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| Zh Mikrobiol Epidemiol Immunobiol, 1979 Sep, (9), 65 - 8 {Cholesterol synthesis by several strains of Escherichia}; Panchishina MV; The results of the study of lipids in the media used for growing different Escherichia strains are presented . Donor plasma with carbon-labeled sodium acetate was used as culture medium . Those strains which induced an increase in cholesterol content in the medium after 48-hour incubation were considered cholesterin-synthetizing . During the growth of these strains the radioactive marker became incorporated into the lipids accumulated in the medium: phospholipids, cholesterol, fatty acids . The degree of this incorporation depended on the dose of labeled sodium acetate and the amount of the inoculated culture . Cholesterol-synthetizing activity of Escherichia is characteristic of only freshly isolated strains. J Exp Zool, 1979 Sep, 209(3), 367 - 76 The origin of yolk-DNA in Xenopus laevis; Opresko L et al.; Xenopus laevis serum and plasma was found to contain an average of 25 microgram DNA/ml . Isolated X . laevis oocytes incubated in medium containing 25 microgram DNA/ml labeled with either 125I, 32P or 14C and from three different sources (bovine, E . coli and X . laevis), incorporated the label at an average rate of 0.11 ng.mm-2.hr-1 . Sucrose gradient fractionation of oocytes revealed that 40-75% of the acid-precipitable label incorporated was associated with the yolk platelets . Additional incubations of oocytes in unlabeled medium demonstrated that the DNA incorporated into the yolk platelets was undergoing turnover; only 20% of the yolk-associated DNA was still present after a one-week incubation . Our data suggest that yolk-DNA arises by the adventitious uptake of DNA present in the maternal serum by vitellogenic oocytes. J Clin Microbiol, 1979 Sep, 10(3), 275 - 8 Biotyping of Escherichia coli by a simple multiple-inoculation agar plate technique; Buckwold FJ et al.; A nine-test system using multiple-inoculation agar plates for biotyping of Escherichia coli is described . Testing of 959 strains resulted in 78 biotypes . On repeated testing, 96% of 182 strains had identical biotypes or differed by only one test . This system provides satisfactory differentiation among strains and is reproducible . Precise standardization of inoculum size is not required . Multiple inoculation allows time and cost-efficient testing of large numbers of strains. Genetika, 1979 Sep, 15(9), 1578 - 87 {Protective action of colicinogenic factor Ib-P9 on Escherichia coli cells defective in known repair functions after ultraviolet irradiation}; Khmel' IA et al.; The presence of the plasmid colicinogenic factor Ib-P9 in Escherichia coli wild type cells is shown to increase bacterial survival after UV irradiation and the action of N-methyl-N'-nitro-N-nitrosoguanidine . The ability of the plasmid to cause the UV protection is observed in uvrA, uvrB, uvrC, polA, recB, recF E . coli strains, but the plasmid does not restore the UV resistance of the mutant cells to the wild type level . The protective effect of the plasmid CoI Ib-P9 depends on the recA+lexA+ genotype of the cells . The inhibition of protein synthesis (amino acid starvation) before and after UV irradiation does not prevent the UV protection by ColIb-P9 . The nature of the plasmid-associated repair functions is discussed. Am J Med Technol, 1979 Sep, 45(9), 787 - 92 Gastroenteritis due to enteropathogenic, enterotoxigenic, and invasive Escherichia coli: A review; Pickering LK; Escherichia coli that produce diarrhea can be divided into three groups: 1) enteropathogenic, 2) enterotoxigenic, and 3) enteroinvasive . The mechanism of disease production by enteropathogenic E . coli is unknown, but these strains are not presently known to be inherently pathogenic, although they may be important as a cause of gastroenteritis in infants . The two known mechanisms of disease production are elaboration of enterotoxin and mucosal invasion . Heat-labile toxin-producing E . coli are the main cause of diarrhea in travelers while heat-stable toxin-producing E . coli are a cause of scours among new-born swine and cattle . Enteroinvasive E . coli have not been shown to be an important cause of diarrhea in the United States . Enteropathogenic, enterotoxigenic, and enteroinvasive E . coli that currently are associated with diarrhea worldwide may each consist of relatively few serotypes different from those associated with out-breaks of diarrhea in the past . This implies a possible new role for sero-typing of E . coli. J Bacteriol, 1979 Sep, 139(3), 961 - 76 Specificity in formation of type II F' plasmids; Hadley RG et al.; Eight new F' plasmids derived from Hfr strains in which F is integrated at the chromosomal element alpha 3 beta 3 have been isolated and subjected to restriction enzyme, hybridization, and electron microscope heteroduplex analysis . Plasmids carrying extensive amounts of bacterial deoxyribonucleic acid were produced even though they were obtained by selection for transfer of lac, which is closely linked to F in the parental Hfr strains . Seven plasmids were type II Flac+ proC+ purE+ plasmids, and one was a type I Flac+ proC+ plasmid . Five of the Flac+ proC+ purE+ plasmids contain approximately 284 kilobases of bacterial deoxyribonucleic acid, which is identical for all five within the resolution of the restriction enzyme analysis . Theses results indicate that type II F' plasmids are the predominant tra+ F' type from this region of the Escherichia coli K-12 chromosome and that the recombination events leading to formation of these plasmids exhibit site specificity. J Bacteriol, 1979 Sep, 139(3), 850 - 8 Cistrons encoding Escherichia coli heat-labile toxin; Dallas WS et al.; The structure and products of the two cistrons encoding the Escherichia coli heat-labile toxin (LT) were studied . The LT deoxyribonucleic acid (DNA) region had been isolated as part of a DNA fragment from the plasmid P307, and this fragment was joined to the cloning vector pBR313 . Deletion mutations of various lengths were introduced into the LT DNA region and into the adjacent DNA sequences . Analysis of the deletions indicated that the maximum size of the LT DNA region was 1.2 x 10(6) daltons . Two proteins of 11,500 daltons and 25,500 daltons had been shown to be encoded by the LT DNA region . The functions of these LT gene products were investigated . The 11,500-dalton protein had an adsorption activity for Y-1 adrenal cells, and this protein was shown to form aggregates of four or five monomers . The 25,500-dalton protein was shown to have an adenylate cyclase-activating activity . The two cistrons encoding for each of the LT proteins have been located on a genetic map of the LT DNA region . Both cistrons are probably transcribed from the same promoter. J Bacteriol, 1979 Sep, 139(3), 835 - 41 Protein K: a new major outer membrane protein found in encapsulated Escherichia coli; Paakkanen J et al.; The protein composition of purified outer membranes of 47 Escherichia coli strains was examined by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis . Of 33 encapsulated strains, all contained an outer membrane protein distinguishable from previously reported proteins . The 14 non-encapsulated strains with one exception lacked this protein . Because of its apparent association with encapsulation (K antigen) we have named it K protein . The protein was purified nearly to homogeneity by chromatography in the presence of detergents, and its composition was determined . Its amino acid composition does not differ significantly from that reported for protein I, another E . coli major outer membrane protein . Furthermore, the N-terminal amino acid sequence of protein K indicates that it is related to protein I. J Bacteriol, 1979 Sep, 139(3), 824 - 34 Properties of Escherichia coli mutants altered in calcium/proton antiport activity; Brey RN et al.; Mutants sensitive to growth inhibition by CaCl2 were found to have alterations in calcium uptake in everted membrane vesicles . These mutations map at different loci on the Escherichia coli chromosomes . A mutation at the calA locus results in vesicles which have two- to threefold higher levels of uptake activity than vesicles from wild-type cells . The calA mutation is phenotypically expressed as increased sensitivity to CaCl2 in a strain also harboring a mutation in the corA locus, which is involved in Mg2+ transport . The calA locus maps very close to purA and cycA at about min 97 . The calB mutation results both in sensitivity to CaCl2 at pH 5.6 and in vesicles with diminished calcium transport capability . The CalB phenotype is also expressed only in a corA genetic background; the calB locus appears to map very near, yet separately from, the calA locus . When the cor+ allele is present, calA and calB mutations still result in a defect in calcium transport in vesicles . In addition, both calC and calD mutations result in vesicles with impaired calcium transport activity . calC is cotransducible with kdp and nagA, whereas calD is cotransducible with proC. J Bacteriol, 1979 Sep, 139(3), 783 - 91 Isolation and characterization of Escherichia coli K-12 mutants unable to induce the adaptive response to simple alkylating agents; Jeggo P; When Esherichia coli cells are exposed to a low level of simple alkylating agents, they induce the adaptive response which renders them more resistant to the killing and the mutagenic effects of the same or other alkylating agents . This paper describes the isolation of one strain that was deficient in mutagenic adaptation and five that were deficient in both mutagenic and killing adaptation, confirming previous suggestions that killing and mutagenic adaptation are, at least to some extent, separable . These six strains have been called Ada mutants . They were more sensitive to the killing and mutagenic effects of N-methy-N'-nitro-N-nitrosoguanidine (MNNG) than the unadapted Ada+ parent . Thus, the adaptation pathway is responsible for circumventing some alkylation-induced damage even in cells that are preinduced . The increase in mutation frequency seen in Ada cells treated with MNNG was the same whether the cells were lexA+ or lexA, showing that the extra mutations found in Ada- strains do not depend upon the SOS pathway . Ada strains accumulated more O6-methyl guanine lesions than the Ada+ parent on prolonged exposure to MNNG, and this supports the idea that O6-methyl guanine is the most important lesion for MNNG-induced mutagenesis . The ada mutations have been shown to map in the 47 to 53-min region of the E . coli chromosome. J Bacteriol, 1979 Sep, 139(3), 1093 - 6 Growth-rate-dependent alteration of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase levels in Escherichia coli K-12; Wolf RE Jr et al.; The levels of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase are subject to metabolic regulation; they increased three- to fivefold with increasing growth rate. J Bacteriol, 1979 Sep, 139(3), 1085 - 8 Interaction between mutant alleles of araC of the Escherichia coli B/r L-arabinose operon; Sheppard DE et al.; Strains were constructed that contain mutational alterations affecting two distinct functional domains within the araC gene protein . The araCi (catabolite repression insensitivity) and araCh (catabolite repression hypersensitivity) mutations were used to alter the catabolite repression sensitivity domain, and mutation to D-fucose resistance was used to alter the inducer binding domain . araCh, D-fucose-resistant double mutants never exhibited constitutive ara operon expression, whereas all of the araCi, D-fucose-resistant double mutants did exhibit constitutivity . When L-arabinose was used as an inducer, most of the double mutants exhibited the sensitivity to catabolite repression associated with the araCi or araCh mutation . However, when D-fucose was used as an inducer, changes in sensitivity to catabolite repression were observed that were attributed to interactions between the two protein domains . The roles of catabolite activator protein and araC gene protein in the induction of the araBAD operon were discussed. J Bacteriol, 1979 Sep, 139(3), 1079 - 81 Effect of transcription on RecBC- and RecF-mediated recombination within the tryptophan operon of Escherichia coli K-12; Cosloy SD; Recombination between tryptophan gene mutations within the trp operon was determined among transductants for an outside linked cysB marker under conditions of repression and derepression . These studies, carried out with recipient strains utilizing the RecBC or RecF pathway, or a combination of these pathways of recombination, demonstrate that transcription of trp genes as regulated by the trp repressor has no significant effect on RecBC- or RecF-mediated recombination within the trp operon. J Bacteriol, 1979 Sep, 139(3), 1049 - 53 Small arrays of electron-dense cylinders in Escherichia coli cells; Bradley DE; Electron microscopy of unstained Escherichia coli cells from cultures kept near 0 degrees C after incubation at 37 degrees C revealed small areas of geometrically arranged electron-dense cylinders . Their morphology, organization, and occurrence are described. Chem Biol Interact, 1979 Sep, 27(1), 1 - 15 Non-random nature of 2-acetylaminofluorene-induced alterations of DNA template capacity; Schwartz EL et al.; Male rats were fed a diet containing 0.03% (w/w) 2-acetylaminofluorene (AAF) and their hepatic DNA was isolated and transcribed with E . coli RNA polymerase . Ingestion of the carcinogen-containing diet for 4 days substantially reduced the template capacity of the isolated DNA . This reduction in template capacity was due to an apparent decreased RNA chain size (up to 50%), with no significant changes in initiation or re-initiation of RNA synthesis . This premature termination of RNA synthesis was accompanied, in some instances, by a reduced rate of RNA chain elongation . When the rats were returned to a basal diet for 7 days following 4 days of AAF ingestion, template capacity and RNA chain size returned to control values . Fractionation of hepatic chromatin on a glycerol gradient revealed that inhibition of DNA template capacity occurs on portions exhibiting characteristics of expressed, as well as those with characteristics of repressed, segments of the genome . In contrast, the DNA isolated from a small, highly condensed chromatin fraction (15% of total chromatin-DNA) showed no significant reduction in total template capacity . Analysis of the fidelity of RNA synthesis on this DNA template was performed by determining the rate of addition of individual nucleotide triphosphates to a growing RNA chain . Large reductions in the rates of adenosine and uridine polymerization were observed while no changes in guanosine or cytidine polymerization were found . This suggests the presence of functionally significant carcinogen-induced modifications of adenine . The inhibition in the rate of adenosine and uridine polymerization was reversed when the animals were placed on a basal diet after AAF ingestion. Proc Natl Acad Sci U S A, 1979 Sep, 76(9), 4370 - 4 Construction and cloning of rat albumin structural gene sequences; Kioussis D et al.; A recombinant plasmid containing a DNA segment complementary to rat liver albumin mRNA has been constructed, cloned, and used to examine the organization of albumin gene . The 18S fraction of total liver poly(A)-containing RNA was copied into a double-stranded cDNA by avian myeloblastosis virus reverse transcriptase and Escherichia coli DNA polymerase I . The cDNA was inserted into the HindIII site of the plasmid pBR322 via the addition of specific oligonucleotide linkers . Recombinant plasmids were screened by hybrid arrest of mRNA translation and hybridization with specific cDNAs . Thereby, a plasmid was identified that contained a 1200-nucleotide insert corresponding to a segment adjacent to the 5'-terminal region of albumin mRNA . The inserted sequence was used as a hybridization probe to detect five EcoRI fragments of genomic DNA which encode albumin mRNA . These were compared to eight EcoRI fragments identified within the rat genome by albumin cDNA . We conclude that the albumin gene (or genes) is interrupted at more than one site in the coding DNA by intervening sequences . Furthermore, we were able to distinguish those fragments that encode the 5' and 3' ends of the mRNA. Antimicrob Agents Chemother, 1979 Sep, 16(3), 247 - 51 Enhancement of post-ultraviolet killing in Escherichia coli K-12 through the action of gyrase inhibitors: evidence for associated gyrase-recBC deoxyribonuclease function; Purdy MA et al.; This work in conjunction with the results presented in an earlier report (M . A . Purdy and K . L . Yielding, Antimicrob . Agents Chemother . 10:182--184, 1976) showed the following . (i) Nalidixic acid and novobiocin could inhibit post-ultraviolet and post-X-ray survival, implicating gyrase function in deoxyribonucleic acid repair . (ii) The inhibition of post-ultraviolet survival requires the action of functional recBC deoxyribonuclease . (iii) Structural changes in the gyrase could (a) cause recBC mutants to exhibit enhancement of post-ultraviolet killing in the presence of novobiocin, (b) increase the ultraviolet sensitivity of recBC mutants, and (c) enhance the thermal lability of a recBCts mutant. Proc Natl Acad Sci U S A, 1979 Sep, 76(9), 4308 - 12 A general priming system employing only dnaB protein and primase for DNA replication; Arai K et al.; Priming of phage phi X174 DNA synthesis is effected simply by dnaB protein and primase when the DNA is not coated by single-strand binding protein (SSB) . The five prepriming proteins (n,n',n'',i, and dnaC protein) required for priming a SSB-coated phi X174 DNA circle are dispensable . The dnaB protein-primase priming system is also active on uncoated phage G4 and M13 DNAs and on poly(dT) . Multiple RNA primers, 10--60 nucleotides long, are transcribed with patterns distinctive for each DNA template . Formation of a stable dnaB protein.DNA complex in the presence of primase and ATP supports the hypothesis that dnaB protein provides a mobile replication promoter signal for primase. Mol Biol (Mosk), 1979 Sep-Oct, 13(5), 1021 - 34 {DNA fragmentation of chicken adenovirus CELO by specific endonucleases R . HpaI, R . EcoRI, R . HindIII}; Denisova TS et al.; The effect of specific endonucleases on DNA chiken adenovirus CELO was studed . It was shown that endonucleases R . HpaI, R . EcoRI and R . HindIII cleaved viral DNA into 5,7 and 9 specific fragments, respectively . The sequence of fragments (physical map) was determined and found to be: D-A-E-C-B for enzyme R . HpaI; B-(EG)-C-A-D-F for enzyme R . EcoRI; H-F-A-C-G-B-D-E-I for enzyme R . HindIII. Gene, 1979 Sep, 7(1), 15 - 31 Cloning the argF gene from Escherichia coli K-12 with simian virus 40; Purchio AF et al.; We have inserted a gene coding for ornithine transcarbamylase (OTCase) from Escherichia coli K-12 into the late gene region of simian virus 40 (SV40) DNA and propagated the hybrid molecules as free episomes or by co-infection with an SV40 tsA helper virus . In the first case, the E . coli argF gene was inserted via the EcoRI and BamHI termini in the late gene region of SV40 and the recombinant molecules were used to transfect monkey kidney cells . The hybrid DNA, which was too large to be encapsidated, was replicated for a short time (14 days) but was eventually lost from the surviving cells . In order to allow the argF gene to be packaged into virions, we purified two SV40 vectors containing large deletions of late gene region sequences . One was a 3325 base pair segment from a HaeII + BamHI digest . The argF gene was joined to both vectors at the BamHI site and these linear molecules were used to transfect monkey cells in the presence of SV40 tsA58 DNA as helper . These hybrid DNAs were replicated and packaged into virions . Late in the lytic infection of monkey cells, polyadenylated, cytoplasmic argF transcripts were detected, but significant translation of these trancripts was not observed. Eur J Biochem, 1979 Sep, 99(3), 499 - 505 Regulation of transcription by DNA-bound non-histone nuclear proteins; Crepin M et al.; Purified non-histone proteins from mouse mammary cells bind specifically to homologous DNA or chromatin . Complexes of non-histone protein with DNA or chromatin, isolated on agarose columns, were transcribed with both Escherichia coli RNA polymerase and RNA polymerase B from calf thymus . The fact that complexing of DNA with non-histone proteins increases transcription by E . coli RNA polymerase but not by RNA polymerase B suggests different mechanisms of transcription by these two enzymes . Similar experiments with mouse and Drosophila chromatin indicate that non-histone proteins specifically stimulate the transcription of mouse chromatin by RNA polymerase B . Non-histone proteins stimulate the transcription of mouse mammary tumor virus sequences in chromatin by RNA polymerase B but not by E . coli RNA polymerase . We conclude that those non-histone proteins bound specifically to chromatin are able to activate the transcription of specific genes by eukaryotic RNA polymerase. Arch Environ Health, 1979 Sep-Oct, 34(5), 372 - 6 Effects of silica dust inhalation on the susceptibility of mice to influenza infection; Zarkower A et al.; Mice were exposed to silica dust for durations of up to 36 wk . At intervals of 15, 21, 27, 33, and 36 wk, the ability of the splenic lymphocytes to respond to T and B cell mitogens were determined, and their ability to resist influenza virus infections was determined after 3 and 20 wk of exposure . These exposures had a depressing effect on the response to T cell mitogens at 21 and 27 wk of silica exposure, but there was no effect on the T cell responses after 15, 33, and 36 wk or on B cell responses after all exposure times . No changes could be detected in the ability of the mice to resist pulmonary influenza virus infections or in the survivors ability to form HI antibodies against this virus. J Med Chem, 1979 Sep, 22(9), 1051 - 5 Synthesis and biological properties of N2-substituted spin-labeled analogues of actinomycin D; Sinha BK et al.; We have synthesized N2-{4-(2,2,6,6-tetramethyl-1-piperidinyloxy)}actinomycin D And the related 1,2-diaminoethane and 1,3-diaminopropane derivatives and evaluated their biological properties . Binding studies with the spin-labeled actinomycin D analogues and DNA were carried out by using circular dichroism, electron spin resonance, and thermal denaturation . These studies have suggested that the derivatives bind to DNA and that their DNA-binding modes are similar but not identical . Spin-labeled actinomycin D derivatives were less potent in inhibiting Escherichia coli DNA-dependent RNA polymerase reaction than actinomycin D and were less toxic to L1210 cells in vitro than the parent compound . Spin-labeled actinomycin D derivatives were more common than the parent compounds against P-388 leukemia cells in vitro with little or no toxicity. Z Naturforsch {C}, 1979 Sep-Oct, 34(9-10), 797 - 804 {Quantitative comparison of ribosome binding sites of twelve nucleotide sequences from Escherichia coli (RNA- and DNA phages) based on triplet patterns (author's transl)}; Kohler E; The molecular structure of ribosome binding sites of ten phage genes and two messengers of Escherichia coli were compared concerning the signation parts which are presumably used by ribosomes for recognition and binding . With a simple calculation based on triplet patterns sofar unknown agreements between all of these sequences were found . In several cases it was shown that agreements between old sequences are easier recognizable if the purine- and pyrimidine bases are put into the triplets instead of the four A, G, C, and U (T) bases . In such cases "homologous" parts of sequences were recognized with more distinctness . This is true in our case for the double triplet (hexaplet) py-pu-pu-pu-pu-(pu) and the binding site triplet py-pu-pu, which are preceding the initiator . These triplets are in specific positions in all twelve sequences which were compared . The different course of the quaternary and the binary conformity curves (diagram 1) may show for the investigated area that the RNA phage gene-part is organized according to the well known quaternary triplet code . On the contrary the phage phi-gene-part seems to be organized according to a more simple, binary triplet sequence of purine and pyrimidine bases . The binary sequence seems to be the more original, the quaternary the derived one. Mol Gen Genet, 1979 Sep, 175(2), 203 - 8 Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis . II . Further evidence for a novel function in error-prone repair; Steinborn G; Uvm mutants of Escherichia coli K12 selected for defective UV reversion induction have previously been reported to differ considerably from the UV-reversion-less recA and lexA mutants with regard to survival or mutagenic response to UV, X-rays and alkylating agents . In the present study, the phenotypic characterization of uvm mutants was extended to investigate several cellular processes which also may be related to or involved in UV mutagenesis . Like recA and lexA mutations, the uvm mutations exhibit highly reduced Weigle reactivation and normal host cell reactivation of UV irradiated phage lambda . But unlike recA and lexA, the uvm mutations do not impair genetic recombination, UV induction of prophage lambda or R plasmid-mediated UV resistance and mutagenesis . These phenotypical characteristics and preliminary results of genetic mapping lend further support to the assumption that the uvm site may be a novel locus affecting, apart from the recA and lexA loci, the error-prone repair pathway in E . coli. Mol Gen Genet, 1979 Sep, 175(2), 135 - 44 Characterization of the dnaA, gyrB and other genes in the dnaA region of the Escherichia coli chromosome on specialized transducing phages lambda tna; Hansen FG et al.; Specialized transducing phages lambda tna (tryptophanase) harboring chromosomal DNA and genetic markers from the dnaA region of the Escherichia coli chromosome were isolated . Transductional analysis showed that some of these tnaA transducing phages carry two genes important in DNA replication, namely the dnaA gene (initiation of chromosome replication) and the gyrB gene (subunit B of DNA gyrase), formerly designated couR . The following clockwise order of genetic markers was found: uhp, gyrB, dnaA, rimA, tnaA, bglB . The gene-protein relationship was established by the determination of the gene products encoded on the chromosomal DNA of the different lambda tna . A 54 kD and a 91 kD polypeptide appear to be coded for by the dnaA and gyrB genes, respectively; the 91 kD protein is encoded on a region in which coumermycin sensitivity maps and is with respect to electrophoretic behavior identical to subunit B of DNA gyrase . The 54 kD protein is encoded on the region in which different independently isolated dnaA(Ts) mutations (dnaA5, dnaA46, dnaA167, dnaA203, dnaA204, dnaA205, dnaA211, dnaA508) are located . Additional genes which code for polypeptides with hitherto unknown functions were identified and mapped . The acriflavin sensitivity mutation acrB1 was found to be an allele of the gyrB gene (see "Note Added in Proof"). Proc Natl Acad Sci U S A, 1979 Sep, 76(9), 4593 - 7 Sigma subunit of Escherichia coli RNA polymerase affects the function of lambda N gene; Nakamura Y et al.; A new class of Escherichia coli mutants, referred to as grn, has been isolated by localized mutagenesis . These mutations affect the sigma subunit of DNA-dependent RNA polymerase (ribonucleoside 5'-triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) by abolishing the expression of the lambda N gene, and they are closely lniked to dnaG in the order dnaG-grn-uxaA . Detailed study of one such mutant, grn1, yielded the following results: (i) grn1 is a single mutation and the mutant cell shows cold-sensitivity in growth; (ii) the Grn phenotype of the mutant can easily be suppressed by secondary mutations in the beta subunit gene of RNA polymerase; (iii) purified holoenzyme of RNA polymerase isolated from the mutant showed an altered salt-dependency in vitro, and the mixed reconstitution of the mutant with the wild-type subunits showed that the sigma subunit of the grn1 mutant is altered; (iv) lambda phage mutants (lambda grg), which overcome the grn mutation, can be classified into two groups, the "nin-deletion" and the "N-mutant" groups (both of these are also able to grow on the previously described groN mutant of Georgopoulos and nusAB of Friedman); (iv) the mutant polymerase transcribed 12S as well as 7S RNA from lambda DNA in the presence of the rho factor in vitro . These results indicate that the grn mutation alters the sigma subunit of RNA polymerase and that the sigma subunit participates in activating the N-mediated antitermination mode of lambda phage transcription. Proc Natl Acad Sci U S A, 1979 Sep, 76(9), 4571 - 5 Transcription promotes recA-independent recombination mediated by DNA-dependent RNA polymerase in Escherichia coli; Ikeda H et al.; The Rpo-mediated recombination of phage lambda takes place independently of the recA function and is promoted by DNA-dependent RNA polymerase of Escherichia coli {Ikeda, H . & Kobayashi, I . (1977) Proc . Natl . Acad Sci . USA 74, 3932--3936} . The crossovers were particularly frequent to the cIII-N and N-cII regions which are transcribed actively . To determine whether the transcription process required for the recombination is the initiation step or the chain elongation step, we have examined the effect of bacterial rho mutation, which affects transcription termination, on the distribution of crossover points in the lambda phage genome . The crossovers in the cII-S interval took place more frequently in rho mutant strains than in wild-type strains . Analysis of lambda mRNA showed that much more O-P-Q mRNA is synthesized in the rho mutant cells than in the wild-type cells and is largely produced by the readthrough from the PR promotor . These results strongly suggest that the chain elongation in transcription plays an essential role in this recombination . Physical analysis of the recombinant phage DNA showed that this recombination is a legitimate type . Models are presented to explain how the transcription complex can promote this recA-independent recombination. Proc Natl Acad Sci U S A, 1979 Sep, 76(9), 4360 - 4 Cloning of the structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12; Henning U et al.; The gene (ompA) for the major outer membrane protein II* from Escherichia coli K-12 has been cloned on a 5-megadalton EcoRI fragment by using phage lambda as vector . The gene is expressed during the lytic cycle of the recombinant phage and the insoluble membrane-bound protein was detected in phage plaques with a simple radioimmunoassay . Transfer of the EcoRI fragment into plasmid pSC101 and expression in a host lacking protein II* led to overproduction of protein II* and decreased production of two other major outer membrane proteins . Expression of the plasmid pSC101-ompA+ in minicells derived from an ompA minicell-producing strain led to synthesis, at high rates, of this protein and massive accumulation of a second cell envelope protein most likely representing the biosynthetic precursor of protein II*. Genetika, 1979 Sep, 15(9), 1543 - 54 {New transducing phage with RNA polymerase beta- and beta'-subunit genes derived from a hybrid phage lambda att80: isolation, genetic analysis and physical mapping}; Mindlin SZ et al.; A hybrid lambda att 80 phage with the genetic structure lambda (A-J) phi 80 (att-int-xis) imm lambda..cI857s7 is shown to be a convenient vector for creating transducing phages . On the one hand, the restriction analysis indicates that it has 3 restriction sites for EcoRI in comparison with 5 and 9 sites for parental phages lambda and phi 80 respectively . On the other hand, its buoyant density is less than that of phage lambda and under centrifugation it is easier separated from the phage transducing particles . When lambda att 80 prophage was excluded from the bfe locus of Escherichia coli, transducing phages with genes of two RNA polymerase beta-subunits (rpoB and rpoC) were isolated . To identify the latter, a convenient genetic test was worked out . A physical map of lambda att 80 drifd 35 transducing phage, carrying rpoB and rpoC genes has been constructed using endonucleases EcoRI and HindIII . A comparison of this map and the corresponding maps of transducing phages lambda drifd 18 and lambda drifd 47, studied earlier, led to the discovery of two integration sites of phage lambda within the locus bfe spaced apart by about 1800 nucleotide pairs . At all the sites both phages (lambda and lambda att 80) have integrated in the locus bfe in the counter clockwise order. J Bacteriol, 1979 Sep, 139(3), 1054 - 7 Gene lon and plasmid inheritance in Escherichia coli K-12; Falkinham JO 3rd; Lon- mutants of Escherichia coli K-12 are deficient in the inheritance of F-plasmids by conjugation . This deficiency is distinct from the conjugation deficiency caused by overproduction of capsular polysaccharide which decreases donor-recipient pair formation. Afr J Med Med Sci, 1979 Sep-Dec, 8(3-4), 115 - 23 The risks of umbilical vessel catheterization in a neonatal intensive care unit; Omene JA et al.; Five hundred and fourteen high-risk neonates who had indwelling umbilical catheters at the neonatal intensive care unit of the University of Benin Teaching Hospital were studied . of these 514 neonates, 122 (23.8%) had their catheters in-situ for longer than 24 h . Of the 122, fifty-four (44%) had positive bacterial cultures from their catheter tips . Seven (5.7%) and four (3.2%) of the 122 neonates studied developed septicaemia and necrotizing enterocolitis respectively . Catheterization for periods in excess of 48 h significantly increased the risk of bacterial colonization . Malposition of umbilical catheter tips include: insertion into the right portal vein (thirty-six cases); superior mesenteric vein (three cases) and the left atrium (four cases) . The complications related specifically to the malposition were: air collection in the hepatic venous system (two cases); cardiac arrest (one case); necrotising enterocolitis (one case) and a case of blanching of the abdominal wall . Because of these complications, the indications for catheterization should be restricted to carefully selected patients and strict aseptic technique be adhered to during the procedure. Int J Radiat Biol Relat Stud Phys Chem Med, 1979 Sep, 36(3), 289 - 300 The nature of the damage to Escherichia coli DNA induced by gamma-irradiation; Bresler SE et al.; Quantitative studies of the number of gamma-induced single-strand breaks (SSBs) and enzyme-labile sites (ELSs) were performed using the model of Col E1 plasmids, which undergo transition from the covalently closed form (CCF) into the open circular form (OCF) during gamma-irradiation of the plasmid-bearing strain E . coli JC 411 . By adding 0.5 MEDTA the repair endonucleases of the cell, which effect the transition of ELSs into SSBs during and after gamma-irradiation, were totally inhibited . It was found thless than 15 per cent of the number of gamma-induced lesions are primarily induced SSBs . About the saditions of direct radiation damage . The conclusions are that (1) the contribution of the direct radiation effect in the cell is greater than that of the indirect effect; (2) the main type of gamma-induced lesions are the ELSs (most of which--more than 75 per cent--are alkali-stable; (3) the enzymatic incision of gamma-induced ELSs into SSBs is effected very quickly, mainly during irradiation; and (4) 0.5 MEDTA is a universal inhibitor of repair processes in cell, including the action of N-glycosidases and endonucleases. Carbohydr Res, 1979 Sep, 74, 301 - 7 The ineffectiveness of analogs of D-galactal as competitive inhibitors of, and substrates for, beta-D-galactosidase from Escherichia coli; Dettinger HM et al.; 2,6-Anhydro-3-deoxy-aldehydo-D-lyxo-hept-2-enose (7) and 2,6-anhydro-3-deoxy-D-lyxo-hept-2-enitol (8) were synthesized as half-chair analogs of D-galactal (1) . As 1 is a strong inhibitor of, as well as a substrate for, beta-D-galactosidase from Escherichia coli, the same properties were expected for 7 and 8; however, both were ineffective . This result, together with those of other authors, allows speculative conclusions on the tight binding of 1 to the enzyme only, when water or an alcohol is bound as a co-substrate. J Bacteriol, 1979 Sep, 139(3), 932 - 9 Use of gene fusions to determine a partial signal sequence of alkaline phosphatase; Sarthy A et al.; We have isolated strains of Escherichia coli in which an amino-terminal portion of the cytoplasmic enzyme beta-galactosidase is replaced by an amino-terminal portion of the periplasmic enzyme alkaline phosphatase . The synthesis of these hybrid proteins is regulated by inorganic phosphate and they are located in the cytoplasm . One of these proteins was purified, and 14 amino acids of the amino-terminal sequence were determined . The first five amino acids, Met-Lys-Gln-Ser-Thr, appear to represent a portion of the signal sequence of the precursor of alkaline phosphatase, and the remaining sequence corresponds to that of beta-galactosidase, beginning at amino acid residue 20 . The approach described here could be used for the analysis of signal sequences of exported proteins and for partial amino acid sequence determination of certain of certain other proteins. J Bacteriol, 1979 Sep, 139(3), 721 - 9 Regulation of expression of the flagellin gene (hag) in Escherichia coli K-12: analysis of hag-lac gene fusions; Komeda Y et al.; Previous studies have defined 28 genes necessary for the synthesis of the flagellar apparatus of Escherichia coli K-12 . This study analyzed the influence of the flagellar genes on the expression of the hag gene (structural gene for flagellin) . To this end, a hag::Mu d(Apr lac) mutant which had the lac genes fused to the promoter of the hag gene was constructed . This allowed the measurement of hag gene expression by detection of beta-galactosidase activity . The following observations were made . (i) The hag gene was expressed constitutively in Fla+ cells . (ii) hag gene expression was positively regulated by flaA, FLAB, flaC, flaD, flaE, flaG, flaH, flaI, flaK, flaL, flaM, flaN, flaO, flaP, flaQ, flaR, flaV, flaW, flaX, flaY, flaZ, flbA, and flbB genes.hag-lac expression was not observed in strains with these fla mutations . (iii) The hag gene was expressed in mutants with flaS, flaT, flaU, and flbC defects . Therefore, these genes were not involved in regulation of hag gene transcription. J Bacteriol, 1979 Sep, 139(3), 1014 - 20 Positive control of ilvC expression in Escherichia coli K-12; identification and mapping of regulatory gene ilvY; Watson MD et al.; The construction of a plasmid carrying the ilvC::lacZ fusion is described . This plasmid provides a convenient source of template deoxyribonucleic acid for use in an in vitro protein-synthesizing system . We screened strains deleted in regions of the ilv cluster for their ability to support ilvC-dependent beta-galactosidase synthesis . The fact that two deletions prevented beta-galactosidase production indicated that ilv-C expression is under positive control . By use of plasmids carrying the positive-control factor structural gene ilvY, we were able to restore protein-synthesizing ability to these strains . These plasmids also enabled us to map ilvY between ilvA and ilvC. Clin Chem, 1979 Sep, 25(9), 1649 - 54 Viable and total cell masses in dental plaque as measured by bioluminescence methods; Robrish SA et al.; Bioluminescence methods have been applied to the measurement of the viable and total cell masses of small samples of dental plaque . The total adenine nucleotide content of dental plaque samples and of a pure culture of bacteria was determined and the adenylate energy charge calculated from this . When a pure culture of bacteria was killed with heat, the adenylate energy charge decreased exponentially with duration of treatment and corresponded with a decrease in the count of viable organisms. Tropenmed Parasitol, 1979 Sep, 30(3), 301 - 7 {Immunofluorescent diagnosis of Entamoeba histolytica trophocoites in preserved stool specimens of patients (author's transl)}; Hess U et al.; A fixative and examination technique is described for identification of Entamoeba histolytica trophocoites in faeces with the aid of the indirect immunofluorescence technique . Magnaforms in bloody-dysenteric specimens and Minutaforms in spontaneous or saline purged specimens give equally good fluorescence . The specifity of the rabbit antiserum against axenically grown E . hist . is good: There is strong positive reaction only with trophocoites of E . hist . Weak crossreactions are encountered with trophocoites of E . coli, E . hartmanni and E . polecki . Negative reactions are encountered with trophocoites of Endolimax nana, Jodamoeba bautschlii, and Dientamoeba fragilis . Amebic cysts, flagellates, leucocytes, epithelial cells and Blastocytis give negative reactions, too. Immunology, 1979 Sep, 38(1), 123 - 7 An examination of the O and K specificity involved in the antibody-induced loss of the K88 plasmid from porcine enteropathogenic strains of Escherichia coli; Linggood MA et al.; The heat-labile K88 antigen, a virulence determinant coded for by a transmissible plasmid, was eliminated from enteropathogenic strains of Escherichia coli by passage through media containing antibodies to the heat stable antigens of an Abbotstown (O149:K91,K88ac) strain . The plasmid-curing activity of O149 antisera was not O-antigen specific as O149, O45, O8 and O138 strains of E . coli could be 'cured' of their K88 plasmids by this technique . The curing activity was differentiated from the O-antibody by gel filtration, the O149 antibodies were eluted in the IgM peak while the curing activity was found in the IgG peak . In view of the lack of O-specificity and the absence of K88 antibodies it appears that antibodies to a common heat-stable antigenic determinant were involved in this phenomenon. Mol Biol (Mosk), 1979 Sep-Oct, 13(5), 1070 - 6 {Enzymatic synthesis and characterization of DNA complementary to ceruloplasmin mRNA from rat liver}; Frolova LIu et al.; Poly(A) containing rat liver 21S RNA homogeneous in polyacrylamide gel electrophoresis under denaturing conditions and stimulating the synthesis of ceruloplasmin in a cell-free proteinsynthesizing system, was used as a template for reverse transcription in the presence of T10 primer and highly purified reverse transcriptase from avian myeloblastosis virus . The cDNA made this way was characterized by means of hybridization kinetics with mRNA, by melting of the hybrids formed and by chain length measurements . To increase the degree of representativity, the ceruloplasmin mRNA was fragmented by mild alkaline treatment, enzymatically polyadenylated and transcribed . The cDNA made was fully characterized and the kinetic complexity measured by hybridization with the mRNA was found to be equal to 2300 nucleotides as compared with the value of 3000 nucleotides is expected from gel electrophoresis data . The observed difference may indicate the presence of repeated sequences in the given mRNA . The sufficient representativitness of the synthesized cDNA and its specificity with respect to ceruloplasmin mRNA allows to use it as a molecular probe to study the ceruloplasmin gene structure. Proc Natl Acad Sci U S A, 1979 Sep, 76(9), 4669 - 73 Requirement for membrane potential in injection of phage T4 DNA; Labedan B et al.; The first stages of infection by phage T4 may be divided into energy-dependent and energy-independent processes . Irreversible adsorption, unplugging, and initial exposure of the DNA terminus may occur at 4 degrees C, or at 37 degrees C in bacteria whose energy-yielding metabolism has been poisoned . DNA injection into the cytoplasm needs higher temperatures and energy from the host cell . The nature of this energy requirements was deduced from the use of metabolic inhibitors . Our results show that T4 DNA injection specifically requires the presence of a protonmotive force across the cytoplasmic membrane of the host . Moreover, the chemical gradient (delta pH) does not appear to be essential, but the membrane potential (delta psi) is required. Proc Natl Acad Sci U S A, 1979 Sep, 76(9), 4544 - 8 A new glnA-linked regulatory gene for glutamine synthetase in Escherichia coli; Pahel G et al.; Mutations in the glnA region of the Escherichia coli chromosome due to Mu prophage insertion result in two phenotypic classes . One class is Gln- and does not synthesize glutamine synthetase{L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2} under any growth condition . The other class produces a low level of glutamine synthetase under all growth conditions and is uncoupled from the regulatory effects of mutations in the glnF and glnD genes . Complementation analysis demonstrates that these two classes of insertions are in different cistrons . From these data we suggest that a regulatory gene, glnG, tightly linked to glnA, mediates both activation and repression of glutamine synthetase synthesis . An analysis of the evidence accumulated to date makes it unlikely that glnG is the only gene in the glnA region involved in the complex system of nitrogen regulation. Science, 1979 Aug 31, 205(4409), 936 - 7 Cytotaxins after the sedimentation behavior of human granulocytes; Catsimpoolas N et al.; Human granulocytes from the peripheral blood of healthy donors were subjected to transient gravity sedimentation analysis in Ficoll density gradient columns (37 degrees C) containing different concentrations of Escherichia coli endotoxin-activated serum and medium 199 . A dramatic serum concentration-dependent dispersion of the cells based on changes in sedimentation velocity was observed as a function of time, using a new optical scanning instrument . The phenomenon was virtually abolished in the presence of cytochalasin B, a known inhibitor of cellular chemotaxis . The width (second statistical moment) of the sedimenting cell distribution increased in a sigmoid fashion as a function of time regardless of cytotaxin concentration . This indicates that a slow and nonlinear response of the granulocytes to the cytotaxins occurs . This new kinetic method should be useful in examining an alternate manifestation of the chemoresponsiveness of phagocytic cells and of cell interactions in general. Biochim Biophys Acta, 1979 Aug 29, 564(1), 31 - 6 The fate of phage lambda DNA in lambda-infected minicells; Witkiewicz H et al.; The fate of phage lambda DNA in lambda-infected Escherichia coli minicells harboring the plasmid ColE1, and in plasmid-free minicells, were studied . Binding of lambda DNA to the minicell membrane, and formation of the supercoiled covalently-closed circular structure has been demonstrated . Phage infection abolishes plasmid DNA synthesis . Only a very slight, non-replicative lambda DNA synthesis occurs, soon after infection . This synthesis is associated with fragments of lambda DNA arising during, or soon after its penetration. Biochim Biophys Acta, 1979 Aug 28, 579(2), 483 - 6 Amino acid sequences of soluble tryptic peptides of chorismate mutase/prephenate dehydratase from Escherichia coli K12; Baldwin GS et al.; The amino acid sequences of 28 soluble tryptic peptides from chorismate mutase/prephenate dehydratase from Escherichia coli K12 have been determined . Together with the four unique cysteine-containing peptides sequenced by Gething and Davidson ((1976) Eur . J . Biochem . 71, 327-336) this accounts for approximately 75% of the total sequence expected for this protein . A high frequency of identify between some of the peptides suggests the possibility of gene duplication during the evolution of the structural gene for the enzyme. Biochim Biophys Acta, 1979 Aug 28, 579(2), 291 - 7 Subunit localizations of zinc(II) in DNA-dependent RNA polymerase from Escherichia coli B; Miller JA et al.; RNA Polymerase holoenzyme and core enzyme from Escherichia coli B have been shown to contain two zinc ions . Flameless atomic absorption spectroscopy of the isolated core subunits indicated that one zinc ion is localized on the beta subunit and the other is bound on the beta' subunit . Atomic fluorescence spectroscopy showed that prolonged dialysis of the metalloenzyme against 0.01 M o-phenanthroline resulted in the removal of both zinc(II) ions with accompanying loss of enzymatic activity . The activity of the apoenzyme was observed to be completely restored by readdition of zinc(II) and partially restored by cobalt(II). Vet Rec, 1979 Aug 25, 105(8), 159 - 64 The pathogenesis of enteric colibacillosis in neonatal unsuckled calves; Pearson GR et al.; The development of pathological lesions in the small intestine of neonatal calves is described . Seven newborn calves were challenged orally with a known enteropathogenic strain of E coli 0101k?(A) and killed at varying times after inoculation . Adhesion of bacteria to the mucosa of the small intestine was observed in all calves . A few organisms were seen in the distal small intestine at three hours after inoculation and thereafter adhesion progressed anteriorly along the intestine in calves killed from six to 36 hours . In these calves pathological changes occurred between six and 12 hours after inoculation . Villi were stunted and thickened and the epithelial surface was irregular . A further calf, anaesthetised from five-and-a-half to 10 hours after inoculation and repeatedly sampled from the distal small intestine, developed similar lesions abruptly at nine hours after inoculation . Villus and crypt lengths in the challenged calves were compared with those in three normal uninoculated control calves. J Biol Chem, 1979 Aug 25, 254(16), 8074 - 82 Purification and properties of L-Aspartate-alpha-decarboxylase, an enzyme that catalyzes the formation of beta-alanine in Escherichia coli; Williamson JM et al.; L-Aspartate-alpha-decarboxylase, an enzyme that catalyzes the production of beta-alanine, has been purified to apparent homogeneity from Escherichia coli . The properties of the enzyme are: (a) pH optimum of 6.8 to 7.5, (b) temperature optimum of 55 degrees C, (c) Km for L-aspartate of 0.16 mM, and (d) molecular weight of 58,000 . The activity of the enzyme is inhibited by reagents (hydroxylamine, phenylhydrazine, and sodium borohydride) that react with carbonyl groups, but no pyridoxal phosphate is present . The compound containing the carbonyl group has been identified as covalently bound pyruvate . Approximately 1 mol of pyruvate was found/mol of enzyme . That the enzyme has a biosynthetic function rather than a catabolic role is indicated by the observations that a mutant (designated as E . coli 99-2) which requires either beta-alanine or pantothenic acid for growth contains only trace amounts of enzyme activity, whereas it is present in substantial amounts in the parent strain (E . coli W) and in a spontaneous revertant of the mutant. J Biol Chem, 1979 Aug 25, 254(16), 7820 - 6 The effect of template secondary structure on vaccinia DNA polymerase; Challberg MD et al.; Vaccinia virus DNA polymerase will utilize a substrate consisting of phi X174 DNA primed with a strand of a unique restriction fragment, but the reaction is inefficient . Examination of the reaction products by alkaline agarose gel electrophoresis revealed a few discrete fragments, each corresponding to an extended primer strand . This result implies that specific barriers exist on the phi X174 template which impede, but do not completely halt, the progress of the enzyme . Only a few per cent of the template molecules were completely copied . Similar findings were reported by Sherman and Gefter using Escherichia coli DNA polymerase II and fd DNA (J . Mol . Biol . (1976) 103, 61-76) . Several observations suggest that the barriers are regions of template secondary structure . Some barriers are more effective than others, and they increase in both effectiveness and number as the temperature is decreased . The same barriers are observed with T4 DNA polymerase, but none are detected with E . coli DNA polymerase I . Finally, the major barriers are located in regions of the phi X174 sequence known to contain hairpin structures of relatively high stability . The exact stopping point of one of the major barriers is within the duplex stem of a hairpin structure . These results show that DNA polymerases are a useful probe of the secondary structure of a single-stranded DNA. J Biol Chem, 1979 Aug 25, 254(16), 7752 - 7 Conformational and ligand binding properties of the isolated domains from the beta 2 subunit of Escherichia coli tryptophan synthetase investigated by the reactivity of their cysteines; Goldberg ME et al.; A mild proteolytic treatment of the dimeric beta 2 subunit of Escherichia coli tryptophan synthetase (L-serine hydrolase (adding indole) EC 4.2.1.20) is known to nick each polypeptide chain into two complementary fragments, F1 and F2 (Hogberg-Railbaud, A., and Goldberg, M.E . (1977) Proc . Natl . Acad . Sci . U.S.A . 74, 442-446) . The reactivity of the cysteines in the isolated or associated fragments is studied and used to characterize the structural and functional properties of these fragments . It is shown that the total number of cysteines, their reactivity to dithiobisnitrobenzoate, and their protection by various ligands are the same in the nicked and intact enzyme, thus demonstrating the close structural analogy between these two proteins . In the isolated F1 fragments two cysteines are reactive and two are buried, thus confirming that this fragments has a compact, globular structure . Various ligands tested fail to produce any modification of the cysteines in the isolated fragments, thus suggesting that none of the fragments alone carries a binding site for the substrates and coenzyme. J Biol Chem, 1979 Aug 25, 254(16), 7529 - 33 Location of the sugar-binding site of L-arabinose-binding protein . Sugar derivative syntheses, sugar binding specificity, and difference Fourier analyses; Newcomer ME et al.; The sugar-binding site of the L-arabinose-binding protein, an essential component of the high affinity L-arabinose uptake system in Escherchia coli, is located deep in a cleft formed by the asymmetric contributions from both of the two similar domains . The site was unambiguously identified with the electron-rich substrate analog 6-bromo-6-deoxy-D-galactose in a difference Fourier analysis . The observation that the original native structure might have been solved with bound L-arabinose necessitated the synthesis of a heavy atom analog, its structure consistent with the known sugar-binding specificity of the protein . Difference Fourier maps (3.5 A) of crystals soaked in 46 mM analog showed a peak 3.5 times background, which is attributed to the -CH2Br moiety of the analog . Superposition of a difference map onto a 2.8-A native electron density map indicated that the difference peak is 6 to 7 A from the reactive single cysteine (Cys-64) and partially coincident with an "extraneous" density found in the native map . This "extraneous" peak was previously attributed to a bound L-arabinose molecule, and its presence accounts for the early failures of difference Fourier analyses of crystals soaked in or co-crystallized with L-arabinose to locate the sugar-binding site. J Biol Chem, 1979 Aug 25, 254(16), 7485 - 7 Differential inhibition of host and viral thymidylate synthetases by folylpolyglutamates; Maley GF et al.; The ability of folate analogues to inhibit host and viral thymidylate synthetases was measured using the corresponding Escherichia coli and T2-phage-induced enzymes . In the absence of Mg2+, 6 x 10(-7) M pteroylhexaglutamate inhibited the T2-phage-induced synthetase by 50%, but at least 100-fold greater levels of this compound were necessary to inhibit the E . coli synthetase by this amount . At 2.5 x 10(-6) M pteroylhexaglutamate, at least 80% inhibition of the T2-phage synthetase could be obtained with little or no inhibition of the E . coli enzyme . The pteroylmonoglutamate was about 2 orders of magnitude less inhibitory towards the T2-phage enzyme than the pteroyltri- to -heptaglutamates . However, upon addition of Mg2+ to the assay mixture, the inhibition produced by pteroylhexaglutamate was essentially reversed, with the E . coli synthetase now increasingly inhibited by this compound and the T2-synthetase only minimally impaired . Methotrexate and N10-formyl-2-amino-4-hydroxyquinazoline, although inhibitory to both enzymes in the presence or absence of Mg2+, did not show this differential selectivity . These results suggest that certain folate analogues may be useful in distinguishing between a host and an infecting organism's thymidylate synthetase and could thus provide an additional means of screening for potential chemotherapeutic agents. J Biol Chem, 1979 Aug 25, 254(16), 7465 - 7 A simple and rapid purification method for Escherichia coli DNA polymerase I; Rhodes G et al.; We report a simple, three-step method for the purification of Escherichia coli DNA polymerase I . Its advantages over other procedures are ease and rapidity, the absence of an autolysis or any high speed centrifugation step, and applicability to large quantities of material . In addition, RNA polymerase can be isolated as a by-product . We have applied this method to purify DNA polymerase both from wild type E . coli cells and from cells bearing a lambda prophage carrying the polA gene (Kelley, W.S., Chalmers, K., and Murray, N.E . (1977) Proc . Natl . Acad . Sci . U.S.A . 74, 5632-5636) . This latter source amplifies the amount of DNA polymerase in the cells by at least 10-fold. J Biol Chem, 1979 Aug 25, 254(16), 7915 - 20 Purification and properties of phosphorylated isocitrate dehydrogenase of Escherichia coli; Garnak M et al.; Phosphorylated NADP+-isocitrate dehydrogenase (EC 1.1.1.42) has been purified to electrophoretic homogeneity from in vivo 32P-labeled Escherichia coli . The cells used as the source of phosphorylated enzyme were harvested 1 h after the addition of 5 mCi of {32P}orthophosphoric acid and 25 mM sodium acetate to cultures grown to early stationary phase on a low phosphate medium with limiting glucose . Double immunodiffusion and autoradiography demonstrated immunological identity between the 32P-labeled NADP+-isocitrate dehydrogenase and the enzyme isolated from glucose-grown E . coli . The phosphoenzyme had an apparent subunit molecular weight of 51,000 as determined by denaturing acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the radioactivity co-electrophoresed with NADP+-isocitrate dehydrogenase activity when purified enzyme was subjected to nondenaturing gel electrophoresis . {32P}Phosphoserine was identified following partial acid hydrolysis of the purified phosphoenzyme. Nucleic Acids Res, 1979 Aug 24, 6(12), 3759 - 84 Cloning of chicken embryo tRNA genes using single stranded nucleosomal DNA highly enriched for tRNA complementary sequences; Wittig B et al.; DNA from chicken embryo nucleosome tetramers (about 760 base pairs in size) was enriched for tRNA genes by RPC-5 chromatography . The enriched DNA was hybridized with chicken embryo total tRNA and the hybridized DNA isolated utilizing a) avidinbiotin interaction, b) diazobenzyloxymethyl paper, and c) high temperature RPC-5 chromatography . The obtained single stranded DNA highly enriched for tRNA complementary sequences was hybridized with total DNA from nucleosome monomers (140--190 base pairs in size) and the excess of non hybridized monomer nucleosome DNA removed by Sepharose 4B chromatography . The hybrid molecules obtained were made fully double stranded by incubation with E . coli DNA polymerase I, DNA ligase, and exonuclease III . DNA was inserted into plasmid pBR322 by G-C joining procedure and the recombinant DNA used to transform the E . coli strain chi 1776 . More than 70% of the transformants obtained hybridize to chicken embryo total tRNA. Biochemistry, 1979 Aug 21, 18(17), 3714 - 23 Mechanism of phenylalanyl-tRNA synthetase of Escherichia coli K . 10 . Modulation of catalytic properties by magnesium; Pimmer J et al.; The association of phenylalanylptRNA and Mg2+ follows a biphasic concentration dependence as indicated by the active site directed fluorescent indicator 2-p-toluidinyl-naphthalene-6-sulfonate . The macroscopic dissociation constants are 0.16 +/- 0.03 and 4.1 +/- mM . The effect of Mg2+ on the association of enzyme and MgATP, on the synergistic binding of MgATP and L-phenylalaninol, and on the pre-steady-state synthesis and pyrophosphorolysis of the enzyme-phenylalanyladenylate complex in the absence and the presence of tRNA Phe has been measured by established equilibrium and stopped-flow techniques using 2-p-toluidinylnaphthalene-6-sulfonate . At 10 mM Mg2+, the association of enzyme and MgATP is biphasic with dissociation constants of 0.25 +/- 0.03 and 9.1 +/- 1.7 mM . At 2 mM Mg2+, a single dissociation constant of 5.0 +/- 0.5 mM is indicated . The coupling constant of the synergistic reaction is 15 at 1 mM Mg2+ and 290 at 10 mM Mg2+ . The Hill constant of the sigmoidal dependence is 3.6 . The strengthening of the synergism is believed to reflect a Mg2+-dependent coupling of the synergistic reactions at the two active sites of the enzyme, the coupling being negligible at 1 mM and maximal at 10 mM Mg2+ . The pre-steady-state rate of adenylate synthesis is accelerated by the presence of Mg2+ . The effect is to decrease the value of the Michaelis-Menten constant of MgATP . Another effect is to increase the rate constant when tRNA Phe is present . At subsaturating {MgATP}, the {Mg2+} dependence of the observed rate constant is hyperbolical in the absence and sigmoidal (Hill constant, 3.5) in the presence of tRNA Phe . The rate of the pyrophosphorolysis is enhanced by a decrease of the Michaelis-Menten constant of MgPPi . The effects on the thermodynamics and kinetics parallel the occupancy of the low-affinity Mg2+-binding sites of the enzyme. Biochem J, 1979 Aug 15, 182(2), 493 - 502 Abnormal ribosome assembly in a mutant of Escherichia coli; Butler PD et al.; The mutant strain, 15--28, of Escherichia coli accumulates ribonucleoprotein ('47S') particles that were previously shown {Markey, Sims & Wild (1976) Biochem . J . 158, 451--456} to be an unusual intermediate in the assembly of 50S ribosomal subunits... Biochem J, 1979 Aug 15, 182(2), 407 - 12 Biosynthesis and turnover of outer-membrane proteins in Escherichia coli ML308-225; Allen RJ et al.; Isolated outer membranes and outer-membrane extracts from Escherichia coli ML308-225 in the early-exponential growth phase contain more protein than do corresponding preparations from late-exponential- or stationary-phase bacteria . Isotope-dilution experiments show that this is due to a loss of protein from the membrane during the exponential growth phase . Inhibition of bacterial growth and protein synthesis stabilizes the outer-membrane-protein concentration . Protein synthesis in the absence of bacterial growth results in higher concentrations of protein in the outer membrane. J Am Vet Med Assoc, 1979 Aug 15, 175(4), 388 - 91 Peritoneal lavage in the horse; Valdez H et al.; Eight horses ranging in age from 4 days to 9 years were treated for peritonitis . Escherichia coli was isolated in four cases and Nocardia sp in one case . In each case, a catheter placed in the peritoneal cavity allowed drainage of a large amount of purulent fluid . Retrograde peritoneal lavage was performed through a Foley catheter or medical tubing, using Ringer's lactate solution containing kanamycin, povidone iodine, or nitrofurazone . All except two horses responded well to repeated lavage. Eur J Biochem, 1979 Aug 15, 99(1), 39 - 47 Iron supply of Escherichia coli with polymer-bound ferricrocin; Coulton JW et al.; Uptake of ferric iron from ferricrocin was studied in Escherichia coli using a polymer-coupled ferricrocin that was unable to penetrate into the cell . Ferricrocinyl polyethylene glycol succinate (Mr 7000 -- 8500) promoted growth of E . coli K-12 AB2847 aroB under iron-limiting conditions . In iron-starved cells, uptake of 55Fe could be demonstrated; the amount of iron accumulated amounted to 10% of that observed with free ferricrocin . The iron supply by ferricrocin bound to polyethylene glycol was strictly dependent upon the functions expressed by the tonA and the tonB genes, as was the iron uptake promoted by free ferricrocin . Polymer-bound ferricrocin protected cells against colicin M and phage T5 by competition for the common tonA-coded outer membrane receptor protein . In addition, the rate of iron transport via the negatively charged ferricrocinyl succinate was as fast as via the neutral ferricrocin molecule . No ligand was found associated with the cells . Penetration of chelator beyond receptor is not necessary for siderophore-mediated iron uptake . It is concluded that sufficient amounts of iron can be released from the polymer complex to satisfy growth requirements. Eur J Biochem, 1979 Aug 15, 99(1), 187 - 201 On the binding of tRNA to Escherichia coli RNA polymerase; Spassky A et al.; The fixation of tRNA to Escherichia coli RNA polymerase has been investigated . Bound and free tRNA have been separated and quantified after filtration through cellulose nitrate filters, centrifugation or sucrose gradients or electrophoresis in polyacrylamide gels . We detect no differences between the fixation of E . coli fMet-tRNAfMet, Met-tRNAmMet or uncharged unfractionated tRNA to RNA polymerase . Tight complexes, with a long residence time, are formed between core enzyme and tRNA with a dissociation constant of less than 1 nM . Complexes exist between tRNA and both monomer and dimer forms of the core enzyme . In the monomer complex, one tRNA is bound per alpha 2 beta beta' unit, whereas in the dimer complex only 0.5 tRNA molecule is fixed per alpha 2 beta beta' unit . In contrast to the core enzyme, very little tRNA fixes tightly to the holoenzyme at salt concentrations greater than 80 mM . At lower salt concentrations tRNA fixation results in a loss of sigma subunit from the holo enzyme to the resulting core enzyme where it binds tightly . DNA fixation reduces the binding of tRNA to RNA polymerase and tRNA fixation reduces the binding of DNA . However, binding of DNA to polymerase is not competitive with binding of tRNA, and ternary complexes between RNA polymerase, DNA and tRNA are shown to exist . Our results are discussed in relation to other studies concerning the effects of tRNA upon RNA polymerase. Eur J Biochem, 1979 Aug 15, 99(1), 105 - 11 Isolation of plasmid-protein complexes from Escherichia coli; Busby S et al.; A procedure is described for the isolation of complexes between pMB9 plasmids and protein from Escherichia coli which are stable during centrifugation on sucrose gradients and are not destroyed in the presence of competitor DNA . The proteins in these complexes have been analysed by dodecyl sulphate/polyacrylamide gel electrophoresis . Only 10 polypeptide species are found in significant quantities, many of which are bound to both the plasmid and host DNA . We have also detected the presence of one protein which binds to a specific DNA sequence inserted in the plasmid. Biochim Biophys Acta, 1979 Aug 14, 547(2), 198 - 210 Localization and characterization of cytochromes from membrane vesicles of Escherichia coli K-12 grown in anaerobiosis with nitrate; Sanchez Crispin JA et al.; Cytochromes b of anaerobically nitrate-grown Escherichia coli cells are analysed . Ascorbate phenazine methosulfate distinguishes low and high potential cytochromes b . Reduction kinetics performed at 559 nm presents a very complex pattern which can be analysed assuming that at least four b-type cytochromes are present . The electron transport chain from formate to oxygen would contain a low potential cytochrome b-556, a cytochrome b-558 associated to the oxidase, and a cytochrome d as the principle oxidase . Cytochrome o is also present, but seems to be functional only at low oxygen concentrations . A cytochrome b-556 associated to nitrate reductase is shown to belong to a branch of the formate-oxidase chain . 2-N-Heptyl-4-hydroxyquinoline-N-oxide affects the reduction kinetics in a very complex way . One inhibition site is in evidence between cytochrome b-558 and cytochrome d; another between the cytochrome associated to nitrate reductase and the nitrate reductase . A third inhibition site is located in the common part of the formate-nitrate and the formate-oxidase systems . Ascorbate phenazine methosulfate is shown to donate electrons near cytochrome b-558. J Chromatogr, 1979 Aug 11, 176(2), 217 - 24 Rapid assay for tryptophanase using reversed-phase high-performance liquid chromatography; Krstulovic AM et al.; A rapid and sensitive high-performance liquid-chromatographic assay for tryptophanase based upon the fluorometric measurement of the enzymatically liberated indole was developed . The total incubation time is 20 min, and the reversed-phase separation is fast (elution time of indole in 8 min) and reproducible . The sensitivity of the method is in the nanomole range . This method was tested in the assay of tryptophanase activity in E . coli, giving an average activity of 6589.6 U/g of cells . Because of its speed, high sensitivity and minimal sample preparation, this method circumvents several problems commonly encountered in standard spectrophotometric methods of analysis. Nucleic Acids Res, 1979 Aug 10, 6(11), 3673 - 84 Release of 7-methylguanine residues whose imidazole rings have been opened from damaged DNA by a DNA glycosylase from Escherichia coli; Chetsanga CJ et al.; Double-stranded DNA containing 7-methylguanine residues whose imidazole rings have been opened, i.e . 2,6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine residues, may be prepared by treatment of DNA with dimethyl sulfate followed by prolonged incubation at pH 11.4 . These substituted formamidopyrimidine residues are actively removed from DNA by a DNA glycosylase present in E . coli cell extracts . The enz;me shows no apparent cofactor requirement and has a molecular weight of about 30 000 . The release of ring-opened 7-methyl-guanine residues is due to a previously unrecognized activity, different from the three known E . coli DNA glycosylases that release uracil, 3-methyladenine, and hypoxanthine from DNA . This enzyme may serve to repair a major secondary alkylation product in DNA . In addition, it may remove nonmethylated purines, whose imidazole rings have been opened, from X-irradiated DNA. Nucleic Acids Res, 1979 Aug 10, 6(11), 3641 - 50 Binding of Escherichia coli ribosomal proteins to 23S RNA under reconstitution conditions for the 50S subunit; Marquardt O et al.; The RNA binding capacity of 50S proteins from E . coli ribosomes has been tested under improved conditions; purified proteins active in reconstitution assays were used, and the binding was studied under the conditions of the total reconstitution procedure for the 50S subunit . The results are: 1) Interaction of 23S RNA was found with 17 proteins, namely L1, L2, L3, L4, L7/L12, L9, L10, L11, L15, L16, L17, L18, L20, L22, L23, L24 and L29 . 2) The proteins L1, L2, L3, L4, L9, L23 and L24 bound to 23S RNA at a level of about one copy per RNA molecule, whereas L20 could bind more than one copy (no saturation was observed at 1.8 copies per 23S RNA), and the other proteins bound 0.2--0.6 copies per RNA . 3) L1, L3, L7/L12 showed a slight binding to 16S RNA, L26 (identical with S20) strong binding to 16S RNA . 4) The binding of L2, L7/L12, L10, L11, L15, L16 and L18 was preparation sensitive, i.e . the binding ability changed notably from preparation to preparation . 5) All proteins bound equally well to 23S RNA in presence of 4 and 20 mM Mg2+, respectively, except L2, L3, L4, L7/L12, L9, L10, L15, L16 and L18, which bound less strongly at 20 mM than at 4 mM Mg2+. Nucleic Acids Res, 1979 Aug 10, 6(11), 3459 - 69 Nucleotide sequence of starfish initiator tRNA; Kuchino Y et al.; The nucleotide sequence of starfish ovary initiator tRNA was determined to be pA-G-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-U-G-G-A-A-G-C-G-U-G-C-U-G-G-G-C-C-C-A-U-t6A-A-C-C-C-A-G-A-G-m7G-D-m5C-C-G-A-G-G-A-psi-C-G-m1A-A-A-C-C-U-C-G-C-U-C-U-G-C-U-A-C-C-AOH . The sequence was determined by a combination of the two different post-labeling techniques . Two-dimensional cellulose thin-layer chromatography was adopted for analysis of 5'-terminal nucleotides of tRNA fragments produced by formamide treatment . The nucleotide sequence of starfish initiator tRNA is very similar to that of mammalian cytoplasmic initiator tRNAs, but has seven different nucleotide residues and two modifications: residue 55 is psi instead of U, and residue 26 is unmodified G instead of m2G. Nucleic Acids Res, 1979 Aug 10, 6(11), 3443 - 58 Rapid print-readout technique for sequencing of RNA's containing modified nucleotides; Gupta RC et al.; A rapid, simple, and highly sensitive method for sequence analysis of RNA was developed, which consists of the following steps: (i) controlled hydrolysis of the RNA by brief heating in water; (ii) (32P)-labeling of 5'-hydroxyl groups of the fragments produced in (i); (iii) resolution of labeled fragments by size on polyacrylamide gels giving the familiar "ladder"; (iv) contact transfer ("print") of the ladder from the gel to a PEI-cellulose thin layer; (v) in situ treatment of the ladder with RNase T2 resulting in the release of 5'-(32P)-labeled nucleoside-3',5' diphosphates; (vi) contact transfer and thin-layer separation of (32P)-labeled nucleotides on PEI-cellulose in ammonium sulfate and ammonium formate solvents; (vii) autoradiography . The chromatographic behavior of the 4 major and 18 modified nucleotides was determined . The positions of major and modified nucleotides in the sequence can be read directly from the separation patterns displayed on X-ray film . As this is the only sequencing method presently available that allows one to display and identify directly the positions in the RNA chain of major and modified nucleotides, no additional procedures are required to analyze the latter. J Biol Chem, 1979 Aug 10, 254(15), 7377 - 87 Role of a membranous sialyltransferase complex in the synthesis of surface polymers containing polysialic acid in Escherichia coli . Temperature-induced alteration in the assembly process; Troy FA et al.; Membrane-associated sialyltransferase complexes of Escherichia coli K-235 catalyze the synthesis of sialyl polymers which remain associated with the cell envelope . Sialyl monophosphorylundecaprenol is an intermediate in the formation of these unique surface structures, and fluidity of the lipid phase is required for the proper function of the enzyme complex (Troy, F.A., Vijay, I.K., and Tesche, N . (1975) J . Biol . Chem . 250, 156-163, 164-170) . In membranes containing an increased unsaturated fatty acid content of the phospholipids, obtained by growing cells at 15 degrees C, synthesis of polysialic acid was uncoupled from synthesis of the sialyl lipid-linked intermediate . Using reconstruction experiments, the importance of the role of an endogenous acceptor in polymer formation was suggested by the unexpected finding that polysialic acid synthesis could be reactivated in inactive membranes by the addition of an exogenous acceptor which contained sialic acid . Concomitant with polymer synthesis was a rapid loss of labeled sialic acid from the lipid phase . The activated sialic acid was shown to be transferred directly to the exogenous acceptor . These results establish: 1) that the temperature-induced alteration in polymer synthesis resulted from the inability of cells grown at 15 degrees C to either synthesize or assemble a functional endogenous acceptor and not from a defect in the synthesis of the sialyltransferase; 2) the intermediate precursor role of lipid-soluble sialic acid in sialyl polymer synthesis; and 3) that the exogenous acceptor served directly as an "acceptor" and not as a catalytic "effector" which stimulated an inactive membrane-enzyme complex . These results are in accord with the possibility that the low temperature-induced derangement in polymer formation is a consequence of the altered lipid structure resulting from the greater unsaturated fatty acid content in the membrane phospholipids . U-14C-labeled exogenous acceptor was isolated from the culture filtrate of cells grown at 37 degrees C and purified to homogeneity by preparative polyacrylamide gel electrophoresis . The pure acceptor was characterized structurally as a homopolymer of sialic acid with a degree of polymerization of approximately 12 . Potassium borohydride reduction of the acceptor prior to complete hydrolysis with neuraminidase established that the polymer possessed a free reducing terminus of sialic acid . Subsequent structural studies showed that these oligomers of sialic acid appeared in the culture filtrate as a result of acid-catalyzed hydrolysis from membrane-associated polysialic acids of about 150 to 200 sialyl residues . Marked diminution of several membrane proteins was observed for cells grown at 15 degrees C . The possible relationship of these alterations to the upward shift in unsaturated lipids and to the loss of a functional endogenous acceptor is currently under study. J Biol Chem, 1979 Aug 10, 254(15), 7123 - 8 Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein; Rock CO et al.; We have used purified preparations of acyl-acyl carrier protein synthetase to prepare pure, native acyl-acyl carrier proteins (acyl-ACP) ranging in chain lengths from C10:0 to C delta 9 18:1 . Factors affecting yield are explored and reaction conditions are presented that yield 0.8 to 0.9 mg of C16:0-ACP/ml of reaction mix . Ohter acyl groups, such as C10:0 and C delta 9 18:1 are poorer substrates and gave correspondingly lower yields . Acyl-Acp synthetase may be recovered from the reaction mixture using blue-Sepharose CL-6B and recycled . ACP and acyl-ACP are separated by hydrophobic chromatography on octyl-Sepharose CL-4B . Mixtures of acyl-ACPs could be resolved according to acyl chain length using octyl-Sepharose CL-4B columns eluted with a 2-propanol gradient . The high resolution obtained using 2-propanol gradients to separate acyl-ACP species suggests that similar techniques would be applicable to the chromatography of protein mixtures on hydrophobic supports. J Biol Chem, 1979 Aug 10, 254(15), 7111 - 5 The effect of chemical modification of 3-(3-amino-3-carboxypropyl)uridine on tRNA function; Friedman S; The minor base 3-(3-amino-3-carboxypropyl)uridine (acp3U) in Escherichia coli tRNAPhe was acylated with the N-hydroxysuccinimide esters of acetic, phenoxy-acetic, and naphthoxyacetic acid, as well as the ester of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-glycine . The derivatives of tRNAPhe formed were all capable of accepting phenylalanine . There were only minor effects on the kinetic parameters of these derivatives for E . coli phenylalanyl-tRNA synthetase . There was no effect on the ability of tRNAPhe to participate in poly(U)- or poly(ACU)-directed polypeptide synthesis or in the poly(U)-stimulated binding to E . coli ribosomes . The rate of photodynamic cross-linking of 4-Srd 8 to Cyd 13 was decreased in tRNAs containing the acetyl and dansyl-glycyl derivatives of acp3U, indicating that acylation of this base may perturb the tertiary structure of the tRNA . This base in tRNAPhe does not appear to play any role in the known biological functions of tRNAPhe. J Biol Chem, 1979 Aug 10, 254(15), 6894 - 903 Both positional isomers of aminoacyl-tRNA's are bound by elongation factor Tu; Alford BL et al.; Six purified Escherichia coli and yeast tRNA's were converted to positionally defined tRNA's terminating in 2'- and 3'-deoxyadenosine; the modified (amino-acyl) tRNA's were compared for their abilities to bind to elongation factor Tu (EF-Tu) in the presence both of GTP and guanylylimidodiphosphate (GMP-P(NH)P) . Formation of aminoacyl-tRNA . EF-Tu . guanine nucleotide ternary complexes was monitored by gel filtration on Sephadex G-100 and Ultrogel ACA 44 columns and also by measurement of the ability of the factor to diminish the rate of chemical hydrolysis of the aminoacyl-tRNA's . The apparent positional specificity of the factor was found to be affected substantially both by the choice of guanine nucleotide and gel filtration resin utilized, but not in any systematic fashion . Likewise, assay of ternary complex formation by diminution of the rate of chemical deacylation failed to reveal any consistent positional preference from one isoacceptor to another . It is worthy of note that each modified aminoacyl-tRNA tested did form a ternary complex with EF-Tu under each of the experimental conditions used for assay, but that in each case the difference in affinity of the factor for isomeric aminoacyl-tRNA's was less than that between either of the modified aminoacyl-tRNA's and the corresponding unmodified species . On the basis of the experiments performed, we conclude that (i) EF-Tu has remarkable conformation flexibility, possibly reflecting its physiological role in recognizing 20 tRNA isoacceptors and (ii) the factor has no obvious preference for a single positional isomer of aminoacyl-tRNA and it is not clear that any preference that might exist could be established convincingly using tRNA's terminating in 2'- and 3'-deoxyadenosine. J Biol Chem, 1979 Aug 10, 254(15), 6880 - 8 Hemoglobin switching in sheep . Synthesis, cloning, and characterization of DNA sequences coding for the beta B, beta C, and gamma-globin mRNAs; Benz EJ Jr et al.; Synthetic double-stranded DNAs (sDNAs) were prepared from sheep globin mRNA templates isolated from reticulocytes producing either hemoglobin B (HbB) (alpha 2 beta B2), HbC (alpha 2 beta C2), or HbF (alpha 2 gamma 2) . These DNAs were inserted into the Eco RI site of plasmid pMB9 by the homopolymer tailing method and used to transform Escherichia coli X1776 to tetracycline resistance . Recombinant clones were identified by colony hybridization and further characterized by molecular hybridization and restriction endonuclease analysis . All plasmids analyzed thus far contained either beta- or gamma-globin DNA sequences . Moreover, sDNAs used for cloning yielded restriction endonuclease fragments consistent with the presence of predominantly beta- or gamma-sDNA, indicating that formation of double-stranded alpha-sDNA proceeds much less efficiently under our conditions than the formation of non-alpha-sDNAs . Three recombinant plasmids, pS beta B2, pS beta C69, and pS gamma 56, were selected for detailed study . These were shown to contain, respectively, beta B-, beta C-, and gamma-DNA sequences by molecular hybridization and by protection of the appropriate cDNAs from S1 nuclease digestion . Each contained all of the restriction endonuclease sites defined for the synthetic sDNAs and protected at least 90% of the sequence length of homologous cDNA . Restriction endonuclease maps of the beta B- and beta C-globin genes were identical at all 12 sites that were mapped, whereas four differences were identified in the gamma gene compared to the two others; three of these corresponded to differences in amino acid sequence of the globins . A method was developed to isolate the anti-mRNA strand of the insert for use as a specific molecular hybridization probe analogous to complementary DNA. J Biol Chem, 1979 Aug 10, 254(15), 6873 - 5 Transfer RNA control of the activation of isomeric tRNATrp's; Alford BL et al.; Previous studies of the homologous aminoacylations of Escherichia coli and yeast tRNATrp's terminating in 2'- and 3'-deoxyadenosine established that E . coli tryptophanyl-tRNA synthetase activates its cognate tRNA preferentially on the 2' position, while the corresponding yeast enzyme utilizes the 3' position on its homologous substrate tRNA . As this seemed to be the only change in positional specificity during evolution, the heterologous activations were investigated in an effort to determine the basis for this change . Remarkably, E . coli tRNATrp terminating in 3'-deoxyadenosine was found to be the preferred substrate for both the E . coli and yeast activating enzymes, while the same tryptophanyl-tRNA synthetase preparations both activated the isomeric yeast tRNATrp's preferentially on the 3' position . Thus, the preferred position of activation was found to be specified by the tRNA rather than the activating enzyme and, additionally, to be due to some process not reflected in initial velocity measurements . The variable utilization of individual modified aminoacyl-tRNA's as substrates in an enzyme-catalyzed deacylation process appears to provide the most likely explanation for the experimental observations. J Biol Chem, 1979 Aug 10, 254(15), 7917 - 202 Control of membrane lipid synthesis in Escherichia coli during growth and during the stringent response; Snider MD; The regulation of phospholipid synthesis in cells of Escherichia coli was studied in vivo during growth and during the stringent response to amino acid starvation . Strains harboring the hybrid plasmid pLC44-14 (Clark, L., and Carbon, J . (1976) Cell 9, 91-99), which had increased levels of glycerophosphate acyltransferase, were used to study the involvement of this enzyme in the control of phospholipid synthesis . In addition, regulation was studied by measuring the levels of three early intermediates of phospholipid synthesis:phosphatidic acid, CDP-diglyceride, and dCDP-diglyceride . The liponucleotides were measured by a new enzymatic method which allows determinations to be made on crude lipid extracts . Results from experiments on growing cells are consistent with regulation of membrane lipid synthesis occurring in fatty acid synthesis or at the level of glycerophosphate acylation, but not at any later step . Experiments on the inhibition of lipid synthesis during the stringent response make it possible to rule out explanations which involve the inhibition of a single enzyme; enzymes both before and after the liponucleotides in phospholipid synthesis must be affected. Biochemistry, 1979 Aug 7, 18(16), 3557 - 63 Mechanism of action of D-serine dehydratase . Identification of a transient intermediate; Schnackerz KD et al.; Static absorbance measurements of D-serine dehydratase from Escherichia coli taken at 2 degrees C show that during the steady-state course of D-serine conversion the absorption maximum of the Schiff base of the cofactor pyridoxal 5'-phosphate (pyridoxal-P) is shifted from 415 to 442 nm . Furthermore, the progress curve of intermediates was monitored by stopped-flow techniques at wavelengths ranging from 320 to 500 nm . A point by point construction of successive spectra from these stopped-flow traces at various time intervals after the start of reaction resulted in a series of consecutive spectra exhibiting two isobestic points at 353 and 419 nm . The half-time of the absorbance changes occurring at 330 and 455 nm was found to be 6.5 ms, suggesting the observation of a single, enzyme-bound intermediate . The spectral data with substrate and inhibitors provide evidence that the intermediate is the Schiff base of alpha-aminoacrylate and pyridoxal-P . The proposed assignment is strongly supported by experiments of apodehydratase with transient-state analogues which exhibit a similar absorbance shift on binding to apoenzyme . Moreover, these results suggest that the phosphate group of the substrate--pyridoxal-P complex serves as the main anchoring point during catalysis . A reaction mechanism of the D-serine dehydratase is presented. Science, 1979 Aug 3, 205(4405), 508 - 11 A relationship between DNA helix stability and recognition sites for RNA polymerase; Vollenweider HJ et al.; The RNA polymerase binding sites on the DNA of (i) the aroE-trkA-spc segment of the Escherichia coli genome, (ii) transposon Tn3, (iii) plasmid ColE1, and (iv) coliphage lambda were mapped by electron microscopy, with the use of the BAC technique; these maps were compared with the maps of the early-melting regions for the same genomes . The results indicate that in all these cases the binding sites for the E . coli RNA polymerase lie preferentially in the early melting regions of DNA . These data indicate that helix stability may be an important feature of the multipartite nature of the promoter structure. Trop Anim Health Prod, 1979 Aug, 11(3), 171 - 4 Cell counts in bulked milk supplies from dairy farms of northern Nigeria; Lombin LH et al.; Somatic cells in 596 milk samples collected from bulked supplies of 3 northern Nigeria dairy farms were counted by an electronic method following a standard method for the preparation of the milk samples . Mean somatic cell counts per ml indicating low level of infection were 158,597, 166,742 and 155,032 for the 3 farms, without any significant differences . Mean somatic cell counts per ml indicating herd mastitis averaged 354,768 +/- 66,348 and the pathogenic organisms isolated were Escherichia coli and Staphylocucus aureus . Counts useful for future regular monitoring of somatic cells in bulked milk supplies in northern Nigeria are presented. J Cell Biol, 1979 Aug, 82(2), 369 - 79 Localization of submembranous cations to the leading end of human neutrophils during chemotaxis; Cramer EB et al.; Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis) . The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe) . The small pore filters permitted pseudopod formation but impeded cell imgration through the filter . Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules . However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis . Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant . In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media . However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen . EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis . X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium . The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant. Med J Zambia, 1979 Aug-Sep, 13(4), 67 - 70 Tubo-ovarian abscess: a review of 46 cases; Chatterjee TK et al.; Forty-six patients with tubo-ovarian ascess are analysed . The abscess developed mostly in young multiparous women soon after menstruation . Coliforms were the main causative organism . The abscess resolved with conservative management in 21 cases and surgical intervention was necessary in 25 . The mortality (4.4%) due to conservative medical and surgical management was nearly half that of radical surgery. Can J Microbiol, 1979 Aug, 25(8), 937 - 9 A new gene involved in expression of fructose-1,6-diphosphate aldolase activity in Escherichia coli; Atherly AG et al.; A new gene, fdaB, has been mapped by transduction and partial diploid analyses and located adjacent to argA at 59.9 min on the Escherichia coli recalibrated linkage map . This gene is involved in expression of fructose-1,6-diphosphate aldolase activity and indirectly in ribosomal RNA synthesis . The temperature-sensitive mutant strain AA-157, containing the defective gene product of of fdaB, accumulates high concentrations of fructose 1,6-diphosphate at the nonpermissive temperature. Genetics, 1979 Aug, 92(4), 1041 - 59 Isolation and characterization of dnaX and dnaY temperature-sensitive mutants of Escherichia coli; Henson JM et al.; Escherichia coli mutants with temperature-sensitive (ts) mutations in dnaX and dnaY genes have been isolated . Based on transduction by phage P1, dnaX and Y have been mapped at minutes 10.4--10.5 and 12.1, respectively, in the sequence dnaX purE dnaY . Both dna Xts36 and Yts10 are recessive to wild-type alleles present on episomes . F13 carries both dnaX+ and Y+; the shorter F210 carries dnaY+, but not X+ . Lambda tranducing phages that carry dnaX+ or Y+ have been isolated, and hybrid plasmids of Col E1 and E . coli DNA from the Clarke and Carbon (1976) collection also carry portions of the dnaX purE dnaY region . Results obtained with the lambda transducing phages and the hybrid plasmids suggest that dnaX is a different gene from the previously characterized dnaZ gene, which is also near minute 10.5--The dnaXts36 mutant, after a shift to 42 degrees, stopped DNA synthesis gradually, and the total amount of DNA increased two-fold . When this mutant was shifted to 44 degrees, the rate of DNA synthesis dropped immediately and the final increment of DNA was only 10% of the initial amount . Replicative DNA synthesis in toluene-treated cells was completely inhibited at 42 degrees and was partially inhibited even at 30 degrees.--When the dnaYts10 mutant was shifted to 42 degrees, DNA synthesis gradually stopped, and the amount of DNA increased 3.6-fold . At 44 degrees, residual DNA synthesis amounted to a two-fold increase . Replicative DNA synthesis in vitro in toluene-treated cells was inactivated after 20 minutes at 42 degrees or by "preincubation" of cells at 42 degrees before toluene treatment.--The dnaX and dnaY products probably function in polymerization of DNA, although participation also in initiation cannot be excluded. Mol Gen Genet, 1979 Aug, 175(1), 53 - 6 IS2-43 and IS2-44: new alleles of the insertion sequence IS2 which have promoter activity; Sommer H et al.; The sequence of two new IS2 alleles with promoter activity (IS2-43 and IS2-44) is reported . The alleles are identical and are formed by a 17 bp tandem duplication in an AT-rich region of IS2 . This created a new RNA polymerase binding site . A mutation was found that increased the frequency of formation of these 17 bp duplications but not of another class of duplications, the "mini-insertions" . This suggested that the mechanisms of formation of the two classes of duplications are different. Mol Gen Genet, 1979 Aug, 175(1), 39 - 44 Construction and expression of a hybrid plasmid containing the Escherichia coli thrA and thrB genes; Cossart P et al.; In vitro recombination techniques were used to clone the Escherichia coli thrA and thrB structural genes in the plasmid vector pBR322 . The chimeric plasmid was analyzed and characterized genetically, by restriction mapping and DNA sequencing . The limited expression of the threonine biosynthetic enzymes in the strain carrying the recombinant plasmid is discussed. Mol Gen Genet, 1979 Aug, 175(1), 31 - 8 Regulation of dihydrofolate reductase synthesis in Escherichia coli; Smith DR et al.; Two clones from the Clarke-Carbon Escherichia coli colony bank were resistant to inhibition by trimethoprim, a potent inhibitor of dihydrofolate reductase . Both clones had elevated levels of dihydrofolate reductase . Furthermore, trimethoprim resistance and elevated enzyme levels were associated with ColE1 plasmids that carried DNA from the trkC ksgA pdxA region of the E . coli chromosome . Plasmid pLC1437a was shown by two criteria to carry the structural gene for dihydrofolate reductase: 1) A partial diploid containing plasmid pLC1437a produced a kinetically-recognizable dihydrofolate reductase that was not present in the parent haploid strain . 2) Plasmid pLC1437a coded for dihydrofolate reductase in vitro . A 1,000 base pair fragment of plasmid pLC1437a containing fol was used as a probe to measure fol mRNA in a mutant strain isolated by Sheldon and Brenner (Molec . gen . Genet . 147, 91-97, 1976) . The mutation in this strain, which results in constitutively-high levels of dihydrofolate reductase and in the inability of the strain to grow at 42 degrees C, is cis dominant (Sheldon and Brenner, 1976) . The results of kinetic hybridization and pulse-labeling experiments indicated that the regulatory mutant produced elevated levels of dihydrofolate reductase in response to an increased rate of synthesis of fol mRNA. Mol Gen Genet, 1979 Aug, 175(1), 13 - 7 Radiation sensitivity of messenger RNA; Ponta H et al.; Messenger RNA function is inactivated by irradiation with ultraviolet light . A unit length mRNA (in bases) is 2-3 times more sensitive than a unit length of DNA (in base pairs) with respect to the inactivation of template function . These data stem from four experimental systems all of which do not repair DNA: the translation of E . coli mRNA in rifampicin-treated cells, of T7 mRNA in infected E . coli, of f2 phage RNA in vivo, and of stable mRNA in chromosomeless minicells . The comparison of relative sensitivities to UV is relevant to the technique of UV mapping of transcription units which enjoys increasing popularity in pro- and eukaryotic genetic research. J Gen Microbiol, 1979 Aug, 113(2), 425 - 7 Specific proline accumulation in an acrA mutant of Escherichia coli K12 grown in salt-hypertonic medium; Nakamura H; Free proline is specifically accumulated in an acrA mutant of Escherichia coli K12 when cultured in a medium containing excess NaCl. J Gen Microbiol, 1979 Aug, 113(2), 297 - 303 Mapping of a new hem gene in Escherichia coli K12; Sasarman A et al.; A new type of haem-deficient mutant was isolated in Escherichia coli K12 by neomycin selection . The mutant, designated SASX38, accumulated uroporphyrin, coproporphyrin and protoporphyrin . Since it possessed normal ferrochelatase activity, it was assumed to be deficient in protoporphyrinogen oxidase activity . The gene affected in the mutant was designated hemG . Mapping of the hemG gene by phage P1-mediated transduction showed that it was located very close to the chlB gene (frequency of cotransduction 78.7%), between the metE and rha markers . This location is distinct from the other known hem loci in E . coli K12. Gann, 1979 Aug, 70(4), 421 - 8 Properties of interferon induced by purified protein derivative of tuberculin in mice sensitized with BCG or cell-wall skeleton of BCG; Takeyama H et al.; Mice sensitized with either BCG or cell-wall skeleton of BCG (BCG-CWS) produced interferon in blood after stimulation with specific antigen, purified protein derivative of tuberculin (PPD) . Both BCG-infected normal (C57BL/6) and thymic nude (BALB/c, nu/nu) mice showed enhanced activity to produce interferon by stimulation with E . coli endotoxin . However, detectable interferon was not produced in athymic nude mice sensitized with BCG or BCG-CWS by stimulation with PPD . Immune-induced interferon (I-IF) produced by BCG-CWS and PPD in mice was different in biological and physicochemical properties from virus-induced interferon . I-IF showed about 100 times more potent L-cell growth inhibitory activity than virus-induced L-cell interferon (L-IF) . Both I-IF and L-IF showed macrophage-activating activity, which renders resting macrphages cytotoxic to L1210 leukemia cells . Antiviral and macrophage-activating activity of interferon preparation was not separated physicochemically in this study. Biokhimiia, 1979 Aug, 44(8), 1377 - 80 {Kinetics of phenylacetamide hydrolysis by immobilized penicillinamidase}; Milman IA et al.; The kinetics of phenylacetamide hydrolysis catalyzed by polyacrylamide gel-immobilized penicillinamidase were studied . The Km and Kp values obtained were compared to the literary data for the specific substrate--benzylpenicillin . It was shown that the type of inhibition by the reaction product was the same, whereas the efficiency of binding of phenylacetic acid depended on the substrate structure. Agents Actions, 1979 Aug, 9(3), 280 - 3 The role of prostaglandins in experimental ocular inflammations; Yamauchi H et al.; Intraocular inflammations (uveitis) were produced in rabbits by intravitreal injection of killed and dried Mycobacterium butyricum or E . coli endotoxin, or paracentesis of the anterior chamber . The increase in permeability of the blood-aqueous barrier and the leucocyte migration into the aqueous humor were observed after these stimuli, although the leucocyte did not migrate after paracentesis . Topically applied indomethacin reduced these inflammatory parameters in the latter two models . However, ththacin, though the leucocyte was not affected by indomethacin, though the leucocyte migration was reduced . On the other hand, prostaglandin-like substances in thces of these substances were detected in the former model . These results indicated that, unlike the increase in the permeability of the blood-aqueous barrier after endotoxin injection andparacentesis, the response after M . butyricum injection is not mediated by prostaglandins . The role of prostaglandins in the leucocyte migration in these ocular inflammations was also discussed. Res Commun Chem Pathol Pharmacol, 1979 Aug, 25(2), 293 - 306 Studies on the mode of action of partially thiolated polycytidylic acid (MPC), a novel type of antineoplastic agent; Ho YK; Partially thiolated polycytidylic acid (MPC), a representative member of the "antitemplate" class of novel chemotherapeutic agents, is a potent inhibitor of the E . coli DNA-dependent RNA polymerase . It inhibited 50% of the enzymic reaction at a concentration of 6 micrometers . Kinetic studies indicated that MPC had no effect on the chain elongation of the transcription process, but it appeared to inhibit the initiation of RNA synthtesis presumably by competing with the DNA template for binding to the RNA polymerase . Binding studies, using a gel filtration method, showed that MPC and the RNA polymerase formed a stable complex which was not dissociated by 0.3 M NaCl . It is inferred that mixed disulfide linkage(s) might have been formed between the enzyme and MPC . The implications of these findings are discussed. Proc Natl Acad Sci U S A, 1979 Aug, 76(8), 4020 - 4 Identification and characterization of a self-regulated repressor of translocation of the Tn3 element; Chou J et al.; Gene fusions that bring expression of the lacZ gene under control of transcriptional and trnaslational signals within the transposable element Tn3 have been used to study regulation of Tn3-specified proteins . A gene encoding a 21,355-Mr peptide that represses translocation of Tn3 and acts at the level oquenced; amber, missense, and cis-dominant (operator-constitutive) point mutations in this gene have been isolated and characterized. Proc Natl Acad Sci U S A, 1979 Aug, 76(8), 3795 - 9 Reconstitution of RNase P activity from inactive RNA and protein; Kole R et al.; RNase P preparations from Escherichia coli can be separated into RNA and protein by chromatography, in buffers containing 7 M urea, on Sephadex G-200, DEAE-Sephadex, or CM-Sephadex columns . Neither RNA nor protein components alone exhibits any RNase activity . RNase P activity can be reconstituted by mixing separated RNA and protein components in buffer containing 7M urea followed by dialysis of this mixture to remove the urea . Of several purified RNAs tried, only M2 RNA, the RNA species found in purified RNase P, is active in the reconstitution experiments. Proc Natl Acad Sci U S A, 1979 Aug, 76(8), 3769 - 73 Ribosome structure: localization of 3' end of RNA in small subunit by immunoelectronmicroscopy; Olson HM et al.; The 3' end of the RNA in the 30S ribosomal subunit of Escherichia coli has been modified by oxidation with sodium periodate and conjugation with the (mono) dinitrophenyl derivative of ethylenediamine . Antibodies, induced with dinitrophenyl-bovine serum albumin, interact with the modified ribosomal subunits . Electron micrographs of negatively stained antibody-subunit complexes show individual ribosomal subunits to which a single antibody molecule is bound and subunit dimers cross-linked by an IgG molecule . The modified 3' terminus has been localized to a single site on the upper portion of the platform region of the 30S subunit . This location is consistent with earlier placements of proteins that react with the 3' end of the RNA. Proc Natl Acad Sci U S A, 1979 Aug, 76(8), 3632 - 6 A DNA primase specified by I-like plasmids; Lanka E et al.; An enzyme has been isolated from Escherichia coli strains harboring the I-like plasmid R64drd11, which is capable of initiating DNA synthesis on the circular, single-stranded DNA of phages phi X174, fd, and G4 . In the conversion of these templates to duplex forms in vitro, the enzyme can substitute for the functions of E . coli dna B-dnaB-dnaC-dnaG proteins, E . coli RNA polymerase, and E . coli dnaG protein, respectively . The enzyme requires all four ribonucleoside triphosphates for optimal activity, although a combination of ATP, CTP, and GTP can almost completely satisfy the rNTP requirement . The enzyme appears to cooperate specifically with DNA polymerases III because single-stranded DNA-dependent synthesis takes place in extracts deficient in DNA polymerases I and II but not in extracts from a dnaZ mutant . Highly purified enzyme preparations consist mostly of two major polypeptides, Mr 140,000 and 180,000, when analyzed by sodium dodecyl sulfate gel electrophoresis . These polypeptides cosediment with the enzyme activity through a glycerol gradient with a sedimentation coefficient of 3.6 S . DNA priming activity in extracts of E . coli strains harboring the mutant plasmids R64drd11 or ColIdrd1, which are derepressed in functions of conjugational DNA transfer, severalfold higher than the activity from strains carrying the corresponding wild-type plasmid . This correlation suggests that the enzyme may play a role in conjugational DNA synthesis. Mutat Res, 1979 Aug, 62(1), 7 - 17 A recA-dependent mutator of Escherichia coli K12: method of isolation and initial characterization; Hombrecher G et al.; A number of mutator strains of E . coli were isolated using histochemical techniques which allow the identification of a single mutator colony on agar plates with as many as 2000 colonies . Several mutators isolated in this way were found by P1-mediated transduction to map to the proA--proB region of the E . coli chromosome . The map position of these mutators is very close to that of the conditional mutator, mutD . However, in contrast to mutD, one of these newly isolated mutators was suppressed in a thermosensitive recA strain at 43 degrees C, but not at 30 degrees C . This mutator mutation has been named mut-8 . Besides being dependent upon recA, mut-8 is also dependent upon growth in enriched medium for the expression of its mutator activity . The mutator activity of mut-8 was found to be recessive to the wild-type allele. Infect Immun, 1979 Aug, 25(2), 603 - 9 Role of Escherichia coli K capsular antigens during complement activation, C3 fixation, and opsonization; Van Dijk WC et al.; Escherichia coli strains with K capsular polysaccharides are relatively resistant to phagocytosis by polymorphonuclear leukocytes, in contrast to E . coli strains without K antigens . This inhibition of phagocytosis is related to an impaired recognition of the K+ strains by the phagocytes due to ineffective opsonization . All five strains without K antigens were readily phagocytized after opsonization in 5% normal serum, compared with no uptake of the K+ strains . Evidence is presented that the decreased opsonization of the K+ strains in normal serum is caused by a low rate of complement activation of the strains, with subsequent absence of C3b fixation or C3d fixation or both to the cell wall of the bacteria . After removal of the K+ antigens by heating of a K+ E . coli strain, the strain was able to activate complement, to bind C3b or C3d or both, and to become opsonized . Complement was then activated via the classical and alternative pathways, which was comparable to the complement consumption by K- E . coli. Eur J Biochem, 1979 Aug 1, 98(2), 567 - 71 Reconstitution of active 30-S ribosomal subunits in vitro using heat-denatured 16-S rRNA; Barritault D et al.; 30-S ribosomal subunits which have been reconstituted using heat-denatured 16-S rRNA can participate in the synthesis of lysosyme in vitro . Therefore all the information contributed by 16-S rRNA to the reconstitution process is carried in the primary sequence of this RNA . The specific protein-synthesizing activity of 30-S subunits reconstituted from 30-S subunit proteins and heat-denatured 16-S rRNA is about one third of that observed if unheated 16-S rRNA is used and is comparable to the activity of 30-S particles isolated after dissociation of 70-S ribosomes in the presence of 0.1 mM Mg2+. Eur J Biochem, 1979 Aug 1, 98(2), 557 - 66 Protein . nucleic-acid reaction kinetics . Theoretical analysis of the binding reaction between DNA and RNA polymerase; Giacomoni PU; This paper presents methods developed in order to analyze experimental results concerning the binding of Escherichia coli DNA-dependent RNA polymerase to DNA at high and at low DNA concentrations, using the filter retention assay . The basis hypotheses, under which the mathematical expressions for describing the kinetics of binding are derived, are as follows . (a) At low DNA concentration: equivalence and independence of the specific binding sites; first-order dependence of the binding reaction on both DNA and protein concentration . (b) At high DNA concentration: equivalence and independence of the non-specific binding sites; no direct transfer or one-dimensional sliding of the protein along the DNA . Comparison between theoretical predictions and experimental results at high DNA concentration will allow one to determine the relative value of the rates of binding of RNA polymerase to different promoters (between 1 and 2 in T5 DNA) . Binding experiments performed at low DNA concentration are reported in this paper: these results and the analysis which is reported allow one to determine the value of the rate constant of formation of non-filterable complexes for the system fd DNA (replicative form) . RNA-polymerase (kappa a = 3.3 X 10(8) M-1 s-1 in 0.1 M NaCl, 0.01 M MgCl2). Mutat Res, 1979 Aug, 64(4), 241 - 8 Methodology for the testing of food dyes for genotoxic activity: experiments with red 2G (C.I . 18050); Haveland-Smith RB et al.; A methodology for investigating genotoxicity of food colours using the fluctuation and DNA-repair assays with bacteria is described . In addition, a liquid repair test, developed to permit incorporation of microsomes and the quantitative estimation of cell viability, has been characterised with a number of positive control agents . Results obtained in these systems suggest that the food colour Red 2G induces repairable DNA damage and base-substitution mutation, but only in the presence of a rat-liver microsomal preparation . The significance of the data in the light of other toxicological information is discussed. J Virol, 1979 Aug, 31(2), 277 - 80 Prophage substitution and prophage loss from superinfected Escherichia coli recA(P1) lysogens; Meurs E et al.; It is shown that the plasmid prophage P1 can be displaced by a superinfecting P1 phage in Escherichia coli recA(P1) lysogens . Six widely separated phage markers were used to distinguish between residual recombination and total substitution . It is further shown that superinfection of recA lysogens can lead to loss of both phage (curing) . These two phenomena, previously reported in Rec+ strains, are thus independent of host recombination and may result from perturbations of some function involved in plasmid maintenance. J Inorg Biochem, 1979 Aug, 11(1), 67 - 76 Effect of acute stress on the absorption and distribution of zinc and on Zn-metallothionein production in the liver of the chick; Sas B et al.; A study has been made of the effects of chloroform inhalation, Escherichia coli endotoxin injection and hydrocortisone injection on the absorption of a single intragastric dose of 65Zn by the chick . Injection of hydrocortisone increased the absorption of the 65Zn by 30-55% in both Zn-deficient and Zn-supplemented chicks . The influence of chloroform and endotoxin was less consistent; the former treatment only increased 65Zn absorption and endotoxin was less consistent; the former treatment only increased 65Zn absorption in Zn-supplemented chicks fed ad libitum whereas endotoxin only increased that in Zn-supplemented chicks on a restricted food intake . Injection of endotoxin increased the hepatic uptake of the absorbed 65Zn in both Zn-deficient and Zn-supplemented chicks, whereas hydrocortisone had a similar effect in the Zn-supplemented birds only . Chloroform inhalation increased hepatic 65Zn uptake in Zn-deficient chicks only . The increase in hepatic Zn concentrations in the stressed chicks was mainly associated with a protein in the cytosol identified as metallothionein . Both endotoxin and hydrocortisone decreased total plasma Zn concentrations in Zn-supplemented and Zn-deficient chicks; chloroform decreased plasma 65Zn content only. J Med Microbiol, 1979 Aug, 12(3), 291 - 302 Assay of the heat-labile enterotoxin of Escherichia coli in infant rabbits; Burgess MN et al.; Infant rabbits were shown to respond to Escherichia coli heat-labile enterotoxin by a consistent increase in intestinal fluid content, which was maximal 5 h after oral dosing . Infant rabbits could be used in a simple quantitative assay for heat-labile E . coli enterotoxin based on the ratios of gut weight to remaining body weight 5 h after oral dosing . Infant rabbits remained responsive to heat-labile enterotoxin up to 14 days of age, after which their gastric pH became low enough to destroy the enterotoxin . Rabbits that had been deprived of food before being dosed had a reduced gastric pH and a reduced response to the enterotoxin . Lincomycin andmitomycin C were found not to increase th e yield of heat-labile enterotoxin from E . coli strain P307. J Bacteriol, 1979 Aug, 139(2), 701 - 4 Conditional-lethal deoxyribonucleic acid ligase mutant of Escherichia coli; Dermody JJ et al.; A new Escherichia coli deoxyribonucleic acid (DNA) ligase mutant has been identified among a collection of temperature-sensitive DNA replication mutants isolated recently (Sevastopoulos, Wehr, and Glaser, Proc . Natl . Acad . Sci . U.S.A . 74:3485-3489, 1977) . At the nonpermissive temperature DNA synthesis in the mutant stops rapidly, the DNA is degraded to acid-soluble material, and cell death ensures . This suggests that the mutant may be among the most ligase-deficient strains yet characterized. J Bacteriol, 1979 Aug, 139(2), 671 - 4 Inhibition of deoxyribonucleic acid repair in Escherichia coli by caffeine and acriflavine after ultraviolet irradiation; Fong K et al.; The effects of caffeine and acriflavine on cell survival, single-strand deoxyribonucleic acid break formation, and postreplication repair in Escherichia coli wild-type WP2 and WP2 uvrA strains after ultraviolet irradiation was studied . Caffeine (0.5 mg/ml) added before and immediately after ultraviolet irradiation inhibited single-strand deoxyribonucleic acid breakage in wild-type WP2 cells . Single-strand breaks, once formed, were no longer subject to repair inhibition by caffeine . At 0.5 to 2 mg/ml, caffeine did not affect postreplication repair in uvrA strains . These data are consistent with the survival data of both irradiated WP2 and uvrA strains in the presence and absence of caffeine . In unirradiated WP2 and uvrA strains, however, a high caffeine concentration (greater than 2 mg/ml) resulted in gradual reduction of colony-forming units . At a concentration insufficient to alter survival of unirradiated cells, acriflavine (2 microgram/ml) inhibited both single-strand deoxyribonucleic acid breakage and postreplication repair after ultraviolet irradiation . These data suggest that although the modes of action for both caffeine and acriflavine may be similar in the inhibition of single-strand deoxyribonucleic acid break formation, they differ in their mechanisms of action on postreplication repair. J Bacteriol, 1979 Aug, 139(2), 608 - 19 ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility; Hashimoto-Gotoh T et al.; Deletion mutants of plasmid ColE1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility . It was observed that (i) a region of ColE1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of ColE1 DNA between base pairs -185 and -572 is essential for the expression of ColE1 incompatibility; (iii) the expression of incompatibility is independent of the ability of the ColE1 genome to replicate autonomously; (iv) plasmid incompatibility is affected by plasmid copy number; and (v) ColE1 plasmid-mediated DNA replication of the lambda phage-ColE1 chimera lambda imm434 Oam29 Pam3 ColE1 is inhibited by ColE1-incompatible but not by ColE1-compatible plasmids. J Bacteriol, 1979 Aug, 139(2), 597 - 607 Isolation and characterization of replication-deficient mutants of ColE1 plasmids; Hashimoto-Gotoh T et al.; Replication-defective mutants of plasmid ColE1 were isolated from a chimeric plasmid formed by ligating a temperature-sensitive replication derivative of pSC101, pHSG1, with a ColE1-Tn3-containing plasmid . The replication-defective ColE1 mutants isolated were all spontaneous deletion mutants that had lost the ColE1 replication origin and regions adjacent to it . The extent of a deletion was determined by analyzing restriction endonuclease-generated deoxyribonucleic acid fragments of the ColE1 plasmid component of the chimeras by both agarose and polyacrylamide gel electrophoresis . None of the chimeras containing the replication-defective ColE1 mutants was able to replicate in the presence of chloramphenicol . The expression of ColE1 incompatibility was either markedly reduced or not detectable in the replication mutants isolated. J Bacteriol, 1979 Aug, 139(2), 515 - 9 Effect of temperature on translocation frequency of the Tn3 element; Kretschmer PJ et al.; The effect of temperature on the translocation frequency of the Tn3 element was investigated . The temperature optimum for translocation of Tn3 was in the range from 26 to 30 degrees C . At temperatures above 30 degrees C, the translocation frequency decreased rapidly and linearly; at 36 degrees C it was only 5% of the frequency observed at 30 degrees C . The duration and reversibility of the temperature effect were utilized to demonstrate a requirement for protein synthesis in the translocation process. J Bacteriol, 1979 Aug, 139(2), 418 - 23 Isolation and partial characterization of protein E, a major protein found in certain Escherichia coli K-12 mutant strains: relationship to other outer membrane proteins; Chai TJ et al.; Escherichia coli outer membrane protein E was purified, and its amino acid composition and N-terminal amino acid were determined . The purified protein was shown to be immunologically and electrophoretically identical to proteins Ic (U . Henning, W . Schmidmayr, and I . Hindennach, Mol . Gen . Genet . 154:293-298, 1977) and e (W . van Alphen, N . van Selm, and B . Lugtenberg, Mol . Gen . Genet . 159:75-83, 1978) . Proteins E, e, and Ic were also immunologically related to E . coli outer membrane protein Ia . Lugtenberg and co-workers (B . Lugtenberg, R . van Boxtel, C . Verhoef, and W . van Alphen, FEBS Lett . 96:99-105, 1978) have shown that electrophoretically identical peptides were generated by cyanogen bromide treatment of proteins E, e, and Ic. J Bacteriol, 1979 Aug, 139(2), 398 - 403 Escherichia coli mutant strain with altered expression of the tryptophan operon: ribonucleic acid synthesis in vitro; Pouwels PH et al.; Ribonucleic acid (RNA) synthesis has been studied in vitro with a partially purified preparation of RNA polymerase from a mutant strain of Escherichia coli with a reduced rate of accumulation of tryptophan RNA (P.H . Pouwels and H.J . Scholten, J . Bacteriol . 139:393-397, 1979) . The incorporation of radioactive label into RNA with polymerase from mutant bacteria is considerably lower than that with the enzyme from wild-type bacteria . These results are explained by the presence in mutant bacteria, but not in wild-type bacteria, of a factor which suppresses the accumulation of RNA . Mutant bacteria contain a factor which renders RNA synthesis with mutant and wild-type RNA polymerase resistant to various inhibitors of RNA synthesis, e.g . rifampin, streptolydigin, and heparin . We conclude that in mutant bacteria a factor is modified which suppresses the accumulation of some RNA species and lowers the sensitivity of RNA polymerase to some transcription inhibitors. J Bacteriol, 1979 Aug, 139(2), 346 - 55 Transposition of ampicillin resistance to an enterotoxin plasmid in an Escherichia coli strain of human origin; McConnell MM et al.; We examined a strain of Escherichia coli, serotype O159.H34, of human origin which produced heat-stable and heat-labile enterotoxins, was resistant to ampicillin, and produced colicin . By conjugation and transformation experiments plasmids coding for enterotoxin production (Ent), enterotoxin production and ampicillin resistance (Ap-Ent), ampicillin resistance (Ap), and colicin production were isolated . Both the Ent and Ap-Ent plasmids were autotransferring and belonged to the F-incompatibility complex . However, the Apr Ent+ transconjugants showed differences in their levels of resistance and in their abilities to propagate F-specific phages and to transfer resistance . The results suggested there was transposition from the small Ap plasmid to the Ent plasmid . The Ap-Ent plasmids were larger than the enterotoxin factor and when treated with restriction endonuclease BamHI showed an additional fragment not present in the enterotoxin plasmid . The insertion of ampicillin resistance probably occurred at different sites on the enterotoxin plasmid, resulting in the observed variation in phenotype. Immunology, 1979 Aug, 37(4), 765 - 75 Immune activation by T-independent antigens: lack of effect of macrophage depletion on the immune response to TNP-LPS, PVP and dextran; Wong DM et al.; Carrageenan, a sulphated polysaccharide, and rabbit anti-mouse macrophage serum, were used to inhibit macrophage function in BALB/c mice as well as to deplete macrophages from spleen cell cultures in an attempt to determine the requirement for macrophages in the immune response to several thymus-independent antigens . Carrageenan inhibited macrophage function and was cytotoxic at low concentrations . The ability of T and B lymphocytes to undergo mitogen-induced proliferation in the presence of PHA and PLS, respectively, was not affected by in vitro exposure of lymphoid cells to carrageenan . BALB/c mice injected with carrageenan demonstrated a suppressed immune response to SRBC, a thymus-dependent antigen, but not to E . coli LPS, polyvinyl-pyrrolidone or dextran B-1355S, all of which are known to be thymus independent antigens . The sensitivity of the in vivo immune response to SRBC after depletion of macrophages by carrageenan treatment was confirmed in vitro using the Marbrook--Diener culture system . The in vitro immune response to TNP-LPS was unaffected by either carrageenan treatment or treatment of BALB/c spleen cells with AMS and complement . The results of experiments which utilized the two anti-macrophage reagents, carrageenan and AMS, both in vivo and in vitro systems, suggest that the immune response to thymus-independent antigens does not require the participation of macrophages. Blood, 1979 Aug, 54(2), 412 - 20 A chemotactic inhibitor produced by blast cells and present in the serum of a patient with acute lymphoblastic leukemia; Blumenfeld W et al.; A chemotactic factor inhibitor (CFI) was found in the serum of a patient with acute lymphoblastic leukemia . The factor was characterized as heat-labile, with activity against complement-dependent chemotactic factors and against the chemotactic factor produced by Escherichia coli . Normal sera and sera from other patients with acute leukemia also demonstrate heat-labile inhibitory activity against complement-dependent chemotactic factors but not against the E . coli-derived chemotactic factor . Supernatants from cultured lymphoblasts of the patient also possessed CFI activity similar in character to that found in his serum . It is suggested that in vivo the lymphoblasts were responsible for the chemotactic defect observed in his serum, presumably by manufacturing and releasing a chemotactic factor inactivator. Can J Biochem, 1979 Aug, 57(8), 1073 - 9 Effect of carbon sources on the rates of cyclic AMP synthesis, excretion, and degradation, and the ability to produce beta-galactosidase in Escherichia coli; Fraser AD et al.; We have determined the rates of adenosine 3',5'-cyclic monophosphate (cAMP) synthesis, excretion, and degradation, and the cAMP pool size in Escherichia coli grown on various carbon sources . We have found that the cAMP pool size increases in approximate proportion to increases in the cAMP synthetic rate . Although the combined rate of excretion and degradation of cAMP is in approximate proportion to the cAMP pool size, no such regular relationship is seen between the cAMP pool size and either the excretion rate or the degradation rate . Using a method which we have developed for determining the cellular efficiency of enzyme production (termed 'cellular' rate), we have reexamined the relationship between cAMP pool size and the rate of beta-galactosidase production . Although there exists an overall trend of increasing rate of beta-galactosidase production with increasing cAMP pool size, large variations in the rates of beta-galactosidase production are seen even under culture conditions which yield similar cAMP pool sizes . This suggests that the intracellular level of cAMP cannot be the unique regulator of beta-galactosidase production. Appl Environ Microbiol, 1979 Aug, 38(2), 290 - 6 Survey of human enterovirus occurrence in fresh and marine surface waters on Long Island; Vaughn JM et al.; A variety of surface water systems, including a lake, a creek, and two marine embayments, were analyzed on a monthly basis for indigenous human enteroviruses and coliform bacteria . Findings are discussed in terms of the probable pollution sources to each system and their relationship to data from previous studies. Proc Natl Acad Sci U S A, 1979 Aug, 76(8), 3755 - 9 Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12; Colbere-Garapin F et al.; A herpes simplex virus DNA fragment that is produced by digestion with BamHI endonuclease and carries the thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene has been cloned in Escherichia coli . A recombinat plasmid, pFG5, has been analyzed extensively and a detailed restriction map is presented . pFG5 DNA efficiently transforms TK- mouse L cells . The TK coding sequence in the cloned fragment has been localized and a smaller recombinant plasmid, pAG0, also carrying an active TK gene, has been constructed to serve as a more convenient vector for transfer, into TK- cells, of genes previously cloned in E . coli. Proc Natl Acad Sci U S A, 1979 Aug, 76(8), 3737 - 41 T4 DNA-delay proteins, required for specific DNA replication, form a complex that has ATP-dependent DNA topoisomerase activity; Stetler GL et al.; Under some conditions, T4 DNA replication requires the products of the DNA-delay genes, genes 39, 52, 58, and 60 . By using an in vitro complementation assay that stimulates DNA replication in T4 39(-)-infected cell extracts, T4 gene 39 protein has been purified . The purified fraction also contains complementing activities for T4 genes 52 and 60 . On sodium dodecyl sulfate/polyacrylamide gel analysis the purified preparation exhibits three protein components: a 51,000-dalton protein corresponding to the product of gene 52, a 64,000-dalton protein corresponding to the product of gene 39, and a 110,000-dalton protein . This purified fraction shows a DNA topoisomerase activity that untwists superhelical DNA in an ATP- and Mg2+-dependent reaction . The analogs adenylyl imidodiphosphate and adenyl {beta, gamma-methylene}diphosphonate cannot be used to replace ATP . The topoisomerase activity is not sensitive to the antibiotics oxolinic acid and novobiocin, known antagonists of Escherichia coli DNA gyrase . The possible relationship among the three polypeptides and their biological activities is discussed. J Biochem (Tokyo), 1979 Aug, 86(2), 325 - 33 Studies on regulatory functions of malic enzymes . IV . Effects of sulfhydryl group modification on the catalytic function of NAD-linked malic enzyme from Escherichia coli; Yamaguchi M; Reactions catalyzed by NAD-linked malic enzyme from Escherichia coli were investigated . In addition to L-malate oxidative decarboxylase activity (Activity 1) and oxaloacetate decarboxylase activity (Activity 2), the enzyme exhibited oxaloacetate reductase activity (Activity 3) and pyruvate reductase activity (Activity 4) . Optimum pH's for Activities 3 and 4 were 4.0 and 5.0, and their specific activities were 1.7 and 0.07, respectively . Upon reaction with N-ethylmaleimide (NEM), Activity 1 decreased following pseudo-first order kinetics . Activity 2 decreased in parallel with Activity 1, while Activities 3 and 4 were about ten-fold enhanced by NEM modification . Modification of one or two sulfhydryl groups per enzyme subunit caused an alteration of the activities . Tartronate, a substrate analog, NAD+, and Mn2+ protected the enzyme against the modification . The Km values for the substrates and coenzymes were not significantly affected by NEM modification . Similarly, other sulfhydryl reagents such as p-hydroxymercuribenzoate (PMB), 5,5'-dithiobis(2-nitrobenzoate) (DTNB), and iodoacetate inhibited the decarboxylase activities and activated the reductase activities to various extents . Modification of the enzyme with PMB or DTNB was reversed by the addition of a sulfhydryl compound such as dithiothreitol or 2-mercaptoethanol . Based on the above results, the mechanism of the alteration of enzyme activities by sulfhydryl group modification is discussed. J Clin Invest, 1979 Aug, 64(2), 381 - 4 Gangliosides sensitize unresponsive fibroblasts to Escherichia coli heat-labile enterotoxin; Moss J et al.; Chemically transformed mouse fibroblasts did not raise their cyclic AMP level in response to Escherichia coli heat-labile enterotoxin . These fibroblasts did, however, incorporate exogenous mono-, di-, and trisialogangliosides . After the uptake of monosialoganglioside galactosyl-N-acetylgalactosaminyl-{N-acetylneuraminyl}-galactosylglucosylceramide (GM1), the cells responded to E . coli heat-labile enterotoxin . The di- and trisialogangliosides were considerably less effective . GM1, the putative cholera toxin (choleragen) receptor, has been implicated previously as the receptor for E . coli heat-labile enterotoxin based on the ability of the free ganglioside to inhibit the effects of toxin . This investigation establishes that the ganglioside, when incorporated into fibroblasts, serves a functional role in mediating the responsiveness to the toxin. J Bacteriol, 1979 Aug, 139(2), 690 - 3 Antipain lethality to Escherichia coli: dependence upon cyclic adenosine 3',5'-monophosphate and its receptor protein; Swenson PA; Antipain kills Escherichia coli K-12 cells in an exponential manner beginning 1 h after its addition . Mutant strains, delta cya and crp, which are unable to synthesize cyclic adenosine 3',5'-monophosphate (cAMP) and the cAMP receptor protein, respectively, are not affected . Addition of cAMP (5 mM) to antipain-treated mutant strains causes killing of delta cya cells, but not crp cells . Thus the lethal effect of antipain is dependent upon cAMP and its receptor protein. J Biochem (Tokyo), 1979 Aug, 86(2), 403 - 11 Studies on stringent control in a cell-free system . Regulation by guanosine-5'-diphosphate-3'-diphosphate of the synthesis of elongation factor Tu; Shibuya M et al.; The biosynthesis of elongation factor Tu (EF-Tu) has been studied in a cell-free system with DNA of the transducing phage lambdarifd18 as a template . It was found that the synthesis of EF-Tu in this system was inhibited by about 60% in the presence of 0.3 to 0.6 mM guanosine-5'-diphosphate-3'-diphosphate (ppGpp) . The syntheses of several ribosomal proteins encoded in this template, i.e . L1, L10, L11, and L7/L12, were also depressed, whereas those of phage lambda proteins were rather enhanced by the addition of ppGpp . By separating the reaction into two steps, i.e., transcription and translation, the effect of ppGpp was shown to occur at the level of transcription . Several analogs, such as guanosine-5'-diphosphate-3'-monophosphate (ppGp) and guanosine-5'-diphosphate (ppG), were without effect . The formation of mRNA for EF-Tu was assessed directly by specific hybridization with pTUA1 DNA carrying tufA gene . The results clearly indicated that the synthesis of tufB . MRNA was severely and selectively inhibited by ppGpp. Appl Environ Microbiol, 1979 Aug, 38(2), 191 - 6 Inhibitory effects of carcinogenic mycotoxins on deoxyribonucleic acid-dependent ribonucleic acid polymerase and ribonuclease H; Tashiro F et al.; Fourteen mycotoxins were tested for inhibitory effects on ribonucleic acid polymerase of rat liver and Escherichia coli and nuclear ribonuclease H of rat liver and Tetrahymena pyriformis . These enzymes were strongly inhibited by (-)-luteoskyrin, (+)-rugulosin, patulin, and PR toxin. Vet Med (Praha), 1979 Aug, 24(8), 483 - 91 {Effectiveness of environmental disinfection with formalin aerosol}; Kubicek K; The effectiveness was tested of preventive disinfection of pig-fattening houses with formalin, applied as aerosol with the Swingfog SN 100 apparatus . The effectiveness of this treatment was evaluated according to the proportion of smears containing lactoso-positive micro-organisms, expressed as a percentage of the total number of smears, and according to the reduction of contamination with E . coli and St . aureus on wooden or aluminium carriers . The effectiveness was found to be good and the treatment is recommended as suitable for the preventive disinfection of stables . However, the following procedure should be strictly observed: Before disinfection the stables must be thoroughly mechanically cleansed, dried, and well closed to be air-tight . The air temperature in these premises should not be lower than 12 degrees C . Formalin (with a minimum content of 40% of formaldehyde) should be applied at a rate of at least 10 ml per 1 m3 of space and the agent should be diluted with water at 1 : 1 ratio prior to use . The aerosol should be produced by the Swingfog SN 100 generator, adjusted to apply about 40 1 of the solution per hour . The maximum space treated from one place should be limited to 500 m3 . The exposure time should be at least 16 hours. Eur J Biochem, 1979 Aug 1, 98(2), 465 - 75 Fluorescent derivatives of yeast tRNAPhe; Wintermeyer W et al.; The preparation of four fluorescent derivatives of tRNAPhe (yeast) and their characterization by chemical, spectroscopic, and biochemical methods is described . The derivatives are prepared by replacing wybutine (position 37 in the anticodon loop) or NaBH4-reduced dihydrouracil (positions 16/17 in the hU loop) with ethidium or proflavine; they are isolated by reversed-phase chromatography (RPC-5) . All tRNAPhe-dye derivatives are aminoacylated by yeast phenylalanyl-tRNA synthetase to at least 80% of the charging capacity of the unmodified tRNAPhe with an unchanged Km (0.2 mucroM) and a V lowered by 30--50% . They exhibit good to excellent activity in the aminoacylation assay from synthetase from Escherichia coli . It is concluded that the insertion of the dyes does not seriously disturb essential elements of the native tRNAPhe structure . The dyes are bound via N-ribosylic linkages . The appearance of isomeric tRNAPhe-ethidium derivatives is attributed to the involvement of the different amino groups of ethidium in the condensation . In addition, there are indications for the existence of alpha and beta anomers of the tRNA-dye compounds . The dyes are rigidly fixed to their position in the tRNA molecule by stacking interactions with the neighboring bases . The ethidium probes show Mg2+-induced changes of the tRNA conformation which are paralleled by changes of the rate of aminoacylation . On the basis of this observation it is hypothesized that conformational flexibility of the tRNA molecule is a functionally important feature of the tRNA structure. Eur J Biochem, 1979 Aug 1, 98(2), 409 - 16 Acidic ribosomal proteins from eukaryotic cells . Effect on ribosomal functions; Sanchez-Madrid F et al.; Precipitation of Saccharomyces cerevisiae ribosomes by ethanol under experimental conditions that do not release the ribosomal proteins can affect the activity of the particles . In the presence of 0.4 M NH4Cl and 50% ethanol only the most acidic proteins from yeast and rat liver ribosomes are released . At 1 M NH4Cl two more non-acidic proteins are lost from the ribosomes . The release of the acidic proteins causes a small inactivation of the polymerizing activity of the particles, additional to that caused by the precipitation itself . The elongation-factor-2-dependent GTP hydrolysis of the ribosomes is, however, more affected by the loss of acidic proteins . These proteins can stimulate the GTPase but not the polymerising activity when added back to the treated particles . Eukaryotic proteins cannot be substituted for bacterial acidic proteins L7 and L12 . We have not detected immunological cross-reaction between acidic proteins from Escherichia coli and those from yeast, Artemia salina and rat liver or between acidic proteins from these eukaryotic ribosomes among themselves. Clin Chem, 1979 Aug, 25(8), 1448 - 52 Improved double-antibody enzyme immunoassay for methotrexate; Al-Bassam MN et al.; We report an enzyme immunoassay procedure for methotrexate measurement that takes less than 3 h to perform . beta-D-Galactosidase (EC 3.2.1.23) from Escherichia coli was conjugated to methotrexate by means of the mixed anhydride reaction . Bound and free labeled drug were separated by a preincubated cubic complex of first and second antibody . The enzyme activity of the bound fraction was measured with o-nitrophenyl-beta-D-galactopyranoside as substrate . The standard curve covered the range 1 to 10 micrograms of methotrexate per liter . One microgram of methotrexate per liter inhibited binding of the tracer by 17% . The assay is specific for methotrexate in the presence of folinic acid (citrovorum factor), folic acid, tetrahydrofolic acid, and other methotrexate metabolities . Intra- and inter-assay CVs were less than 5 and 10%, respectively . Results obtained with this enzyme immunoassay method agreed well with those obtained with an established radioimmunoassay method. J Gen Microbiol, 1979 Aug, 113(2), 267 - 74 Altered phospholipid composition in mutants of Escherichia coli sensitive or resistant to organic solvents; Clark DP et al.; Mutants of Escherichia coli with altered resistance to low molecular weight organic solvents were isolated . Solvent-resistant mutants showed a decrease in the ratio of phosphatidylethanolamine to the anionic phospholipids (phosphatidylglycerol and cardiolipin) relative to the wild-type, whereas solvent-sensitive strains showed an increase . Reversion studies on representative mutants demonstrated that the phenotypic response to solvents and the changes in phospholipid composition were genetically associated . The fatty acid and lipopolysaccharide compositions of the various mutants showed no significant differences from the parental strain . The lesions in two of the solvent-sensitive mutants (DC7 and DC9) and one of the resistant mutants (DC11) were mapped by cotransduction with phage P1 and shown to lie very close to the pss locus at 56 min on the Escherichia coli map. Proc Natl Acad Sci U S A, 1979 Aug, 76(8), 3751 - 4 Reverse transcriptase pauses at N2-methylguanine during in vitro transcription of Escherichia coli 16S ribosomal RNA; Youvan DC et al.; A restriction fragment strand complementary to a sequence near the 3' end of Escherichia coli 16S rRNA has been used to prime reverse transcriptase (avian myeloblastosis virus RNA-directed DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) . In addition to transcripts that were extended to the 5' end of the RNA, two major transcription intermediates were observed . These discrete-sized cDNA intermediates are the result of a kinetic barrier imposed by monomethylation of the amino group on guanine that participates in base-pairing . Both major transcription intermediates correspond to attenuation at the known positions of N2-methylguanine (m2G) in the rRNA sequence . The relaxation time for elongation of the cDNA through m2G is approximately 3 min . No other major kinetic pauses were observed in the 1340 bases transcribed. J Bacteriol, 1979 Aug, 139(2), 683 - 5 New maltose Blu mutations in Escherichia coli K-12; Roehl RA et al.; Mutations in the genes pgi, pfkA, and ptsG resulted in a maltose Blu phenotype in Escherichia coli K-12, bringing the number of known Blu alleles to six . The Blu phenotype, as visualized by staining with iodine vapor, is a convenient mutant isolation technique. Appl Environ Microbiol, 1979 Aug, 38(2), 181 - 90 Hyperproduction of tryptophan by Escherichia coli: genetic manipulation of the pathways leading to tryptophan formation; Tribe DE et al.; Conversion of glucose and ammonium salts into tryptophan by mutants of Escherichia coli was examined as part of a feasibility study on the manufacture of tryptophan . This involved construction, largely by transduction, or a variety of multiple-mutation strains with defined genotypes . By comparing the properties of these strains, we were able to define in biochemical terms several changes that significantly enhance process productivity, namely (i) release of the first enzyme of the common pathway of aromatic biosynthesis and the first enzyme of the tryptophan pathway (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and the anthranilate aggregate, respectively) from inhibition by end products, (ii) blockage of the diversion of chorismate to phenylalanine and tyrosine biosynthesis, and (iii) presence of highly elevated tryptophan pathway enzyme levels, such as result from interference with both repression and attenuation, combined with gene amplification . By using strains carrying appropriate mutations to effect all of these changes, high values of specific productivity were obtained in bath culture (approximately 80 mg/g {dry weight} per h) . Furthermore, a pronounced decay in the level of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase activity was implicated as a cause of declining process producitivity during stationary phase, emphasizing the value of having derepressed levels of this enzyme. Biochim Biophys Acta, 1979 Jul 26, 563(2), 422 - 31 Binding sites for ribosomal proteins S8 and S15 in the 16 S RNA of Escherichia coli; Zimmermann RA et al.; A fragment of the 16 S ribosomal RNA of Escherichia coli that contains the binding sites for proteins S8 and S15 of the 30 S ribosomal subunit has been isolated and characterized . The RNA fragment, which sediments as 5 S, was partially protected from pancreatic RNAase digestion when S15 alone, or S8 and S15 together, were bound to the 16 S RNA . Purified 5 S RNA was shown to reassociate specifically with protein S15 by analysis of binding stoichiometry . Although interaction between the fragment and protein S8 alone could not be detected, the 5 S RNA selectively bound both S8 and S15 when incubated with an unfractionated mixture of 30-S subunit proteins . Nucleotide sequence analysis demonstrated that the 5 S RNA arises from the middle of the 16 S RNA molecule and encompasses approximately 150 residues from Sections C, C'1 and C'2 . Section C consists of a long hairpin loop with an extensively hydrogen-bonded stem and is contiguous with Section C'1 . Sections C'1 and C'2, although not contiguous, are highly complementary and it is likely that together they comprise the base-paired stem of an adjacent loop. Biochim Biophys Acta, 1979 Jul 26, 563(2), 356 - 64 Assays for the fidelity of DNA polymerases in cell-free extracts of Escherichia coli are complicated by contaminating nucleoside triphosphatases; McGarva D et al.; In the presence of DNA and a divalent cation, an enzyme activity in cell-free extracts of Escherichia coli readily hydrolyses dATP to dADP . dGTP is degraded to a smaller extent, dCTP and dTTP being hardly affected . The artificial template primers poly(dC) . oligo(dG) and poly(dT) . oligo(dA) are also effective cofactors for this triphosphatase activity . As a consequence, assays measuring the misincorporation, by cell-free extracts, of dATP and dGTP into these defined templates are difficult to interpret, since the triphosphate substrate is being rapidly degraded during the polymerase reaction . A partial characterization of the dATPase activity was performed, demonstrating that the optimal conditions for its activity resemble those commonly used for assaying polymerase activity . Thus in crude extracts both polymerase and dATPase compete for the same substrate . The inclusion of an ATP-generating system in the reaction mixture maintains the levels of deoxynucleoside triphosphates and changes the kinetics of misincorporation of dAMP into poly(dC) . oligo(dG) . No reproducible difference in such misincorporation has been found between lysates prepared from tif-1 cells grown at either permissive or restrictive temperature. Biochim Biophys Acta, 1979 Jul 26, 563(2), 508 - 17 Poly(2-fluoroadenylic acid) . The role of basicity in the stabilization of complementary helices; Broom AD et al.; The polymerization of 2-fluoroadenosine 5'-diphosphate by polynucleotide phosphorylase to give high molecular weight poly(2-fluoroadenylic acid), poly(fl2A), is described . Both the single-stranded and double-stranded (acid) forms of poly(fl2A) exhibit strikingly similar ultraviolet and circular dichroism spectra to those of poly(A), and the enzymatic polymerization rates and thermal hyperchromicities of the two polymers are also very similar . However, the pKa of poly(fl2A) for protonation at N-1 is 2.9 compared to 5.9 for poly(A) under similar conditions . Poly(fl2A) forms a triple-stranded helix with poly(U) which has ultraviolet and cd spectra very reminiscent of poly(A) . 2 poly(U), but no conditions could be found which permitted the formation of a double helix . In the Escherichia coli ribosome system poly(fl2A) codes for the synthesis of polylysine, as does poly(A), although the rate and extent of incorporation were less in the former case . The role of basicity of adenine N-1 in these interactions is discussed. Nucleic Acids Res, 1979 Jul 25, 6(10), 3289 - 304 Characterization of an improved in vitro DNA replication system for Escherichia coli plasmids; Conrad SE et al.; A modified in vitro replication system has been characterized and used to catalogue the host proteins required for the replication of plasmid RSF1030 . These extracts differ from systems described previously in that endogenous DNA is removed . Replication in vitro therefore requires an exogenouos RSF1030 . Synthesis in the in vitro system faithfully mimics in vivo replication with respect to the products synthesized, effects of specific inhibitors, and requirements for RNA polymerase and DNA polymerase I . In addition, we find that proteins encoded by dnaB, dnaC, dnaG, dnaI, dnaP and polC (DNA polymerase III), are required for in vitro plasmid synthesis . The product of dnaA is not required . Extracts prepared from E . coli mutants deficient in in vitro replication can be complemented by addition of purified proteins or of extracts carrying the wild type protein. Nucleic Acids Res, 1979 Jul 25, 6(10), 3267 - 87 Characterizing wild-type and mutant promoters of the tetracycline resistance gene in pBR313; Rodriguez RL et al.; By employing a system of RNA polymerase binding and restriction endonuclease digestion, we demonstrate that the region in and around the Hind III site of pBR313 and pBR322 is the promoter for the tetracycline (Tc) resistance gene(s) . Furthermore, it is shown that this region was transferred intact from pSC101 during the construction of the latter plasmids . The in vitro insertion of a few base pairs at the Hind III site produces a series of "down" promoter mutations in which the level of in vivo Tc resistance is reduced . Sequence analysis of the various promoter mutations revealed significant base pair rearrangements in the region between -40 and -12 of the promoter . While these base alterations do not appear to affect the firm binding of RNA polymerase, they do affect the ability of mutant promoters to initiate transcription . These observations suggest that the region from -40 to -12, previously designated as the "recognition region", is actually involved in the process of initiation of transcription. J Biol Chem, 1979 Jul 25, 254(14), 6667 - 72 A fluorescent derivative of ribosomal protein S18 which permits direct observation of messenger RNA binding; Kang C et al.; 70 S Escherichia coli ribosomes were reacted with the fluorescent dye N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid for 10 min under mild conditions . The resulting ribosomes were fully active . 30 S subunits isolated from these particles were also fully active . They contain approximately 0.7 eq of fluorescent dye . Nearly all of it is attached to protein S18 . Competitive reaction with N-ethylmaleimide implies that the fluorescent dye is located at cysteine 10 of the protein . The labeled 30 S particles will recombine with 50 S subunits to form stable 70 S particles . Thus the procedures we have developed allow the large scale preparation of an active fluorescent conjugate of the 70 S ribosome . The fluorescence of the 70 S particles is sensitive to the binding of mRNA, showing both quenching and a shift in emission spectra . Thus it affords a simple way to quantitate mRNA binding directly . In pilot studies without tRNA, the binding constant of the initiation triplet codon adenylyl-(3' leads to 5')-uridylyl-(3' leads to 5')-guanosine to 70 S ribosome was found to be an order of magnitude larger than that of polyuridylic acid. J Biol Chem, 1979 Jul 25, 254(14), 6397 - 401 Relative efficiency of anticodons in reading the valine codons during protein synthesis in vitro; Mitra SK et al.; Using a protein synthesizing in vitro system programmed with MS 2-RNA, the relative efficiency (in the presence of each other) of valine tRNAs with the anticodons U*AC (U* represents 5-oxyacetic acid uridine monophosphate), GAC, and IAC to read the valine codons was investigated . An anticodon which can read all three positions of the codon according to the rules of Watson-Crick base-pairing and the wobble hypothesis is an order of magnitude more efficient than an anticodon which misreads the codon by reading only the first two positions and presumably disregards the third nucleotide of the codon . There are two seeming exceptions to this behavior: the anticodon U*AC reads the codon GUU quite efficiently and IAC is as effective as U*AC in reading the codon GUG . The significance of these exceptions is evaluated with respect to the organization and evolution of the genetic code. J Biol Chem, 1979 Jul 25, 254(14), 6222 - 5 R plasmid dihydrofolate reductase with subunit structure; Smith SL et al.; Dihydrofolate reductase, specified by the type II plasmid of a trimethoprim-resistant Escherichia coli, was purified 40-fold to homogeneity using a combination of gel filtration, DEAE-Sephacel chromatography, and hydrophobic chromatography . The final product shows a single protein band on polyacrylamide gel electrophoresis and has a specific activity of 1.0 unit/mg . The molecular weight of the purified enzyme is 36,000 as determined both by gel filtration and Ferguson analysis of polyacrylamide gel electrophoresis . In contrast, a single polypeptide with a molecular weight of 8,500 was observed on sodium dodecyl sulfate-gel electrophoresis . These experiments suggest that, unlike any bacteria or vertebrate dihydrofolate reductase previously examined, the type II R plasmid reductase is a tetramer composed of four identical subunits . A partial amino acid sequence determination shows no heterogeneity of the subunits and also no clear homology with any reductase sequence previously reported. Nucleic Acids Res, 1979 Jul 25, 6(10), 3305 - 21 Sequence analysis of cloned cDNA encoding part of an immunoglobulin heavy chain; Rogers J et al.; The recombinant plasmid pH21-1 consists of mouse-derived complementary DNA (cDNA) in the E . coli plasmid pMB9 . The mouse insertion has been completely sequenced, and encodes the CH3 domain and half the CH2 domain of the immunoglobulin gamma1 heavy chain . The predicted amino acid sequence differs at several positions from that previously published for this protein . The pattern of codon usage resembles that in some other eukaryotic messenger RNAs . A computer program has been used to predict the optimum secondary structure for the mRNA encoding the CH3 domain and the inter-domain junction. Biochemistry, 1979 Jul 24, 18(15), 3363 - 71 Interaction between wheat germ RNA polymerase II and adenovirus 2 DNA . Evidence for two types of stable binary complexes; Seidman S et al.; Transcription of Adenovirus 2 DNA (Ad 2 DNA) by wheat germ RNA polymerase II in vitro satisfies criteria that have been used to establish that Escherichia coli or coliphage transcription in vitro is initiated at true promoters . (1) Wheat germ RNA polymerase forms highly stable complexes at specific sites on Ad 2 DNA, with a Kassoc of (4--5) X 10(10) M-1 . (2) Electron microscopic visualization of enzyme bound to Ad 2 DNA reveals the location of eight strong binding sites, at least five of which appear to correspond to promoters that have been identified in studies of Ad 2 transcription in vivo {Evans, R . M., Fraser, N., Ziff, E., Weber, J., Wilson, M., & Darnell, J.E . (1977) Cell 12, 733--739; Berk, A.J., & Sharp, P.A . (1977) Cell 12, 45--55; Weinmann, R., & Aiello, L . O . (1978) Proc . Natl . Acad . Sci . U.S.A . 75, 1662--1666} . (3) Transcription of Ad 2 DNA from preformed complexes with wheat germ polymerase is capable of escaping the action of rifamycin AF/013 and is relatively resistant to polyriboinosinic acid . In addition, our results are consistent with a two-state model for the interaction of wheat germ RNA polymerase with Ad 2 DNA, indicating that the mechansisms of transcription initiation and promoter-site selection in eucaryotes may be very similar to mechanisms elucidated in procaryotic systems. Mol Gen Genet, 1979 Jul 24, 174(3), 261 - 7 Structure of the malB region in Escherichia coli K12 . III . Correlation of the genetic map with the restriction map; Raibaud O et al.; A correlation between the genetic and physical maps of the malB region was obtained by performing a restriction cleavage analysis of DNA's carrying various genetically characterized malB deletions . This also allowed to localize the boundaries between malF and malE, malE and malK, mal K and lamB on the restriction map . The genetic map is not grossly distorted with respect to the physical map. Mol Gen Genet, 1979 Jul 24, 174(3), 249 - 59 Structure of the malB region in Escherichia coli K12 . II . Genetic map of the malE,F,G operon; Silhavy TJ et al.; Starting with a strain containing a malK-lacZ fusion, a series of lambda plaque-forming phages which carry varying amounts of the malE,F operon have been isolated . We have used these phages to construct a deletion map of the malE,F operon . The construction of this deletion map has led to the identification of a new gene, malG . The malG gene is located distal to malF . The malG gene product is a protein required for the active transport of maltose and maltodextrins. Mol Gen Genet, 1979 Jul 24, 174(3), 241 - 8 Structure of the malB region in Escherichia coli K12 . I . Genetic map of the malK-lamB operon; Raibaud O et al.; A series of deletions, Mu insertions and point mutations affecting the malK-lamB operon have been isolated . They were used to establish a deletion map of this operon, which could be divided in 27 intervals, with 16 in malK and 11 in lamB . One interesting feature of this map is the lack of randomness in the distribution of Mu insertions in the lamB gene; by using data published elsewhere on the physical length of the deletion intervals it can be concluded that about 25% of these Mu insertions are clustered in a segment representing 2 to 8% of the gene . This map is presently being used to study the biosynthesis, structure, and function of the lamB product, which is an outer membrane protein involved in the transport of maltose and maltodextrin, and which in addition constitutes the receptor for phage lambda. Biochemistry, 1979 Jul 24, 18(15), 3219 - 23 Reversible modification of Escherichia coli ribosomes with 2,3-dimethylmaleic anhydride . A new method to obtain protein-deficient ribosomal particles; Pintor-Toro JA et al.; Treatment of Escherichia coli ribosomes with the protein reagent 2,3-dimethylmaleic anhydride is accompanied by inactivation of polypeptide polymerization and by dissociation of ribosomal proteins . Regeneration of the modified amino groups at pH 6.0 is followed by reactivation and reconstitution of the ribosomes . Prior to regeneration of the amino groups, ribosomal particles and split proteins can be separated by centrifugation, which allows the preparation of new protein-deficient particles . The ribosomal particles obtained by three successive treatments with 2,3-dimethyl-maleic anhydride at a molar ratio of reagent to ribosome equal to 16,000 lack proteins S1, S2, S3, S5, S10, S13, S14, L7, L8, L10, L11, L12, and L20 and have lost part of proteins S4, L1, L6, L16, and L25 . This new procedure to obtain protein-deficient ribosomal particles is mild and might be useful to dissociate other protein-containing structures in addition to ribosomes. Biochemistry, 1979 Jul 24, 18(15), 3232 - 42 Enzymic polyadenylation of 5S ribosomal ribonucleic acid and synthesis of a complementary deoxyribonucleic acid; Ackerman S et al.; The 5S ribosomal RNA has been isolated, pure and intact, from rat liver (5 mg of 5S RNA from 150g of liver) . The 5S RNA serves as a primer for calf thymus poly(A) polymerase with 20% of the efficiency of (Ap)3A . Bacterial 5S RNA and transfer RNA also serve as primers; rat liver 18S and 28S ribosomal RNAs support poly(A) synthesis poorly . Neither the 5S RNA primer nor the appended poly(A) tract is nicked or degraded by poly(A) polymerase, and initiation of poly(A) tracts on 5S RNA primers continues throughout the reaction period . The rate of initiation is dependent on the enzyme concentration; the ATP concentration affects the rate of elongation . The polyadenylated material increases in size over time, with the largest material reaching a size of 6.8 S in 5 h, corresponding to an appended poly(A) tract of 140 nucleotides . Using polyadenylated 5S RNA, oliog(dTY as primer, and avian myeloblastosis virus reverse transcriptase, we synthesized DNA complementary to 5S RNA . The complementary DNA has an apparent molecular weight (in alkaline sucrose gradients) of 4.3 X 10(4) . Base composition analysis and nearest-neighbor analysis of the DNA are as expected for a complement of 5S RNA, indicating that the entire 5S sequence is copied . The complementary DNA hybridizes to 5S RNA with a R0t1/2 of 8.9 X 10(-4) mol.s.L-1 . No hybrid is formed with Escherichia coli 16S and 23S ribosomal RNA, E . coli 5S ribosomal RNA, yeast transfer RNA, rat liver transfer RNA, or rat liver 18S and 28S RIBOSOMAL RNA . The Tm of the 5S RNA:5S DNA hybrid in 15 mM NaCl containing 1.5 mM sodium citrate is 74 degrees C, 2.5 degrees C below the theoretical melting temperature of a DNA duplex of 60% G + C . Analysis of the hybrid in buoyant density gradients also indicates that hybridization is both specific and precise . The complementary DNA anneals to calf thymus, rat liver, and salmon sperm DNAs but not to E . coli DNA . Annealing of 5S cDNA to calf thymus DNA with a C0t1/2 of 2.1 suggests that there are several thousand 5S RNA genes in the calf thymus genome (haploid) . At least that number of 5S RNA genes is present in the salmon sperm genome. Res Exp Med (Berl), 1979 Jul 20, 175(3), 233 - 8 {Disseminated intravascular coagulation (DIC) after endotoxin infusion into the common bile duct of rabbits (author's transl)}; Ishikawa A et al.; Endotoxin from E . coli was infused into the distally ligated common bile duct of rabbits under the static pressure of 25 cm H2O . Fibrinogen, soluble fibrin monomer complexes, antithrombin III, leukocyte and platelet counts were estimated before, and 2, 4, and 6 h after endotoxin infusion . All parameters were found significantly changed 2 h after endotoxin infusion . While fibrinogen level, AT III, leukocyte and platelet counts decreased after the endotoxin infusion the amount of SFMC increased . The change of hematological parameters showed a pattern characteristic of disseminated intravascular coagulation (DIC) . In accordance with this microclots in the glomeruli of the kidneys could be demonstrated in all endotoxin-treated animals by pathological study . The findings suggest that by endotoxin infusion into the common bile duct, as a focal origin, DIC can be produced. Biochim Biophys Acta, 1979 Jul 19, 555(1), 56 - 66 Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12; Janoff AS et al.; Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43 degrees C were labelled with the fatty acid spin probe 5-doxyl stearate . Electron spin resonance spectroscopy revealed broad thermotropic phase changes . The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature . The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth . As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20 degrees C, but in a liquid crystalline state when cells were grown at 37 and 43 degrees C . In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures . Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs. C R Seances Acad Sci D, 1979 Jul 16, 289(3), 367 - 70 {Protein kinase activity in Escherichia coli}; Manai M et al.; When growing E . coli in a minimal medium, at least four proteins from the soluble fraction and one ribosome-associated protein are found phosphorylated at the level of their threonine and serine residues. Mol Gen Genet, 1979 Jul 13, 174(2), 221 - 4 Construction of an HpaI and HindII plasmid vector allowing direct selection of transformants harboring recombinant plasmids; Schumann W; The construction of the vector plasmid PKN80 is described, which can be used as HpaI or HindII cloning vehicle with direct selection on transformants harboring hybrid plasmids . pKN80 carries the EcoRI.C fragment of phage Mu DNA coding for a killing function which is efficiently expressed upon transformation of pKN80 into Mu-sensitive bacteria . Cloning of DNA fragments at the single HpaI site of pKN80 results in insertional inactivation of the killing function . Whereas religated pKN80 molecules yielded only a few transformants, the transformation efficiency had been increased by a factor of at least ten when HpaI fagments of lambda DNA were added to the linearized vector prior to ligation . More than 90% of the transformants tested containted hybrid plasmids. Mol Gen Genet, 1979 Jul 13, 174(2), 135 - 47 Genetic analysis of the inter-relationship between plasmid replication and incompatibility; Meacock PA et al.; The relationship between replication control and plasmid incompatibility has been investigated using a composite replicon, pPM1, which consists of the pSC101 plasmid ligated to another small multicopy plasmid, RSF1050 . Since pPM1 can utilise the replication system of either of the two functionally distinct components, propagation of the composite plasmid can occur in the presence of a mutation of one of its moieties . Such mutants are detected by their inability to rescue the composite plasmid under conditions not permissive for replication of the other moiety . Mutations in incompatibility functions can be detected by the failure of the composite replicon to exclude co-existing plasmids carrying a replication system identical to the one on pPM1 . The inability of the composite plasmid to replicate at 42 degrees in a host synthesizing temperature-sensitive DNA polymerase I, which is required by the RSF1050 replication system, was used to isolate pPM1 mutants defective in replication of the pSC101 component . Mutants defective in the incompatibility functions of pSC101 were obtained by selecting derivatives that allow the stable coexistence of a second pSC101 replicon in the same cell . Analysis of these two classes of mutants indicates that plasmids selected for defective pSC101 replication ability nervertheless retain pSC101 incompatibility . In contrast, plasmid mutants that have lost incompatibility functions were found always to be defective in replication ability. Mol Gen Genet, 1979 Jul 13, 174(2), 117 - 26 The effects of an Escherichia coli dnaAts mutation on the replication of the plasmids colE1 pSC101, R100.1 and RTF-TC; Frey J et al.; The rate of replication of the plasmids colE1, pSC101, R100.1 and pAR132 (an RTF-TC derivative of the drug resistance factor R100.1) has been investigated directly by DNA:DNA hybridization . These rates have been compared, in a dnaAts strain, to that of various markers of the host chromosome at permissive and non-permissive temperatures . Chromosome initiation in the dnaAts strain stops rapidly after a shift to the non-permissive temperature, but plasmids R100.1 and pAR132 do not seem to be affected directly and continue replication for some time . The colE1 replication rate undergoes a large increase after the temperature shift, followed by a rapid decrease to a very low level 25 min after the shift . In contrast pSC101 replication stops immediately after the shift . ColE1 is able to replicate in an integratively suppressed dnaAts strain at 42 degrees C whereas pSC101 stops replication immediately under these conditions . We conclude that R100.1 and its derivative RTF-TC can replicate without a functional dnaA product; that colE1, while affected by a shift in temperature in a dnaAts strain, does not directly require dnaA; and that the plasmid pSC101 has an absolute requirement for dnaA . The absolute requirement of pSC101 for dnaA in the integratively suppressed Hfr strain provides a useful system for further investigation of the dnaA function. Mol Gen Genet, 1979 Jul 13, 174(2), 127 - 33 Characterization of a plasmid mutation affecting maintenance, transfer and elimination by novobiocin; Taylor DE et al.; We have isolated a mutant H group plasmid temperature-sensitive for plasmid maintenance . Unlike the wild type plasmid (pSD114), the mutant (pDT4) was eliminated at 37 degrees C and also at 30 degrees C after novobiocin treatment . The mutant plasmid interfered with host cell growth at the non-permissive temperature . Conjugative transfer of the mutant was reduced at 30 degrees C compared to the wild-type plasmid . Introduction of a coumermycin-novobiocin resistance DNA gyrase (cou) mutation into Escherichia coli prevented pDT4 elimination by novobiocin but did not affect the temperature-sensitive phenotype . The evidence indicates that the mutant plasmid used bacterial DNA gyrase for replication . Models to account for the behaviour of this unusual mutant are discussed. Science, 1979 Jul 13, 205(4402), 160 - 6 Cellular applications of 31P and 13C nuclear magnetic resonance; Shulman RG et al.; High-resolution nuclear magnetic resonance (NMR) studies of cells and purified mitochondria are discussed to show the kind of information that can be obtained in vivo . In suspensions of Escherichia coli both phosphorus-31 and carbon-13 NMR studies of glycolysis and bioenergetics are presented . In rat liver cells the pathways of gluconeogenesis from carbon-13-labeled glycerol are followed by carbon-13 NMR . In the intact liver cells cytosolic and mitochondrial pH's were separately measured by phosphorus-31 NMR . In purified mitochondria the internal and external concentrations of inorganic phosphate, adenosine diphosphate, and adenosine triphosphate were determined by phosphorus-31 NMR while the pH difference across the membrane was measured simultaneously. Nucleic Acids Res, 1979 Jul 11, 6(9), 3025 - 40 Preparation of triple-block DNA polymers using recombinant DNA techniques; Selsing E et al.; The construction of several recombinant plasmid derivatives containing novel triple-block DNA sequence insertions is described . The protocol for these constructions involves synthesis of a heterogenous mixture of block oligomer duplexes, : formula: (see text), using pancreatic deoxyribonuclease and terminal transferase . The synthetic duplexes were mixed with linearized and dG-tailed vectors and the DNA mixture used to transform E . coli . Triple-block sequences of the type dGidAjdCk.dGkdTjdCi, characterized by DNA sequencing, were inserted into the Bam HI site of pBR322 and next to the lac wild-type and UV5 promoter regions in pRW26 and pRW28 . Similarly, sequences were inserted into the Sma I site of pACYC189 and could be excised by cleavage with Sma I since the procudure regenerates the recognition site . The approach provides a technique for the synthesis of a large family of defined sequence triple-block polymers in essentially unlimited amounts . Although these inserts contain sequences which have the potential for forming stable hairpin structures, the recombinant plasmids are stable and appear to replicate normally. Nucleic Acids Res, 1979 Jul 11, 6(9), 3161 - 76 Specificity and mode of cleavage of the pH 4.0 endonuclease from adenovirus type 2 - infected KB cells; Padmanabhan R et al.; Adenovirus type 2 or lambda DNA was digested with the pH 4.0 endonuclease, purified from adenovirus 2-infected KB cells . The enzyme produces a limit digest of approximate size in the range of 140-210 base pairs long . The termini of the DNA fragments generated by the endonuclease digestion had 3'-P and 5'-OH groups . The 3' and 5' end groups of the products were analyzed . Our data indicate that 3' end group was a purine (68-76%), dA occuring about twice the frequency of dG . The 5' end group was either dG or dC with equal frequency . Data obtained by treatment of the 5' labeled endonuclease product of lambda DNA with single-strand specific S1 nuclease from Asperigillus oryzae or exonuclease VII from Escherichia coli indicated that the majority of the products had a short 5' protruding ends . The mode of cleavage of this endonuclease seems to be through initial formation of several single-strand breaks with some base specificity . If these breaks are at close proximity on opposite strands, double-stranded fragments with protruding ends are generated. J Biol Chem, 1979 Jul 10, 254(13), 6128 - 37 Enzymological characterization of DNA polymerase alpha . Basic catalytic properties processivity, and gap utilization of the homogeneous enzyme from human KB cells; Fisher PA et al.; This report describes the results of our initial enzymological characterization of a homogeneous preparation of DNA polymerase alpha that we have purified from cultured human KB cells . Although the enzyme is most reactive with duplex DNA substrates that contain short gaps (optimally activated) in incubations that require Mg2+, the polymerase possesses the intrinsic capacity to copy the initiated ribohomopolymer template, (A)-n, (dT)-200, at low rates in the presence of Mn2+ . Because of the preponderance of DNA polymerase alpha in actively multiplying vertebrate cells, it is probable that this low level of activity comprises the majority of the ribopolymer copying activity that can be detected in crude tissue extracts . The presence of contaminating or associated deoxyribonuclease activities can be excluded from the purified enzyme to levels of 10(-4) to 10(-7) of the polymerase activity . The mechanism of polymerization on activated DNA under optimum conditions is moderately processive, with 11 +/- 5 nucleotides incorporated per polymerization cycle . The polymerase is unable to work at nicks or at short gaps of approximately 20 to 30 nucleotides in length, and it measures a surprisingly invariant effective template length on optimally activated DNA and on DNA molecules that have been gapped to varying extents with Escherichia coli exonuclease III . In the "Appendix" we present an amplification of the theoretical formulation of Bambara et al . (Bambara, R . A., Uyemura, D., and Choi, T . (1978) J . Biol . Chem . 253, 413--423) that permits the use of DNA polymerases with significant associated 3' leads to 5'-exonuclease activities for the accurate measurement of average template lengths (gap sizes) and titration of usable 3'-hydroxyl primer termini in gapped, duplex DNA substrates. Biochemistry, 1979 Jul 10, 18(14), 2990 - 6 Escherichia coli phosphoenolpyruvate dependent phosphotransferase system . Copurification of HPr and alpha 1-6 glucan; Dooijewaard G et al.; A rapid, high-yield procedure has been developed for the purification of HPr from the Escherichia coli phosphoenolpyruvate dependent phosphotransferase system . During this procedure, the protein copurifies with a 2500-dalton homopolysaccharide which we have identified as alpha 1-6 glucan . The results of steady-state kinetic measurements of the phosphotransferase activity demonstrate that the polysaccharide works as an activator of the phosphotransferase system probably at the level of the HPr:P-E1 complex or the P-HPr:E11 complex. Biochemistry, 1979 Jul 10, 18(14), 2980 - 4 Nucleophile in the active site of Escherichia coli galactose-1-phosphate uridylyltransferase: degradation of the uridylyl-enzyme intermediate to N3-phosphohistidine; Yang SL et al.; The {32P}uridylyl-enzyme intermediate form of Escherichia coli galactose-1-P uridylyltransferase can be converted to a {32P}phosphoryl-enzyme by first cleaving the ribosyl ring with NaIO4 and then heating at pH 10.5 and 50 degrees C for 1 h . After alkaline hydrolysis of the {32P}phosphoryl-enzyme the major radioactive product is N3-{32P}phosphohistidine . A lesser amount of 32Pi is also produced as a side product of the hydrolysis of N3-{32P}phosphohistidine . No N1-phosphohistidine, N-phospholysine, or phosphoarginine can be detected in these hydrolysates . It is concluded that the nucleophile in galactose-1-P uridylyltransferase to which the uridylyl group is bonded in the uridylyl-enzyme intermediate is imidazole N3 of a histidine residue . This degradation procedure should have general applicability in the degradation and characterization of nucleotidyl-proteins. J Biol Chem, 1979 Jul 10, 254(13), 5926 - 33 The structure of lipopolysaccharide from an Escherichia coli heptose-less mutant . III . Two fatty acyl amidases from Dictyostelium discoideum and their action on lipopolysaccharide derivatives; Rosner MR et al.; Two fatty acyl amidases have been partially purified from the slime mold, Dictyostelium discoideum . Their action on lipopolysaccharide derivatives, especially Compound I, has been studied . Amidase I removes specifically the beta-hydroxymyristyl group, which is present on the amino group adjacent to the C-1 phosphate . The product, Compound V, is then a substrate for Amidase II, which removes the remaining beta-hydroxymyristyl group from the amino group in the distal glucosamine ring to give Compound VI . Compound I itself is resistant to Amidase II . Thus, the two enzymes show a high degree of structural specificity . The structure of lipopolysaccharide from the E . coli K-12 mutant is concluded in the light of studies reported in this and the accompanying papers, and this structure is discussed in relation to other bacterial lipopolysaccharides. J Biol Chem, 1979 Jul 10, 254(13), 5868 - 77 Purification of hog liver isomerase . Mechanism of isomerization of 3-alkenyl and 3-alkynyl thioesters; Miesowicz FM et al.; A hog liver enzyme that catalyzes the reversible conversion of 3-acetylenic fatty acyl thioester to (+)-2,3-dienoyl fatty acyl thioester has been purified to homogeneity . The enzyme is not inhibited by the allenic product that it generates . The same homogenous enzyme catalyzes the conversions of 3-cis- or 3-trans-acyl Coenzyme A derivatives to 2-trans-acyl-CoA derivatives . Four forms of the isomerase differing in charge (pI = 6.57, 6.83, 7.01, and 7.27) have been separated by isoelectric focusing . Ultracentrifugation and sodium dodecyl sulfate-gel electrophoresis indicate that each of these enzyme forms is dimeric and composed of two 45,000-dalton subunits . With 3-acetylenic substrates, all enzyme forms exhibit broad specificity for chain length (C6 to C12) and for the thioester moiety (N-acetylcysteamine (NAC), pantetheine, or CoA) . The 3-cis and 3-trans olefinic substrates are active only in the form of their coenzyme A derivatives; their NAC thioesters inhibit competitively . Mechanistic studies favor an isomerization pathway by way of carbanion intermediates . The acetylene-allene isomerase described here and the reported crotonase-catalyzed hydration of allenic thioesters (Branchini, B.R., Miesowicz, F.M., and Bloch, K . (1977) Bioorg . Chem . 6, 49-52) may be responsible for the degradation of naturally occurring acetylenic and allenic acids. J Biol Chem, 1979 Jul 10, 254(13), 5781 - 6 Total synthesis of a tyrosine suppressor tRNA gene . XV . Synthesis of the promoter region; Sekiya T et al.; By use of polynucleotide kinase and polynucleotide ligase, the 10 deoxyoligonucleotide segments, whose syntheses have been described in accompanying papers, have been joined to form the 62-nucleotide-long DNA corresponding to the promoter region of an Escherichia coli suppressor tRNA gene . The following sequence in the joining reactions was used to obtain error-free and optimal yields of the products: 1) joining of Segment P-1 to P-3 in the presence of Segment P-2; 2) joining of Segments P-4 to P-7 to form Duplex {P4-7}; 3) joining of Segments P-8 to P-10 to Duplex {P4-7} to form Duplex {P4-10}; and finally, 4) joining of P-(1 + 3) and P-2 to Duplex {P4-10} to form the total promoter Duplex {P}. J Biol Chem, 1979 Jul 10, 254(13), 5765 - 80 Total synthesis of a tyrosine suppressor transfer RNA gene . XIV . Chemical synthesis of oligonucleotide segments corresponding to the terminal regions; Belagaje R et al.; Chemical syntheses of the two dodecanucleotides d(T-C-A-A-C-G-T-A-A-C-A-C) and d(A-C-G-T-T-G-A-G-A-A-A-G), the two undecanucleotides d(T-T-T-A-C-A-G-C-G-G-C) and d(T-G-T-A-A-A-G-T-G-T-T), the decanucleotide d(A-G-T-C-C-G-A-A-A-G), and the nonanucleotide d(A-A-T-T-C-T-T-T-C) are described . These deoxyribo-oligonucleotide segments, excluding the decanucleotide, represent the DNA duplex corresponding to the previously determined nucleotide sequence -30 to -51 of the promoter region of the gene for the tyrosine suppressor tRNA (Sekiya, T., Gait, M.J., Norris, K., Ramamoorthy, B., and Khorana, H.G . (1976) J . Biol . Chem . 251, 4481-4489) and include the EcoRI restriction endonuclease sequence at the appropriate 5'-end . The nona- and decanucleotide along with the previously synthesized deoxyribo-oligonucleotide segments 25 to 27 (Ramamoorthy, B., Lees, R.G., Kleid, D., and Khorana, H.G . (1976) J . Biol . Chem . 251, 676-694) together represent the DNA duplex corresponding to the natural nucleotide sequence 121 to 142 of the region adjoining the C-C-A end of the tyrosine tRNA gene and, in addition, a run of nine nucleotides which include the EcoRI restriction enzyme sequence at the 5'-end . The syntheses used protected mono- and oligonucleotides and stepwise condensation methods . A noteworthy feature of the present syntheses was the use of reverse phase high pressure liquid chromatography for the rapid and efficient separation of synthetic reaction mixtures. J Biol Chem, 1979 Jul 10, 254(13), 5713 - 7 A noncycling activity assay for the omega subunit of Escherichia coli RNA polymerase; Hansen UM et al.; We describe a new method for quantitatively assaying the omega subunit of Escherichia coli RNA polymerase . The assay is based on the ability of RNA polymerase holoenzyme to catalyze the continuous synthesis of the dinucleotide pApU on a poly{d(A-T)} . poly{d(A-T)} template when supplied with AMP and UTP as substrates . Core enzyme, lacking omega subunit, catalyzed this reaction at a rate less than 1% that of holoenzyme . The omega subunit was not released from the enzyme/DNA complex during dinucleotide synthesis . Using this assay, a titration of a fixed concentration of core enzyme was observed with increasing concentrations of added omega subunit . Below a 1:1 omega:core ratio the measured activity increased linearly with omega concentration, whereas above a 1:1 ratio the activity remained constant . An immediate application of the assay is in determining the concentration of active omega, or equivalently of active holoenzyme, in any RNA polymerase preparation. J Biol Chem, 1979 Jul 10, 254(13), 5609 - 12 In vivo transcription of rRNA operons in Escherichia coli initiates with purine nucleoside triphosphates at the first promoter and with CTP at the second promoter; de Boer H et al.; 32P-labeled RNA was isolated from growing Escherichia coli and the 5' end nucleoside triphosphates of rRNA were analyzed after hybridization to various DNA fragments derived from one of the rRNA operons, rrnE . The results show that there are two transcription start sites for each rRNA operon in vivo, one initiating with ATP (or GTP), and the second initiating with CTP . Transcription from the first site was observed to be stronger than that from the second site . These two sites correspond to the two promoters identified in the previous in vitro studies, indicating that the two promoters are used in vivo . These results also demonstrate previously unrecognized transcription initiation with CTP in growing E . coli cells. J Biol Chem, 1979 Jul 10, 254(13), 5906 - 17 Structure of the lipopolysaccharide from an Escherichia coli heptose-less mutant . I . Chemical degradations and identification of products; Rosner MR et al.; The structure of lipopolysaccharide from a heptose-less mutant of Escherichia coli K-12 has been investigated . Lipopolysaccharide isolated from 32P-labeled cells was treated with mild alkali to yield two separable components: {OH-LPS}-I (approximately 70%) and {OH-LPS}-II (approximately 30%) . Mild acidic treatment of {OH-LPS}-I gave mainly a product which was identified as (4-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine 1-phosphate (Compound I) . Further acidic hydrolysis of both {OH-LPS}-I and {OH-LPS}-II yielded as the main product (4-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine (Compound II) . The structures of the above products were deduced by a combination of compositional analyses, sensitivity to phosphomonoesterase, rates of hydrolysis of the phosphate groups and alkali-catalyzed beta elimination of the phosphate residues following appropriate oxidation of hydroxyl groups . These studies together with work reported in the accompanying papers have led to the identification of two species of lipopolysaccharide in the E . coli strain both of which contain a single glucosamine dissacharide unit but differ in having monosubstituted phosphate or pyrophosphate groups at the glycosidic position . Each species of lipopolysaccharide also appeared to be heterogeneous with respect to the number of esterified fatty acyl groups. J Biol Chem, 1979 Jul 10, 254(13), 5787 - 801 Total synthesis of a tyrosine suppressor transfer RNA gene . XVI . Enzymatic joinings to form the total 207-base pair-long DNA; Sekiya T et al.; The total synthesis of a 207-base pair-long DNA, which is biologically functional as a tyrosine suppressor transfer RNA gene, has been completed . The synthesis involved the enzymatic joining of the previously synthesized duplexes . Thus, the duplex corresponding to the promoter region {P} (Sekiya, T., Brown, E.L., Ramamoorthy, B., Fritz, H.-J., Gait, M.J., Lees, R.G., Ryan, M.J., Khorana, H.G., and Norris, K.E . (1979) J . Biol . Chem . 254, 5781-5786) was jointed to Duplex {I} (Caruthers, M.H., Kleppe, R., Kleppe, K., and Khorana, H.G . (1976) J . Biol Chem . 251, 658-666) to form {P + I} . Separatively, Duplex {III + IV + Vb} was prepared from the previously described Duplexes {III}, {IV}, and {Vb} . (Loewen, P.C., Miller, R.C., Panet, A., Sekiya, T., and Khorana, H.G . (1976) J . Biol . Chem . 251, 642-650; Sekiya, T., Besmer, P., Takeya, T., and Khorana, H.G . 1976) J . Biol . Chem . 251, 634-641; Ramamoorthy, B., Lees, R.G., Kleid, D., and Khorana, H.G., (1976) J . Biol Chem . 251, 676-694) . The product {P + I}, was joined to Duplex {II} (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H.G . (1976) J . Biol . Chem . 251, 651-657) and then to {III + IV + Vb} without isolation of the intermediates . In all the above joinings, the duplexes carried 32P-labeled phosphate groups at the appropriate 5'-ends . The total DNA and the intermediate duplexes were all characterized by their relative mobilities in electrophoresis on polyacrylamide gel slabs, by nearest neighbor analysis, and by degradation to 5'-nucleotides of radioactively labeled joined products . Two succeeding papers describe the transcription in vitro and the suppressor activity in vivo, of the synthetic gene now described. J Biol Chem, 1979 Jul 10, 254(13), 5754 - 63 Total synthesis of a tyrosine suppressor transfer RNA gene . XIII . Synthesis of deoxyribopolynucleotide segments corresponding to the nucleotide sequence -1 to -29 in the promoter region; Belagaje R et al.; Chemical syntheses of two tridecanucleotides, d(G-C-A-T-C-A-T-A-T-C-A-A-A) and d(G-C-G-T-C-A-T-T-T-G-A-T-A), and three undecanucleotides, d(G-G-A-A-G-C-G-G-G-G-C), d(T-G-A-T-G-C-G-C-C-C-C), and d(T-G-A-C-G-C-G-C-C-G-C), are described . These deoxyribo-oligonucleotide segments together represent the DNA duplex corresponding to the previously determined nucleotide sequence -1 to -29 of the promoter region of the tyrosine tRNA gene (Sekiya, T., van Ormondt, H., and Khorana, H.G . (1975) J . Biol . Chem . 250, 1087-1098) . Chemical syntheses used the principles of stepwise addition of protected mono- and oligonucleotides to the 3'-hydroxyl end of growing oligonucleotide chains . The desired condensation products were isolated by solvent extraction methods in the case of di- and trincleotides and by anion exchange chromatography in the case of longer chains . All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling at the 5'-ends with a {32P}phosphate group. J Biol Chem, 1979 Jul 10, 254(13), 5802 - 16 Total synthesis of a tyrosine suppressor transfer RNA gene . XVII . Transcription, in vitro, of the synthetic gene and processing of the primary transcript to transfer RNA; Sekiya T et al.; Primer- and promoter-dependent transcription of the synthesis gene had been studied . Primer-dependent transcription gave, as a major product, an end-to-end transcript which was strand-specific . The transcript was characterized rigorously by two-dimensional separation and analysis of the oligonucleotides formed on digestion with T1-RNase and pancreatic RNase and by nearest neighbor analyses of the oligonucleotides obtained when different alpha-32P-labeled ribonucleoside triphosphates were used as substrates . Minor products accompanying the major transcript were characterized similarly . The major transcript, when treated with an Escherichia coli S-100 extract, was processed to the tRNATyr with correct 5'- and 3'-ends . The nucleolytic cleavages occurring at the 3'-end were characterized . In promoter-dependent transcription, transcription of a restriction fragment containing phi80psu+III gene and the synthetic gene with and without the promoter were compared . Transcription of the synthetic gene was promoter-dependent and strand-specific, the initiation of transcription occurring at the same point as previously found in vivo . Although the synthetic gene contains only 16 base pairs corresponding to the natural sequence following the C-C-A end, processing of the transcript at the 3'-end occurred normally, the endonucleolytic cleavage being followed by exonucleolytic cleavages . The products of promoter-dependent transcription were completely characterized . An examination of the base modifications of the primary transcript during treatment of the latter with E . coli S-100 extract showed couplete modification of uridine to pseudouridine and partial methylation of uridine to ribosylthymine in TpsiCG sequence and partial formation of pseudouridine in the anticodon loop . However, hardly any formation of 2'-O-methylguanosine or of 2-methylthio-6-isopentenyl adenosine could be detected. Biochemistry, 1979 Jul 10, 18(14), 3171 - 8 Arginyl-tRNA synthetase from Escherichia coli K12 . Purification, properties, and sequence of substrate addition; Charlier J et al.; Arginyl-tRNA synthetase from Escherichia coli K12 has been purified more than 1000-fold with a recovery of 17% . The enzyme consists of a single polypeptide chain of about 60 000 molecular weight and has only one cysteine residue which is essential for enzymatic activity . Transfer ribonucleic acid completely protects the enzyme against inactivation by p-hydroxymercuriben zoate . The enzyme catalyzes the esterification of 5000 nmol of arginine to transfer ribonucleic acid in 1 min/mg of protein at 37 degrees C and pH 7.4 . One mole of ATP is consumed for each mole of arginyl-tRNA formed . The sequence of substrate binding has been investigated by using initial velocity experiments and dead-end and product inhibition studies . The kinetic patterns are consistent with a random addition of substrates with all steps in rapid equilibrium except for the interconversion of the cental quaternary complexes . The dissociation constants of the different enzyme-substrate complexes and of the complexes with the dead-end inhibitors homoarginine and 8-azido-ATP have been calculated on this basis . Binding of ATP to the enzyme is influenced by tRNA and vice versa. Biochemistry, 1979 Jul 10, 18(14), 2996 - 3001 Escherichia coli phosphoenolpyruvate dependent phosphotransferase system . NMR studies of the conformation of HPr and P-HPr and the mechanism of energy coupling; Dooijewaard G et al.; 1H and 31P nuclear magnetic resonance investigations of the phosphoprotein intermediate P-HPr and the parent molecule HPr of the E . coli phosphoenolpyruvate dependent phosphotransferase system (PTS) show that HPr can exist in two conformations . These conformations influence the protonation state of the reactive histidine residue, thereby determining the reaction pathway in the phosphoryl group transfer step . A general mechanism is proposed for the energy-coupling process in the PTS. Mol Gen Genet, 1979 Jul 2, 174(1), 75 - 88 Tandem and inverted repeats of arginine genes in Escherichia coli: structural and evolutionary considerations; Charlier D et al.; Duplications of arg genes produced in the Rec+ and in the recA genetic backgrounds are shown by heteroduplex analysis to be strictly tandem at the level of resolution of this technique . The formation of these particular rearrangements therefore does not require the inclusion of transposons or other sequences of an appreciable size in their final structure . Duplications of short segments (about 2,000 nucleotides) appear unexpectedly stable when compared with duplications of longer segments (about 10,000 nucleotides) . One of the structures analyzed displays two inversely repeated argE genes rearranged into an artificial divergent operon . The bearing of this observation on the origin of bipolar operons, of "mirror-image" map symmetries and on the production of inverted repeats in general, is discussed. Mol Gen Genet, 1979 Jul 2, 174(1), 25 - 32 Characterization of 10S RNA: a new stable rna molecule from Escherichia coli; Ray BK et al.; When cells of Escherichia coli are labeled with 32Pi for long periods of time and the cell content is subjected to electrophoresis in polyacrylamide gels, an RNA band appears which is about 10S in size . This band seems to contain three conformers . After treatment with formamide only a single band appears in this region of the gel, which contains 550 nucleotides as determined from its mobility . The complexity of the fingerprint of this material, after digestion with T1-RNase, is in agreement with the size as determined by the mobility, this confirming that indeed it is a single molecule . Composition of the T1-oligonucleotides was determined by digesting the T1-generated oligonucleotides with pancreatic RNase and T2-RNase . The quantitative and qualitative analysis of these digestions suggest that 10S RNA contains 609 nucleotides . The molecule contains, besides the four regular bases, one copy per molecule of the modified base pseudouridine . 10S RNA cannot be processed by cell extracts to tRNA-sized molecules and does not bind significantly to ribosomes, hence it is unlikely to be a tRNA precursor or an mRNA. Mol Gen Genet, 1979 Jul 2, 174(1), 11 - 5 Genetic analysis of a mutation affecting ribosomal protein S1 in Escherichia coli; Ono M et al.; Ribosomal protein S1 from a newly isolated Escherichia coli mutant has a molecular weight of about 54,000 which is smaller than the wild type S1 (M.W . 65,000) . The isoelectric points of the smaller and the wild type S1 species are similar in the gel electrophoresis system of O'Farrell (1975) . Genetic analyses by Hfr conjugation and P1 phage transduction indicate that the mutation affecting S1 (rpsA) is located close to the serC gene {20 min on the E . coli genetic map of Bachmann et al . (1976)}, with a co-transduction frequency of 61% . The most probable gene order is serC-rpsA-cmlB. Morphol Igazsagugyi Orv Sz, 1979 Jul, 19(3), 183 - 91 {Transformation of the epithelial cells of the kidney tubules in experimental malacoplakia}; Ormos J et al.; As an effect of administration of the extract of E . coli in the kidney of rats phagolysosomal reaction has been observed, not only in the granulomatous tissue characteristic of malacoplakia, but in the epithelial cells of the proximal tubules . The latter became similar to the histiocytes of malacoplakis i.e . Hansemann's cells . The transformation of the tubular epithelium in severe cases of malacoplakia was followed by necrosis . In a later phase tubular atrophy came into being, which did not differ from the atrophy caused by other agents, although epithelial cells contained numerous residual body . Authors believe, that granular tubular cells of megalocytic interstitial nephritis--regarded as malacoplakia of the renal cortex--originate from tubular epithelium. Infect Immun, 1979 Jul, 25(1), 463 - 6 Cytochalasin B does not inhibit ingestion of Chlamydia psittaci by mouse fibroblasts (L cells) and mouse peritoneal macrophages; Gregory WW et al.; Cytochalasin B did not inhibit ingestion of Chlamydia psittaci by either mouse fibroblasts (L cells) or mouse peritoneal macrophages in concentrations that produced distinctive morphological changes and inhibited phagocytosis of polystyrene latex beads and Escherichia coli K-12. Inflammation, 1979 Jul, 3(3), 203 - 14 Chemotactic factor inactivator release from rat leukocytes; Hunt JD et al.; Rat neutrophils, alveolar macrophages, and peritoneal macrophages each released chemotactic factor inactivator (CFI) activities under conditions of endocytosis (uptake of opsonized zymosan particles and preformed immune complexes) . CFI activities in supernatant fluids from phagocytizing leukocytes were found for the chemotactic factors from the third (C3) and the fifth (C5) components of complement and for the bacterial chemotactic factor (present in culture supernatant fluids from Escherichia coli.) CFI activity could also be demonstrated in homogenates obtained from disrupted leukocytes . CFI release from intact leukocytes was dependent on the duration of incubation of leukocytes with the phagocytic stimulus . No quantitative relationships were noted between the amount of CFI activity and the amount of beta-glucuronidase in phagocytic supernatant fluids released from leukocytes . The release of CFI activities from phagocytizing leukocytes may represent a regulatory ("turn-off") mechanism for the inflammatory response. Genetics, 1979 Jul, 92(3), 741 - 7 Escherichia coli K-12 auxotrophs induced by insertion of the transposable element Tn5; Shaw KJ et al.; The sites of insertion of the transposable kanamycin-neomycin resistance-determining element, Tn5, in the E . coli K-12 chromosome were assessed in a collection of over 300 auxotrophs . Although mutations in at least 45 different cistrons were obtained, the distribution of insertion sites was not completely random: proA or proB; cysG; and cysH, cysD or cysC mutants were found in excess. Avian Dis, 1979 Jul-Sep, 23(3), 682 - 7 Prophylactic and therapeutic activity of Rofenaid-40A in an experimental Escherichia coli airsac infection in chickens; Maestrone G et al.; Rofenaid at a 0.02% dose level in feed was effective prophylactically and therapeutically against an experimentally induced Escherichia coli airsac infection in chickens . The activity of Rofenaid compared very favorably with that of the approved dose level of NF-180 . Furthermore, the prophylactic use of Rofenaid did not interfere with the therapeutic efficacy of NF-180. Am J Vet Res, 1979 Jul, 40(7), 991 - 8 Equine Escherichia coli endotoxemia: comparison of intravenous and intraperitoneal endotoxin administration; Burrows GE; Certain physiologic and hematologic data were determined in ponies given Escherichia coli endotoxin by three routes: single IV dose, single intraperitoneal (IP) dose, and multiple IP boluses . In all ponies, the reaction was characterized by weakness, depression, peripheral circulatory abnormalities, and pyrexia . The pyrexia was more severe and was sustained in the ponies given multiple IP bolus endotoxin . Changes in packed cell volume, peripheral blood neutrophil, lymphocyte, and thrombocyte counts, and blood glucose were noticed in the three groups . Blood lactate and beta-glucuronidase values were determined and increases occurred only in the two IP endotoxin administration groups . A fibrinogen increase was observed in only the multiple IP bolus group . Attempts were made to correlate the lactate and beta-glucuronidase values with the severity and prognosis of the endotoxemia response . In general, the single IV bolus and, to a lesser extent, the single IP bolus endotoxin produced abrupt but transient responses . The multiple IP bolus endotoxin administration produced a more gradual and sustained response, which was more closely comparable with a clinical gastrointestinal disease problem than the other routes of administration produced. Am J Vet Res, 1979 Jul, 40(7), 1048 - 9 Heat extraction of animal plasma in preparation for endotoxin testing with the limulus amebocyte lysate test; Berg JN et al.; A heat-extraction procedure for endotoxin extraction from animal plasma has been developed for use with the limulus amebocyte lysate test . The procedure is simple to use, is accurate, and has shown good sensitivity with blood from several animal species. Res Vet Sci, 1979 Jul, 27(1), 133 - 4 Heat-labile enterotoxin antibodies in calves; Dobrescu L; The presence of antibodies to Escherichia coli heat-labile enterotoxin (LT) was measured in serum samples collected from 91 calves . Of these animals, 87 per cent had detectable levels of anti-LT in a seroneutralisation test performed in the Y1 adrenal cell time. J Gen Microbiol, 1979 Jul, 113(1), 189 - 93 Denaturation map of the ColE1-Km plasmid pCR11; Coggins LW; The denaturation map of EcoRI-digested pCR11, a ColE1-Km plasmid, is described . The 2.0 kilobase ColE1-derived segment contains an adenine+thymine rich site in the colicin immunity gene region . In the 7.2 kilobase kanamycin resistance region, the transposon Tn903 consists of an adenine+thymine rich 0.98 kilobase kan gene region flanked by a guanine+cytosine rich 1.09 kilobase inverted duplication. J Gen Microbiol, 1979 Jul, 113(1), 165 - 8 Mutants of Escherichia coli K12 permeable to haemin; McConville ML et al.; Mutants of Escherichia coli which require 5-aminolaevulinic acid (5-ALA), the first intermediate of haem biosynthesis, do not respond to haemin and porphyrins . The probable explanation of the lack of response is that E . coli may be impermeable to haemin and porphyrins . Mutants are described which responded to haemin and porphyrins as well as to 5-ALA . Indirect evidence is presented that the mutants were permeable to haemin . The mutants showed other phenotypic changes, and resembled some mutants which are known to have changes in the cell envelope. J Gen Microbiol, 1979 Jul, 113(1), 155 - 64 Isolation of haemin-requiring mutants of Escherichia coli K12; McConville ML et al.; Fifty-five haemin-requiring mutants were isolated from haemin-permeable mutants . According to their growth responses to haem precursors and their patterns of porphyrin accumulation, the 55 mutants fell into three groups which were judged to have defects in 5-aminolaevulinate dehydratase, ferrochelatase, and uroporphyrinogen III cosynthase or uroporphyrinogen decarboxylase . In mutants of the group deficient in 5-aminolaevulinate dehydratase, the mutations were adjacent to lac, and evidence is presented that the mutations were in hemB and were commonly deletions extending into proC. Proc Natl Acad Sci U S A, 1979 Jul, 76(7), 3279 - 83 Subunit stoichiometry and molecular weight of the pyruvate dehydrogenase multienzyme complex from Escherichia coli; Angelides KJ et al.; The molar ratio of the component enzymes of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was found to be 1.8:1.7:1{pyruvate decarboxylase (E1):dihydrolipoyl transacetylase (E2):dihydrolipoyl dehydrogenase (E3)} . This ratio was determined by measuring the Coomassie blue staining of the constituent enzymes after sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis . The above ratio is the average of four separate experiments with two different enzyme preparations . The average molecular weights of the individual enzymes were found to be 96,000, 76,000, and 55,000 for E1, E2, and E3, respectively, by sodium dodecyl sulfate and sodium dodecyl sulfate/8 M urea polyacrylamide gel electrophoresis and by column chromatography in 6 M guanidine . HCl . The molecular weight of E2 was reduced to 33,000-36,000 after extensive reduction and alkylation with iodoacetamide . The molecular weights of the complex, E1, and E3 were found to be 4,800,000, 182,000, and 104,000, respectively, with low-angle laser light scattering . Both E1 and E3 are dimeric under the conditions employed . If octahedral symmetry is assumed for the E2 core, a polypeptide chain ratio of 24:24:12 (E1:E2:E3) is in good agreement with the measured molar ratio of component enzymes and the molecular weight of the pyruvate dehydrogenase complex. Proc Natl Acad Sci U S A, 1979 Jul, 76(7), 3266 - 70 Evidence from ultraviolet absorbance measurements for a codon-induced conformational change in lysine tRNA from Escherichia coli; Moller A et al.; From experiments with equilibrium dialysis it was concluded earlier that formation of the codon-anticodon complex triggers a conformational change in the tertiary structure of tRNAPhe from Escherichia coli . A similar conformational transition is demonstrated here in the poly(A)/tRNALys system . C-G-A or C-G-A-A was used as a probe for the conformational transition in tRNA . These probes bound to tRNAPhe and tRNALys more strongly in the presence of the corresponding codons than in the absence . In order to verify these data by an independent method, the decrease in absorbance at 300 nm that occurs on formation of the codon-anticodon complex in tRNALys (which contains 2-thio-5-methylaminomethyluridine, s2mam5U) was used . The binding constants for formation of A3 . tRNALys (Ka = 2.4 . 10(4) M-1) and A4 . tRNALys (Ka = 2.5 . 10(5) M-1) are very close to those obtained by equilibrium dialysis . In the presence of C-G-A the apparent binding constant of A3 to tRNA was raised 10-fold to 2.5 . 10(-5) M-1 . It was calculated that the constant for the binding of C-G-A to the binary complex A3 . tRNALys is approximately 2 . 10(4) M-1, whereas binding to the free tRNA is lower than 10(3) M-1 . Under appropriate conditions binding of A3 to tRNALys can be induced directly by the addition of C-G-A . These data demonstrate that codon-anticodon complex formation induces a conformational change in the tRNA that as a consequence allows the binding of a trinucleoside diphosphate, presumably to the T-psi-G region. Proc Natl Acad Sci U S A, 1979 Jul, 76(7), 3233 - 7 Contacts between Escherichia coli RNA polymerase and thymines in the lac UV5 promoter; Simpson RB; I have identified those 5 positions of thymines in the lac UV5 promoter that lie close to bound Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) . Although ultraviolet irradiation of DNA with 5-bromouracil substituted in place of thymine normally cleaves the DNA at the bromouracils, a protein bound to the DNA can perturb these cleavages at those locations at which the protein lies close to the bromine . In the lac promoter most of these contacts lie in three regions . Four contacts lie in the region where transcription initiates; four lie in the "Pribnow box," which is located about 10 base pairs upstream from the initiation site; and three more lie in the "-35 region," located about 35 base pairs upstream from the initiation site . The "Pribnow box" and the "-35 region" are regions whose sequences are partially conserved between promoters and in which most promoter mutations are located; thus, contacts in these two regions probably represent sites of sequence-specific recognition by RNA polymerase. Proc Natl Acad Sci U S A, 1979 Jul, 76(7), 3144 - 8 Physical map of the recA gene; Sancar A et al.; We have cloned the recA gene of Echerichia coli K12 and some of its restriction fragments on the plasmid cloning vehicle pBR322 . The recA gene was mapped with regard to the restriction sites of EcoRI, BamHI, Pst I, Hha I, Hae III, HinfI, and Taq I restriction endonucleases . The recA promoter was localized by the binding of RNA polymerase to restriction fragments . The initiation point of transcription of recA mRNA and the direction of transcription were determined from in vitro transcription of recA gene fragments and from analysis of the polypeptides made in maxicells that contain plasmids carrying only part of the recA gene. Int J Pept Protein Res, 1979 Jul, 14(1), 27 - 33 Synthesis of model compounds relevant to the active-site-directed inactivation of L-asparaginase by 5-diazo-4-oxo-L-norvaline; Chang PK et al.; Earlier work has shown that 5-diazo-4-oxo-L-norvaline (DONV) irreversibly inactivates the L-asparaginase from E . coli by formation of a covalent bond in the region of the active site . Model compounds have been prepared to study this acid-labile covalent bond tentatively assigned to a serine or possibly a threonine residue in a decapeptide isolated from 14C-DONV-inactivated enzyme . Appropriately blocked DONV was found to alkylate methanol, and the hydroxyl function of blocked serine or threonine in the presence of boron trifluoride . The labile beta-ketoethers thus formed were reduced to the more stable beta-hydroxyethers . Facile lactonization of these 5-substituted-4-hydroxy-L-norvalines was observed . The diastereoisomers of both the lactonized and open forms of 5-methoxy-4-hydroxy-L-norvaline and related 4-hydroxy-L-2-amino acids of similar length were distinguishable on the amino acid analyzer . The beta-hydroxyethers derived from serine and threonine were hydrolyzed with acid and yielded the expected cleavage products . When the beta-ketoether was reduced by sodium borohydride prior to deblocking, in addition to the beta-hydroxyether, N-blocked amino alcohols were also formed, yielding a complex mixture of products. Can J Comp Med, 1979 Jul, 43(3), 321 - 7 The effects of repeated administration of Escherichia coli lipopolysaccharides to ponies; Burrows GE; Repeated exposure of ponies in Escherichia coli endotoxin resulted in attenuation of the packed cell volume, beta-glucuronidase, capillary refill time and neutrophil responses usually accompanying endotoxin administration . An overall decrease in severity of clinical response including reduced mortality was also apparent in ponies with repeated endotoxin exposure . Modification of the febrile response was not observed in any of the experimental groups. Can J Comp Med, 1979 Jul, 43(3), 237 - 42 Prenatal immunization against experimental enteric colibacillosis in piglets; Gay CC; To establish a model for the study of prenatal immunization against enteric colibacillosis a proportion of the litters of seven sows were immunized in utero 18 to 22 days before term by intra-amniotic and intramuscular injection of Escherichia coli antigen and the litters challenged at birth with either homologous or heterologous strains . Protection against homologous challenge was demonstrated in some but not all vaccinated piglets . The study was severely compromised by the occurrence of intrauterine death in a significant proportion of vaccinated piglets. Mutat Res, 1979 Jul, 61(2), 163 - 79 Repair promoted by plasmid pKM101 is different from SOS repair; Goze A et al.; In E . coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria . UV-induced reactivation per se was less effective . Bacteria with pKM101 showed no alteration in their division cycle . Plasmid pKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair . Plasmid pKM101 protected E . coli bacteria from UV damage but slightly sensitized them to X-ray lesions . Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional . Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased . Plasmid pKM101 repaired potentially lethal DNA lesions, although wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions. Mutat Res, 1979 Jul, 61(2), 153 - 62 Spontaneous mutagenesis in Escherichia coli strains lacking 6-methyladenine residues in their DNA: an altered mutational spectrum in dam- mutants; Glickman BW; The mutational spectrum at the lacI locus in a dam-4 strain of Escherichia coli was examined . The observed 20-fold increase in spontaneous mutagenesis in a dam- strain was found to be due to base substitutions, primarily transitions, which had increased 140-fold . Using the trpE997 mutation it was found that the dam mutations also resulted in an increase in frameshift mutagenesis . The mutational spectrum of dam- strains was similar to that found with strains carrying the mutH, mutL, mutS and uvrE mutations thought to result in a defect in the repair of mismatched bases . These results are taken to be consistent with, and to support the hypothesis that, dam- strains are deficient in a post-replicative error-avoidance pathway which allows the directed elimination of mismatch lesions by a mechanism in which parental strands are recognized by their level of DNA methylation. J Biochem (Tokyo), 1979 Jul, 86(1), 161 - 5 Distribution of phospholipid molecular species in outer and cytoplasmic membrane of Escherichia coli; Ishinaga M et al.; The outer membrane of Escherichia coli K-12 contained a smaller proportion of phospholipid molecular species with two unsaturated fatty acyl chains than did the cytoplasmic membrane . Proportions of phospholipid molecular species in the outer and cytoplasmic membranes changed in response to temperature changes . As the temperature increased, the content of 1-palmitoyl-2-cis-9,10-methylenehexadecanoyl species increased . Translocation of phospholipids from the cytoplasmic membrane to the outer membrane and synthesis of various molecular species were observed. J Biochem (Tokyo), 1979 Jul, 86(1), 1 - 10 Phosphoenolpyruvate carboxylase of Escherichia coli . Effect of proteolytic modification on the catalytic and regulatory propties; Kameshita I et al.; Phosphoenolpyruvate carboxylase from Escherichia coli W was treated with ten proteases, and the effects of the treatments on the enzyme activity and sensitivity to effectors were investigated . Proteases such as trypsin, alpha-chymotrypsin, papain, and subtilisin inactivated the enzyme, whereas elastase, carboxypeptidase Y and leucine aminopeptidase had no effect on the enzyme activity . Elastase and carboxypeptidase Y, however, inactivated the enzyme in the presence of 1 m urea . Subtilisin and alpha-chymotrypsin caused not only inactivation of the enzyme but also a significant desensitization to the effectors . DL-Phospholactate, a potent competitive inhibitor, markedly protected the enzyme from inactivation by subtilisin but did not protect it from desensitization to the effectors . Acetyl-CoA, fructose 1, 6-bisphosphate, and GTP-the allosteric activators--protected the enzyme from subtilisin inactivation, while laurate, the other allosteric activator, accelerated the inactivation . These activators did not protect the enzyme from desensitization to themselves . In contrast, modification with subtilisin in the present of l-aspartate, the allosteric inhibitor, caused an apparent transient activation of the enzyme . The enzyme modified in the presence of L-aspartate retained its sensitivity to L-aspartate, but the sensitivities to the other effectors were reduced to about one-half their initial values . Based on these results, a possible mode of desensitization of the enzyme by subtilisin modification and the possible existence of a multiplicity of conformational states of the enzyme, induced upon binding with the various effectors, are discussed. Infect Immun, 1979 Jul, 25(1), 337 - 44 Endotoxin toxicity in rats with 6-sulfanilamidoindazole arthritis; Miller ML et al.; Seven oral administrations of 6-sulfanilamidoindazole (6-SAI) to 10- to 12-month-old rats sensitized the animals to endotoxin, with dosages as small as 2.5 microgram causing death in 80% of animals . Endotoxin in a dosage of 3,000 microgram was not lethal for nonmedicated control animals . 6-SAI-treated 1-month-old rats were not as sensitive to endotoxin as aged animals . The sulfonamide-induced sensitivity to endotoxin could not be passively transferred and could not be explained by blockade of the reticuloendothelial system or impairment of endotoxin detoxification . 6-SAI administration was associated with both depletion of liver glycogen and lowering of blood glucose concentration without changes in blood lactic acid concentration . Disseminated intravascular coagulation is believed to be involved in the pathogenesis of shock and death as evidenced by: (i) concomitant decreases in plasma fibrinogen concentration and elevations in fibrin degradation products after endotoxin challenge; (ii) protection against lethal actions of endotoxin by pretreatment with heparin . Treatment of 6-SAI-medicated rats with glucocorticoids before endotoxin challenge protected the animals against lethal doses of endotoxin and prevented deposition of fibrin thrombi in the glomerular capillaries. Infect Immun, 1979 Jul, 25(1), 127 - 32 Effects of age, ambient temperature, and heat-stable Escherichia coli enterotoxin on intestinal transit in infant mice; Moon HW et al.; Some interrelationships among age, ambient temperature, intestinal transit, and enterotoxigenic Escherichia coli infection were studied in an infant mouse model . The transit of dye in the small intestine was accelerated during the response to heat-stable E . coli enterotoxin . Transit in the small intestine of normal mice accelerated with increased age (from less than 17 h to 8 days old) and accelerated with increased ambient temperature (from 25 to 37 degrees C) . Transit was more rapid in the jejunum than in the ileum throughout the range of experimental conditions studied . E . coli strains that do not produce any of the pili known facilitate intestinal colonization were cleared from the small intestine more rapidly at 37 degrees C than at 25 degrees C . This clearance was thought to be due to accelerated transit at the higher temperature . In contrast, a strain of E . coli that produces K99 (pili previously shown to facilitate intestinal colonization in other species) was not cleared from the small intestine and colonized more intensively at 37 degrees C than at 25 degrees C . Intensified colonization by this strain was thought to be due to increased production of K99 at the higher temperature . It was suggested that sluggish intestinal transit may also be characteristic of the neonates of other species and be one of the factors predisposing them to intestinal colonization by enteropathogens . It was speculated that this predisposition may be enhanced if the neonates are chilled . However, the effect of ambient temperature on intestinal transit in homeothermic neonates such as pigs, calves, and humans may be different from that in mice because neonatal mice are poikilothermic. Gene, 1979 Jul, 6(3), 251 - 64 Expression of the yeast galactokinase gene in Escherichia coli; Citron BA et al.; In Saccharomyces cerevisiae the genes for three of the enzymes involved in galactose metabolism are tightly linked near the centromere of chromosome II (Douglas and Hawthorne, 1964) . However, the molecular mechanisms which control the expression of these genes are not well understood . A DNA fragment containing at least one of these yeast genes, the galactokinase gene (gal1), has been joined to the bacterial plasmid pBR322 and maintained in an Escherichia coli strain that carries a deletion in its own galactokinase gene, galK . The presence of the yeast gene was demonstrated by (i) complementation of the E . coli galactokinase deletion, (ii) by hybridization of the cloned DNA fragment to restriction enzyme digests of total yeast DNA and (iii) by assaying for yeast galactokinase activity in bacterial cell extracts . The yeast DNA fragment is 4700 base pairs long, and enables the host E . coli K-12 strain to grow in minimal medium containing galactose as the sole carbon source with a generation time of 14.3 h . The yeast galactokinase activity in the bacterial extracts is 0.7% of the bacterial galactokinase activity found in wild-type E . coli fully induced with fucose. Zh Mikrobiol Epidemiol Immunobiol, 1979 Jul, (7), 30 - 4 {Integration of the RP4 factor with the chromosome in Escherichia coli K12 and the formation of 2 types of Hfr-strains}; Smirnov SP et al.; The mutant RP4ts12, derived from the R-factor RP4 and thermosensitive in replication, is incorporated into the chromosome A3dna(ts) of E . coli K12, thus suppressing dnaA mutation . The integration of this factor into the chromosome leads to the formation of Hfr strains of two types: the strains of the first type transfer plasmid markers to recipient cells earlier than to chromosomal ones; the strains of the second type transfer plasmid markers to recipient cells after chromosomal ones . During conjugation the R-factor integrated into the chromosome dissociates from chromosomal DNA introduced into the recipient cell and becomes autonomous. Mol Biol (Mosk), 1979 Jul-Aug, 13(4), 955 - 9 {Oligoadenylate 4-(N-2-chloroethyl-N-methylamino) benzylidene 5'-phosphamides complex formation with DNA by equilibrium dialysis}; Karpova GG et al.; Alkylating derivatives of oligoadenylate 5'-phosphamides CIRCH2NH(pA)n n=4-7 attach to complementary regions of denatured E . coli DNA . Apparent binding constants Kapp are evaluated from plots of binding at 0 degrees obtained by equilibrium dialysis . Kapp of CIRCH2NH(pA)7 is shown to be similar to that of (Ap)5ARCl-benzylidene derivatives of hexaadenylate; Kapp both of them differ from Kapp of hexaadenylate only slightly . Under saturation conditions CIRCH2(Ap)7 binds to 24 sites per 10 kilobases of E . coli DNA . Macrostructure of denatured DNA is changed in the course of binding and number of burned complementary sites becomes exposed to following binding with the oligomer. Mol Biol (Mosk), 1979 Jul-Aug, 13(4), 845 - 53 {Transcription of synthetic oligonucleotides}; Belova NV et al.; The decadeoxynucleotides d(pTTC)3 and d(CCATCTTTT) transcription by E . coli RNA-polymerase was studied . The nucleotide composition of d(pTTC)3 transcript was shown to be consistent with that of the template, but RNA-product was several times longer than the template . With nonanucleotide d(CCATCTTTT) the poly(A) synthesis was observed . This fact may be attributed to the reiteration on the TTTT-cluster . When using the oligonucleotide primers d(pGGA) and r(pAAAA) complementary to the templates d(pTTC)3 and d(CCATCTTTT), accordingly, the nucleosidetriphosphates concentration being reduced and the RNA-polymerase "holo" being replaced by "core", the considerable decrease in the unhomogeneity of the transcript length and in the length itself was found . With d(pTTC)3 as a template the length of RNA-product was found to be of 24-25 nucleotides and with d(CCATCTTTT) it was of 18-19 nucleotides . The sequence of RNA transcribed from both templates in the presence of primers was in accordance with the structure of templates. Mol Biol (Mosk), 1979 Jul-Aug, 13(4), 811 - 21 {Interaction of oligophosphates of pyridoxal with certain enzymes of polynucleotide synthesis}; Mamaeva OK et al.; The interaction of pyridoxal, pyridoxal-5'-mono-, di- and triphosphate with certain enzymes of polynucleotide synthesis (DNA-dependent RNA polymerase, DNA-dependent DNA polymerase I and polynucleotide phosphorylase from Escherichia coli and terminal deoxyribonucleotide transferase from calf thymus) was studied . All compounds tested was found to be reversible and competitive inhibitors of these enzymes . The reduction of the enzyme-inhibitor complex with NaBH4 gives rise to the complete irreversible inhibition of the enzymes under study . The comparison of the inhibition constants for pyridoxal and its phosphorylated derivatives with those for mono-, di- and triphosphates of nucleosides was carried out for the enzymes . The results obtained suggest that the modified epsilon-amino-group of lysine residue should be localized at the catalytic site in the vicinity of the pyrophosphate binding area of an enzyme. Mol Biol (Mosk), 1979 Jul-Aug, 13(4), 798 - 803 {Physico-chemical characteristics of ribosomal proteins L10 and L11}; Gudkov AT et al.; The procedure of isolation and purification of ribosomal proteins L10 and L11 from the 50S subparticle of E . coli ribosomes is described . Sedimentation data, spectra of circular dichroism and the results of microcalorimetric studies are given for these proteins . It has been shown that in the range of the concentrations studied protein L11 does not associate in solution . Protein L10 has a tendency to self-association at increased concentrations (over 1.5 mg/ml) . Protein L10 also forms a complex with the intact ribosomal protein L7 . From the data presented it follows that protein L7 binds to protein L10 in a dimer form and the regions of L7 responsible for its dimerization participate in binding . It has been also shown that the proteins in solution have a rather low thermostability . An assumption is made on the stabilization of proteins within the ribosomes or in the complex with other ribosomal proteins. Mol Biol (Mosk), 1979 Jul-Aug, 13(4), 788 - 97 {Influence of the modification of Phe-tRNA synthetase from Escherichia coli by lysine- and arginine-specific reagent on the ionic interactions of the enzyme with tRNA Phe}; Gorshkova NI et al.; The influence of modification of Phe-RSase from E . coli MRE-600 by lysine- and arginine-specific reagent 2,4-pentandione on the Phe-RSase.tRNAPhe interactions was investigated . It was shown that modification of Phe-RSase with 2,4-pentandion leads to a decrease of the aminoacylation rate without any influence on the value of Km for tRNAPhe in this reaction and only a slight increase of the value of Kdiss for Phe-RSase.tRNAPhe complex . The log Km (Km-1)--ionic strength dependence for native enzyme and log Kdiss (K-1diss) for native enzyme and two forms modified on arginine and lysine residues were investigated . Results were interpreted quantitatively by Debye--Huckel approximation for two spherical macroions and by Daune approximation assuming that the region of tRNA implicated in ionic interactions is locally a cylindrical polyelectrolyte . It was shown that there are 2-4 electrostatic contacts in Phe-RSase.tRNAPhe interactions in limits of both approximations; modification of arginine residues in Phe-RSase doesn't change the number of electrostatic contacts, modification of lysine residues leads to an increase in the number of contacts . It was assumed that there are lysine residues in Phe-RSase essential for the tRNAPhe recognition . The possibility of participation of negative amino acid residues in electrostatic interactions with tRNAPhe is not excluded. Mol Biol (Mosk), 1979 Jul-Aug, 13(4), 752 - 60 {Nature of the heterogeneity of the 30S ribosomal subunits in vitro . II . Two types of inactivation of the 30S subunits of Escherichia coli ribosomes}; Peshin NN et al.; The influence of concentration of monovalent cations on the binding constant of Phe-tRNAPhe to 30S.poly(U) complex was studied . Two types of inactivation of the 30S subunits by ammonium ions at the low magnesium concentration (1 mM) were found . The first type of inactivation was observed at high concentrations of NH4+ ions (from 0.5 to 1.5 M) and due to the dissociation of ribosomal proteins from 30S subunits . This inactivation only decreased the binding constant of Phe-tRNAPhe to 30S.poly(U) complex up to 50 times but all 30S subunits were equally achieved in Phe-tRNAPhe binding . This type of inactivation was reversible, addition of S-proteins restored the association constant to the original value . At low concentration of NH4+ ions (below 100 mM) about half of the 30S subunits is irreversibly inactivated (the binding constant of Phe-tRNAPhe decreased below detectable level) probably as a result of conformational changes in ribosomal RNA . Both types of inactivation of the 30S subunits can take place during the preparation of isolated subunits of ribosomes. Eur J Biochem, 1979 Jul, 98(1), 149 - 54 Recognition of tRNA Trp by initiation factors from Escherichia coli; Leon M et al.; Binding of acetyl or formyltryptophanyl-tRNA Trp from Escherichia coli or beef liver to E . coli ribosomes is strongly stimulated by E . coli initiation factors and requires GTP . The N-acylated tryptophan is puromycin reactive . Polypeptide chain initiation with acetyltryptophan dependent on poly(U,G) has been demonstrated and is highly dependent on added initiation factors . tRNA Trp appears, therefore, to share some structural features with tRNAfMet of significance to the process of polypeptide chain initiation. Eur J Biochem, 1979 Jul, 97(2), 463 - 9 The ternary 5-S RNA complex of proteins L 18 and L 25 . A small-angle X-ray scattering titration study; Osterberg R; The 5-S RNA (A) and the proteins L 18 (B) and L25 (C) from Escherichia coli ribosomes form a ternary complex of the type ABC with a stepwise stability constant, log K111 approximately equal to 6.5 . This is indicated from X-ray scattering titrations recorded at 21 degrees C in ribosomal reconstitutional buffer . When the ternary ABC complex forms there is only a limited change in the scattering curve compared to that of 5-S RNA, indicating that 5-S RNA does not undergo a major conformational change during the complex formation . The increase in the radius of gyration from 3.61 nm (5-S RNA) to 3.95 nm (ABC complex) as well as the experimental scattering curve can be explained by models where it is assumed that the elongated L 18 and L25 models are quite far from the electron density centre and where the protein molecules interact mainly with the minor arms of the supposed Y-shaped 5-S RNA molecule. Eur J Biochem, 1979 Jul, 97(2), 435 - 43 The structure of a transcriptional unit on colicin E1 plasmid; Morita M et al.; In an RNA-synthesizing system in vitro, a low-molecular-weight RNA consisting of about 110 residues (RNA-I) was efficiently synthesized on DNA of colicin E 1 plasmid (ColE1) and its deletion derivatives . The promoter site for RNA-I was analysed by testing the RNA polymerase-binding ability and template activity of restriction fragments; it was mapped in the region between the replication initiation site and the colicin immunity gene of ColE1 . The direction of transcription was determined by hybridization tests to the separated strands of the template . The DNA region directing RNA-I was sequenced, and RNA-I was assigned on the sequence based on the nearest-neighbour data of RNA . The sequences of its promoter and terminator regions were also deduced . Although the function of this small RNA species is unknown, a unique secondary structure could be constructed from its sequence and sensitivity to RNase. J Infect Dis, 1979 Jul, 140(1), 114 - 8 Serologic responses to somatic O and colonization-factor antigens of enterotoxigenic Escherichia coli in travelers; Deetz TR et al.; To improve the retrospective diagnoses of enterotoxigenic Escherichia coli (ETEC) as a cause of travelers' diarrhea, as well as to determine the presence of colonization-factor antigens in these infections, a study of serologic responses to antigens of ETEC was done . Paired sera from 60 United States students in Cholula, Puebla, Mexico, were analyzed for rises in titer of antibody to heat-labile toxin, eight somatic antigen O serogroups associated with ETEC, and two colonization-factor antigens, CFA/I and CFA/II . Only 9% had a response to O antigens, while 20% had responses to the colonization-factor antigens . Response to the colonization-factor antigens correlated significantly with response to the heat-labile toxin and with culture evidence of ETEC infection . Serologic studies confirmed that colonization-factor antigen has a role in naturally acquired cases of travelers' diarrhea and that it can be used as an additional determinant of infection with ETEC. J Bacteriol, 1979 Jul, 139(1), 8 - 12 Novel acriflavin resistance genes, acrC and acrD, in Escherichia coli K-12; Nakamura H; Acriflavine-resistant mutants were isolated from an acriflavine-sensitive (acrA) strain of Escherichia coli K-12 and then tested for temperature sensitivity of cell division . Genetic analysis characterized two new genetic loci, acrC and acrD . The former was mapped between tonA and proA, and the latter between the origin of genetic transfer of HfrH and serB . acrC and acrD mutants could divide but did not initiate a new round of deoxyribonucleic acid (DNA) replication at 43 degrees C . DNA synthesis of the acrC mutant cells ceased after a period of time following temperature shift-up, and thereafter DNA degradation occurred . However, cell mass continued to increase for a long time at the nonpermissive temperature . On the other hand, DNA synthesis of the acrD mutant cells ceased soon after the shift-up, and the cell mass did not appreciably increase during the prolonged incubation. J Bacteriol, 1979 Jul, 139(1), 54 - 63 Expression of the cloned uvrB gene of Escherichia coli: mode of transcription and orientation; Pannekoek H et al.; The Escherichia coli uvrB gene, located on a 1.5-megadalton EcoRI (fragment F, derived from transducing phage lambda b2att2 {lambda b2cI857intam6 delta (bioAB)bio-FCD+uvrB+}, has been cloned in the unique EcoRI site of several "relaxed" plasmids, i.e., pMB9, pBR322, and pBH20 (= ;BR322, including the lac regulatory elements {K . Itakura, T . Hirose, R . Crea, A . D . Riggs, H . L . Heyneker, F . Bolivar, and H . W . Boyer, Science 198:1056--1063, 1977}y . Expression of the uvrB gene, both on pMB9 and on pBH20, occurs only when fragment F has one particular orientation . Cloning of this fragment on pBR322 in either orientation does not allow expression of the uvrB gene . Transcription of this gene on pNP5 ( = pMB9 uvrB) is shown to be dependent on a pMB9 promotor that is located on a 0.22-megadalton EcoRI-HindIII fragment . Using plasmid pBH20 as a vector, we could demonstrate that expression of the uvrB gene is under control of the lac promotor-operator region . From deoxyribonucleic acid-deoxyribonucleic acid hybridization experiments with lambda pgal8 deoxyribonucleic acid and restriction fragments of pNP5 deoxyribonucleic acid it could be shown that the uvrB gene is transcribed clockwise on the chromosome. J Bacteriol, 1979 Jul, 139(1), 48 - 53 Expression of the cloned uvrB gene of Escherichia coli: dependency on nonsense suppressors; Pannekoek H et al.; Recombinant plasmid pNP5, consisting of plasmid pMB9 on which the uvrB gene is cloned, fully complements for the defects due to chromosomal uvrB mutations in the presence of the amber suppressor sup-6 or supF . Correndonuclease II activity was also completely restored in in UvrB strains containing both plasmid pNP5 and amber suppressor sup-6, as compared with the parental UvrB+ strain . It is shown that the amber mutation which interferes with the expression of the cloned uvrB gene is located outside this gene . Apparently, the amber mutation exerts a polar effect on uvrB expression that is relieved by sup-6 or supF . Introduction of a rho mutation into suppressor-free UvrB strains, harboring pNP5, did not relieve the polarity caused by the amber mutation. J Bacteriol, 1979 Jul, 139(1), 311 - 5 Isolation and characterization of a mutant ColE1 plasmid that allows constitutive colicin E1 synthesis; Ohkubo H et al.; It has been possible to isolate a ColE1 mutant which synthesizes colicin E1 constitutively . This result shows that there must be a gene(s) responsible for the regulation of colicin E1 synthesis. J Bacteriol, 1979 Jul, 139(1), 176 - 84 Regulation of the biosynthesis of aminoacyl-transfer ribonucleic acid synthetases and of transfer ribonucleic acid in Escherichia coli . VI . Mutants with increased levels of glutaminyl-transfer ribonucleic acid synthetase and of glutamine transfer ribonucleic acid; Cheung A et al.; Spontaneous revertants of a temperature-sensitive Escherichia coli strain bearing a thermolabile glutaminyl-transfer ribonucleic acid (tRNA) synthetase have been selected for growth at 45 degrees C . Among 10 revertants still containing the thermolabile enzyme, 2 interesting strains were found . One strain has a fivefold elevated level of the thermolabile glutaminyl-tRNA synthetase; the genetic locus, glnR, responsible for this effect maps at min 24, far from glnS, the structural gene of the enzyme . In the other strain the levels of tRNA Gln and several other tRNAs are twice as high as in the parental strain; the locus responsible, glnU, maps at min 59.5 on the E . coli map. J Bacteriol, 1979 Jul, 139(1), 165 - 75 Regulation of the biosynthesis of aminoacyl-transfer ribonucleic acid synthetases and of transfer ribonucleic acid in Escherichia coli . V . Mutants with increased levels of valyl-transfer ribonucleic acid synthetase; Baer M et al.; Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase were selected for growth at 40 degrees C . Of these, a large number still contain the thermolabile valyl-tRNA synthetase . Three of these revertants contained an increased level of the thermolabile enzyme . The genetic locus, valX, responsible for the enzyme overproduction, is adjacent to the structural gene, valS, of valyl-tRNA synthetase . Determination (by radioimmunoassay) of the turnover rates of valyl-tRNA synthetase showed that the increased level of valyl-tRNA synthetase is due to new enzyme synthesis rather than decreased rates of protein degradation. J Bacteriol, 1979 Jul, 139(1), 1 - 7 Role of exonucleases V and VIII in adenosine 5'-triphosphate- and deoxynucleotide triphosphate-dependent strand break repair in toluenized Escherichia coli cells treated with X-rays; Waldstein EA; The repair of X-ray-induced strand breaks was studied in permeabilized Escherichia coli recBC cells deficient for the adenosine 5'-triphosphate (ATP)-dependent exonuclease V and in recBC sbcA cells that possess the ATP-independent exonuclease VIII . It is shown that repair induced by additon of ATP does not take place in recBC and recBC sbcB cells and is limited in recBC sbcA cells . ATP-dependent repair is nevertheless observable if together with ATP a mixture of deoxynucleotide monophosphates is supplied to the cells . These data fit with the assumption that in wild-type cells ATP-dependent repair involves exonuclease V-induced deoxyribonucleic acid degradation and rephosphorylation of the degradation products which are reused for deoxyribonucleic acid polymerase I-dependent break closure . Repair in the presence of deoxynucleotide triphosphates rejoins a similar fraction of breaks in all strains tested irrespective of the amount of postirradiation degradation resulting from exonuclease V and exonuclease VIII activities . Thus, exonuclease V is dispensable for deoxynucleotide triphosphate-dependent repair, i.e., does not "clean" the ends of breaks produced by X-irradiation . ATP- and deoxynucleotide triphosphate-dependent repair are not additive and seem to repair the same population of deoxyribonucleic acid molecules damaged by X-irradiation. Chem Biol Interact, 1979 Jul, 26(2), 197 - 205 Mutagenicity of methyl-, ethyl-, propyl- and butylnitrosourea towards Escherichia coli WP2 strains with varying DNA repair capabilities; Garner RC et al.; Methyl- (MNUA), ethyl- (ENUA), propyl- (PNUA) and butylnitrosourea (BNUA) have been tested for toxicity and mutation in a liquid suspension assay towards Escherichia coli WP2 and some of its repair deficient derivatives . A comparison of survival rates after nitrosourea exposure between WP2 and WP2 uvrA showed no difference between the two strains but a consistent difference in potency between the various nitrosoureas studied . Toxicity increased in the order MNUA less than PNUA less than ENUA less than BNUA . ENUA and PNUA induced a greater number of trp+ revertants in both strains than did MNUA and BNUA, particularly at low survival rates . None of these differences in biological potency could be accounted for by differences in rates of hydrolysis . ENUA, PNUA and BNUA were non-mutagenic towards WP2 lexA, WP2 recA and WP2 uvrA lexA, whereas MNUA did induce mutations . Ethyl methanesulphonate (EMS) was able to mutate WP2 lexA . These results are discussed in the light of current theories regarding the mechanism of action of these compounds. Surgery, 1979 Jul, 86(1), 41 - 8 Mechanisms of the adjuvant effect of hemoglobin in experimental peritonitis . V . The significance of the coordinated iron component; Lee JT Jr et al.; Adjuvant effects of hemoglobin, methemoglobin, hematin, and ferric nitrilotriacetate (FENTA) on the lethality of E . coli peritonitis in rats were compared . The functional importance of coordinated iron was affirmed by the findings that: (1) hematin simulated the hemoglobin effect when administered on an iron-equivalent basis and (2) hematoporphyrin was inactive at the same levels as hematin . The effects of hemoglobin and methemoglobin were virtually identical, suggesting that the oxidation state of the metallic center is immaterial, and analyses of peritoneal contents during lethal peritonitis promoted by either adjuvant revealed insignificant interconversions of these compounds . Saturation of systemic iron-binding capacity could not be detected during lethal E . coli--hemoglobin peritonitis and deliberate saturation of systemic transferrin by infusions of intravenous FENTA did not enhance the adjuvant effect of hemoglobin . The adjuvant effect of intraperitoneally administered FENTA was effectively nullified by simultaneous intraperitoneal deferoxamine injection, but the same maneuver had no effect on hemoglobin potency . Thus the adjuvant effect of hemoglobin in experimental peritonitis is functionally dependent on the iron component but cannot be explained by a non-heme iron flux . These characteristics suggest that adverse interactions of coordinated iron species with host defense chemistry will be fruitful subjects for future study. J Immunol, 1979 Jul, 123(1), 87 - 91 Aging increases expression of LPS-induced autoantibody-secreting B cells; Meredith PJ et al.; Escherichia coli lipopolysaccharide (LPS), a polyclonal B cell activator, has been employed to achieve in vitro stimulation of autoantibody-secreting B cells in young adult and aged mice of long-lived strains as assayed in a hemolytic plaque technique to syngeneic mouse erythrocytes . Aged 21- to 24-month-old C57BL/6J and (C57BL/10Sn x C3H/HeDiSn)F1 mice were found to express 3 to 4 times as many LPS-induced plaque-forming cells (PFC) to autologous erythrocytes than did younger 6-month-old animals . With the use of cyclophosphamide (CY), a significant enhancement of auto-PFC production in young mice occurred, approaching levels found in non-CY-treated old mice . Thus, autoreactive clones of lymphocytes exist in the spleens of young adult mice, but under normal circumstances produce little autoantibody . The situation in aged members of these strains, therefore, does not seem to involve an actual increase in numbers of autoreactive B cells, but may possibly involve some form of deregulation, permitting increased age-related expression of autoreactive lymphocyte clones. Res Vet Sci, 1979 Jul, 27(1), 52 - 8 The aetiology of diarrhoea in pigs weaned at two days of age; Barrow PA et al.; When pigs are weaned at two days of age large numbers of Excherichia coli appear in the anterior gut and the incidence of diarrhoea rises . The two phenomena do not appear to be directly related because the strains of E coli isolated are not serotypes previously found to be associated with neonatal pig scouring . Representative strains of the non-enteropathogenic serotypes did not produce enterotoxin and did not adhere to small intestine brush borders . Moreover when antibiotics were fed to eliminate E coli from the gut, the pigs still scoured . Rotavirus was detected in the gut contents and gut epithelium of scouring pigs and a bacteria-free filtrate of gut contents produced diarrhoea when administered to germ-free pigs . It is suggested that rotavirus may be one of the causes of the scouring seen shortly after weaning pigs at two days of age. J Gen Virol, 1979 Jul, 44(1), 255 - 60 Protection of mice against infection with wild-type Mengo virus and an interferon sensitive mutant (IS-1) by polynucleotides and interferons; Stebbing N; Single-stranded polynucleotide preparations {tRNA, poly(rI) plus poly(ho5C)-copolymer} which protect mice against picornavirus infections without inducing interferon, protected mice equally against infection with an interferon-sensitive mutant (IS-1) of Mengo virus and with wild-type virus (IS+) . Poly(rI) . poly(rC), and mouse macrophage interferon {i.e . serum from mice treated with poly(rI) . poly(rC)} protected mice equally against infections with the two viruses, but fibroblast interferon protected better against infection with the interferon-sensitive mutant than with the wild-type virus . These and other results indicate that: Mengo virus had a genetic locus affecting sensitivity to fibroblast but not macrophage interferon; these two types of interferon have different mechanisms of action against Mengo virus infections in mice; Mengo virus genes controlling sensitivity to fibroblast interferon may modulate disease since infection in vivo induces only fibroblast interferon; the antiviral activity of the single-stranded polynucleotides is unlikely to be mediated by induction of either macrophage or fibroblast interferon. Proc Natl Acad Sci U S A, 1979 Jul, 76(7), 3194 - 7 Cyclic AMP as a modulator of polarity in polycistronic transcriptional units; Ullmann A et al.; The degree of natural polarity in the lactose and galactose operons of Escherichia coli is affected by adenosine 3',5'-cyclic monophosphate (cAMP) . This effect, mediated by the cAMP receptor protein, is exerted at sites distinct from the promoter . Experiments performed with a mutant bearing a thermosensitive rho factor activity indicate that cAMP relieves polarity by interfering with transcription termination . Conflicting results in the literature concerning the role of cAMP receptor protein and cAMP in galactose operon expression can be reconciled by the findings that cAMP stimulates the expression of operator distal genes without significantly affecting the proximal genes . Therefore, it appears necessary to reevaluate the classification of the galactose operon as exhibiting cAMP-mediated catabolite repression at the level of transcription initiation. Eur J Biochem, 1979 Jul, 98(1), 203 - 14 Studies on the methylation of cytoplasmic ribosomal RNA from cultured higher plant cells; Cecchini JP et al.; The methylation of cytoplasmic ribosomal RNA of cultured sycamore cells (Acer pseudoplatanus L.) was investigated . Labelled 17-S and 26-S rRNA were prepared from cells that had been incubated with either {32P}phosphate, {Me-3H}methionine or {Me-14C}methionine . Ion-exchange resin chromatography of 0.3 M KOH or 1 M HCl hydrolysates and two-dimensional chromatographic analyses of phosphodiesterase plus phosphatase digests of 17-S and 26-S rRNA were performed . 17-S and 26-S rRNA contain 49 and 91 methyl groups per molecule, respectively . These values were verified in sevemral ways . The high degree of methylation of sycamore rRNA, particularly for the 26-S rRNA, contrasts with the situation in all other investigated organisms . Several methylated bases were identified . 7-Methylguanine and 5-methylcytosine both occur in 17-S and 26-S rRNA . N6-Methyladenine and N6,N6-dimethyladenine are restricted to the 17-S rRNA while 3-methyluracil and 1-methyladenine occur in the 26-S rRNA . One hypermodified uridine was also tentatively identified in the small rRNA . In 17-S rRNA, there is one copy of 7-methylguanine, N6-methyladenine and hypermodified uridine and two copies of N6,N6-dimethyladenine . 3-Methyluracil, 1-methyladenine and 5-methylcytosine occur twice, twice and three times, respectively, in 26-S rRNA . 7-Methylguanine and 5-methylcytosine are only in submolar amounts in the 26-S and 17-S rRNA, respectively . There are 40 +/- 2 and 83 +/- 3 2'-O-methylriboses per 17-S and 26-S rRNA molecule, respectively . In addition to the four 2'-O-methylnucleosides, one 2'-O-methylpseudouridine is present in the 17-S rRNA . Several lines of evidence argues for a non-random distribution of the methylriboses . In particular, one and seven Nm-Nm-Np structures occur in the 17-S and 26-S rRNA, respectively . The data are discussed comparatively with the methylation pattern of Escherichia coli, yeast and HeLa cell rRNA. J Bacteriol, 1979 Jul, 139(1), 320 - 2 Orientation of the guanine operon of Escherichia coli K-12 by utilizing strains containing guaB-xse and guaB-upp deletions; Vales LD et al.; Temperature induction of an Escherichia coli strains with lambda cI1857 integrated in the guaB gene has been used to produce strains containing chromosomal deletions extending into the xse and upp genes . By utilizing strains containing these deletions, it has been possible to order the genes in the guanine operon with respect to the xseA and upp genes . The order of the genes in this region is glyA-hisS-xseA-guaO-guaB-guaA-purG-upp-purC. Proc Natl Acad Sci U S A, 1979 Jul, 76(7), 3411 - 5 Regulation of ribosomal protein synthesis in Escherichia coli by selective mRNA inactivation; Fallon AM et al.; In an Escherichia coli strain lysogenic for lambda spc2 transducing phage, an extra copy of ribosomal protein (r-protein) genes in the spc and alpha operons are carried on the phage chromosome . Expression of genes in the spc operon in this merodiploid strain was compared with that in a control "haploid" strain carrying lambda trkA phage . It was found that the synthesis rate of spc mRNA, relative to other reference mRNA in the merodiploid strain, is about 2-fold higher than that in the control strain; yet, no dosage effect was observed in the synthesis rate of r-proteins in the spc or alpha operon . The spc mRNA was found to be more rapidly degraded in the merodiploid strain than in the control strain, and its steady-state amount, relative to reference mRNA, was only slightly higher in the merodiploid strain than in the control strain . Thus, E . coli cells have the ability to regulate the rate of r-protein synthesis regardless of the rate of transcription of r-protein genes, presumably by inactivation of the mRNA followed by its degradation . A model is proposed which involves selective inactivation of r-protein mRNA by a feedback mechanism . The model can explain coordinated synthesis of r-proteins and other observations related to selective expression of certain alleles in diploid strains. J Bacteriol, 1979 Jul, 139(1), 185 - 94 Levels of major proteins of Escherichia coli during growth at different temperatures; Herendeen SL et al.; The adaptation of Escherichia coli B/r to temperature was studied by measuring the levels of 133 proteins (comprising 70% of the cell's protein mass) during balanced growth in rich medium at seven temperatures from 13.5 to 46 degrees C . The growth rate of this strain in either rich or minimal medium varies as a simple function of temperature with an Arrhenius constant of approximately 13,500 cal (ca . 56,500 J) per mol from 23 to 37 degrees C, the so-called normal range; above and below this range the growth rate decreases sharply . Analysis of the detailed results indicates that (i) metabolic coordination within the normal (Arrhenius) range is largely achieved by modulation of enzyme activity rather than amount; (ii) the restricted growth that occurs outside this range is accompanied by marked changes in the levels of most of these proteins; (iii) a few proteins are thermometer-like in varying simply with temperature over the whole temperature range irrespective of the influence of temperature on cell growth; and (iv) the temperature response of half of the proteins can be predicted from current information on their metabolic role or from their variation in level in different media at 37 degrees C. J Biochem Biophys Methods, 1979 Jul, 1(3), 145 - 51 13C NMR analysis of methionine sulfoxide in protein; Cohen JS et al.; The 13C epsilon NMR signal of methionine sulfoxide is 22.6 ppm downfield from that of methionine . This affords a method by which the extent of methionine oxidation can be determined in intact protein . We demonstrate the utility of this approach with beta-galactosidase enriched with 13C in its methionine methyls. Am J Vet Res, 1979 Jul, 40(7), 971 - 3 Prolactin and colibacillus-induced fluid transport in ligated intestinal segments; Mulloy AL et al.; Two experiments, using the ligated intestinal segment technique, were conducted to determine whether the pituitary hormone prolactin (PRL) could reduce Escherichia coli-induced fluid loss into the small intestine of 2- to 3-week-old pigs . Inoculation of 10(6) to 10(8) enteropathogenic E coli organisms into ligated jejunal segments caused a significant accumulation of luminal fluid within 12 hours . In the first experiment, intraluminal inoculation with 0.5 mg of ovine PRL along with the bacteria did not have any effect on fluid accumulation . Systemic IV treatment of the animals with 1.0 mg of ovine PRL at 3-hour intervals, beginning either immediately after or 9 to 10 hours before intestinal ligation, did not significantly (P less than 0.05) reduce fluid accumulation as compared with control animals . In the second experiment, IM administration of 100 microgram of thyrotropin-releasing hormone (TRH) at 3-hour intervals, beginning 6 hours before intestinal ligation, significantly (P less than 0.05) increased circulating PRL concentrations, as measured by radioimmunoassay . However, TRH treatment did not reduce the accumulation of luminal fluid in E coli-inoculated segments. Eur J Biochem, 1979 Jul, 97(2), 615 - 21 Chemical evidence for a codon-induced allosteric change in tRNALys involving the 7-methylguanosine residue 46; Wagner R et al.; {32P}TRNALys, from Escherichia coli, was modified with kethoxal, in the presence and absence of the oligonucleotide codon (A)4 . The presence of the codon resulted in a faster modification rate of the tRNA at three guanine sites which were identified by a diagonal fingerprint method . A large increase in the modification rate occurred at the 7-methylguanosine residue 46 (m7G-46) in the presence of the codon: weakly enhanced modification was observed at G-15 and G-57 . It is concluded that the formation of a codon-anticodon complex induces, primarily, a conformational change involving disruption of the m7G-46 from the m7G-46 . G-22 . C-13 base triple . Subsequently, the guanines of G-15 and G-57, in the D and T loops, respectively, become slightly more reactive, suggesting a weak tendency for these two interacting arms to unfold . The results are interpreted in terms of an equilibrium between two main conformers, and a third minor one; the possible significance of these conformers in protein biosynthesis, is considered. J Bacteriol, 1979 Jul, 139(1), 19 - 31 Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm; Bassford PJ Jr et al.; We have employed the technique of gene fusion to fuse the LacZ gene encoding the cytoplasmic enzyme beta-galactosidase with the malE gene encoding the periplasmic maltose binding protein (MBP) . Strains were obtained which synthesize malE-lacZ hybrid proteins of various sizes . These proteins have, at their amino terminus, a portion of the MBP and at their carboxyl terminus, enzymatically active beta-galactosidase . When the hybrid protein includes only a small, amino-terminal portion of the MBP, the hybrid protein residues in the cytoplasm . When the hybrid protein contains enough of the MBP to include an intact MBP signal sequence, a significant portion of the hybrid protein is found in the cytoplasmic membrane, suggesting that secretion of the hybrid protein has been initiated . However, in no case is the hybrid protein secreted into the periplasm, even when the hybrid protein includes almost the entire MBP . In the latter case, the synthesis and attempted export of the hybrid protein interferes with the export of at least certain normal envelope proteins, which accumulate in the cell in their precursor forms, and the cell dies . These results suggest that a number of envelope proteins may be exported at a common site, and that there are only a limited number of such sites . Also, these results indicate that it is not sufficient to simply attach an amino-terminal signal sequence to a polypeptide to assure its export. J Bacteriol, 1979 Jul, 139(1), 13 - 8 Sites within gene lacZ of Escherichia coli for formation of active hybrid beta-galactosidase molecules; Brickman E et al.; We describe the genetic analysis of 21 Escherichia coli strains in which the amino-terminal sequence of beta-galactosidase has been removed and replaced by an amino-terminal sequence from one or another of the proteins involved in maltose transport . Genetic mapping of the lacZ end of these fused genes indicates that only those fusions in which fewer than 41 amino acids are removed from the amino-terminal sequence of beta-galactosidase result in enzymatically active molecules . Within the region between amino acid 17 and amino acid 41 there are at least four or five sites where enzymatically active hybrid proteins can be formed. J Immunol, 1979 Jul, 123(1), 442 - 6 Specific inhibition of the antibody-mediated activation of a defective beta-D-galactosidase by circulating activating epitope-binding molecules; de Macario EC et al.; Activation of a defective Escherichia coli beta-D-galactosidase by specific activating antibody is inhibited competitively by a molecule with immunoglobulin properties but devoid of activating capacity . This molecule is found in the serum of nonimmunized rabbits and is no longer detectable after beta-D-galactosidase administration, but can be demonstrated in rabbits injected with antigens other than the enzyme . The data show that the inhibitory molecule recognizes and interacts specifically with the activating epitope of the activatable enzyme and that, although unable to activate the latter, it competes with the activating antibody and inhibits activation. Infect Immun, 1979 Jul, 25(1), 121 - 6 Immunization of calves against enterotoxigenic colibacillosis by vaccinating dams with purified K99 antigen and whole cell bacterins; Acres SD et al.; Pregnant cattle were either vaccinated subcutaneously with (i) a suspension of purified Escherichia coli K99 pili, (ii) a Formalin-killed whole cell bacterin containing enterotoxigenic E . coli strain B44 (O9:K30;K99:H-), or (iii) a bacterin containing six different strains of bovine enterotoxigenic E . coli (multiple-strain bacterin), or were left as nonvaccinated controls . After birth, calves were allowed to nurse their dams and, at 12 to 14 h of age, were challenged orally with 10(11) cells of enterotoxigenic E . coli strain B44 . Colostral antibody titers were determined against K99, K30, and O9 antigens of B44 . In the nonvaccinated control group, 9 of 10 calves developed diarrhea and died within 24 to 72 h . Similarly, all six calves in the multiple-strain bacterin group developed diarrhea and four died . In contrast to calves in the two groups mentioned above, calves nursing cows vaccinated with either purified K99 or the homologous whole cell bacterin were protected against fatal diarrhea . There was a highly significant correlation (P less than 0.0005) between protection against fatal diarrhea and K99, but not K30 or O9 colostral antibody titers . Vaccination of cows with either purified pili or whole cell preparations containing sufficient K99 antigen may provide a means of preventing enterotoxigenic colibacillosis in calves. J Bacteriol, 1979 Jul, 139(1), 161 - 4 Reduction of methionine sulfoxide to methionine by Escherichia coli; Ejiri SI et al.; L-Methionine-dl-sulfoxide can support the growth of an Escherichia coli methionine auxotroph, suggesting the presence of an enzyme(s) capable of reducing the sulfoxide to methionine . This was verified by showing that a cell-free extract of E . coli catalyzes the conversion of methionine sulfoxide to methionine . This reaction required reduced nicotinamide adenine dinucleotide phosphate and a generating system for this compound . The specific activity of the enzyme increased during logarithmic growth and was maximal when the culture attained a density of about 10(9) cells per ml. Biochemistry, 1979 Jun 26, 18(13), 2810 - 4 Isoleucyl transfer ribonucleic acid synthetase . Competitive inhibition with respect to transfer ribonucleic acid by blue dextran; Moe JG et al.; The inhibitory effects of blue dextran and a small dye molecule derived from it (F3GA-OH) on the steady-state reaction catalyzed by Escherichia coli isoleucy-tRNA synthetase have been studied . Blue dextran gave uncompetitive inhibition with respect to Mg.ATP, mixed inhibition with respect to L-isoleucine, and competitive inhibition with respect to tRNA . The small dye molecule (F3GA-OH) was also competitive with respect to tRNA . These inhibition patterns were not consistent with the bi-uni-uni-bi Ping Pong mechanism generally accepted for aminoacyl-tRNA synthetases . They were consistent with a mechanism in which a second L-isoleucine is bound after isoleucyl-AMP synthesis and before transfer of the isoleucyl moiety to tRNA . Enzyme-bound L-isoleucine lowered the affinity of the enzyme for blue dextran approximately fivefold, a value comparable to the ninefold lowering of the enzyme's affinity for tRNA upon binding L-isoleucine . The affinity of the synthetase for F3GA-OH (K1 = 1.0 X 10(-7) M) is approximately fivefold higher than its affinity for blue dextran (K1 = 5.3 X 10(-7) M) . These results indicate that blue dextran and its derivatives may be useful for kinetic and physical studies of polynucleotide binding sites on proteins as well as NAD and ATP sites. Biochemistry, 1979 Jun 26, 18(13), 2804 - 10 Isoleucyl transfer ribonucleic acid synthetase . Steady-state kinetic analysis; Moe JG et al.; A steady-state kinetic analysis was conducted of the overall aminoacylation reaction catalyzed by isoleucyl-tRNA synthetase . The patterns of Lineweaver-Burk plots obtained indicated that tRNA adds to the enzyme only after isoleucyl adenylate formation and pyrophosphate release . These kinetic patterns were consistent with the bi-uni-uni-bi Ping Pong mechanism generally accepted for this aminoacyl-tRNA synthetase, but they could also be accommodated by a mechanism in which a second molecule of L-isoleucine added to the enzyme between isoleucyl adenylate formation and aminoacylation of tRNA {Fersht, A.R., & Kaethner, M.M . (1976) Biochemistry 15, 818} . The values of the kinetic parameters favor the latter mechanism . The results of this kinetic analysis indicated that the affinity of isoleucyl-tRNA synthetase for Mg.ATP was enhanced upon binding of L-isoleucine and vice versa . It also indicated that the affinity of the enzyme for L-isoleucine is decreased upon binding tRNA and vice versa . The values of dissociation constants calculated for each of the substrates by this study generally compared well with those determined by other authors using a variety of kinetic and equilibrium methods. Biochemistry, 1979 Jun 26, 18(13), 2896 - 904 Magnesium ion dependent rabbit skeletal muscle myosin guanosine and thioguanosine triphosphatase mechanism and a novel guanosine diphosphatase reaction; Eccleston JF et al.; The mechanism of the Mg2+-dependent myosin subfragment 1 catalyzed hydrolysis of GTP and 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate (thioGTP) has been investigated by rapid-reaction techniques . The myosin was isolated from rabbit skeletal muscle . The steady-state intermediate of these reactions consists pre-dominantly of a protein-substrate complex unlike the myosin subfragment 1 ATPase reaction which has a protein-products complex as the principal steady-state component . The mechanism of GTP hydrolysis catalyzed by subfragment 1 has other marked differences from the ATPase mechanism . The second-order rate constant of binding of GTP to subfragment 1 is tenfold greater than that for GDP binding . The dissociation rate constant of GDP from subfragment 1 is 0.06 s-1 compared with the subfragment 1 catalytic center activity for GTP hydrolysis of 0.5 s-1 at pH 8.0 and 20 degrees C . This shows that GDP bound to subfragment 1 forms a complex which is not kinetically competent to be an intermediate of the GTPase mechanism . GDP is hydrolyzed in the presence of subfragment 1 to GMP and Pi . The subfragment 1 GTPase mechanism has a nuber if features in common with that of the elongation factor Tu GTPase of the protein biosynthetic system of Escherichia coli. Nucleic Acids Res, 1979 Jun 25, 6(8), 2717 - 29 Studies on the binding of the ribosomal protein complex L7/12-L10 and protein L11 to the 5'-one third of 23S RNA: a functional centre of the 50S subunit; Dijk J et al.; The RNA binding sites of the protein complex of L7/12 dimers and L10, and of protein L11, occur within the 5'-one third of 23S RNA . Binding of the L7/12-L10 protein complex to the 23S RNA is stimulated by protein L11 and vice-versa . This is the second example to be established of mutual stimulation of RNA binding by two ribosomal proteins or protein complexes, and suggests that this may be an important principle governing ribosomal protein-RNA assembly . When the L7/12-L10 complex is bound to the RNA, L10 becomes strongly resistant to trypsin . Since the L7/12 dimer does not bind specifically to the 23S RNA, this suggests that L10 constitutes a major RNA binding site of the protein complex . Only one of the L7/12 dimers is bound strongly in the (L7/12-L10)-23S RNA complex; the other can dissociate with no concurrent loss of L10. Nucleic Acids Res, 1979 Jun 25, 6(8), 2697 - 705 Entrapment of plasmid DNA in liposomes; Dimitriadis GJ; The entrapment of plasmid DNA (pMB9) and high molecular weight DNA into large unilamellar liposomes is described . The entrapment of DNA is specific and due to encapsulation of DNA into the aqueous compartment of liposomes . The entrapped Dna, resistant to deoxyribonuclease treatment, could be reisolated from liposomes intact and, as has been shown by transformation assay, it remains biologically active . The advantages of our method and possible applications are discussed. J Biol Chem, 1979 Jun 25, 254(12), 5548 - 54 Influence of A-T content on the fractionation of DNA restriction fragments by RPC-5 column chromatography; Patient RK et al.; The properties of the fractionated Hae III fragments of pRZ2 DNA (Patient, R.K., Hardies, S.C., and Wells, R.D . (1979) J . Biol . Chem . 254, 5542-5547) were studied in an effort to determine why several of the fragments bind more tightly to RPC-5 than expected on the basis of their length . The purified fragments were analyzed for their nucleotide composition by direct determination of their constituent mononucleotides and by analytical CsCl and Cs2SO4 density gradient analyses . A-T-rich fragments elute at higher salt concentrations than fragments of equivalent size which are not A-T-rich . In addition, denaturation mapping studies by electron microscopy indicate that an A-T-rich run within an otherwise G-C-rich fragment can give rise to delayed elution . At least one other factor influences the separation of DNA restriction fragments by RPC-5 chromatography . Some of the fragments in this digest which elute later than predicted from their size either contain known genetic regulatory sites or bind regulatory proteins. J Biol Chem, 1979 Jun 25, 254(12), 5542 - 7 Identification of regions of pRZ2 which have delayed elution behavior on RPC-5 column chromatography; Patient RK et al.; DNA restriction fragments generally elute from RPC-5 in order of their size . However, some fragments elute later than predicted . Chromatographic studies were performed on five different restriction digests (Hae III, Hha I, Alu I, Taq I, and Hae III + HindII) of pRZ2 DNA in an effort to localize the regions which have the delayed properties . Also, the magnitude of the delay was quantitated in each case . Most of the delayed fragments were localized in one major (931 bp) and one minor (approximately 210 bp) region of the genome . The fragments exhibiting a greater extent of delay were in the major region . The results described herein and in the following paper show that, in most cases, this effect can be explained by the base composition, or sequence of the fragments, or both. J Biol Chem, 1979 Jun 25, 254(12), 5483 - 7 Role of the spoT gene product and manganese ion in the metabolism of guanosine 5'-diphosphate 3'-diphosphate in Escherichia coli; Johnson GS et al.; Addition of divalent ion chelating agents picolinic acid, 1,10-phenanthroline, or quinoline-2-carboxylic acid to wild type, relA, or relX, but not spoT strains of Escherichia coli increases the levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) . Poorly chelating analogs of these agents and a larger and more highly charged chelating agent, ethylene glycol bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid are ineffective . Mn2+ reverses the increase in ppGpp . The increase in ppGpp in wild type cells can be explained by an inhibition of degradation . In spoT cells the response is more complex; ppGpp does not increase although degradation is completely inhibited . The lack of increase in spoT cells suggests a role for spoT in synthesis of ppGpp in addition to its known role in degradation . Growth of both spoT+ and spoT cells is inhibited following chelator addition . This suggests that growth inhibition is through a mechanism not directly involving ppGpp . The results of this study provide evidence in intact cells for a role for Mn2+ and the spoT gene product in ppGpp degradation, and provide further evidence for an involvement of spoT and possibly divalent ions in ppGpp synthesis. J Biol Chem, 1979 Jun 25, 254(12), 5128 - 34 Chemical modification of lactose repressor protein using N-substituted maleimides; Brown RD et al.; Lactose repressor protein has been modified with N-ethylmaleimide, two N-maleimide spin labels, and an N-maleimide fluorophore . The reaction with repressor cysteine residues has been characterized . Approximately 2 of the 3 eq of cysteine/repressor monomer are reactive toward these reagents . Repressor cysteines are reactive toward these reagents in the order cysteine 140 greater than or equal to cysteine 107 greater than cysteine 281 . The reaction is sulfhydryl-specific . Comparison of chemical modification data obtained in this laboratory using a variety of sulfhydryl-specific reagents has been used to assess chemical features of individual cysteine environments . Effects of the maleimide reagents on biological activity have been determined . Only the fluorophore N-(3-pyrene)maleimide has significant effect; this agent selectively perturbs repressor's ability to bind to operator DNA . This result suggests that regions of protein structure surrounding 1 or more of the cysteine residues possess determinants required for normal operator DNA binding. J Biol Chem, 1979 Jun 25, 254(12), 5257 - 63 Size and molecular parameters of adenosine triphosphatase from Escherichia coli; Paradies HH et al.; The Mg2+- and Ca2+-stimulated ATPase (bacterial coupling factor) has been investigated in solution with different independent techniques . The molecular weight of the five-subunit enzyme was found to be 345,000 +/- 5,000 by means of light scattering, 350,000 by sedimentation equilibrium experiments, and 358,000 by means of small-angle x-ray scattering . The radius of gyration was found to be 41.9 A, the volume 7.39 x 10(5) A3, and the surface to volume ratio 5.5 x 10(-2) A-1 from small-angle x-ray scattering measurements of the enzyme in solution . The degree of hydration was found to be 0.62 ml of H2O/g of ATPase . The translational diffusion coefficient was determined to be 3.47 x 10(-7) cm2 s-1 by means of inelastic light scattering . The distribution of the scattered intensity near the origin appears to be bimodal, suggesting that the ATPase molecule is composed of spherical parts bound together by a flexible polypeptide chain . The largest dimension of the ATPase in solution is 120.0 A, determined from the pair distribution function. J Biol Chem, 1979 Jun 25, 254(12), 5562 - 6 The purification and sequence of a temperature-sensitive tryptophan tRNA; Eisenberg SP et al.; Escherichia coli can be temperature-sensitive due to a lesion in the gene for tRNATrp (Yanofsky, C., and Soll, L . (1977) J . Mol . Biol . 113, 663-677) . Purification of tRNATrp from this strain (temperature-sensitive tRNATrp) was achieved by one of two methods . Either a combination of benzoylated DEAE-cellulose column chromatography and two-dimensional polyacrylamide gel electrophoresis, or hybridization to plasmid DNA covalently bound to cellulose (this is a recombinant plasmid carrying the gene for tRNATrp) and electrophoresis of the eluted material on a 10% polyacrylamide gel, produced isotopically pure tRNA . The sequence of the temperature-sensitive tRNATrp was determined by standard methods . We find that the sequence differs from that of wild type tRNATrp by a single residue; G in position 7 (G7) in wild type tRNATrp is A7 in temperature-sensitive tRNATrp . This base difference results in one less base pair in the CCA stem of the temperature-sensitive species . The effect of this base change in the in vitro and in vivo properties of tRNATrp (presented elsewhere (S.P . Eisenberg and M . Yarus, manuscript in preparation.)) are discussed. Biochim Biophys Acta, 1979 Jun 20, 563(1), 171 - 80 Sedimentation behavior of chloroplast ribosomes from Chlamydomonas reinhardtii; Margulies MM et al.; The identity of peaks generated by chloroplast ribosomes of Chlamydomonas reinhardtii were determined by zone velocity sedimentation on sucrose density gradients, and analysis of distribution of ribosomal RNAs in the gradients . The sedimentagion coefficient of the principal peak was 66-70 S (usually 69 S), in good agreement with previously reported values for chloroplast ribosomes of C . reinhardtii, and other organisms . The fast sedimenting side of the 69 S peak contained an excess of chloroplast large subunit . When ribosome dissociation was prevented by sedimentation at low velocity, by aldehyde fixation, or by the presence of nascent polypeptide chains, the principal peak had a sedimentation coefficient of about 75 S . Thus the 69 S peak was an artifact caused by dissociation during centrifugation . Peaks that contained chloroplast ribosomal RNAs were also observed at '60 S' and '45 S' when chloroplast ribosomes were centrifuged unfixed at high velocity . The amounts of '60 S' and '45 S' components were decreased by centrifugation at low speed, or fixation, but sedimentation coefficients remained unchanged . The '60 S', and '45 S' components were identified as large, and small subunits of chloroplast ribosomes, respectively . The artifacts produced by centrifugation of chloroplast ribosomes, are similar to the artifacts produced by centrifuging ribosomes of Escherichia coli . Similar explanations appear to apply to both . We concluded that the 69 S chloroplast ribosome peak occurs because of dissociation of 'tight' couples, and incomplete separation of subunits . Subunit peaks (60 S and 45 S) arise from free subunits, and/or from dissociation of 'loose' couples. Biochim Biophys Acta, 1979 Jun 20, 563(1), 17 - 27 Escherichia coli mut T1 . II . Consequences of modification on the association of DNA with the cell membrane; Campana T et al.; 1 . Two isogenic strains of Escherichia coli, K-12 which differ by mutator gene character (mut T1) have been studied . This characteristic caused introduction of a high frequency of undirectional transversions, A-T leads to -CG, into the DNA of the strain harboring it . 2 . It had been previously shown that the presence of this gene is accompanied by an alteration of a cell membrane component . Now, the nuclease susceptibility of DNA associated with membrane/DNA/DNA polymerase complexes is reported . DNA of mut T1 membranes is more sensitive towards exogenous nuclease than DNA of membrane complexes from the wild type mut+ strain . 3 . Auto-digestion of this DNA by endogenous nuclease associated with the membrane complex is, also, more severe in preparations derived from mut T1 than from the wild-type strain, mut+, but to a lesser extent than observed with exogenous nucleases . 4 . Nuclease susceptibility of mut+ membrane bound DNA is markedly influenced by the growth state of the cell . The nuclease susceptibility of membrane bound DNA from mut T1 cells, however, shows no differences between stationary and log states . 5 . These differential sensitivities may be due to conformational changes in the membrane introduced as a pleiotrophic consequence of an altered membrane protein . A pertinent role of this protein in a modified replication/repair complex is an attractive suggestion, especially in the context of the mutator character of this strain. Biochim Biophys Acta, 1979 Jun 20, 563(1), 163 - 70 The effect of magnesium starvation on the dissociation of ribosomal proteins from Escherichia coli K-12 ribosomes; Adachi K et al.; The effect of magnesium starvation upon the fate of individual ribosomal proteins was studied in Escherichia coli . During a 21 h incubation in the absence of Mg2+ the 30 S subunit was more susceptible to degradation, retaining an average 31.9% of its ribosomal proteins as compared to 40.0% for the 50 S subunit . An examination of those 50-S proteins dissociated to a lesser extent than the average value (L1, L2, L3, L7, L10, L13, L16, L17, L19, L21, L22, L23, and L29) revealed that, with the exception of L16, all were classified by Dohme and Nierhaus {5} as tightly bound . Of the ribosomal proteins dissocated during magnesium starvation only five were reincorporated (and these to a minimal degree) during recovery of cells in a medium containing Mg2+ . These studies suggest that ribosomal proteins once released from the ribosome particles during magnesium starvation are not reutilized in the assembly of new subunits.
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