNAPhe from yeast can be photocross-linked to poly(U) on Escherichia coli ribosomes . The photoreaction occurs at the wybutine base situated next to the 3' side of the anticodon . The kinetics and efficiency of crosslinking of AcPhe-Phe-tRNA are the same at both the acceptor site and the peptidyl site . Therefore, the orientation of wybutine with respect to the mRNA is similar in both the pretranslocational and posttranslocational states . AcPhe-Phe-tRNA crosslinked at the acceptor site can still be translocated to the peptidyl site, demonstrating that tRNA and mRNA are transported together . The experiments support a model of translocation in which the conformation of the anticodon loop of tRNA is similar in both the peptidyl site and the acceptor site.

Infect Immun, 1980 Sep, 29(3), 1125 - 33
Cloning and expression of a deoxyribonucleic acid fragment that encodes for the adhesive antigen K99; van Embden JD et al.; Deoxyribonucleic acid fragments of the naturally occurring conjugative K99 plasmid were cloned into vectors pBR322 and pBR325 . The smallest deoxyribonucleic acid segment obtained that still expressed K99 was 4.5 megadaltons in size . With regard to the serological, adhesive, and morphological properties, no differences in the nature of the K99 antigen was observed between Escherichia coli strains carrying recombinant plasmids and those carrying pRI9901 . Furthermore, the regulation of K99 expression from the recombinant plasmid deoxyribonucleic acid was similar to that from pRI9901 . the low level of K99 expression in E . coli K-12 compared with natural K99-producing isolates seems to be host specific.

J Bacteriol, 1980 Sep, 143(3), 1534 - 7
Lactose operon transcription from wild-type and L8-UV5 lac promoters in Escherichia coli treated with chloramphenicol; Hirschel BJ et al.; In cells treated with chloramphenicol and the inducer isopropyl-beta-D-thiogalacto-pyranoside, messenger ribonucleic acid transcription from the wild-type lac promoter was not detected . Transcription occurred from the mutant UV5-L8 promoter . The transcripts were of variable length; some included the whole Z gene . No major site of transcription arrest within the Z gene was apparent.

J Bacteriol, 1980 Sep, 143(3), 1307 - 17
tif-dependent induction of colicin E1, prophage lambda, and filamentation in Escherichia coli K-12; Tessman ES et al.; To help understand how the tif-1 mutation of the recA gene of Escherichia coli confers adenine activability on the recA protein, we used the fact that cytidine plus guanosine inhibits induction of prophage lambda and cell filamentation in a tif-1 mutant, and that adenine reverses this inhibition . We varied the amount of adenine in agar plates containing a fixed amount of cytidine and scored for survivors of three different tif-dependent lethal induction processes . Much more adenine was required for cell killing when cytidine was present than when it was absent . Therefore adenine does not override cytidine inhibition, but instead appears to compete with it for a site of action which may be on the recA protein . The competition is not at the cell transport level . Our results lead to a model in which the tif form of the recA protein is an allosteric enzyme that binds both negative and positive modulators . By varying the adenine-cytidine ratio of the medium it is possible to control the degree of induction in a tif-1 cell . For the three different tif-dependent inductions studied here, least adenine was required for lambda induction and most for lethal filamentation, presumably reflecting requirements for different amounts of activated recA protein in each process . Varying the adenine-cytidine ratio revealed two stable intermediate stages in lambda induction, as well as a stage of colicin E1 induction in which the cells produced colicin without cell death . The rate of filament formation could be similarly controlled . Experiments with tif (ColE1, lambda) gave evidence of a competition between colicin repressor and lambda repressor for activated recA protein.

J Bacteriol, 1980 Sep, 143(3), 1265 - 74
Kinetics and regulation of cell-free alkaline phosphatase synthesis; Pratt C; Regulation of alkaline phosphatase (EC 3.1.3.1) synthesis in a cell-free system from Escherichia coli has been observed . Synthesis from transducing phage deoxyribonucleic acid templates carrying phoA+ occurred in S30 fractions from wild-type or alkaline phosphatase-constitutive mutants . It did not occur in S30) fractions from alkaline phosphatase-negative mutants (phoB) . The hybrid gene phoA-lacZ was also subject to phoB control, implying that phoA transcription is regulated . The yield of active alkaline phosphatase per phoA+ gene copy from cell-free synthesis was similar to that of beta-galactosidase . Alkaline phosphatase activity took longer to appear than beta-galactosidase activity . Synthesis of alkaline phosphatase subunits was not delayed, suggesting that a minimum number of subunits are synthesized before formation of active alkaline phosphatase occurs.

J Bacteriol, 1980 Sep, 143(3), 1223 - 33
Effects of starvation for potassium and other inorganic ions on protein degradation and ribonucleic acid synthesis in Escherichia coli; St John AC et al.; Starvation of Escherichia coli for potassium, phosphate, or magnesium ions leads to a reversible increase in the rate of protein degradation and an inhibition of ribonucleic acid (RNA) synthesis . In cells deprived of potassium, the breakdown of the more stable cell proteins increased two- to threefold, whereas the hydrolysis of short-lived proteins, both normal ones and analog-containing polypeptides, did not change . The mechanisms initiating the enhancement of proteolysis during starvation for these ions were examined . Upon starvation for amino acids or amino acyl-transfer RNA (tRNA), protein breakdown increases in relA+ (but not relA) cells as a result of the rapid synthesis of guanosine-5'-diphosphate-3'-diphosphate (ppGpp) . However, a lack of amino acyl-tRNA does not appear to be responsible for the increased protein breakdown in cells starved for inorganic ions, since protein breakdown increased in the absence of these ions in both relA+ and relA cultures, and since a large excess of amino acids did not affect this response . In bacteria in which energy production is restricted, ppGpp levels also rise, and protein breakdown increases . The ion-deprived cultures did show a 40 to 75% reduction in adenosine-5'-triphosphate levels,l similar to that seen upon glucose starvation . However, this decrease in ATP content does not appear to cause the increase in protein breakdown or lead to an accumulation of ppGpp . No consistent change in intracellular ppGpp levels was found in relA+ or relA cells starved for these ions . In addition, in relX mutants, removal of these ions led to accelerated protein degradation even though relX cells are unable to increase ppGpp levels or proteolysis when deprived of a carbon source . In the potassium-, phosphate-, and magnesium-deprived cultures, the addition of choramphenicol or tetracycline caused a reduction in protein breakdown toward basal levels . Such findings, however, do not indicate that protein synthesis is essential for the enhancement of protein degradation, since blockage of protein synthesis by inactivation of a temperature-sensitive valyl-tRNA synthetase did not restore protein catabolism to basal levels . These various results and related studies suggest that the mechanism for increased protein catabolism on starvation for inorganic ions differs from that occurring upon amino acid or arbon deprivation and probably involves an enhanced susceptibility of various cell proteins (especially ribosomal proteins) to proteolysis.

Plasmid, 1980 Sep, 4(2), 155 - 69
Morphological and serological relationships of conjugative pili; Bradley DE; It is now known that conjugative pili are determined by representative plasmids for all incompatibility groups in Escherichia coli K-12 . They fall into three basic morphological groups, which are described: thin flexible, thick flexible, and rigid filaments or rods . The main thrust of this study, however, has been the use of immune electron microscopy to survey pili of all established incompatibility groups for serological cross-reactions . Morphologically identical thin flexible pili were determined by plasmids of the I complex, as well as IncB and IncK . Immune electron microscopy revealed two unrelated serotypes typified by Ia and I2 pili; K and B pili belonged to the first serotype . Thick flexible pili were determined by plasmids of Inc groups C, D, the F complex, H1, H2, J, T, V, X, com9, the single plasmid F0 lac, and the unclassified plasmid R687 . Serological tests showed that C pili were related to J pili, H1 pili to H2 pili, com9 pili to F0 lac pili, and R687 pili to D pili, the remainder being unrelated . Rigid pili were determined by plasmids of Inc groups M, N, P, W, and by the unclassified plasmids R775, RA3, and pAr-32 . The only relationship detected was between RA3 and pAr-32 pili . No cross reactions were found between pili of the three different morphological groups.

Eur J Biochem, 1980 Sep, 110(1), 279 - 88
Affinity chromatography on immobilised nucleotides . The synthesis, specificity and applications of immobilised inosine 5'-monophosphate; Clonis YD et al.; The synthesis and characterisation of two IMP analogues, 8-(6-aminohexyl)-ionosine 5'-monophosphate, Ahx8IMP, and inosine 2',3'-O-{1-(6-aminohexyl)-levulinic acid amide}-acetyl 5'-monophosphate, (AhxLvn)2',3'IMP, is described . These analogues were attached to CNBr-activated agarose through the terminal amino group of the spacer molecule . The immobilised-IMP analogues displayed specificity for the inosine-nucleotide-dependent enzyme, IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.2.1.14) but not for the NAD+-dependent enzymes, L-alanine and L-acetate dehydrogenases . Escherichia coli IMP dehydrogenase could be eluted biospecifically from immobilised 8-substituted and ribose-substituted IMP adsorbents with IMP, XMP and GMP . Multiple peaks of enzyme activity in the elution profiles were interpreted in terms of aggregation of the enzyme . A protocol for the large-scale purification of E . coli IMP dehydrogenase is proposed . Homogeneous enzyme of specific activity 9.1 units/mg was obtained in 50% overall yield, representing 14 mg pure protein from a 20-1 culture of E . coli . The two IMP analogues were inactive as substrates in the IMP dehydrogenase reaction.

Infect Immun, 1980 Sep, 29(3), 908 - 13
Inhibition of Escherichia coli heat-stable enterotoxin by indomethacin and chlorpromazine; Greenberg RN et al.; Purified heat-stable enterotoxin (ST) from a procine strain of enterotoxigenic Escherichia coli activates quanylate cyclase in particulate fractions of rat intestinal tissue and induces fluid accumulation in suckling mice . These effects of ST were examined in the presence of either indomethacin or chlorpromazine . We also examined the effects of these two drugs on fluid accumulation in suckling mice induced by the 8-bromo analog of cyclic guanosine monophosphate . Either indomethacin or chlorpromazine reduced ST activation of guanylate cyclase . Both drugs also reduced intestinal fluid accumulation in suckling mice that resulted from submaximal doses of ST (both P < 0.001) . However, there was no reduction in fluid secretion by either drug when a maximally effective dose of ST was used, suggesting that inhibition of fluid secretion by both drugs can be overcome by increasing the ST dose and that a threshold level of guanylate cyclase activity results in maximal secretory response . Both drugs also reduced basal guanylate cylase activity in rat intestinal tissue and fluid secreton in suckling mice . Chlorpromazine also reduced intestinal secretion mediated by 8-bromo cyclic guanosine monophosphate (P < 0.001) . These findings indicate that chlorpromazine interferes with the effects of ST both before and after its activation of guanylate cyclase, whereas indomethacin interfers with ST only before its activation of guanylate cyclase.

Plasmid, 1980 Sep, 4(2), 175 - 83
Cloning the Tra1 region of RP1; Watson J et al.; The Tra1 region of RP1 from a derivative with Tn7 inserted into the kanamycin resistance determinant was cloned, using EcoRI, into the multicopy vector plasmid pBR325 . For one orientation of the cloned fragment the resultant chimeric plasmid was very frequently lost from the cell, but in the other orientation it was much more stable and also compatible with RP1 . Complementation by the stable chimeric plasmid, pED800, of a series of RP1 tra mutants showed that the mutations of all those retaining sensitivity to the P-specific phages PRR1, Pf3, and PR4, or only to PR4, mapped in the Tra1 region, while only 2 out of 20 amber mutations leading to full P-specific phage-resistance did so.

Plasmid, 1980 Sep, 4(2), 170 - 4
Cloning of Lac+ BamHI fragment into transposon Tn3 and transposition of the Tn3{lac} element; Manis J et al.; By use of recombinant DNA techniques, we have inserted the lac+ operon into a transposon (Tn3) . We constructed the recombinant in such a way that the essential step in assaying for transposition consisted of screening for bacteria with a thermostable Lac+ phenotype . Our results showed that transposition of the Tn3{lac+} element occurred and that its frequency was derepressed compared to frequencies reported by others for wild-type Tn3 transposition.

Plasmid, 1980 Sep, 4(2), 123 - 9
Persistence and spread of a chloramphenicol resistance-mediating plasmid in antigenic types of Escherichia coli, pathogenic for piglets; Jorgensen ST et al.; All chloramphenicol-resistant Escherichia coli strains isolated from piglets in the State veterinary Serum Laboratory, Copenhagen, in 1974-1975 harbored plasmids of IncFII group with largely the same resistance markers . Two strains from 1978 carried plasmids with similar characters . Restriction enzyme analysis of DNA from these plasmids with restriction endonucleases EcoRI, BglII, and PstI shows that the Cm plasmids are extremely closely related; but the patterns obtained (particularly from PstI digests) enable the classification of the plasmids into groups . These bear a strong relation to time and place of isolation so that plasmids isolated on the same farm belong to the same group even when their host strains are of different antigenic types . It is concluded that these plasmids have evolved from a single plasmid.

Biochim Biophys Acta, 1980 Aug 26, 609(1), 84 - 96
Specific sequences within single-stranded regions in the sea urchin embryo genome; Wortzman MS et al.; Naturally occurring single-stranded regions in native DNA isolated from sea urchin embryos at the morula stage were removed by digestion with S1 nuclease . Renaturation experiments show that this nuclease removes a portion of the repetitive sequences renaturing between Cot 10(-2) and 20 including about two-thirds of the histone genes . The latter was shown by hybridization of S1-treated morula DNA to either sea urchin 9 S polysome RNA (containing histone mRNA) or to Escherichia coli plasmid DNA carrying sea urchin histone genes . Hybridization of sea urchin DNA to strand-separated recombinant histone gene DNA shows that it is the histone gene antisense strand that is missing from the gapped regions of the native DNA from morulae . Present and previous data support the conclusion that a portion of the single-stranded regions are not in random positions in the embryo genome and do not result from the unwinding of DNA in vivo at replication sites.

Biochim Biophys Acta, 1980 Aug 26, 609(1), 197 - 200
Thermoplasma acidophilum histone-like protein . Partial amino acid sequence suggestive of homology to eukaryotic histones; Searcy DG et al.; Thermoplasma acidophilum is a freeliving mycoplasma-like organism that has a small basic protein tightly bound to its DNA . The N-terminal sequence of this protein has been determined . It has a distanct but statistically significant homology to eukaryotic histones H2A, H3, and to Escherichia coli protein HU.

Biochim Biophys Acta, 1980 Aug 26, 609(1), 53 - 60
Translation of poly-A RNA from rat liver in vitro . Evidence for a high molecular weight subunit of tyrosine aminotransferase; Wagner H et al.; Poly-A RNA extracted from the rat liver was translated in a cell-free wheat germ system and a rabbit reticulocyte lysate . The subunit of tryptophan pyrrolase precipitated by specific antiserum after synthesis in vitro has the same molecular weight as the corresponding subunit derived from the rat liver . With specific antiserum prepared against tyrosine aminotransferase, however, a radioactive protein from both the in vitro assays was precipitated with an about 5% higher molecular weight than the tyrosine aminotransferase subunit precipitated from rat liver . The immunological evidence and the comparison of the specific peptide patterns prepared by cyanogen bromide treatment showed that the in vitro product corresponds to tyrosine aminotransferase . Various concentrations of potassium or spermidine used in the wheat germ translation system did not alter the size of the enzyme subunit synthesized . The run of the tyrosine aminotransferase purified form the rat liver in the SDS-polyacrylamide gel electrophoresis was not influenced by treatment with Escherichia coli alkaline phosphatase . The possibility is discussed that the larger enzyme synthesized in vitro represents a precursor molecule which is cleaved proteolytically in vivo.

Nucleic Acids Res, 1980 Aug 25, 8(16), 3721 - 8
Site-specific DNA-affinity chromatography of the lac repressor; Herrick G; To test the feasibility of site-specific DNA-affinity chromatography, E . coli lac repressor was bound to an operator-containing DNA column, and in parallel to a non-operator DNA column . Salt gradient elution shows: 1) elution from non-operator DNA was near 250mM KCl or NaCl; interpretation of this result suggests the usefulness of the procedure for studying salt-dependence of DNA-protein affinities; 2) elution from operator-containing DNA was delayed (average elution = 1000mM salt), demonstrating a feasibility of site-specific DNA-affinity chromatography, if one provides a sufficiently favorable ratio of specific to non-specific DNA binding sites; 3) repressor eluted from operator-containing DNA over a very broad salt range, which may represent chromatography-generated repressor heterogeneity.

J Biol Chem, 1980 Aug 25, 255(16), 7858 - 62
S-acetonyl-CoA . A nonreactive analog of acetyl-CoA; Rubenstein P et al.; We have synthesized S-acetonyl-CoA from CoASH and 1-bromoacetone . This thioether-containing structural analogue of acetyl-CoA is a potent competitive inhibitor, with respect to acetyl-CoA, of citrate synthase, phosphotransacetylase, and carnitine acetyltransferase . This analog will not activate Escherichia coli phosphoenolpyruvate carboxylase or rat liver pyruvate carboxylase, two enzymes which require acetyl-CoA as an obligate activator . Furthermore, acetonyl-CoA will not compete with acetyl-CoA for binding to these enzymes showing the apparent absolute requirement of these two enzymes for a thioester group on the activating ligand . S-Acetonyl-CoA should be a useful reagent in the investigation of acetyl-CoA-requiring processes.

J Biol Chem, 1980 Aug 25, 255(16), 7583 - 8
T4 ribonucleotide reductase . Physical and kinetic linkage to other enzymes of deoxyribonucleotide biosynthesis; Allen JR et al.; This laboratory has described a multienzyme aggregate from T4 phage-infected Escherichia coli which seems to participate in deoxyribonucleotide biosynthesis and efficient delivery of DNA precursors to the replication apparatus . This paper describes improved methodology for isolation of this aggregate, and we present three lines of evidence supporting a role for ribonucleoside diphosphate reductase in functioning of the presumed complex . 1) Ribonucleoside diphosphates are readily incorporated into DNA as deoxyribonucleotides in an in situ DNA-synthesizing system from T4 phage-infected cells . 2)Ribonucleotide reductase is associated with the complex, as shown by co-sedimentation of reductase activity with other activities in the multienzyme aggregate we have described . 3)Ribonucleotide reductase is kinetically coupled to at least four other enzymes involved in a sequential pathway . The aggregated enzymes catalyze the five-step conversion of uridine diphosphate to deoxythymidine triphosphate with but a brief lag before dTTP production reaches its maximal rate . These studies have also confirmed the existence of dCTPase-dUTPase and dCMP deaminase activities in the putative complex.

Nucleic Acids Res, 1980 Aug 25, 8(16), 3553 - 64
Brain tubulin and actin cDNA sequences: isolation of recombinant plasmids; Ginzburg I et al.; Rat brain mRNA enriched for tubulin and actin sequences was used to prepare double stranded cDNA . A library of recombinant clones was constructed by inserting the dsDNA into the Pst1 site of pBR322 plasmid and transformation of E . coli chi 1776 host . Clones bearing sequences coding for tubulin and actin were identified and characterized.

J Biol Chem, 1980 Aug 25, 255(16), 7521 - 4
Molecular cloning of DNA complementary to bovine growth hormone mRNA; Miller WL et al.; We have cloned DNA complementary to mRNA coding for bovine growth hormone (bGH) . Double-stranded DNA complementary to bovine pituitary mRNA was inserted into the Pst I site of plasmid pBR322 by the dC x dG tailing technique and amplified in E . coli x 1776 . A recombinant plasmid containing bGH cDNA ws identified by hybridization to cloned rat growth hormone cDNA . It contains the entire coding and 3'-untranslated regions and 31 bases in the 5'-untranslated region . Nucleotide sequence analysis determined the sequence of the 26-amino acid signal peptide and confirmed the published amino acid sequence of the secreted hormone at all but 2 residues . Codon usage is nonrandom, with 81.7% of the codons ending in G or C . The nucleotide sequence of bGH mRNA is 83.9% homologous with rat GH mRNA and 76.5% homologous with human GH mRNA, while the respective amino acid sequence homologies are 83.5% and 66.8%.

J Biol Chem, 1980 Aug 25, 255(16), 7665 - 72
On the cloning of eukaryotic total poly(A)-RNA populations in Escherichia coli; Norgard MV et al.; A total clone bank of cDNAs synthesized from mouse liver poly(A)-RNA was constructed in Escherichia coli K-12 RR1 using the plasmid pBR322 . Sequences of cDNAs were inserted into the PST-I site of pBR322 by the "G-C-tailing" method . Bulk preparations of these cDNA sequences were obtained by treatment of the resultant total hybrid plasmid population with PST-I . Aliquots of this cDNA material were then labeled with 32P by "nick translation" using E . coli DNA polymerase I for the preparation of hybridization probes . Experiments involving the back-hybridization of these probes to the total hybrid-plasmid clone bank population revealed that virtually all of the liver poly(A)-RNA sequences were represented in the total clone bank . The long term stability of these sequences residing in RR1 host cells was then examined through extensive serial passage of the initial total clone cell population culture . The results showed: 1) that the small percentage of natural pBR322 molecules (containing no cDNA inserts) usually present in such total clone bank preparations do not outgrow the respectively hybrid specie of plasmids in these populations: 2) few, if any, cDNA sequences are completely lost; and 3) some redistribution of the abundant and more unique DNA sequences probably does occur . The use of total clone hybrid-plasmid populations (constructed from poly(A)-RNA isolated from different tissues) described here should allow the identification of tissue-specific RNA sequences through the use of cross-hybridizaton techniques.

Lancet, 1980 Aug 23, 2(8191), 398 - 401
Human insulin produced by recombinant DNA technology: safety and hypoglycaemic potency in healthy men; Keen H et al.; Human insulin synthesised by recombinant DNA technology was compared with highly purified porcine insulin in healthy men . Intracutaneous injection over a wide range of concentrations of both insulins into five subjects gave rise to no local reactions over a 48 h period . The glycaemic response to standard subcutaneous injection at high and low dose levels was measured with both insulins in each of six men . Plasma glucose decrement with the two insulins was similar but human insulin was perhaps slightly more potent than porcine insulin at the low dose, and slightly less so at the high . The glycaemic response to the isulins, each infused intravenously at high and low concentrations for 1 h in a further six subjects, showed a similar trend . Depression of glycaemia with human insulin slightly exceeded that with porcine insulin at the low concentration infusion and fell slightly short of it at the high . Genetically synthesised human insulin seems to be safe and effective in man . Its dose-response relationship may differ from that of porcine insulin.

Clin Chim Acta, 1980 Aug 19, 105(3), 367 - 76
Intestinal alkaline phosphatase: an immunoprecipitation method for the determination in feces; Lehmann FG et al.; An immunoprecipitation method for the determination of human intestinal alkaline phosphatase (I-AP) in feces is described using anti-human I-AP IgG . Antibodies to human I-AP are monospecific after absorption with human placental alkaline phosphatase and show no cross-reactions with human liver alkaline phosphatase or alkaline phosphatase from E . coli . Normal controls demonstrate a logarithmic normal distribution of I-AP activity in feces . The standard deviation of the intra- as well as of the inter-assay variation is 11.7% . This method represents a simple quantitative non-invasive in vivo assay for brush border damage under experimental and/or clinical conditions.

Biochemistry, 1980 Aug 19, 19(17), 4087 - 90
Small-angle X-ray studies of the quaternary structure of the lac repressor from Escherichia coli; Pilz I et al.; The quarternary structures of the lac repressor molecule from Escherichia coli and its tetrametic core, which can be derived from it by proteolytic cleavage, were studied in dilute solutions by small-angle X-ray scattering . The dimensions and general shape of the lac repressor and of the tetrameric core are reported . The core itself appears to be an elongated structure, and in the intact repressor the headpieces are located at its ends . The results ar derived from model calculations and from the following molecular parameters determined from the scattering curve and the pair distance distribution function: for lac repressor, radius of gyration 5.30 +/- 0.02 nm, radius of gyration of the cross section 2.20 +/- 0.03 nm, maximum diameter 18.0 +/- 0.5 nm, hydrated volume 329 +/- 20 nm3, relative molecular mass 149 000 +/- 15 000, for tetrameric core, radius of gyration 4.92 +/- 0.02 nm, radius of gyration of the cross section 2.24 +/- 0.03 nm, maximum diameter 16.0 +/- 0.5 nm, hydrated volume 278 +/- 15 nm3, relative molecular mass 120 000 +/- 10 000.

Biochemistry, 1980 Aug 19, 19(17), 4031 - 43
Determination by cadmium-113 nuclear magnetic resonance of the structural basis for metal ion dependent anticooperativity in alkaline phosphatase; Otvos JD et al.; Cadmium-113 nuclear magnetic resonance (113Cd NMR) has been used to probe the binding characteristics of 113Cd2+ to the three classes of metal binding sites in Escherichia coli alkaline phosphatase to help elucidate the molecular origin of the metal ion dependent "half-sites" reactivity exhibited by this dimeric Zn2+ metalloenzyme {Otvos, J.D., Armitage, I.M., Chlebowski, J.F., & Coleman, J.E . (1979) J . Biol . Chem . 254, 4707-4713} . In the absence of phosphate, the first two 113Cd2+ ions added to the apodimer give rise to a single 113Cd resonance (169 ppm), indicating selective binding to the pair of symmetrically disposed A sites . Resonances arising from additional 113Cd2+ bound to the B and C sites cannot be observed; B- and/or C-site occupation also renders the A-site 113Cd resonance undetectable . Both these observations have been attributed to severe chemical exchange broadening in the A-, B-, and C-site 113Cd signals induced by an unknown modulation process(es) . Interestingly, covalent phosphorylation of the active-site serine residues abolishes this exchange modulation, allowing three separate resonances to be detected and assigned to 113Cd2+ located at each of the three classes of metal binding sites in the enzyme . By varying the metal composition of the phosphorylated enzyme, we have characterized the correlations that exist between the chemical shifts ana intensities of these 113Cd resonances and the metal occupancies of the A, B, and C sites in the individual subunits . This information has allowed us to conclude that the half-sites phosphorylation of the Cd2 2+ enzyme is accompanied by a slow migration of half the Cd2+ originally located at the A sites to the B sites on the phosphorylated subunits . The driving force for this metal redistribution, which at equilibrium leaves half the subnits devoid of metal ion and thereby incapable of binding phosphate, is apparently the dramatic stabilization of the complex of Cd2+ with the B sites, which was demonstrated to occur in those subunits that become phosphorylated . From the kinetics of both phosphorylation and metal redistribution in Cd2 2+ enzyme, we suggest that population of the A and B sites in a subunit, rather than the A site alone, constitutes the minimum requirement for induction of catalytic function in alkaline phosphatase . The spin relaxation properties of the enzyme-bound 113Cd2+ ions are also briefly discussed.

Biochemistry, 1980 Aug 19, 19(17), 4021 - 30
Characterization of the properties of the multiple metal binding sites in alkaline phosphatase by carbon-13 nuclear magnetic resonance; Otvos JD et al.; Carbon-13 nuclear magnetic resonance (13C NMR) of Escherichia coli alkaline phosphatase labeled biosynthetically with beta,beta-{gamma-13C}dideuteriohistidine has been used to determine the number and identity of the histidine residues that participate in metal ion coordination at the three classes of binding sites in this dimeric Zn2+ metalloenzyme . Detailed 13C NMR titrations of the apoenzyme with 113Cd2+ and Mg2+, in conjunction with parallel 13 Cd NMR measurements {Otvos, J.D., & Armitage, I.M . (1980) Biochemistry (third of three papers in this issue)}, permitted the assignment of four histidine residues as ligands to the "catalytic", or A site, metal ions, two coordinated via their N pi imidazole nitrogens and two via N pi . In addition, a fifth histidyl ligand, coordinated through N pi, was shown to be located at the "structural", or B, sites on the dimer . The "regulatory", or C, sites do not contain histidyl metal ligands . Unambiguous identification of the three histidines coordinated to metal ion via N pi was provided by the observation of resolved 113Cd-13C spin-spin coupling (3J = 12-19 Hz) in their gamma-carbon resonances . Once assigned, the 13C resonances of the five histidyl metal ligands were used to monitor the relative affinities of the A, B, and C sites for Cd2+ and Zn2+ . At pH 6.3, Cd2+ was found to bind to the A sites at least 10 times tighter than to the B or C sites, which have roughly equal affinities . In marked contrast, Zn2+ was found to have similar affinities for the A and B sites at both pH 6.3 and 8.0 . The affinity of the C sites for Zn2+ and Mg2+ was shown to be at least an order of magnitude lower . The binding constants of all three sites for Cd2+ and Zn2+ are greater than 10(5) M-1 . Evidence is also presented that suggests that the A, B, and C sites may be located in close proximity to one another in the monomers.

Biochemistry, 1980 Aug 19, 19(17), 4011 - 21
Characterization of the histidine residues in alkaline phosphatase by carbon-13 nuclear magnetic resonance; Otvos JD et al.; beta,beta-{gamma-13C}Dideuteriohistidine has been biosynthetically incorporated into alkaline phosphatase from Escherichia coli and utilized as a nonperturbing 13C nuclear magnetic resonance (NMR) probe of the environments of the histidine residues in this zinc metalloenzyme . The 13C NMR spectrum of the labeled enzyme exhibits 9 separate resonances arising from the 10 histidine residues located in each of the symmetrically disposed subunits of the dimer . The excellent resolution and large chemical shift range (14 ppm) displayed by these signals are direct consequences of the sensitivity of the histidine gamma-carbon chemical shift to the ionization state and tautomeric form of the imidazole side chains and the coordination of several of these to metal ion . The environments of the individual histidine residues were inferred by investigating the chemical shift responses of their 13C resonances to enzyme metal composition, pH, and inhibitor binding . Additional information concerning their motional freedom was obtained from spin relaxation measurements which were analyzed in terms of the contributions expected from intramolecular 13C-1H and 13C-14N dipolar relaxation and chemical shift anisotropy . The combined results indicate that 4 of the 10 histidines, the only ones that titrate with pH, are surface residues located relatively remote from the active site . Of the six nontitrating residues, one appears to be buried in a solvent-inaccessible region of the protein . Three others are almost certainly involved in metal ion ligation to active-site metal ion(s), two via their N pi nitrogen atoms and the other via N pi . The spectral characteristics of the remaining two histidine residues strongly suggest they are also located at or near the active site . One or both may also participate in metal ion coordination, although the current evidence for this is inconclusive.

Mol Cell Biochem, 1980 Aug 16, 31(3), 177 - 96
Autogenous and post-transcriptional regulation of RNA polymerase synthesis; Ishihama A et al.; The regulation of gene expression was studied, for the Escherichia coli rpoBC operon, which includes the genes, rpoB and rpoC, for the beta and beta subunits of RNA polymerase, and rplJ and rplL, for the two proteins, L10 and L7/12, of the 50S ribosome . The gene organization agrees well with the accumulated observations indicating the coordinate synthesis of RNA polymerase and ribosomes under various growth conditions for wild-type E . coli cells . On the other hand, the differential regulation of the two essential components observed under restrictive growth conditions, after addition of various drugs or with certain mutants, in particular those carrying mutations in the RNA polymerase genes, was found to take place through two novel regulation systems: The transcriptional termination at an internal attenuation site and the two autogenous and posttranscriptional controls, being specific for the two ribosomal protein genes and the two RNA polymerase subunit genes, respectively . The majority of the transcription initiated from the promoter rpoP beta terminates at an attenuator site between the promoter-proximal rplJL and the promoter-distal rpoBC genes . The frequency of the attenuation seems to control the relative level of RNA polymerase synthesis to that of ribosomes . The expression of rpoBC genes is subject to an autogenous regulation, in which both RNA polymerase holoenzyme and alpha 2 beta complex function as regulatory molecules with repressor activity . The autogenous regulation was found to operate at post-transcriptional step(s), probably at the level of translation . During the study on the regulation of RNA polymerase synthesis, we noticed that the rpoBC operon contained another autogenous regulation circuit, in which the synthesis of L10 and L7/12 was specifically repressed by the L10-L7/12 complex . Molecular mechanisms and physiological meanings of the novel regulations are discussed.

Mol Cell Biochem, 1980 Aug 16, 31(3), 147 - 64
Double-stranded RNA; Libonati M et al.; High molecular weight, fully double-stranded RNA (dsRNA) has been recognized as the genetic material of many plant, animal, fungal, and bacterial viruses (Diplornaviruses): virusspecific dsRNA is also found in cells infected with single-stranded RNA viruses . DsRNA has identified in a variety of apparently normal eucaryotic cells and is associated with the "killer" character of certain strains of Saccaromyces cerevisiae.

Experientia, 1980 Aug 15, 36(8), 916 - 8
Active transport of {32P}thiamine diphosphate in Escherichia coli; Nishimune T et al.; Active uptake of {32P}thiamine diphosphate by E . coli was analyzed using an improved method of gel filtration chromatography . The radioactive coenzyme was accumulated without dephosphorylation . From this result it was concluded that thiamine kinase is not involved in the membrane transport of thiamine in E . coli.

Nucleic Acids Res, 1980 Aug 11, 8(15), 3319 - 33
Repetitive DNA sequences near three human beta-type globin genes; Coggins LW et al.; Five repetitive DNA sequences, of average length 259 bp, have been identified in the intergenic regions which flank three human beta-tupe globin genes . A pair of inverted repeat sequences, separated by 919 bp, was found 1.0 kb to the 5' side of the epsiln-globin gene . Each contains a homologous Alu I site . Another repetitive sequence, with the same orientation as the inverted repeat sequence closest to the epsilon-globin gene, lies about 2.2 kb to the 5' side of the delta-globin gene . A pair of inverted repeat sequences, with the same relative orientations as the other pair and separated by about 800 bp, was found about 1.5 kb to the 3' side of the beta-globin gene.

Philos Trans R Soc Lond B Biol Sci, 1980 Aug 11, 290(1040), 395 - 407
Prospects for new viral vaccines; Marmion BP; Animal virology has made outstanding contributions to preventive medicine by the development of vaccines for the control of infectious disease in man and animals . Cost-benefit analysis indicates substantial savings in health care costs from the control of diseases such as smallpox, poliomyelitis, yellow fever and measels . Areas for further development include vaccines for influenza (living, attenuated virus), the herpes group (varicella: cytomegalovirus), respiratory syncytial virus, rotavirus and hepatitis A, B, and non A/non B . The general options for vaccine formulation are discussed with particular emphasis on approaches with the use of viral genetics to 'tailor make' vaccine viruses with defined growth potential in laboratory systems, low pathogenicity, and defined antigens . Current progress with the development of an inactivated hepatitis B vaccine is reviewed as a case study in vaccine development . The impact of recent experiments in cloning hepatitis B virus DNA in E . coli on the production of a purified viral polypeptide vaccine is assessed.

J Biol Chem, 1980 Aug 10, 255(15), 7276 - 80
Mechanism of propylamine-transfer reactions . Kinetic and inhibition studies on spermidine synthase from Escherichia coli; Zappia V et al.; Spermidine synthase (EC 2.5.1.16) purified from Escherichia coli has been subjected to a kinetic analysis including initial velocity and substrate analogs inhibition studies . Evidence is reported for a ping-pong mechanism, indicating that a propylaminated form of the enzyme is an obligatory intermediate in the reaction mechanism . S-Adenosyl(5')-3-methylthiopropylamine exerts a competitive substrate inhibition by combining with the improper stable enzyme form, while putrescine does not show any inhibitory effect . In order to investigate the substrate binding sites, new sulfonium-deaminated analogs of S-adenosyl(5')-3-methylthiopropylamine have been synthesized and assayed as substrates and as inhibitors of the reaction . The replacement of the amino group of adenine, or propylamine moiety of the sulfonium compound by the hydroxyl group, or both, resulted in a complete loss of activity as substrate . On the other hand, the deaminated analogs exert a competitive inhibition with respect to putrescine . On the basis of these results and in analogy with methyltransfer reactions, three recognition sites for S-adenosyl(5')-3-methylthiopropylamine on propylamine transfer enzymes are proposed.

J Biol Chem, 1980 Aug 10, 255(15), 7079 - 81
Sequence homology between the tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Escherichia coli and hemerythrin from Sipunculida; Herrmann KM et al.; The first enzyme of the common aromatic biosynthetic pathway in Escherichia coli, the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, contains iron as an integral part of the polypeptide chain, and the enzyme shows an absorption maximum around 350 nm (McCandliss, R.J., and Herrmann, K.M . (1978) Proc . Natl . Acad . Sci . U . S . A . 75, 4810-4813) . These two properties are also found in hemerythrin, the oxygen carrier of certain marine invertebrates . The amino acid sequence of residues 10 to 18 of the enzyme from E . coli, His-Ile-Thr-Asp-Glu-Gln-Val-Leu-Met, is highly homologous to the sequence of residues 54 to 62 of hemerythrin from Phascolopsis gouldii, His-Phe-Leu-Asn-Glu-Gln-Val-Leu-Met . His54 and Glu58 of hemerythrin have previously been identified through x-ray and protein sequence analysis as iron ligands . We suggest that residues 10 to 18 of the E . coli enzyme represent part of the iron binding fold in this protein, and that His10 and Glu14 are iron ligands.

Biochim Biophys Acta, 1980 Aug 7, 614(2), 583 - 90
Comparison of the mechanisms of two distinct aldolases from Escherichia coli grown on gluconeogenic substrates; Scamuffa MD et al.; Escherichia coli grown on gluconeogenic compounds as carbon sources produced two chemically and physically distinct types of fructose-1,6-biphosphate aldolases (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphatelyase, EC 4.1.2.13), while these bacteria produced only a single enzyme when grown on glucose or fructose . We have investigated this enzyme in several strains of Escherichia coli (Crookes, K-12, and B) grown on glucose, fructose lactate, pyruvate, alanine and glycerol by comparing chemical properties and mechanisms of action . Comparison of these mechanisms was accomplished by following the fate of 18O in the keto position of fructose 1,6-bisphosphate during the aldolase catalyzed cleavage reaction . The results show that the two enzymes have different mechanisms of action and are consistent with a Schiff-base mechanism for the one which was induced by gluconeogenic substrates and metal-chelate mechanism for the constitutive enzyme.

Biochim Biophys Acta, 1980 Aug 7, 614(2), 339 - 42
A study of the enzymatic inactivation of chloramphenicol by highly purified chloramphenicol acetyltransferase; Thibault G et al.; We report the purification of chloramphenicol acetyltransferase (acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) by a two-step procecdure involving chromatography on a Sepharose 4B-reduced chloramphenicol matrix and DEAE-Sephadex A-50 . This procedure resulted in a 120-fold purification with 50% recovery of the enzyme . Only one band of enzyme activity was present after electrophoresis on polyacrylamide gel . The enzyme is active over a broad pH range, maximal activity being observed near pH 7.6 . Both chloramphenicol 1-acetate of chloramphenicol 3-acetate were found to be very stable in Tris-maleate buffer at pH 6.09 with negligible interconversion . The incubation at pH 6.0 of chloramphenicol 1-acetate with the purified chloramphenicol acetyltransferase yielded chloramphenicol 1,3-diacetate . These data indicate that the enzyme acetylates specifically at the 3-hydroxy position and the diacetylation is possible only because of non-enzymatic interconversion of chloramphenicol 3-acetate to chloramphenicol 1-acetate at higher pH values.

Biochim Biophys Acta, 1980 Aug 7, 614(2), 446 - 58
Soluble arylsulfatases of human brain and some characteristics of the brain-specific arylsulfatase Bm; Lakshmi S et al.; The brain-specific arylsulfatase Bm (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was demonstrable in human and monkey brain . Arylsulfatases A, B and Bm were separated employing DEAE-cellulose chromatography . There was a distinct difference in the proportion of the sulfatases in infant and adult human brain . Arylsulfatase Bm after concanavalin A-Sepharose chromatography showed the property of binding to Sephadex G-200 totally . Several dissociating agents failed to elute the enzyme from the bound form . Under similar conditions arylsulfatase A did not show any binding to Sephadex . On treatment with Escherichia coli alkaline phosphatase adult human brain arylsulfatase Bm but not arylsulfatase A was converted into a less acidic, presumably dephosphorylated form that did not bind to DEAE-cellulose . Monkey brain arylsulfatase Bm showed a similar susceptibility to E . coli phosphatase treatment . Inorganic phosphate and serine phosphate but not mannose 6-phosphate could inhibit this dephosphorylation . There were differences in the susceptibilities to alkaline phosphatase treatment of the arylsulfatase Bm from infant and adult human brain . Endogenous phosphatase also seemed to have a role on the phosphorylated state of arylsulfatase Bm.

Biochemistry, 1980 Aug 5, 19(16), 3786 - 91
Magnesium-dependent interaction of 30S ribosomal subunits with antibodies to N6, N6-dimethyladenosine; Politz SM et al.; The modified nucleoside N6, N6-dimethyladenosine occurs in Escherichia coli 16S ribosomal RNA only in two successive positions near its 3' end . Antibodies directed against dimethyladenosine were induced with a nucleoside-albumin conjugate . As measured by second antibody precipitation of immune complexes, antidimethyladenosine antibodies bound 30S ribosomal subunits, ribosomal core particles, and ribosomal RNA which contain dimethyladenosine but showed little cross-reactivity with RNA or ribosomal subunits from a kasugamycin-resistant mutant which lacks dimethyladenosine . Antibody binding to ribosomal subunits was strongly influenced by the concentration of magnesium ion in the reaction medium and by the prior treatment of the subunits . Functionally active 30S subunits showed a striking binding optimum at 2-4 mM Mg2+; this optimum disappeared if the subunits were inactivated by dialysis against low concentrations of magnesium ion . Instead, the inactivated subunits showed a gradual increase in antibody binding as the magnesium ion concentration was raised to 20 mM; binding of 16S ribosomal RNA or subribosomal core particles from 30S subunits gave qualitatively similar curves, with no evidence of a low {Mg2+} optimum . The stability of antibody-subunit complexes was also found to depend upon subunit conformation and magnesium ion concentration; the half-life of an inactivated subunit-antibody complex (15 mM Mg2+) averaged 130 min, while active subunit-antibody complexes (3 mM Mg2+) had an average half-life of 70 min . More of the immune complexes with inactivated subunits were found to survive sucrose gradient sedimentation (relative to active subunits), and the concentration of subunits needed to halve antibody binding of {3H}-N6, N6-dimethyladenosine was lower with inactivated subunits . The results suggest that the antibody binding optimum seen with active subunits at 2-4 mM Mg2+ represents a dynamic aspect of the three-dimensional ribosomal subunit structure; a site near the 3' end of the RNA is involved, and both the availability of the modified nucleoside to an antibody probe and the stability of the resulting complexes are involved.

Biochim Biophys Acta, 1980 Aug 4, 600(2), 581 - 4
Apparent pyridoxine transport mutants of Escherichia coli with pyridoxal kinase deficiency; Yamada RH et al.; By nitrosoguanidine treatment of a vitamin B-6 auxotroph (KG980) of Escherichia coli, mutants were isolated that require for growth markedly higher concentrations of pyridoxine than the parent strain . One of the mutants, strain HN1, exhibited a severely reduced ability to take up extracellular pyridoxine . Besides, cell-free extracts of HN1 showed an extremely low activity to phosphorylate pyridoxine compared to that of KG980 . These findings together with other results suggest that phosphorylation of pyridoxine is essential for the concentration uptake of the vitamin.

Lancet, 1980 Aug 2, 2(8188), 222 - 4
Use of antisera for identification of enterotoxigenic Escherichia coli; Merson MH et al.; The usual methods for identifying enterotoxigenic Escherichia coli (ETEC) strains involve testing for production of heat-labile enterotoxins . To simplify the identification of ETEC, antisera against common ETEC O serogropus were used to identify ETEC in the stools from 618 patients with acute diarrhoea and dehydration (greater than or equal to 5% loss of body-weight) receiving treatment at a hospital in Dacca, Bangladesh . Compared with enterotoxin testing the antisera had a sensitivity of 64%, a specificity of 96%, and a predictive accuracy of 89% . These antisera may be useful in the identification ETEC in clinical laboratories which are unable to perform toxin testing and should be evaluated in other geographical areas.

Biophys J, 1980 Aug, 31(2), 215 - 28
Dielectric analysis of Escherichia coli suspensions in the light of the theory of interfacial polarization; Asami K et al.; Dielectric measurements of Escherichia coli suspensions were carried out over a frequency range from 10 kHz to 100 MHz, and marked dielectric dispersions having characteristic frequency of approximately 1 MHz were observed . On the basis of the cell model that a spheroid is covered with two confocal shells, a dielectric theory was developed to determine accurately four electrical parameters for E . coli cells such as the conductivity of the cell wall, the dielectric constant of the cell membrane, and the dielectric constant and the conductivity of the protoplasm . The observed data were analyzed by means of the procedure based on the dielectric theory to yield a set of plausible electrical parameters for the cells . By taking account of the size distribution of the cells and a dielectric relaxation of the protoplasm, the observed dispersion curves were successfully reconstituted by the present theory.

Biokhimiia, 1980 Aug, 45(8), 1457 - 62
{Heterogeneity of DNA-methylases from Escherichia coli CK cells}; Nikol'skaia II et al.; The existence of five DNA methylases, differing in their specificity towards the methylated sequence, in the cells of E . coli CK has been demonstrated . Two enzymes methylate cytosine to form 5'-methylcytosine, while three enzymes methylate adenine to form 6'-methylaminopurine . A method consisting in simultaneous isolation of five individual enzymes, which involves gel-filtration of the protein fraction with 0.3-0.6 M ammonium sulfate on Biogel A-05AM, is proposed.

J Gen Microbiol, 1980 Aug, 119(2), 527 - 33
Effect of thymine deprivation on the restoration of DNA synthesis in ultraviolet-irradiated Escherichia coli B/r Hcr+; Brozmanova J et al.; The influence of a prior period of thymine and amino acid deprivation on the restoration of DNA synthesis and on the fraction of cells surviving after u.v . irradiation and subsequent incubation with chloramphenicol in Escherichia coli B/r Hcr+ has been studied . Thymine and amino acid deprivation stimulated post-irradiation DNA synthesis and increased the fraction of surviving cells if, in the period between deprivation and u.v . irradiation, protein synthesis occurred . It is concluded that proteins induced by thymine starvation participated in the repair of u.v.-irradiated cells.

J Gen Microbiol, 1980 Aug, 119(2), 327 - 31
Effect of tif expression, irradiation of recipient and presence of plasmid pKM101 on recovery of a marker from a donor exposed to ultraviolet light prior to conjugation; von Wright A et al.; To detect the effect of the postulated inducible error-prone repair system ('SOS repair') on the bacterial chromosome, an Hfr Escherichia coli strain JC5088 recA was u.v.-irradiated immediately before mating it with recipients in which SOS repair was supposed to be functioning through tif expression, u.v . irradiation or the presence of plasmid pKM101 . The recombinant yields of these crosses were compared with those obtained in corresponding crosses with recipients in which SOS repair either was not induced or was totally eliminated by the lexA mutation . No difference in marker recovery efficiency could be detected between these two sets of recipients and thus no induced repair process acting on donor DNA could be demonstrated . The possible reasons for this finding are discussed.

J Clin Microbiol, 1980 Aug, 12(2), 193 - 8
Hemolytic activity in enterotoxigenic and non-enterotoxigenic strains of Escherichia coli; DeBoy JM 2nd et al.; We screened 223 strains of Escherichia coli belonging to serotypes previously associated with the production of enterotoxin for hemolytic activity, using horse erythrocytes in liquid and in agar media . Thirty-eight were hemolytic . They belonged to nine different serotypes; most (65.8%) belonged to one serotype, O6: H- . Additionally, all 38 strains were specifically assayed for a filterable, heat-labile hemolytic activity previously associated with a hemolysin plasmid . A comparison of hemolytic activity and enterotoxicity showed that none of 32 strains hemolytic in both media was enterotoxigenic; 28 of the 32 expressed heat-labile hemolytic activity . Four of the six strains hemolytic in only one of the media were enterotoxigenic; none of these six expressed heat-labile hemolytic activity . Of 223 strains, 176 that were of human origin and isolated in the United States were further assayed for three traditionally plasmid-mediated characteristics: heat-labile enterotoxin, heat-stable enterotoxin, and colonization factors . The interrelationships of these characteristics, including hemolytic activity, may reflect varying degrees of plasmid compatibility.

J Bacteriol, 1980 Aug, 143(2), 934 - 41
Pleiotropic effects of alkaline phosphatase regulatory mutations phoB and phoT on anaerobic growth of and polyphosphate synthesis in Escherichia coli; Zuckier G et al.; The appearance during anaerobiosis of spontaneous phoT phoB double mutants in a phoT background is described . During both exponential growth and stationary phase, selection against the phoT mutants relative to the wild type was evident . This reduction in viability of phoT mutants was suppressed in phoT phoB double mutants . Sensitivity to anaerobiosis was shown to be correlated with polyphosphate overproduction . A possible pleiotropic function of phoT and phoB is suggested.

J Bacteriol, 1980 Aug, 143(2), 726 - 30
Regulation of fatty acid degradation in Escherichia coli: dominance studies with strains merodiploid in gene fadR; Simons RW et al.; Strains stably merodiploid in the 25-min region of the chromosome of Escherichia coli were constructed and used in dominance tests between various wild-type and mutant alleles of the fadR gene . Whereas the monoploid fadR+ and fadR strains were inducible and constitutive, respectively, for the enzymes involved in fatty acid degradation (fad), merodiploids with at least one fadR+ allele were inducible . This observation was true whether the fadR+ allele resided on the main chromosome or on the episome . These results show that fadR+ is trans dominant to fadR, and they are consistent with the proposal that the fadR gene product is a repressor protein . Complementation tests were also performed by constructing 24 merodiploids harboring fadR alleles on both the main chromosome and the episome . All of these fadR/fadR diploids were able to utilize the noninducing substrate decanoate as sole carbon source, suggesting that only one polypeptide is encoded by the fadR gene.

J Bacteriol, 1980 Aug, 143(2), 720 - 5
Elevated levels of glyoxylate shunt enzymes in Escherichia coli strains constitutive for fatty acid degradation; Maloy SR et al.; Mutants of Escherichia coli K-12 constitutive for the synthesis of the enzymes of fatty acid degradation (fadR) have elevated levels of the glyoxylate shunt enzymes, isocitrate lyase and malate synthase . A temperature-sensitive fadR strain has high levels of glyoxylate shunt enzymes when grown at elevated temperatures but has low, inducible levels of glyoxylate shunt enzymes when grown at low temperatures . The increased activity of glyoxylate shunt enzymes did not appear to be due to the degradation of intracellular fatty acids in fadR strains or differences in allosteric effectors in fadR versus fadR+ strains . These studies suggest that the fadR gene product may be involved in the regulation of the glyoxylate operon.

J Bacteriol, 1980 Aug, 143(2), 652 - 60
Cloning of deoxyribonucleic acid regions encoding a heat-labile and heat-stable enterotoxin originating from an enterotoxigenic Escherichia coli strain of human origin; Yamamoto T et al.; A heat-labile and heat-stable enterotoxin (LT+ ST+) plasmic (62.7 kilobases in size) was isolated from an enterotoxigenic Escherichia coli human strain, H10407, and used for analysis of the LT+ and ST+ deoxyribonucleic acid (DNA) regions . A DNA segment containing the LT+ and ST+ DNA regions, which consisted of two restriction endonuclease EcoRI fragments (E1 and E2), was inserted into the cloning vehicle ColE1::Tn5 by EcoRI digestion and subsequent ligation . Further cloning experiments localized the LT+ DNA region on a 5.1-kilobase restriction endonuclease PstI fragment present over the junction between the E1 and E2 fragments, as seen in the original LT+ ST+ plasmid, and the ST+ DNA region on a 1.5-kilobase PstI fragment present in either the E1 or E2 fragment . A change in the relative orientation of the E1 and E2 fragments resulted in altered levels of LT production . The relative orientation of the ColE1::Tn5 fragment to the E1 and E2 fragments also markedly influenced both LT and ST production levels . The LT+ ST+ E1-E2 region contained two unique DNA sequences consisting of a DNA segment flanked by inverted repeats which were readily distinguished from each other by size . The cloned ST+ PstI fragment was structurally very similar to one of these unique DNA sequences present in the LT+ ST+ E1-E2 region.

J Bacteriol, 1980 Aug, 143(2), 594 - 602
Reversal by trypsin of the inhibition of active transport by colicin E1; Dankert J et al.; The time course for inhibition of proline transport and irreversible loss of cell viability after treatment with colicin E1 was measured as a function of temperature between 13 and 33 degrees C, using a thermostatted flow dialysis system . Complete inhibition of proline transport at 33 and 13 degrees C occurred in 0.5 min and 3 to 5 min, respectively, after addition of colicin E1 at an effective multiplicity of about 4 . At these times, the fractional cell survival, assayed by dilution directly from the flow dialysis vessel into trypsin, ranged from 35 to 80%, with viability always greater than 50% at the lower incubation temperatures . Further studies were carried out at 15 degrees C . Complete inhibition of proline transport, which required 2 to 3 min, occurred much more rapidly at 15 degrees C than did the decay of trypsin rescue, which required 10 to 15 min to reach a survival level of 10 to 20% . The direct addition of trypsin to the flow dialysis vessel, after an addition of colicin E1 that caused complete inhibition of proline or glutamine transport, resulted in restoration of net transport . The restored level was typically about 40% of the control rate, and was very similar to the fractional cell viability measured after incubation in trypsin in the same vessel . It is concluded that trypsin can restore active transport to a significant fraction of a cell population in which transport has been initially inhibited by colicin E1.

J Bacteriol, 1980 Aug, 143(2), 561 - 8
Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region; Platz A et al.; Recombinant plasmids containing all or part of the genetic region of Escherichia coli coding for the two subunits of ribonucleoside diphosphate reductase (proteins B1 and B2) were constructed with the aid of the multicopy plasmid pBR322 . Two of these plasmids (pPS1 and pPS2) appeared to carry both a regulator and the complete structural information for the enzyme and, after transformation of E . coli, directed a 10- to 20-fold overproduction of both proteins B1 and B2 . The other plasmids (pPS101 and pPS201) carried structural information for only protein B2 . Cells carrying pPS1 and pPS2 showed a 5- to 500-fold increased resistance against the drug hydroxyurea . This establishes that in E . coli the inhibition of deoxyribonucleic acid synthesis by hydroxyurea is fully explained by its action on ribonucleotide reductase.

J Bacteriol, 1980 Aug, 143(2), 1090 - 4
Spontaneous emergence of an Hfr strain with a cit plasmid from natural isolates of citrate-positive Escherichia coli bovine origin; Ishiguro N et al.; From citrate-utilizing (Cit+) Escherichia coli strain C53 of bovine origin, strains C53A and C53B were obtained . Upon mating with recA+ but not with recA mutant recipients of K-12, C53A produced chromosomal recombinants at quite high frequencies, leading to the following conclusions: (i) C53A is an Hfr strain; (ii) the site of integration of the Cit plasmid (IncH1) is between metA (89 min) and ara (1 min); (iii) the direction of chromosome transfer is clockwise; and (iv) the plasmid-associated determinants are transferred as the terminal markers . A transductant of a dnaA(Ts) strain, CRT46, which acquired Cit determinants from a recombinant, SG13, was also an Hfr strain similar to SG13, and thermoresistant due to suppressive integration . On the other hand, unstable C53B did not produce recombinants, but the frequency of RecA-independent transfer of the Cit plasmid was high, indicating that the Cit plasmid (IncH1) exists autonomously in C53B . Attempts to isolate an Hfr strain from C53B failed.

J Bacteriol, 1980 Aug, 143(2), 1073 - 6
Functional relationship between parts of the replication region of plasmid ColE1; Inselburg J; The inhibition of plasmid ColE1 replication caused by a deletion of the ColE1 plasmid replication origin has been previously reported (T . Hashimoto-Gotoh and J . Inselburg, J . Bacteriol . 139:597-619) . Evidence is presented showing that restoration of the deleted nucleotide sequence in the precise relationship it normally has to the rest of the replication region is essential for restoration of ColE1 replication capability to the deletion mutant.

J Bacteriol, 1980 Aug, 143(2), 1060 - 2
Protein synthesis results in guanosine-5'-diphosphate-3'-diphosphate synthesis in Escherichia coli minicells; Nothling R et al.; Minicells of Escherichia coli DS410 synthesized guanosine-5'-diphosphate-3'-diphosphate and guanosine-5'-triphosphate-3'-diphosphate when synthesizing proteins in response to phage T7 infection . The guanosine-5'-diphosphate-3'-diphosphate synthesis was found to be relA+ dependent and inhibited by chloramphenicol.

J Bacteriol, 1980 Aug, 143(2), 1049 - 53
Functional analysis of minichromosome replication: bidirectional and unidirectional replication from the Escherichia coli replication origin, oriC; Meijer M et al.; Replicating molecules of minichromosomes pCM959 and pOC24 were analyzed by electron microscopy . Replication of pCM959 proceeded bidirectionally from the replication origin, oriC, in about 60% of the molecules; the rest of the molecules replicated unidirectionally in either direction . pOC24, in which deoxyribonucleic acid to the right (clockwise) of the oriC segment is deleted, seemed to replicate predominantly unidirectionally counterclockwise from oriC.

J Bacteriol, 1980 Aug, 143(2), 1046 - 8
Runaway replication of plasmid R1 is not caused by loss of replication inhibitor activity of gene cop; Molin S et al.; The replication control functions of a mutant of plasmid R1 that replicates without control at temperatures above 35 degrees C have been analyzed . Although the mutations have not been mapped precisely, the data indicate that the gene (cop) previously identified on the wild-type plasmid (S . Molin and K . Nordstrom, J . Bacteriol . 141:111-120, 1980) as being responsible for expressing a trans-acting replication inhibitor, as well as for incompatibility of plasmid R1, is not affected in this mutant . Thus, the conditional lack of replication control observed in this plasmid mutant presumably is not caused by the loss of inhibitor activity of the cop gene.

J Bacteriol, 1980 Aug, 143(2), 1031 - 2
Role of recBC nuclease in Escherichia coli transformation; Hoekstra WP et al.; In Escherichia coli transformation with linear donor deoxyribonucleic acid, the recBC pathway is functional, but genetic analysis shows that the recBC nuclease is deleterious to linear deoxyribonucleic acid.

J Bacteriol, 1980 Aug, 143(2), 1025 - 8
Ultraviolet light induction of recA protein in a recB uvrB mutant of Escherichia coli; Bockrath RC et al.; Full and persistent ultraviolet light induction of recA protein with minimal bacterial deoxyribonucleic acid degradation implicates blocked replication sites rather than degradation products as the inducing signals.

Appl Environ Microbiol, 1980 Aug, 40(2), 386 - 90
Cell envelope damage in Escherichia coli caused by short-term stress in water; Zaske SK et al.; Physiological and morphological changes in Escherichia coli exposed to oligotrophic natural waters and reagent grade water were studied . Several lines of evidence indicated that short-term exposure in water causes cellular envelope damage . Increasing susceptibility to lysozyme, lag time before cell division, and injury as defined by differential counts on selective and nonselective media occurred with exposure time . Electron micrographs of injured cells showed morphological changes in cell envelope.

Appl Environ Microbiol, 1980 Aug, 40(2), 309 - 12
Variability in gas production by Escherichia coli in enrichment media and its relationship to pH; Meadows PS et al.; Variability in gas production in multiple subcultures of Escherichia coli was assessed in two selective enrichment media and in lactose peptone water . Considerable variability occurred with all media at 37 and 44 degrees C . Addition of buffer increased gas production and decreased variability . The relationships between pH, growth, and gas production were complex . In buffered media, viable counts increased by 269 x 10(6) to 382 x 10(6)/U of pH fall, whereas in unbuffered media, they increased by 9.45 x 10(6) to 30.37 x 10(6)/U of pH fall . In buffered and unbuffered media, pH fell as gas production rose . However, variability in gas production among individual subcultures was not associated with changes in pH.

Appl Environ Microbiol, 1980 Aug, 40(2), 231 - 4
Effects of pesticides on the fatty acid and phospholipid composition of Escherichia coli; Rosas SB et al.; Cells of Escherichia coli contained an altered phospholipid and fatty acid composition when grown in the presence of some pesticides . Whereas parathion increased the concentration of all phospholipid species without changes in their polar head groups . DDT (dichlorodiphenyltrichloroethane) decreased the proportion of neutral serine-derived phosphatides and dieldrin decreased the proportion of negatively charged phospholipids . The saturated/unsaturated plus cyclopropane fatty acid ratio was increased in all cases . The changes suggested that cells adapted their membrane lipids to compensate for the presence of pesticides in the environment.

Can J Biochem, 1980 Aug, 58(8), 655 - 9
The formation of phosphatidylethanolamine from diglyceride by extracts of Escherichia coli; Proulx P et al.; Extensively dialyzed cell-free homogenates or washed particulate fractions, of Escherichia coli in the presence of added CTP, Mg2+, serine, and rac-glycerol 3-phosphate, incorporated {32P}phosphatidic acid into phosphatidylglycerol and cardiolipin but not into phosphatidylethanolamine . {14C}Phosphatidic acid could be incorporated by these preparations into phosphatidylethanolamine in the absence of added CTP and Mg2+ . CDP-diglyceride did not significantly affect the formation of {14C}phosphatidylethanolamine from {14C}phosphatidic acid . 14C-labeled diglyceride was also readily incorporated into phosphatidylethanolamine in the absence of added cofactors by both homogenate and particulate fraction . Serine stimulated the incorporation somewhat whereas CTP + Mg2+ diminished this conversion slightly because of concurrent {14C}phosphatidic acid and {14C}phosphatidylglycerol formation . CDP-diglyceride did not significantly affect the conversion of 14C-labeled diglyceride to phosphatidylethanolamine . Dialyzed 14C-labeled cytosol fractions of E . coli, obtained from cells grown in medium containing {14C}serine, transferred some of their label to phosphatidylethanolamine in the presence of fresh particulate fraction . This transfer was stimulated by added diglyceride . The results indicate that phosphatidylethanolamine can be synthesized from exogenous diglyceride by a pathway which does not require CDP-diglyceride as an intermediate but which likely makes use of an endogenous cofactor supplying phosphorylethanolamine and (or) phosphorylserine.

Biochem Soc Trans, 1980 Aug, 8(4), 413 - 5
Processing of exported proteins in Escherichia coli; Randall LL et al.; The mechanism of export of protein in E . coli can be summarized in terms of the 'signal hypothesis' . The proteins are synthesized on membrane-bound polyribosomes in the form of precursors, which carry N-terminal extensions of amino acids, the 'signal' . The proteins are vectorially transferred through the membrane during synthesis and the signal sequence is removed to generate the mature protein . The basic principle is established and it is now important to elucidate the molecular mechanism of export . We have attempted to detail the proteolytic removal of the signal . We have shown that the precursors are processed post-translationally, and we have data suggesting that two cleavages may be involved . It appears that processing is not necessary to activate the mature proteins . Why then is the signal removed? Perhaps the answer will shed light on the other yet unanswered questions: what is the energy source for the translocation? how does the cell differentiate between the two classes of exported proteins, those in the periplasm and those in the outer membrane? The next few years should see the resolution of these questions.

Mutat Res, 1980 Aug, 72(1), 31 - 42
Killing of Escherichia coli K-12 by near-ultraviolet radiation in the presence of hydrogen peroxide: role of double-strand DNA breaks in absence of recombinational repair; Hartman PS et al.; Near-ultraviolet (NUV) radiation killing of Escherichia coli K-12 can be enhanced by a sub-lethal concentration of hydrogen peroxide . This can be divided into a "RecA-dependent" and "RecA-independent" synergistic killing action . Stationary phase wild-type and 8 closely related repair-deficient mutants were examined for their NUV sensitivities in the presence and absence of H2O2 . All exhibited the "RecA-independent" synergism; i.e., H2O2 enhanced NUV lethality when RecA repair was not operating . The "RecA-independent" synergism did not result from destruction of repair enzymes . Very few DNA--protein crosslinks could be detected following NUV plus H2O2 treatment . However, double-strand (DS) DNA breaks were produced, apparently by conversion of closely spaced single-strand (SS) breaks on opposite strands . The correlation between DS-break formation and lethality in wild-type and a polA mutant indicates that the RecA-independent synergistic killing results from the conversion of SS into lethal DS breaks.

Proc Natl Acad Sci U S A, 1980 Aug, 77(8), 4592 - 6
Primary structure of major outer membrane protein II (ompA protein) of Escherichia coli K-12; Chen R et al.; The amino acid sequence of major outer membrane protein II (ompA protein) from Escherichia coli K-12 has been determined . The transmembrane polypeptide consists of 325 residues, resulting in a molecular weight of 35,159 . The transmembrane part of the protein is located between residues 1 and 177 . In this part of the protein a predominantly lipophilic 27-residue segment exists that perhaps spans the membrane in a mostly alpha-helical conformation, or a 19-residue stretch of this segment might traverse the membrane linearly . Inside the outer membrane a sequence -Ala-Pro-Ala-Pro-Ala-Pro-Ala-Pro- exists that, analogous to the -Cys-Pro-Pro-Cys-Pro- sequence in the hinge region of immunoglobulin, could assume the conformation of a polyproline helix . Computer analysis did not reveal a clear overall pattern of internal homology in the protein; besides the -Ala-Pro- repeat, only one local area (two adjacent dodecapeptide segments) shows some repetitiveness . The same analysis did not produce evidence for internal homology in the previously determined sequence of outer membrane protein I (porin) nor was any marked resemblance detected between transmembrane proteins I and II.

J Biochem (Tokyo), 1980 Aug, 88(2), 379 - 87
Augmentation of glycogen synthesis under stringent control in Escherichia coli; Taguchi M et al.; When Escherichia coli strain CP78 (rel+) was starved for isoleucine by the addition of valine, the amount of glucose in polymeric form in the cells increased markedly compared to that of the control cells . In contrast, this phenomenon was not seen in strain CP79 (rel-) . The increase in CP78 was shown to be due to the increase of glycogen . These results indicate that glycogen synthesis was augmented under stringent control . This was confirmed using other isogenic pairs of rel+ and rel- strains starved for other amino acids . When the cultivation temperature of strains 10B601 (rel+) and 10B602 (rel-) possessing temperature-sensitive valyl-tRNA synthetase was shifted from 30 degrees C to 40 degrees C, no difference was observed in the response of glycogen synthesis between the two strains . These results indicate that protein synthesis was necessary for the augmentation of glycogen synthesis and that guanosine 5'-diphosphate 3'-diphosphate did not exert its effect through stimulation of the activity of pre-existing enzyme(s) involved in glycogen synthesis . These conclusions were supported by the results of experiments using chloramphenicol and rifampicin . The rates of glucose utilization of CP78 and CP79 were decreased to nearly the same extent by valine addition . This suggests that the regulation site of glycogen synthesis under stringent control resides in a step after the transport of glucose by the phosphotransferase system.

Poult Sci, 1980 Aug, 59(8), 1947 - 8
Antibody production and Escherichia coli resistance in socially stable flocks of dwarf and nondwarf chickens; Marsteller FA et al.; Dwarf and normal pullets from lines that had undergone selection for divergence in juvenile body weight were maintained in cages as socially stable flocks . Antibody titers were measured at 6 and 14 weeks of age and an Escherichia coli challenge was administered at 16 weeks of age . There was a significant line by dwarf genotype interaction with dwarfs having lower antibody titers than normal pullets in the low weight line and higher antibody titers in the high weight line . The mortality of dwarfs from the low weight line to an Escherichia coli challenge was much higher than that of any other group . These results were consistent with those from previous experiments where the Escherichia coli challenge was given to hens in single cages.

J Med Microbiol, 1980 Aug, 13(3), 463 - 8
Colony incompatibility among strains of Escherichia coli isolated during an outbreak of gastroenteritis in one ward; Bettelheim KA; Strains of Escherichia coli, isolated during an outbreak of infantile gastroenteritis in one ward were serotyped and tested for incompatibility against each other on semisolid agar . Serotypes commonly isolated in various parts of the world from various habitats showed a greater incompatibility than more rarely isolated serotypes, suggesting that the former may have a selective advantage and be more able to displace other serotypes in a given habitat.

J Pediatr Surg, 1980 Aug, 15(4), 527 - 30
Fluid resuscitation in live Escherichia coli shock in puppies; Benner JW et al.; The infusion of live E . coli in the puppy produces a severe and usually lethal model of pediatric septic shock with characteristic reduction in cardiac output and blood pressure . Hemodynamic abnormalities are partially reversed with fluid resuscitation alone, and large volumes, supplemented with albumin well within tolerated fluid loads, produce the most pronounced corrections of these derrangements . Certain metabolic changes appear to be unique to the young animal and their correction is less successful regardless of resuscitation regimen . These findings suggest that the initial fluid management of the bacteremic pediatric patient should include Ringer's lactate and 5% albumin at approximately 105% of patient's blood volume . Further resuscitation fluid, as well as the role of steroids and antibiotics, remain work currently being carried out in our laboratory.

Eur J Biochem, 1980 Aug, 109(1), 291 - 9
Tetranucleotides as effectors for the binding of initiator tRNA to Escherichia coli ribosomes; Schmitt M et al.; Oligonucleotides such as G-A-G-G, which are complementary to the C-U-C-C region at the 3' end of 16-S RNA, inhibit the R17-RNA-dependent binding of the initiator tRNA (fMet-rRNA) to 30-S ribosomal subunits . However, if phage RNA is replaced by A-U-G, the same oligonucleotides stimulate the binding of fMet-tRNA to the 30-S subunits . This indicates that the formation of the RNA x RNA hybrid acts as a positive control signal for the selection of the initiator tRNA by the 30-S-subunit x mRNA complex . Tetranucleotides of the type A-U-G-N (where N = A, G, C or U) stimulated the IF-2-dependent binding of fMet-tRNA to the 30-S subunit more effectively than A-U-G, with A-U-G-R better than A-U-G-Y (where R is a purine nucleoside and Y is a pyrimidine nucleoside) . Since the 3'-terminal adenosine in A-U-G-A can be replaced by 6-deamino-adenosine, a stacking type of interaction between U-33 of tRNA and N of A-U-G-N should additionally stabilize the codon-anticodon complex . The situation is strictly reversed for 70-S ribosomes where A-U-G is the best codon followed by A-U-G-U, A-U-G-C, A-U-G-G and A-U-G-A . Replacement of GTP by guanosine 5'-{beta, gamma-methylene}triphosphate (GuoPP{CH2}P} results in A-U-G-A becoming more efficient than A-U-G as the codon for the binding of fMet-tRNA to 70-S ribosomes . This indicates that IF-2 and GTP hold the anticodon of the fMet-tRNA in a conformation capable of binding to a tetranucleotide codon . GTP hydrolysis and release of IF-2 from the 70-S ribosome results in a change of the tertiary structure of fMet-tRNA as a consequence of which the initiator tRNA reassumes the conformation which preferentially binds to A-U-G.

Lab Invest, 1980 Aug, 43(2), 165 - 71
Effect of endotoxin on rat liver . Analysis of acid phosphatase isozymes in the liver of normal and endotoxin-treated rats; Hirata K et al.; Lysosomal enzymes in parenchymal and sinusoidal cells in rat liver were sequentially studied following administration of endotoxin and correlated with fine structural changes in liver cells . In normal rat liver, there are three types of acid phosphatase isozymes, two present only in parenchymal cells and the other specifically in sinusoidal cells . The parenchymal acid phosphatase isozymes hydrolyze preferentially AMP and CMP, while the sinusoidal isozyme hydrolyzes phenylphosphate . After an intraperitoneal injection of an LD50 dose of endotoxin, the activity of the sinusoidal acid phosphatase isozyme reached a maximum at 1 hour, and was followed by an elevation of the activity of the isozymes derived from parenchymal cells . Within 1 hour after the injection, morphologic changes were most prominent within sinusoids, including swelling of the Kupffer cells and accumulation of polymorphonuclear leukocytes, platelets, and fibrin clumps on and around the Kupffer cells . Degenerative changes in parenchymal cells were observed following the initial reactions within sinusoids . The results of the present study are compatible with the hypothesis that the hepatic parenchymal cell damage induced by endotoxin is mediated by the initial changes within sinusoids, rather than by the direct aciton of endotoxin on the parenchymal cells.

Chem Biol Interact, 1980 Aug, 31(2), 151 - 66
Nitrofuran induced mutagenesis and error prone repair in Escherichia coli; Bryant DW et al.; Nitrofuran derivatives are a class of compounds which exhibit mutagenic and cytotoxic effects in bacteria and mammalian cells in tissue culture after metabolic activation by endogenous nitroreductases . The relationship between mutation and induction of pleiotropic error-prone repair functions (the 'SOS' system) in bacteria following exposure to nitrofurans was examined . A variety of nitrofurans were found to induce protein X, the recA+ protein, which is characteristic of error-prone repair . Furthermore, induction of the 'SOS' system depended on reductive activation of the mutagen . The use of a mutant thermally inducible for error-prone repair functions (tif-1) provided direct examination of bacteria exposed to non-lethal doses of nitrofuran . These results distinguish between mutants which arose from directly induced base mispairing and those which occur only after induction of error-prone repair functions . The mutational activity of AF2 (furylfuramide) was almost entirely dependent on the induction of error-prone repair since very few tryptophan revertants were detected in conditions which did not induced the 'SOS' repair system . We present a model for mutation induction by nitrofurans in bacteria.

Chem Biol Interact, 1980 Aug, 31(2), 133 - 50
Formation and excision of nitrofuran-DNA adducts in Escherichia coli; Wentzell B et al.; When Escherichia coli were incubated with the strong mutagen 2{14C}2-amino-4-(5-nitro-2 furyl)-thiazole (ANFT) radioactivity became tightly and presumably covalently bound to DNA . Hydrolysis of the DNA with nucleases yielded low molecular weight radioactive material . The bound radioactivity was associated with at least two functionally and chemically distinct adducts . One of these was rapidly removed in uvr+ E . coli while the other was more persistent . Analysis of enzymatic hydrolysates on a Dowex AG 50W-X4 column showed that the 'excisable adducts' were chromatographically different from most of the persistent ones . ANFT caused daughter-strand gaps when the DNA of treated cells was replicated, provided this DNA contained excisable adducts . In situations where removal of these adducts was complete no gaps were found in newly synthesized DNA . 3-{14C}2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2) (another strongly mutagenic nitrofuran) became bound to the DNA of E . coli WP2 uvrA to a slightly greater extent than did {14C}ANFT . In contrast, {14C}nitrofurazone (the semicarbazide of 5-nitro-2-furaldehyde) a much weaker mutagen, gave considerably less binding . With AF2 and ANFT there was roughly the same relation between the amount of adduct formed and the subsequent yield of daughter strand gaps when the DNA replicated while with nitrofurazone the yield of gaps per adduct was somewhat lower . Incubation in vitro of {14C}ANFT with DNA in the presence of an E . coli nitrofuran reductase preparation also resulted in the binding of 14C to DNA.

Biokhimiia, 1980 Aug, 45(8), 1481 - 7
{In vitro binding of 70S ribosomes to membrane structures of Escherichia coli}; Orlov VG et al.; It was found that the maximal disattachment of the ribosomes from the membrane structures is observed upon their treatment with 10 mM tris-HCl buffer, pH 7.5, containing 250 mM sucrose, 750 mM KCl, 5 mM magnesium acetate and 1 mM EDTA or puromycin . The most effective attachment of ribosomes to the membrane occurs in 10 mM tris-HCl buffer, pH 7.5, containing 5% sucrose and Mg2+ . The increase of Mg2+ concentration in the medium from 0.5 mM up to 1 mM results in a 2-fold increase of the ribosomes bound to the membranes . The concentration of the ribosomal material involved in the reaction is very essential for ribosome binding to the membranes . The amount of ribosomes bound to the membranes increases proportionally to the increase of the ribosome concentration in the reaction mixture.

J Bacteriol, 1980 Aug, 143(2), 809 - 15
Multiple electrophoretic forms of methyl-accepting chemotaxis proteins generated by stimulus-elicited methylation in Escherichia coli; Boyd A et al.; The tsr and tar genetic loci of Escherichia coli determine the presence in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of methyl-accepting chemotaxis proteins (MCPs) I and II, respectively, each of which consists of a distinct group of multiple bands . Synthesis of the tsr and tar products was directed in ultraviolet-irradiated bacteria by lambda transducing phages . The addition of appropriate chemotactic stimuli to these cells resulted in the appearance of additional, faster migrating electrophoretic forms of the Tsr and Tar polypeptides which disappeared upon removal of the stimulus . The stimulus-elicited forms comigrated with component bands of the corresponding MCPs . These results indicate that methylation itself caused shifts in electrophoretic mobility and hence led to the observed MCP band patterns . The number of Tsr species suggested that there were at least three methylated sites on the Tsr polypeptide . The conclusion that methylation generates multiplicity was supported by the results of experiments in which the tsr product was synthesized in mutant bacteria defective in specific chemotaxis functions concerned with methylation or demethylation of MCPs . Thus, the presence of a cheX defect blocked the stimulus-elicited appearance of faster migrating forms of the tsr product; conversely, the presence of a cheB defect resulted in a pronounced shift toward these forms in the absence of a chemotactic stimulus.

J Bacteriol, 1980 Aug, 143(2), 789 - 800
Use of bio-lac fusion strains to study regulation of biotin biosynthesis in Escherichia coli; Barker DF et al.; The technique developed by Casadaban (M . J . Casadaban, J . Mol . Biol . 104: 541-555, 1976) has been employed to construct Escherichia coli K-12 derivatives in which the genes determining lactose utilization are fused to the regulatory region of the biotin operon . Fusions of the lac genes to either arm of this divergently transcribed operon have been isolated . When the operon is derepressed, expression of the lac genes is sufficient to permit growth on lactose minimal medium . Repressing conditions prevent growth on lactose . This property of bio-lac fusion strains, as well as the ease of determining the level of operon expression by assaying beta-galactosidase, was used for the isolation and characterization of mutants defective in repression . Preliminary analyses of several newly isolated regulatory mutants are presented . For the several birA mutants examined, there appeared to be no direct correlation between effects on minimum biotin requirement and alterations in repressibility, suggesting a possible dual function for the gene . Parallel attempts to obtain fusions of lac to bioH were unsuccessful, indicating lack of direct biotin control at the bioH locus.

J Bacteriol, 1980 Aug, 143(2), 613 - 20
Regulation of aromatic amino acid biosynthesis in Escherichia coli K-12: control of the aroF-tyrA operon in the absence of repression control; Camakaris H et al.; Evidence was found which indicated that a mutation in gene trpS affected the rate of synthesis of tyrosine-repressible 3-deoxy-D-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase . The effect was found to occur independently of repression mediated by the tyrR gene product, and it was not due to a change in growth rate, nor was it a manifestation of the stringent response . It is proposed that in the proximal region of the aroF-tyrA operon there is an attenuator site controlled by the level of charged tryptophanyl-transfer RNA . In addition, it was demonstrated that starvation for certain amino acids led to degradation of tyrosine-repressible DAHP synthetase, but not phenylalanine-repressible DAHP synthetase, and supplementation with the missing amino acid led to an increased rate of synthesis of tyrosine-repressible DAHP synthetase during subsequent growth.

J Bacteriol, 1980 Aug, 143(2), 921 - 5
Use of gene cloning to determine polarity of an operon: genes carAB of Escherichia coli; Crabeel M et al.; A gene-cloning approach was used to determine the transcription polarity of the carbamoylphosphate operon (carAB) of Escherichia coli . In agreement with the accompanying paper (J . Bacteriol . 143:914-920, 1980), our results lead to the conclusion that carA is the proximal gene of the carAB operon.

J Bacteriol, 1980 Aug, 143(2), 703 - 9
tif-Stimulated deoxyribonucleic acid repair in Escherichia coli K-12; Castellazzi M et al.; Bacterial survival is significantly increased after ultraviolet irradiation in tif sfi cells, provided that the thermosensitive tif mutation has been expressed at 41 degrees C before irradiation . This tif-mediated "reactivation of ultraviolet irradiated bacteria" needs de novo protein synthesis, as is the case for the tif-mediated reactivation of ultraviolet-irradiated phage lambda . However, in striking contrast to the phage reactivation process, this tif-mediated reactivation is no longer associated with mutagenesis . It also requires the presence of the uvrA+ excision function . These results strongly suggest the existence in Escherichia coli K-12 of a repair pathway acting on bacterial deoxyribonucleic acid which is inducible, error free, and uvr dependent.

J Bacteriol, 1980 Aug, 143(2), 661 - 7
Deletion mapping and heterogenote analysis of a mutation responsible for osmosis-sensitive growth, spectinomycin resistance, and alteration of cytoplasmic membrane in Escherichia coli; Yamagata H et al.; Lambda transducing phages carrying Escherichia coli deoxyribonucleic acid of various lengths from the aroE-rpsL region were lysogenized into the F'3 plasmid and were used for heterogenote analysis of YM101, a sucrose-dependent, spectinomycin-resistant mutant of E . coli . Three characteristics of the mutant strain, resistance to spectinomycin, sucrose dependence of growth, and lack of I-19 protein in the cytoplasmic membrane, were shown to be the result of a mutation in a region designated delta 53-spcl . This region extends over 3.6-kilobase pairs and is located within a cluster of ribosomal genes . The mutation is recessive to the wild-type allele.

Mutat Res, 1980 Aug, 72(1), 57 - 62
Kinetics of induction and decay of error-prone DNA repair activity in Escherichia coli after treatment with nalidixic acid; Sarkar SN et al.; Inducible error-prone DNA repair activity was detected by infecting nalidixic acid-pretreated E . coli cells with UV-irradiated phage phi X174 . Induction and decay kinetics of reactivation very much resembled that of mutagenesis of the UV-damaged phage . Repair as well as mutagenic activity increased for about 30 min . The maximal error-prone repair capacity, which was induced in the cell during the 30 min nalidixic acid treatment, rapidly died out during subsequent cell growth in absence of nalidixic acid . Induction of this repair mode was not observed in a recA- mutant . In the presence of nalidixic acid plus rifampicin both repair and mutagenic effects were abolished.

J Biochem (Tokyo), 1980 Aug, 88(2), 525 - 32
Process of attachment of phi X174 parental DNA to the host cell membrane; Azuma J et al.; The phi X174-DNA membrane complex was isolated from Escherichia coli infected with phi X174 am3 by isopycnic sucrose gradient centrifugation followed by zone electrophoresis . The phi X174 DNA-membrane complex banded at two positions, intermediate density membrane fraction and cytoplasmic membrane fraction, having bouyant densities of 1.195 and 1.150 g/ml, respectively . Immediately after infection with phi X147, replicating DNA was pulse-labeled and then the incorporated label was chased . The radioactivity initially recovered in the intermediate density membrane fraction migrated to the cytoplasmic membrane fraction . The DNAs from both complexes sedimented mainly at the position of parental replicative form I (RFI) . The phi X174 DNA-membrane complex contained a speficic membrane-bound protein having a molecular weigth of 80,000 which is accumulated in the host DNA-membrane complex . These results suggest that when phi X174 DNA penetrated into cells in the early phase of infection, single-stranded circular DNA was converted to parental RFI at a wall/membrane adhesion region and migrated to the cytoplasmic membrane fraction, where the parental RF could serve as a template in the replication of progeny RF.

J Infect Dis, 1980 Aug, 142(2), 199 - 204
Role of suppressor cells in experimental pyelonephritis; Smith JW; The effect of suppressor cells on the synthesis of antibody and development of antigen-responsive lymphocytes was studied in rabbits with pyelonephritis produced with Escherichia coli O6:K13:H1 . The responsiveness of splenic lymphocytes to phytohemagglutinin (PHA), which was reduced early in infection, was restored by preincubation of cells with indomethacin . The response of lymphocytes to E . coli antigen was also suppressed early . Indomethacin increased response to PHA but did not affect response to antigen . Rabbits given indomethacin from days 1-3 of infection had a delay in suppressor cell activity until day 9, but this early inhibition had no effect on synthesis of immunoglobulin or antibody or on serum levels of IgM or IgG . Thus, activity of splenic suppressor cells in pyelonephritis can be diminished by indomethacin, an inhibitor of prostaglandin synthetase, but the suppressor cells do not appear to influence the immune response or activities of bone marrow-derived lymphocytes such as antibody formation in experimental pyelonephritis.

Arch Surg, 1980 Aug, 115(8), 975 - 8
Infected aneurysms of the abdominal aorta; Scher LA et al.; Infected aneurysms of the abdominal aorta are relatively uncommon, but potentially lethal if improperly managed . Two case reports emphasize the importance of the preoperative aortogram for accurate diagnosis . We stress the principles of total excision of infected tissue and revascularization in uninfected tissue planes . A useful vascular reconstructive technique consisting of unilateral axillofemoral bypass and an ilioiliac anastomosis was used in both patients.

J Bacteriol, 1980 Aug, 143(2), 825 - 33
Hemolysis determinant common to Escherichia coli hemolytic plasmids of different incompatibility groups; de la Cruz F et al.; By using cloned deoxyribonucleic acid fragments from the hemolysis determinant of the hemolytic plasmid pHly152 as hybridization probes, a deoxyribonucleic acid segment of about 3.8 megadaltons was identified as a common sequence in several hemolytic (Hly) plasmids of Escherichia coli belonging in four different incompatibility groups . This segment contained the genetic information for the synthesis and secretion of the extracellular toxin alpha-hemolysin of E . coli . With the exception of pSU5, representing a composite plasmid, one part of which seems to be very similar to pHly152, the overall sequence homology of these Hly plasmids with pHly152 seems to be rather restricted . However, the Hly plasmid pSU316 showed sequence homology with pHly152 that did not extend beyond the hemolysis determinant . The two other plasmids, pSU233 and pSU105, also shared homology with pHly152 in the hemolysis determinant as well as in various other parts of this plasmid which did not seem to be directly linked to the hemolysis determinant . This suggests that the hemolysis determinant has spread to presumably unrelated plasmids of E . coli.

J Bacteriol, 1980 Aug, 143(2), 761 - 71
Restriction map of the Escherichia coli malA region and identification of the malT product; Raibaud O et al.; A series of plaque-forming lambda h80 transducing phages carrying various portions of the malA region were isolated . A 5,800-base pair HindIII-EcoRI DNA fragment from one of these phages was cloned into pBR322 and shown to contain malT, which is the positive regulator gene of the maltose regulon, and most of malP, the structural gene for maltodextrin phosphorylase . A restriction map of the HindIII-EcoRI fragment was established, and it was correlated with the genetic map of the malA region (i) by mapping deletions which had been generated in vitro on the plasmid and (ii) by locating on the restriction map a DNA insertion of known genetic position . A 600-base pair HincII-HaeII segment was shown to contain all or part of the promoters for malT and malP, which are known to be transcribed in opposite directions . Strains carrying gene malT on a plasmid synthesized a 94,000-dalton polypeptide which was not produced by identical strains carrying similar plasmids in which malT was partially deleted . Estimates of the size of the malT gene support the conclusion that the 94,000-dalton polypeptide is the malT product.

J Bacteriol, 1980 Aug, 143(2), 680 - 92
Specificity in the formation of delta tra F-prime plasmids; Hadley RG et al.; Twenty-three independent delta tra F-prime plasmids from three different Escherichia coli K-12 sublines were isolated from Hfr strains whose points of origin coincided with the IS3 element alpha 3 beta 3 or alpha 4 beta 4 in the lac-purE region of the E . coli chromosome . Electrophoretic analysis of plasmid deoxyribonucleic acid digested with EcoRI and hybridization analysis of plasmid deoxyribonucleic acid digested with BglII revealed that at least 14 of these plasmids were formed by processes involving specific bacterial and F loci . Two of the specific bacterial loci involved in delta tra F-prime formation were located at approximately 3.3 and 11.7 min on the E . coli chromosomal map . Two of the delta tra F-prime plasmids contained bacterial deoxyribonucleic acid with circularization endpoints that mapped very near the termini of the IS2 element that is normally located between lac and proC.

J Virol, 1980 Aug, 35(2), 488 - 97
Frequent site-specific deletion of coliphage lambda murine sarcoma virus recombinants and its use in the identification of a retrovirus integration site; McClements WL et al.; Stocks of hybrid lambda phages carrying the complete integrated provirus of either m1 or HT1 Moloney murine sarcoma virus, as well as flanking host sequences, frequently contain significant numbers of phages carrying a specific deletion . This deletion arises from a recombination event between the terminally repeated sequences in the provirus that deletes the unique Moloney murine sarcoma virus sequences bracketed by the terminally repeated sequences . Physical mapping has shown that the deletion phage retains one complete copy of the terminally repeated sequence and the flanking mink host sequences . One such deletion, lambdaHT1r+, was used to characterize a mink genomic DNA sequence that contains an HT1 Moloney murine sarcoma virus integration site . This integration site sequence from normal mink cells was also cloned into phage lambda . An analysis of the heteroduplexes between the integration site and the lambdaHT1r+ deletion indicated that no major rearrangement of host sequences occurred upon integration of the Moloney murine sarcoma provirus.

J Virol, 1980 Aug, 35(2), 436 - 43
Molecular mechanism for the capture and excision of the transforming gene of avian sarcoma virus as suggested by analysis of recombinant clones; Yamamoto T et al.; Structural analysis of two cDNA clones, derived from reverse transcripts of avian sarcoma virus 21S mRNA's, reveals unusual features in the organization and expression of the integrated avian sarcoma virus (ASV) proviral DNA and predicts a mechanism for recombination events that will lead to either the capture or the excision of the transforming gene of this virus . The latter is supported by our observation that there is an extensive homologous region on either side of the transforming gene that will allow site-specific deletion or integration to occur . Comparison of the clone derived from the src-specific 21S mRNA coding for the transforming gene product to that derived from the env-specific 21S mRNA coding for the envelope glycoprotein show that the common c region present at the 3' terminus of the ASV genome is 326 bases long . Within this c region are nucleotide sequences that may play key roles in the life cycle of this virus . These regulatory sequences include (i) probable promoter sites for the initiation of transcription, (ii) a polyadenylation signal, and (iii) a sequence that is complementary to the 3' termini of both the env and the src regions, which will allow the generation of transformation-defective deletions.

Gene, 1980 Aug, 10(3), 237 - 47
Isolation of recombinant plasmids and phage carrying the lexA gene of Escherichia coli K-12; Little JW; The lexA gene of Escherichia coli K-12 was cloned from the plasmid pLC44-14 into pBR322 . Plasmids carrying lexA+ were selected by their ability to complement a recessive tsl mutation, which is believed to be a mutation in lexA . The smallest lexA+ recombinant plasmid, pJL21, contained an EcoRI-PstI fragment 2.9 kilobases (kb) in length; two larger plasmids also contained this fragment, and genetic material to one or both sides of the EcoRI-PstI fragment . Plasmids homologous to pJL21, but carrying a dominant mutation, lexA3, or one of three recessive amber mutations in lexA, termed spr, were also isolated . To clone the EcoRI-PstI fragment onto a lambda vector, the PstI end was first converted to an EcoRI end by attachment of a 100-base pair PstI-EcoRI fragment isolated from the plasmid ColE1; the resultant EcoRI fragment was then cloned into the lambda vector lambda gt4 . A restriction map of pLC44-14 was obtained for nine restriction enzymes . The orientation of this map was determined relative to the E . coli genetic map by complementation of the gene ubiA+ and by comparison with restriction enzyme digests of another plasmid, pLC11-9, which carries dnaB, a gene closely linked to lexA, but does not carry lexA.

Gene, 1980 Aug, 10(3), 185 - 93
Histone genes from Xenopus laevis: molecular cloning and initial characterization; Moorman AF et al.; Histone DNA sequences, were detected in Eco RI fragments of total Xenopus laevis DNA, by hybridization with 32P-labeled h22-DNA, a histone gene repeat unit of the sea urchin Psammechinus miliaris . The about 6 kb-size class, which was found to hybridize, was subsequently integrated into the E . coli plasmid pCR1 . A clone was isolated that contains a 5.8 kb EcoRI fragment hybridizing with h22-DNA . A physical map was constructed using the restriction endonucleases BamHI, PstI, HincII, BglII, XbaI, PvuII, XhoI, AvaI, SmaI, HinfI and HpaII . The fragment was not cleaved by KpnI, AvaI, SalI and HindIII . Using this restriction map we were able to determine the gene order by hybridization with purified gene probes derived from h22-DNA . The gene order was found to be H3, H4, H2A and H2B . The localization of the H1 gene was not possible, probably due to its greater evolutionary divergence . Part of the sequence of the H3-gene is presented providing unambiguous evidence on the identity, map position and polarity of this gene.

Proc Natl Acad Sci U S A, 1980 Aug, 77(8), 4880 - 4
Control of gene expression by a mobile recombinational switch; Berg DE; Transposable recombinational switches may play important roles in the evolution of bacterial populations by increasing flexibility in the control of expression of particular genes and thereby maintaining heterogeneity in clones of cells growing in a uniform environment . Experiments reported here show that Tn5-112, a deletion derivative of kanamycin-resistance transposon Tn5, can function as such a mobile recombinational switch . The internal deletion in Tn5-112 removes transcription termination signals and permits transcription initiated within the element to continue into nearby bacterial genes . Consequently, in one orientation Tn5-112 stimulates distal gene expression, whereas in the other orientation the normal polarity imposed by wild-type Tn5 intervenes and distal gene expression is not stimulated . Because Tn5-112 contains terminal inverted repeats, intramolecular recombination can invert the Tn5-112 element and alter gene expression . Tn5-112 is transposition deficient . Its mobility derives from the recessive nature of the transposition deficiency and, in this study, from the possibility of homologous recombination which permits its placement in either orientation at any site occupied by nother Tn5 element.

Proc Natl Acad Sci U S A, 1980 Aug, 77(8), 4602 - 6
Apurinic/apyrimidinic endonucleases in repair of pyrimidine dimers and other lesions in DNA; Warner HR et al.; The characteristics of the nicks (single-strand breaks) introduced into damaged DNA by Escherichia coli endonucleases III, IV, and VI and by phage T4 UV endonuclease have been investigated with E . coli DNA polymerase I (DNA nucleotidyltransferase) . Nicks introduced into depurinated DNA by endonuclease IV or VI provide good primer termini for the polymerase, whereas nicks introduced into depurinated DNA by endonuclease III or into irradiated DNA by T4 UV endonuclease do not . This result suggests that endonuclease IV nicks depurinated DNA on the 5' side of the apurinic site, as does endonuclease VI, whereas endonuclease III has a different incision mechanism . T4 UV endonuclease also possesses apurinic endonuclease activity that generates nicks in depurinated DNA with low priming activity for the polymerase . The priming activity of DNA nicked with endonuclease III or T4 UV endonuclease can be enhanced by an additional incubation with endonuclease VI and, to a lesser extent, by incubation with endonuclease IV . These results indicate that endonuclease III and T4 UV endonuclease (acting upon depurinated and irradiated DNA, respectively) generate nicks containing apurinic/apyrimidinic sites at their 3' termini and that such sites are not rapidly excised by the 3' leads to 5' activity of DNA polymerase I . However, endonuclease IV or VI apparently can remove such terminal apurinic/apyrimidinic sites as well as cleave on the 5' side of the unnicked sites . These results suggest roles for endonucleases III, IV, and VI in the repair of apurinic/apyrimidinic sites as well as pyrimidine dimer sites in DNA . Our results with T4 UV endonuclease suggest that the incision of irradiated DNA by T4 UV endonuclease involves both cleavage of the glycosylic bond at the 5' half of the pyrimidine dimer and cleavage of the phosphodiester bond originally linking the two nucleotides of the dimer . They also imply that the glycosylic bond is cleaved before the phosphodiester bond.

Br J Surg, 1980 Aug, 67(8), 565 - 7
The value of polymixin B in endotoxaemia due to experimental obstructive jaundice and mesenteric ischaemia; Ingoldby CJ; The role of polymixin B in endotoxaemia due to obstructive jaundice and mesenteric ischaemia has been examined . Rats made jaundiced by common bile duct ligation were challenged with intragastric endotoxin . Rats given polymixin B infusions had improved survival (11/15) compared with controls (4/14) . Mesenteric ischaemia was induced in rats by clipping the mesenteric artery . Limulus assay revealed marked endotoxaemia in controls . Polymixin B infusions reduced the degree of endotoxaemia . Pre-treatment with non-absorbable intestinal antibiotics markedly reduced endotoxaemia . Polymixin B infusions gave no advantage of survival (3/10) compared with controls (2/10), but all animals pre-treated with intestinal antibiotics survived (12/12) . The difference in results found between obstructive jaundice and mesenteric ischaemia may be due to different degrees of endotoxaemia . Polymixin B may have a place in the prophylaxis of endotoxaemia in clinical obstructive jaundice.

Eur J Biochem, 1980 Aug, 109(2), 457 - 61
Molecular cloning, refined physical map and heterogeneity of methylation sites of papilloma virus type 1a DNA; Danos O et al.; The entire genome of human papilloma virus type 1a was cloned in Escherichia coli using the plasmid pBR322 as vector . The integrity and the homogeneity of the viral DNA thus obtained was confirmed by restriction endonucleases analysis . Viral DNA isolated from a single wart was partially methylated at only one out of the four HpaII sites, d(C-C-G-G) . Recognition sites for Bg/I, Bg/II, PstI and PvuII restriction endonucleases were located on the cloned genome.

Cell, 1980 Aug, 21(1), 127 - 39
Characteristics of an SV40-plasmid recombinant and its movement into and out of the genome of a murine cell; Hanahan D et al.; A bacterial plasmid carrying the early region of SV40 (pOT) has been stably established in high molecular weight (hmw) DNA of mouse L cells by selection for the herpes virus thymidine kinase (tk) gene . DNA blotting has demonstrated that most cell lines contain multiple discrete copies of pOT, generally with an intact SV40 early region . No free copies of pOT have been detected . Both pOT and tk sequences may be amplified up to 20-200 copies of the SV40 early region . In contrast to the uniform staining pattern normally observed in SV40-transformed lines, indirect immunofluorescence using antiserum to the SV40 T antigen has demonstrated that the expression of the early region is heterogeneous in these cell lines . This fraction expressing T is characteristic of a given cell line, and varies from 0 to 99% positive . Several pOT cell lines have been fused to simian cells, and replicating low molecular weight DNAs were isolated from the heterokaryons . Transformation of E . coli with this DNA demonstrates that pOT can be rescued from hmw DNA in L cells and reestablished as a plasmid in E . coli . Excision is generally precise when pOT is introduced to the murine cells as supercoiled molecule, and imprecise when pOT is introduced in linear form.

Biull Eksp Biol Med, 1980 Aug, 90(8), 205 - 8
{Atypical incompatibility of the F-like factors of pA22-4, pAP39 and pA41 genetic transfer with the F-group incompatibility plasmids}; Pekhov AP et al.; A study was made of compatibility of three F-like factors of the genetic transfer (pAP22-4, pAP39, pAP41) identified in the cells of serologically typed E . coli strains with F-group incompatibility reference plasmids . The factors of pAP22-4 and pAP41 transfer are partly incompatible with groups FII, FIII, FIV, and FI, FIV, respectively, while the factors of pAP39 transfer are completely incompatible both with groups FI and FIV plasmids.

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