Proc Natl Acad Sci U S A, 1981 Mar, 78(3), 1773 - 7 Depurination causes mutations in SOS-induced cells; Schaaper RM et al.; Introduction of apurinic sites into phi X174 am3 DNA leads to loss of biological activity when measured in a transfection assay . For single-stranded DNA, approximately one apurinic site constitutes a lethal hit; for double-stranded (RFI) DNA, approximately 3.5 hits per strand are lethal . When the reversion frequency of am3 DNA is measured, no increase due to depurination is observed above the background level . However, a large increase in reversion frequency is observed when the same DNA is assayed by using spheroplasts derived from bacteria previously exposed to UV light . The results suggest that apurinic sites are impediments to a replicating DNA polymerase; however, nucleotides can be incorporated opposite these sites under SOS-induced conditions . We estimate the frequency of mutagenesis per apurinic site to be less than 1 in 1400 in normal spheroplasts and 1 in 100 in SOS-induced spheroplasts. Proc Natl Acad Sci U S A, 1981 Mar, 78(3), 1485 - 9 Activation of plasminogen to plasmin by a protease associated with the outer membrane of Escherichia coli; Leytus SP et al.; Preparations of outer membrane of two strains of Escherichia coli contain a protease that can activate the serum zymogen plasminogen to the active protease plasmin . The amount of plasmin formed is proportional to the membrane concentration . The kinetics of plasminogen activation are linear and obey the Michaelis--Menten rate equation . The Km(app) for the activation of dog plasminogen by E . coli outer membrane preparations is similar to the Km(app) for the activation of dog plasminogen by human urokinase . The E . coli enzyme is active in a membrane-associated form, as opposed to a secreted or soluble form, and is most likely a serine protease because it is inhibited by diisopropyl fluorophosphate . It is also inhibited from activating plasminogen by p-nitrophenyl-p-guanidinobenzoate and aprotinin . Analysis of the activation of plasminogen by the E . coli enzyme by NaDodSO4/polyacrylamide gel electrophoresis showed that the cleavage of plasminogen to plasmin was as specific as that exhibited in the activation of plasminogen to plasmin by urokinase . Possible in vivo roles for this plasminogen activator in E . coli outer membranes are discussed. J Gen Microbiol, 1981 Mar, 123(Pt 1), 187 - 91 Molecular cloning in plasmid pBR322 giving altered expression of the tetracycline resistance gene; Bastin M; The two HindIII fragments of polyoma virus DNA were cloned in the HindIII site of plasmid pBR322, a site located in the RNA polymerase promoter involved in the expression of tetracycline resistance . Although insertion of foreign DNA into this site did not always result in the complete loss of tetracycline resistance, Escherichia coli K12 strain chi 1776 harbouring recombinant plasmids exhibited reduced growth properties in liquid culture with tetracycline and could easily be differentiated from bacteria transformed by non-recombinant plasmids . The formation of plasmid multimers increased the resistance to tetracycline at the level of the induction period, presumably as a result of a gene dosage effect. Biull Eksp Biol Med, 1981 Mar, 91(3), 347 - 50 {Compatibility between F-like genetic transfer factors}; Reshetnikova VN et al.; A study has been made of compatibility among four F-like factors of genetic transfer (pAP22-4, pAP38, pAP39 and pAP41) labeled separately by transpozones Tn1 and Tn9 . It has been established that pAP38 transfer factor is compatible with plasmids pAP22-4, pAP39 and pAP412, while pAP41 transfer factor is compatible with plasmids pAP22-4 and pAP38 but is incompatible with plasmid pAP39. Antimicrob Agents Chemother, 1981 Mar, 19(3), 487 - 9 Filament formation of Fusobacterium nucleatum cells induced by mecillinam; Onoe T et al.; Subinhibitory concentrations of mecillinam transformed Fusobacterium nucleatum cells into a marked filament form, quite different from a spherical form demonstrated in Escherichia coli. Gene, 1981 Mar, 13(2), 203 - 8 Cloning of Epstein-Barr virus (EBV) DNA fragments in pBR32 and Charon3A; Yano S et al.; Restriction fragments of Epstein-Barr virus (EBV; B95-8) DNA were cloned in the Tc gene of pBR322 (HindIII-F, -G, -I, -J, -K, -L, and -M) and in Charon3A (EcoRI-GI and -G2) . Altogether these cloned fragments covered 39% of the entire viral genome . The cloned EcoRI-G2 fragment of EBV (B95-8) DNA was shown to contain, in addition to HindIII-J, two more HindIII-fragments : HindIII-M, which had not been located on the linkage map of the viral genome (Given and Kieff, 1978) and HindIII-N, which had been unrecognized up to now . The utility of this cloning method is discussed in regard to the detailed mapping of a viral genome and large-scale production of the viral DNA. Gene, 1981 Mar, 13(2), 173 - 84 In vivo correlation between DNA supercoiling and transcription; Kano Y et al.; The superhelical density of pMT plasmid DNA in Escherichia coli cells was measured as a function of the transcriptional activity, which was reduced by treatment with coumermycin or oxolinic acid . Superhelicity was assayed by agarose gel electrophoresis of DNA extracted from cells gently lysed by sarkosyl . Coumermycin treatment reduced the proportion of supercoiled plasmid DNA in concert with a decrease in the rate of plasmid-coded synthesis of trp mRNA, implying a correlation between supercoiling of DNA and its suitability for transcription . On the other hand, the oxolinic acid-induced loss of supercoiled plasmid DNA was relatively small, while concomitant inhibition of trp mRNA synthesis was very severe . Treatment of the cells with these two drugs never removed all of supertwists from the pMT plasmids carried. Eur J Biochem, 1981 Mar, 114(3), 517 - 24 Transfer RNA precursors are accumulated in Escherichia coli in the absence of RNase E; Ray BK et al.; A temperature-sensitive Escherichia coli mutant, which contains a heat-labile RNase E, fails to produce 5-S rRNA at a non-permissive temperature . It accumulates a number of RNA molecules in the 4-12-S range . One of these molecules, a 9-S RNA, is a precursor to 5-S rRNA {Ghora, B . K . and Apirion, D . (1978) Cell, 15, 1055-1056} . These molecules were purified and processed in a cell-free system . Some of these RNA molecules, after processing, give rise to products the size of transfer RNA, but not to 5-S-rRNA . Further characterization of the processed products of one such precursor molecule shows that it contains tRNA1Leu and tRNA1His . RNase E is necessary but not sufficient for the processing of this molecule to mature tRNAs in vitro . The accumulation of such tRNA precursors in an RNase E mutant cell and the obligatory participation of RNase E in its processing indicate that RNase E functions in the maturation of transfer RNAs as well as of 5-S rRNA. Proc Natl Acad Sci U S A, 1981 Mar, 78(3), 1619 - 23 Effects of methylation on a synthetic polynucleotide: the B--Z transition in poly(dG-m5dC).poly(dG-m5dC); Behe M et al.; We have compared the behavior in solution of the synthetic polynucleotide poly(dG-m5dC).poly(dG-m5dC) with that of the unmethylated polynucleotide poly(dG-dC).poly(dG-dC) . In solutions containing high concentrations of salt, poly(dG-dC).poly(dG-dC) is known to exhibit altered circular dichroic and absorption spectra correlated with formation of a left-handed Z DNA structure . Poly(dG-m5dC).poly(dG-m5dC) behaves similarly, but the spectral transition from the B to the Z form occurs at much lower salt concentrations, close to usual physiological conditions . Divalent and polyvalent ions are particularly effective: The B--Z transition of poly(dG-m5dC).(dG-m5dC) can be induced at a Mg2+ concentration three orders of magnitude lower than that required for the unmethylated polymer . We have also studied mixed copolymers containing both dC and m5dC . Our results suggest that the sequence m5dC-dG, which occurs in eukaryotic DNA, can have a disproportionately large effect on the B--Z transition. Proc Natl Acad Sci U S A, 1981 Mar, 78(3), 1436 - 40 Conservation of the primosome in successive stages of phi X174 DNA replication; Low RL et al.; Synthesis of a complementary strand to match the single-stranded, circular, viral (+) DNA strand of phage phi X174 creates a parental duplex circle (replicative form, RF) . This synthesis is initiated by the assembly and action of a priming system, called the primosome {Arai, K . & Kornberg, A (1981) Proc . Natl . Acad . Sci . USA 78, 69-73; Arai, K., Low, R . L . & Kornberg, A . (1981) Proc . Natl . Acad . Sci . USA 78, 707-711} . Of the seven proteins that participate in the assembly and function of the primosome, most all of the components remain even after the DNA duplex is completed and covalently sealed . Remarkably, the primosome in the isolated RF obviates the need for supercoiling of RF by DNA gyrase, an action previously considered essential for the site-specific cleavage by gene A protein that starts viral strand synthesis in the second stage of phi X174 DNA replication . Finally, priming of the synthesis of complementary strands on the nascent viral strands to produce many copies of progeny RF utilizes the same primosome, requiring the addition only of prepriming protein i . thus a single primosome, which becomes associated with the incoming viral DNA in the initial stage of replication, may function repeatedly in the initiation of complementary strands at the subsequent stage of RF multiplication . These patterns of phi X174 DNA replication suggest that a conserved primosome also functions in the progress of the replicating fork of the Escherichia coli chromosome, particularly in initiating the synthesis of nascent (Okazaki) fragments. Proc Natl Acad Sci U S A, 1981 Mar, 78(3), 1416 - 20 Contacts between DNA gyrase and its binding site on DNA: features of symmetry and asymmetry revealed by protection from nucleases; Morrison A et al.; DNA gyrase supercoils DNA by passing one DNA segment through another by means of a reversible double-strand break at specific DNA sites . We determined the nucleotide sequence of two highly preferred gyrase binding sites and analyzed the grip of gyrase on the DNA by using protection from nuclease attack . The DNA-breakage site of gyrase was centered in about 50 base pairs (bp) of DNA that was completely protected from DNase I and flanked by DNA regions cut at average intervals of 9.9 bases . The same pattern of protection from DNase I was observed with topoisomerase II', an enzyme that shares structural homology with gyrase . The gyrase site of DNA breakage was off-center in the 140 bp of DNA protected from exonuclease III digestion . ATP or inhibitors of gyrase had little specific effect on DNase I protection . On addition of a nonhydrolyzable analogue of ATP, previously stable barriers to exonuclease III were invaded and new barriers appeared . We discuss a detailed model uniting these results with previous data on gyrase structure and mechanism. Proc Natl Acad Sci U S A, 1981 Mar, 78(3), 1381 - 5 Mapping a eukaryotic promoter: a DNA sequence required for in vivo expression of adenovirus pre-early functions; Osborne TF et al.; This study defines a DNA sequence upstream from the mRNA cap site required for in vivo expression of the adenovirus 2 pre-early region . The adenovirus 2 pre-early region and flanking sequences were cloned in Escherichia coli plasmid pBR322 . Derivatives of the plasmid lacking portions of the upstream viral DNA sequence were constructed . An assay was devised to test the ability of these plasmid DNAs to complement an adenovirus 5 mutant with a deletion in the pre-early region . Plasmids that retained at least 38 base pairs upstream from the mRNA cap site had complementing activity similar to that of the original plasmid, which contains 229 base pairs of upstream viral sequence . However, plasmids retaining 23 or fewer base pairs of viral sequence upstream from the cap site had significantly reduced complementing activity . These results indicate that a portion of the adenovirus 2 sequence between 23 and 38 base pairs upstream from the mRNA cap site is required for expression of the pre-early region . This interval includes the Goldberg-Hogness box-like sequence T-A-T-T-T-A-T-A. J Bacteriol, 1981 Mar, 145(3), 1374 - 85 Adenylate energy charge in Escherichia coli CR341T28 and properties of heat-sensitive adenylate kinase; Glembotski CC et al.; Escherichia coli strain CR341T28 will not grow at temperatures above 34 degrees C in liquid medium, and the adenylate kinase of this strain is heat sensitive . When a culture was shifted from a permissive (30 degrees C) to a nonpermissive (36 degrees C) temperature, the adenylate energy charge fell from 0.9 to 0.2, with a concurrent decrease in the number of viable cells and in the specific activity of adenylate kinase . When cultures of the temperature-sensitive strain were grown at temperatures above 30 degrees C, the adenylate energy charge, the specific activity of adenylate kinase, and the growth rate were lower than the corresponding parameters for the parental strain . By isotopic labeling of the adenine nucleotides in vivo, it was determined that increasing growth temperatures between 30 and 34 degrees C for the heat-sensitive strain resulted in a decrease in the adenosine triphosphate-to-adenosine monophosphate and adenosine triphosphate-to-adenosine diphosphate ratios . Between 26 and 30 degrees C the adenosine triphosphate-to-adenosine diphosphate ratio was essentially normal in the temperature-sensitive strain, but the adenosine triphosphate-to-adenosine diphosphate ratio was decreased . The adenylate ratios in the parental strain did not change between 30 and 34 degrees C . The adenylate kinase mass action ratio for each strain was essentially constant under all growth conditions . When assayed at 30 degrees C, the affinities of the enzyme from the mutant strain were somewhat lower than those of the parent adenylate kinase . The mutant enzyme also did not exhibit the substrate inhibition that was observed at high adenosine monophosphate concentrations with the parental enzyme . An increase in the assay temperature from 30 degrees to 40 degrees C had little or no effect on the Km values determined for the parental adenylate kinase, but caused the Km values determined for the mutant adenylate kinase to increase by a factor of two or more. J Bacteriol, 1981 Mar, 145(3), 1305 - 9 Homology between Escherichia coli plasmids ColE1 and p15A; Bird RE; The location and extent of the homology between plasmids ColE1 and p15A were determined by analysis of heteroduplexes formed between them as well as with a related plasmid, pBR322, and by hybridization of radioactive deoxyribonucleic acids to restriction fragments of p15A and ColE1 . The homology between the plasmids contained the entire region of ColE1 required for its replication as well as an additional 400 base pairs downstream from the origin of replication . This region on p15A, which was 980 +/- 43 base pairs, started at 0.1 of the molecular length from one end formed by cleavage with the restriction endonuclease BglI and extended to 0.54 of the molecular length from the same end . Restriction cleavage maps for the enzymes BglI, HpaI, HaeII, HaeIII, and HincII are also presented. J Bacteriol, 1981 Mar, 145(3), 1273 - 80 Purification and characterization of cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase; Ray PH et al.; Cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) was purified 2,300-fold from frozen Escherichia coli B cells . The enzyme catalyzed the formation of CMP-KDO, a very labile product, from CTP and KDO . No other sugar tested could replace KDO as an alternate substrate . Uridine 5'-triphosphate at pH 9.5 and deoxycytidine 5'-triphosphate at pH 8.0 and 9.5 could be used as alternate substrates in place of CTP . CMP-KDO synthetase required Mg2+ at a concentration of 10.0 mM for optimal activity . The pH optimum was determined to be between 9.6 and 9.3 in tris(hydroxymethyl)aminomethane-acetate or sodium-glycine buffer . This enzyme had an isoelectric point between pH 4.15 and 4.4 and appeared to be a single polypeptide chain with a molecular weight of 36,000 to 40,000 . The apparent Km values for CTP and KDO in the presence of 10.0 mM Mg2+ were determined to be 2.0 X 10(-4) and 2.9 X 10(-4) M, respectively, at pH 9.5 . Uridine 5'-triphosphate and deoxycytidine 5'-triphosphate had apparent Km values of 8.8 X 10(-4) and 3.4 X 10(-4) M . respectively, at pH 9.5. J Bacteriol, 1981 Mar, 145(3), 1257 - 65 Reexamination of the genome size of myxobacteria, including the use of a new method for genome size analysis; Yee T et al.; The genome sizes of two myxobacteria, Myxococcus xanthus and Stigmatella aurantiaca, were measured by renaturation analysis and also by a new method involving the quantitation of individual restriction fragments . In contrast to several previous reports, which indicate that M . xanthus has a genome size which is three to four times that of Escherichia coli, the present measurements indicated that the M . xanthus genome is only about 24 to 53% larger than that of E . coli . S . aurantiaca had a genome size nearly identical to that of M . xanthus . Of possible significance is the fact that the renaturation curves of M . xanthus and S . aurantiaca deoxyribonucleic acid both exhibited significant fractions which renatured with rapid, unimolecular kinetics . However, we were unable to establish that these fractions represented inverted repeats of repetitive sequences. Vopr Med Khim, 1981 Mar-Apr, 27(2), 207 - 10 {Influence of S-methylmethionine on the metabolism of nucleic acids in Escherichia coli}; Lebenka AIu et al.; The influence of S-methylmethionine (SMM) on metabolism of nucleic acids in E . coli MRE-600 has been studied . The influence of SMM was compared with that of methionine . Addition of SMM to the medium at the concentration of 2.7 . 10(-5) M increased the incorporation of 5-methyl-3H-thymidine into DNA by about 20% and the incorporation of 2-14C-uridine into the total RNA of the cells by about 47% . The electrophoretic separation of RNA in polyacrylamide gels showed that SMM stimulated the incorporation of the label into 4 S, 5 S, 16 S and 23 S RNA with different intensity . The highest stimulation of the label incorporation has been observed in 16 S RNA. Cell Biophys, 1981 Mar, 3(1), 89 - 104 A general interactive model for B cell activation II . Experimental verification; Rosenspire AJ et al.; The general interactive model described in the previous paper was subjected to experimental testing . We examined the high affinity anti-TNP plaque-forming cell response of cultures of murine lymphocytes exposed to lipopolysaccharide and trinitrophenylated lipopolysaccharide (TNP--LPS) . From our model we predicted the effects of the addition of free LPS on the dose response to TNP--LPS, the effect of the addition of Polymyxin B to cultures stimulated with TNP--LPS, and the effect on the response to TNP--LPS preparations with different haptenation ratios . The results obtained were consistent with the predictions of the general interactive model. Gene, 1981 Mar, 13(2), 145 - 52 Cloning of alpha 2u globulin cDNA using a high efficiency technique for the cloning of trace messenger RNAs; Kurtz DT et al.; An extremely high-efficiency technique is described for cloning double-stranded (ds) cDNAs in Escherichia coli . The method, which uses two synthetic oligonucleotide linkers rather than one, results in approx . 200--500 recombinant clones per ng of ds cDNA . This technique was used to clone a cDNA comprising 95% of the full length of the mRNA of alpha 2u globulin, a male rat liver protein, which represents approx . 1% of hepatic messenger RNA . The cloned probe was applied to study the complex hormone controls of alpha 2u globulin mRNA in male and female rats. J Gen Microbiol, 1981 Mar, 123(Pt 1), 27 - 37 Dual control of the gua operon of Escherichia coli K12 by adenine and guanine nucleotides; Mehra RK et al.; The gua operon of Escherichia coli K12 comprises structural genes for the two enzymes, IMP dehydrogenase and GMP synthetase, required for the biosynthesis of GMP from IMP . The specific activities of these enzymes were measured in various purine auxotrophs . GuaA and guaB mutants (guanine-specific) were depressed under conditions of growth limitation by guanine but were repressed by excess guanine . This suggests that formation of the enzymes is normally controlled by a guanine nucleotide . Derepression of the operon in purine-starved pur mutants depended on the type of mutant and on whether adenine or guanine was provided . A purA strain (adenine-specific) and strains with early blocks in purine biosynthesis (purF and purD) did not derepress . PurE or purC strains {5'-phosphoribosyl-5-aminoimidazole (AIR)-accumulating} derepressed only 4-fold . The operon was repressed in purH strains {5'-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICAR)-accumulating} grown with limiting guanine or hypoxanthine, but derepressed by growth with limiting adenine . Two mutants (purA guaA and purA guaB) which can neither synthesize AMP and GMP de novo, nor interconvert them, were isolated . The specific activity of IMP dehydrogenase in one of these strains grown with different concentrations of guanine and adenine revealed that adenine induces tha gua operon whereas guanine represses it . Intracellular purine nucleotide pools wee measured in a purH mutant repressed (guanine-grown) and derepressed (adenine-grown) for IMP dehydrogenase . The guanylate pool was similar under the two growth conditions; however the adenylate pool of the adenine-grown bacteria was two to three times greater than that of the guanine-grown cells . A dual mechanism for regulating expression of the gua operon, involving induction by AMP and repression by GMP, is proposed. Biochim Biophys Acta, 1981 Feb 26, 652(2), 303 - 13 Factor-dependent dissociation of wheat germ ribosomes; Goldstein LA et al.; Ribosome dissociation factor has been found in wheat germ acetone powder extracts . Further purification of the crude extract by pH an ammonium sulfate fractionations, DEAE-cellulose and CM-Sephadex column chromatography has resulted in the separation of two active fractions . The possibility that ribosome dissociation activity exhibited by either fraction is due to protease or nuclease is considered unlikely, based on results of experiments involving ribosome dissociation kinetics, subunit structural integrity, and treatment with a serine protease inhibitor . Wheat germ ribosome dissociation factor is not species-specific . Dissociation factor from both fractions will promote the dissociation of Escherichia coli 70-S as well as Artemia salina 80-S ribosomes . Although both dissociation factor activities show the same dependence on K+ and Mg2+ for optimal activity, the two activities exhibit significant differences in their sensitivity to sulfhydryl reagents and heat, and in their dependence on incubation temperature for activity . Certain properties of both factors suggest that neither factor is initiation factor eIF-3; however, the possibility that one or both factors are subunits of initiation factor eIF-3 remains to be determined. Mol Cell Biochem, 1981 Feb 26, 35(1), 3 - 10 On heterogeneity of DNA methylases from Escherichia coli SK cells; Nikolskaya II et al.; The presence of E . coli SK cells of five different DNA-methylases differing in specificity to the methylated sequence is documented has been proven . Two enzymes methylate cytosine with the formation of 5'-methylcytosine and three enzymes methylate adenine with formation of 6'-methylaminopurine . A method for simultaneous isolation of the five individual enzymes including gel filtration on Biogel A-0.5 M is proposed . The direct evidence has been presented showing that the additional methylation test in our method modification actually can discriminate between enzymes differing in sensitive sites. Biochim Biophys Acta, 1981 Feb 26, 652(2), 294 - 302 The interaction of RNA polymerase and DNA . Effects on the helix-coil transition and light scattering; Reisbig RR et al.; To characterize the interactions of RNA polymerase with DNA, we have investigated the thermal transition of poly{d(A-T} bound to RNA polymerase from Escherichia coli and the aggregation properties of the enzyme with DNA . The melting curve of the DNA-enzyme complex demonstrates a sharply lowered melting temperature for part of the DNA, whereas for another fraction the double helix is stabilized . This indicates that the DNA-binding site of RNA polymerase serves two functions: (1) to disrupt the double helix at one point, and (2) to maintain the duplex form at other points . The aggregation of DNA and RNA polymerase has been monitored by turbidity measurements, and conditions have been delineated under which aggregation is minimized . Holoenzyme added to double-stranded DNA or single-stranded DNA has little or no tendency to aggregate under most conditions . Core enzyme, on the other hand, aggregate extensively with double-stranded DNA, the only under conditions of low salt (10 mM KCl), without Mg2+, or at high salt (300 mM KCl), with or without Mg2+, can this aggregation be eliminated . Core enzyme also does not aggregate in the presence of single-stranded DNA . These aggregation properties are interpreted as evidence for more than one DNA-binding site on RNA polymerase. Biochim Biophys Acta, 1981 Feb 26, 652(2), 256 - 60 Iodination of Escherichia coli ribosomal protein L18 abolishes its 5 S RNA binding activity; Fanning TG et al.; Iodination of Escherichia coli ribosomal protein L18 inactivated the 5 S RNA binding activity of the protein . Complete activity loss occurred at a 4-fold molar excess of iodine to L18 . Tyrosine was found to be the reactive amino acid . L18, prebound to 5 S RNA, was inactivated at a much slower rate than unbound L18 . Treatment of L18 with tetranitromethane also resulted in an inactivation of the protein . However, much larger amounts of tetranitromethane, compared to iodine, were necessary to achieve inactivation (50% activity loss at a 600-fold molar excess of tetranitromethane to L18). Biochim Biophys Acta, 1981 Feb 26, 652(2), 274 - 82 Purification and properties of DNA polymerase from Mycobacterium tuberculosis H37Rv; Hiriyanna KT et al.; DNA polymerase has been purified approximately 2000-fold from Mycobacterium tuberculosis H37Rv . The purified preparation was homogeneous by electrophoretic criteria and has a molecular weight of 135 000 . The purified enzyme resembles Escherichia coli polymerase I in its properties, being insensitive to sulfhydryl drugs and possessing 5',3'-exonuclease activity in addition to polymerase and 3',5'-exonuclease activities . However, it differs from the latter in its sensitivity to higher salt concentration and DNA intercalating agents such as 8-aminoquinoline . The polymerase exhibited maximal activity between 37--42 degrees C and pH 8.8--9.5 . The polymerase was stable for several months below 0 degree C . However, the 5',3'-exonuclease activity was more labile . The effects of different metal ions, polyamines and drugs on the polymerase activity are presented. Nucleic Acids Res, 1981 Feb 25, 9(4), 993 - 1004 Topography of the C . coli 5S RNA-protein complex as determined by crosslinking with dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate; Fanning TG et al.; 5S RNA-protein complexes were prepared in vitro using partially purified E . coli 5S RNA and total E . coli 70S ribosomal proteins . The complexes were isolated from sucrose gradients and shown to contain proteins L5, L18, L25 and a fourth protein not heretofore characterized and designed L31 . The complexes were treated with the crosslinking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate . Both reagents gave identical patterns of crosslinked proteins when analyzed by one-dimensional polyacrylamide/dodecylsulfate gel electrophoresis . Dimers of L5-L31', L5-L18 and L18-L18 and a trimer containing L5, L18 and L31' were identified by diagonal polyacrylamide/dodecylsulfate gel electrophoresis of the proteins crosslinked with dimethyl-3,3'-dithiobispropionimidate . No crosslinking was detected between L25 and the other three proteins. J Biol Chem, 1981 Feb 25, 256(4), 2010 - 5 In vitro thermal inactivation of a temperature-sensitive sigma subunit mutant (rpoD800) of Escherichia coli RNA polymerase proceeds by aggregation; Lowe PA et al.; A temperature-sensitive mutant sigma subunit (rpoD800) purified from Escherichia coli was inactivated in vitro by temperatures in excess of 37 degrees C whereas wild type sigma remained stable up to 49 degrees C . Both temperature-sensitive and wild type sigma formed multimeric aggregates upon thermal inactivation which were visualized by electron microscopy as polymeric chains . Conditions favoring sigma monomer (low sigma concentration and binding to core polymerase) protected temperature-sensitive sigma from heat inactivation . Full activity was recovered from inactivated temperature-sensitive sigma aggregates by incubation in a buffer containing 6 M guanidine HCl and subsequent removal of denaturant by dilution . Both wild type and temperature-sensitive sigma recovered full activity levels, retaining their characteristic thermal inactivation temperatures after denaturation in 6 M guanidine HCl and renaturation . Transcription of T4 DNA by RNA polymerase containing the rpoD800 mutant sigma subunit remained undiminished for 10 min after shift up to 46 degrees C but was almost completely inhibited within the following 10 to 15 min. J Biol Chem, 1981 Feb 25, 256(4), 1738 - 47 Effect of a single amino acid substitution on Escherichia coli dihydrofolate reductase catalysis and ligand binding; Baccanari DP et al.; The two isozymes of dihydrofolate reductase (Forms 1 and 2) from, a Trimethoprim-resistant strain of Escherichia coli (RT500) were separated and purified to homogeneity using a simple procedure based on differential elution from a Methotrexate affinity column . The complete amino acid sequence of the Form 2 isozyme was determined, and it differs from that of Form 1 in only one position . Residue 28 is arginine in Form 2 and leucine in Form 1 . However, the isozymes differ greatly in their binding and kinetic properties . Equilibrium dialysis studies showed the Trimethoprim dissociation constants of Form 2 are about 50-fold greater than those of Form 1 in both the binary complex and the ternary complex with NADPH . Similarly, the Methotrexate dissociation constant of Form 2 is about 10-fold greater than that of Form 1 . The two isozymes also differ in their turnover numbers at pH 7 (Form 1 is 10-fold more active) and inhibition by divalent cations . Form 1 is extremely sensitive to BaCl2 (50% inhibition at 0.5 mM), whereas Form 2 is much less sensitive (50% inhibition at 60 mM) . In the presence of 10 mM BaCl2, Form 1 has the functional characteristics of Form 2 . Its turnover number is decreased, its Trimethoprim Ki is increased, and the shape of its pH-activity profile is identical with that of Form 2 . The x-ray structures and amino acid sequences of several bacterial dihydrofolate reductases indicate that Asp-27 is important in inhibitor binding and may be involved in catalysis . The present data provide kinetic evidence for this hypothesis, and it is proposed that almost all the unusual characteristics of Form 2 are the direct result of a charge interaction between Arg-28 and Asp-27 . A similar interaction between Ba2+ and the Asp-27 of Form 1 can result in an enzyme complex that is kinetically similar to Form 2. J Biol Chem, 1981 Feb 25, 256(4), 1809 - 15 L-threonine dehydrogenase . Purification and properties of the homogeneous enzyme from Escherichia coli K-12; Boylan SA et al.; L-Threonine dehydrogenase, which catalyzes the conversion of L-threonine to aminoacetone + CO2 presumably via the intermediate formation of alpha-amino-beta-ketobutyrate, has been purified to apparent homogeneity from extracts of a mutant of Escherichia coli K-12 which has constitutively derepressed levels of the enzyme . Three fractionation steps were used including controlled heat denaturation, DEAE-Sephadex chromatography, and blue dextran-Sepharose affinity chromatography . The purified enzyme migrated as a single band, coincident with dehydrogenase activity, when electrophoresed on polyacrylamide gels at pH 8.0 and 9.5 . Electrophoresis in 1% sodium dodecyl sulfate also showed one band and a single schlieren peak was seen during sedimentation velocity centrifugation . The enzyme has an apparent molecular weight of 140,000 +/- 4,000 as determined by sucrose density and sedimentation equilibrium centrifugation . Based on electrophoresis in 1% sodium dodecyl sulfate, sedimentation equilibrium centrifugation in 6 M guanidine.HCl, and cross-linking with dimethyl suberimidate, the molecule is a tetramer consisting of identical (or nearly identical) subunits with Mr approximately equal to 35,000 . L-Threonine dehydrogenase is specific for NAD+ or NAD+ analogs and utilizes L-threonine, D-allothreonine, or L-threonine amide as the best substrates . In 50 mM Tris.HCl buffer (pH 8.4) and 37 degrees C, the Km values for L-threonine and NAD+ are 1.43 and 0.19 mM, respectively . The enzyme has a pH optimum of 10.3, is activated by Mn2+, and shows a substantial loss of activity when treated with certain sulfhydryl-reacting reagents. Nucleic Acids Res, 1981 Feb 25, 9(4), 753 - 67 Enzymatic synthesis, ligation, and restriction of DNA containing deoxy-4-thiothymidine; Hofer B et al.; Phage fd RF I DNA1 about 90% substituted by deoxy-4-thiothymidine (s4Td) in the codogenic strand was synthesized by the simultaneous actions of DNA polymerase I and DNA ligase . While the rate of DNA synthesis was considerably reduced, the yield the rate of DNA synthesis was considerably reduced, the yield was not affected in the presence of s4TdTP . The conversion of RF II to RF I DNA by DNA ligase was even improved . This effect seems to be related with an altered ratio of affinity of polymerase and ligase for the s4Td-containing substrate . The presence of the base analogue in the DNA was verified independently by chromatographic and spectroscopic methods . The modified genome could be cleaved by restriction endonucleases Hpa II (C/CGG)d and Taq I (T/CGA)d . A number of the fragments produced showed altered mobilities under the conditions of polyacrylamide gel electrophoresis. J Biol Chem, 1981 Feb 25, 256(4), 1903 - 9 Spatial proximity of two divalent metal ions at the active site of S-adenosylmethionine synthetase; Markham GD; S-Adenosylmethionine synthetase from Escherichia coli is shown to require 2 divalent metal ions/enzyme subunit for maximal enzymatic activity . In the absence of substrate, the tetrameric enzyme binds 1 Mn(II) ion/subunit, whereas in the presence of a nucleotide substrate, adenylylimidodiphosphate, or the product pyrophosphate, there are two Mn(II)-binding sites/subunit . Electron paramagnetic resonance spectra of Mn(II) bound to the enzyme reveal a spin exchange interaction between 2 Mn(II) ions in complexes of enzyme and Mn(II) which also contain adenosylmethionine, K+, and either pyrophosphate or imidotriphosphate . Since a spin exchange interaction requires orbital overlap between the 2 ions, the metal ions must be bound close to one another, and they may share a common ligand. J Biol Chem, 1981 Feb 25, 256(4), 1896 - 902 Functional inactivation of lac alpha-peptide mRNA by a factor that purifies that Escherichia coli RNase III; Shen V et al.; Using RNA-directed synthesis of the alpha-peptide of beta-galactosidase as an assay, a factor was purified that inactivated further function of the mRNA . In the presence of Ca2+ ions to inhibit most nuclease activity, inactivation of mRNA occurred during incubation with ribosomes or with a 1 M KCl wash of ribosomes . The inactivation activity required Mg2+ ions, and purified as a single factor which did not bind to DEAE-cellulose, but bound reversibly to phosphocellulose . The factor eluted from Sephadex G-150 with an apparent molecular weight of about 43,000 . Purified 700-fold, it showed no detectable exonuclease activity, and little or no cleavage of a variety of single-stranded substrates, including full length lac operon mRNA; but repurified inactivated mRNA was still inactive for protein synthesis . The factor did not inhibit poly(U)-directed polyphenylalanine synthesis . When proteins isolated from the ribosomal wash were individually tested, highly purified RNase III, which purifies in the same way and has the same size, also inactivated lac mRNA . The ribosomal wash from an RNase III- strain showed little if any activity compared to that from an isogenic RNase III+ strain . The possibility of a site-specific inactivating cleavage of mRNA by RNase III at or near the 5' end is considered. J Biol Chem, 1981 Feb 25, 256(4), 1636 - 42 Purification and properties of an endodeoxyribonuclease from nuclei of bovine small intestinal mucosa; Nakayama J et al.; An endodeoxyribonuclease has been purified from nuclei of bovine small intestinal mucosa to a homogeneous state by a procedure involving affinity chromatography on heparin-agarose . The endonuclease, which was found to be bound to chromatin, has a pH optimum of 5.4 . It requires Mn2+ or Co2+ for activity and its maximum activity with Mg2+ is about 80% of that with Mn2+ . Its activity is strongly inhibited by sulfhydryl-blocking agents, and by ethidium bromide . The enzyme does not attack RNA and is inhibited by it . Its isoelectric point is 8.5 +/- 0.1, and its molecular weight is 49,000 +/- 3,000, determined by sucrose gradient sedimentation and gel filtration on Sephadex G-100 . Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two nonidentical subunits with molecular weights of 30,000 and 23,000 . The enzyme catalyzes the endonucleolytic cleavage of circular duplex ColE1 DNA via single strand scissions from the initial stage of degradation . The average size of the limit products of native phage T7 or ColE1 DNA is about 2,000 to 1,500 base pairs, estimated by neutral sucrose gradient sedimentation or agarose gel electrophoresis . The enzyme degrades denatured DNA about 20 times faster than native DNA . The products contain 5'-phosphoryl and 3'-hydroxyl termini, and all four deoxymononucleotides are present in almost equal amounts at the 5'-termini. Nucleic Acids Res, 1981 Feb 25, 9(4), 935 - 46 Use of a cloned double stranded cDNA coding for a major androgen dependent protein in rat seminal vesicle secretion: the effect of testosterone in gene expression; Mansson PE et al.; The abundant class of poly(A+)RNA {poly(A+)RNA11S} from rat seminal vesicle was used to synthesize ds-cDNA11S . The ds-cDNA11S was inserted and cloned into the Pst I site of pBR-322 using E . coli RR1 as host . Colony filter hybridization and restriction mapping was used to demonstrate that a 620 NTP long insert in a plasmid clone (pSV2) represents the almost full length structural gene coding for a precursor to the seminal vesicle secretion protein IV (SVS IV) . The entire insert was sequenced and the coding region was matched with the known amino acid sequence . Most of the signal peptide sequence was derived from the DNA sequence . The insert in pSV2 was labelled and used to study the effect of testosterone on the accumulation of mRNA SVS IV . Administration of testosterone to castrated rats resulted in the induction of mRNA SVS IV from a few molecules per cell to levels of over 100,000 after 96 h of hormone treatment. J Biol Chem, 1981 Feb 25, 256(4), 1954 - 9 The evolution of alpha-fetoprotein and albumin . I . A comparison of the primary amino acid sequences of mammalian alpha-fetoprotein and albumin; Gorin MB et al.; The amino acid sequence of mouse alpha-fetoprotein has been deduced from the nucleotide sequence of its mRNA and three chimeric plasmids containing overlapping segments of its cDNA . A comparison of the amino acid sequence with that of either human and bovine albumin reveals in each case a 32% conservation of primary sequence . In addition, using the regularly spaced positions of cystine bridges, a 2-dimensional structure was generated, which revealed the presence of 3 closely related domains within alpha-fetoprotein . The structures of these domains are identical with the triplicated domains previously observed in several mammalian albumins . These homologies lend strong circumstantial evidence to the proposal that these two proteins arose in evolution as the consequence of a duplication in a common tripartite ancestral gene. Thromb Haemost, 1981 Feb 23, 45(1), 65 - 7 Plasma prekallikrein and endotoxemia in liver cirrhosis; van Vliet AC et al.; In liver disease low prekallikrein levels may be found which has been suggested to be due to diminished synthesis . However, it may also be due to endotoxemia accompanying liver disease . To study the last possiblity prekallikrein, endotoxins and Normotest were determined in 18 cirrhosis patients . The relation between the prekallikrein concentration (after 15 min activation) and the Normotest was significant (r = + 0.72, P less than 0.001) . Endotoxemia was only found in the more severe forms of liver disease (Normotest below 60%) . During endotoxemia the prekallikrein levels were significantly lower than when no endotoxins were present in the blood of the same patients . The Normotest did not differ significantly in these patients in relation to the presence or absence of endotoxins . The activation of prekallikrein was slower in the more severe forms of liver disease . This might be due to reduced levels of factor XII and high molecular weight kininogen . In conclusion the reduced prekallikrein level in liver cirrhosis may be due to both diminished synthesis and endotoxemia . In the more severe forms of liver disease the time necessary to activate prekallikrein is increased. Nature, 1981 Feb 19, 289(5799), 694 - 6 No need for a new membrane model; Jahnig F; Schindler et al . recently reported lateral diffusion measurements in reconstituted membranes of phospholipid (PL), lipoplysaccharide (LPS) and Escherichia coli matrix protein (P), using the technique of fluorescence recovery after photobleaching (FRAP) . Evaluation of their data led Schindler et al . to conclude that the fluid mosaic model is an inadequate description of the membrane and to propose a new membrane model . Their conclusion was based on identifying the fluid mosaic model with a particular binding behaviour of LPS to matrix protein . I present here a more general model for the association of membrane components, and demonstrate the use of lateral diffusion data in elucidating membrane structure . The data of Schindler et al . are shown to be reasonably interpretable on the basis of an association of LPS and matrix protein, which obviates the necessity for postulating a new membrane model. Biochemistry, 1981 Feb 17, 20(4), 977 - 82 Methyl-accepting chemotaxis proteins of Escherichia coli: methylated at three sites in a single tryptic fragment; Chelsky D et al.; The location of the sites of methylation of the methyl-accepting chemotaxis proteins (MCP) of Escherichia coli has been investigated by trypsin digestion and mapping of the peptides . The two principal MCPs, MCP I and MCP II, were found to have very similar methyl-labeled peptide patterns . Each produced three major species of methylated peptide . Both MCPs migrate on high-resolution sodium dodecyl sulfate--polyacrylamide slab gels a a cluster of discrete protein bands . Each band produced one of the three major methylated peptides as the unique or dominant species . Progressive demethylation of the least acidic peptide produced, sequentially, the intermediate and most acidic peptides before complete loss of the methyl label . This is consistent with a single peptide backbone carrying from one to three methyl groups . The possible significance of three methyl-accepting sites in a single tryptic peptide is discussed. Biochemistry, 1981 Feb 17, 20(4), 874 - 9 Kinetics of substrate, coenzyme, and inhibitor binding to Escherichia coli dihydrofolate reductase; Cayley PJ et al.; Reduced nicotinamide adenine dinucleotide phosphate (NADPH), folate, dihydrofolate, and the inhibitors trimethoprim and methotrexate bind rapidly and reversibly to both dihydrofolate reductase isoenzymes isolated from Escherichia coli RT500 . The coenzyme and substrates appear to bind to only one of the mixture of two forms of the isoenzyme present at equilibrium, while the inhibitors bind to both forms . The proportions of the two forms are different for the two isoenzymes and are pH dependent in each case . The measured association rate constants for substrates and inhibitors lie in the range (1--2) x 10(-7) M-1 s-1 at 25 degrees C but are unlikely to be diffusion controlled . The rate constant for NADPH binding is 2 x 10(6) M-1 s-1 . The formation of binary complexes takes place through a multistep mechanism . A minimum of three steps is required to explain the kinetic results . An equilibrium between two or more forms of the enzyme--ligand complex governs the overall dissociation . The stability of this equilibrium is largely responsible for the tighter binding of inhibitors relative to substrate or coenzyme and also for the different binding strengths of inhibitors to the isoenzymes. Biochemistry, 1981 Feb 17, 20(4), 868 - 73 Kinetics and protein subunit interactions of Escherichia coli phosphatidylserine decarboxylase in detergent solution; Rizzolo LJ; Phosphatidylserine decarboxylase from Escherichia coli, an intrinsic membrane protein, catalyzes the conversion of phosphatidylserine to phosphatidylethanolamine . The physical and kinetic properties of the purified enzyme were studied in several detergents under assay conditions . The active form of the enzyme is an oligomer, probably a trimer, and the enzyme activity was unaffected by the concentration of the nonionic poly(oxyethylene) ether detergent present in the assay medium, so long as the detergent micelle/substrate mole ratio was less than one . When this ratio was greater than one, the detergent acted as an inhibitor by competing with enzyme-containing micelles for substrate . The zwitterionic and bile salt detergents that were tested inactivated the enzyme by dissociating the oligomer . The native, Triton X-100 solubilized, enzyme was modified with a cross-linking reagent . Activity of the cross-linked enzyme was retained after the Triton X-100 was replaced by a zwitterionic sulfobetaine detergent and conformed to the same kinetic model as with the poly(oxyethylene) ether detergents . The cross-linked enzyme was also active when solubilized by the bile salt detergents although the activity did not conform to any simple kinetic model . These data indicate that the oligomer is the active form of the enzyme under assay conditions and that certain nondenaturing detergents can inactivate this enzyme by dissociating the enzyme complex. Biochemistry, 1981 Feb 17, 20(4), 861 - 7 Detection of ligand-induced conformational changes in phenylalanyl-tRNA synthetase of Escherichia coli K10 by laser light scattering; Holler E et al.; The diffusion constant of phenylalanyl-tRNA synthetase has been measured by laser light scattering under conditions of complex formation with Mg2+, L-phenylalanine, MgATP, tRNAPhe, modified tRNAPhe, tRNAPhe (yeast), and noncognate tRNA . The diffusion constant (pH 7.5, 20 degrees C) of the free enzyme is (2.85 +/- 0.005) x 10(-7) cm2 s-1, of the enzyme . Mg2+ complex (2.40 +/- 0.05) x 10(-7) cm2 s-1 and of the enzyme . Mg2+ . tRNAPhe complex (2.95 +/- 0.06) x 10(-7) cm2 s-1 . The effect of tRNAPhe is only seen when the enzyme is saturated with Mg2+ . The smaller substrates exhibit no effect besides a small increase of the value of the diffusion constant under conditions where the enzyme-phenylalanyladenylate is synthesized . Of the noncognate tRNATyr and tRNAIle, the latter is able to associate with the enzyme, causing the value of the diffusion constant to increase . tRNAPhe (yeast) and tRNAhvPhe (photo-cross-linked tRNAPhe) exhibit similar effects . The observed variation of the diffusion constant is attributed to conformational changes of the enzyme . The opposite effects of Mg2+ and tRNAPhe are interpreted as an expansion and recontraction, respectively, of the enzyme molecule . In several cases, the effects were used to follow a titration of the enzyme with a ligand . Dissociation constants were calculated from the resulting titration curves, yielding values which are in agreement with those obtained by other techniques . It is established by comparison that of the two possible binding sites for each Mg2+ and tRNAPhe the diffusion constant reflects occupation of only a single class of sites. Biochemistry, 1981 Feb 17, 20(4), 823 - 9 High-resolution nuclear magnetic resonance studies of the Lac repressor . 2 . Partial analysis of the aliphatic region of the Lac repressor headpiece spectrum; Ribeiro AA et al.; The 360-MHz 1H NMR spectrum of native lac repressor headpiece (HP-51 or HP-59) contains a large number (greater than 30%) of aliphatic side-chain methyl and backbone alpha-CH resonances and three of four aromatic tyrosine multiplet resonances shifted to high-field chemical shift positions, indicating the presence of extensive folded structure . Denaturation leads to loss of the NMR chemical shift differences . Resonance identifications of the 27 methyl-possessing amino acids in HP-59 have been made by using resolution enhancement, double-resonance, and difference spectra . There are three firmly assigned methyl resonances and 21 pairwise identifications of methyl resonances in HP-51 . Comparison of HP-51 and HP-59 allows identification of four additional methyl groups in amino acid residues 52--59 . The sequence HP-50--59 is not essential to maintain the structure of HP-59, but it is of interest itself as the flexible hinge portion connecting HP to the tetrameric core of whole repressor. Biochemistry, 1981 Feb 17, 20(4), 818 - 23 High-resolution nuclear magnetic resonance studies of the Lac repressor . 1 . Assignments of tyrosine resonances in the N-terminal headpiece; Ribeiro AA et al.; The DNA binding site of the lac repressor protein has been implicated to lie within the N-terminal 51 amino acid fragment termed headpiece (HP-51 or LR-51) . High-resolution NMR suggests that isolated HP-51 retains most of the secondary and tertiary structure which it has in the whole repressor . Four of the eight tyrosines of repressor are in HP-51 . 1H NMR spectra (360 MHz) over the aromatic region of native HP-51 show that the four tyrosines are nonequivalent with an unusual distribution of chemical shifts . Denaturation leads to loss of these chemical shift differences . Homonuclear decoupling and a two-dimensional autocorrelated spectrum allow unequivocal pairing of resonances from Tyr A at 6.99 and 6.79 ppm, Tyr B at 6.98 and 6.39 ppm, Tyr C at 6.70 and 6.54 ppm, and Tyr D at 6.39 and 6.33 ppm . The 2,6 protons are low field of the 3,5 protons for each Tyr residue . Selective chemical modification with nitration reagents allows assignments of Tyr A to Tyr-47, Tyr B to Tyr-7, Tyr C to Tyr-12, and Tyr D to Tyr-17 in HP-51 . All four tyrosines are essential for maintaining the structure of the isolated headpiece, and Tyr-7, -12, and -17 appear to be stacked. Biochemistry, 1981 Feb 17, 20(4), 784 - 92 Urea-induced unfolding of the alpha subunit of tryptophan synthase: evidence for a multistate process; Matthews CR et al.; The urea-induced unfolding of the alpha subunit of tryptophan synthase from E . coli was monitored by optical spectroscopy and by urea-gradient gel electrophoresis . Three independent lines of evidence support the conclusion that one or more stable intermediates are present in this process: (i) Satisfactory fits of the equilibrium unfolding transitions obtained from difference spectroscopy at 286 nm and circular dichroism spectroscopy at 222 nm require a model which involves a stable intermediate in addition to the native and unfolded forms . (ii) Kinetic studies of the change in the extinction coefficient at 286 nm show that while the unfolding is well described by a single exponential change the refolding kinetics are complex . The nature of the dependence of the refolding kinetics on the initial concentration of urea supports the conclusion that at least one stable intermediate exists . (iii) The patterns obtained from urea-gradient gel electrophoresis experiments on the alpha subunit show that at least one and possibly two stable intermediates are involved; the intermediates have markedly different degrees of compactness . A kinetic model for the folding of the alpha subunit, consistent with all of these results, can be formulated. Biochemistry, 1981 Feb 17, 20(4), 750 - 4 Identification of the cis-thymine glycol moiety in oxidized deoxyribonucleic acid; Frenkel K et al.; 5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) is formed in DNA by reaction with oxidizing agents and as a result of ionizing and near-ultraviolet radiation . We describe a rapid purification of cis-5,6-dihydroxy-5,6-dihydrothymine and cis-5,6-dihydroxy-5,6-dihydrothymidine (cis-thymidine glycol) and their use as markers in identifying the thymine glycol moiety in oxidized DNA . Both glycols were prepared by oxidation of {14C}thymine and -thymidine with KMnO4 followed by purification on Sephadex LH-20 (LH-20) . {3H}DNA was oxidized with KMnO4 and the thymidine glycol in DNA identified by enzymatic digestion of the DNA followed by cochromatography of the digest with marker {14C}thymidine glycol on LH-20 . The cis conformation of the glycol was confirmed by the change in the elution pattern when borate rather than water was used as eluent . Alkaline hydrolysis of a mixture of {14C}thymine glycol and oxidized {3H}DNA followed by trichloroacetic acid precipitation and LH-20 chromatographic analysis of the neutralized supernatant yielded a complex pattern of radioactive degradation products with coincidence of one 14C marker- and one {3H}-DNA-derived peak . All applied radioactivity was recovered . This methodology should be useful in determining thymine glycol content of irradiated DNA and in elucidating the mechanism by which these altered residues are removed from cellular DNA by repair enzymes. Biochemistry, 1981 Feb 17, 20(4), 1020 - 5 Structural analysis of ribosomal protein L7/L12 by the heterobifunctional cross-linker 4-(6-formyl-3-azidophenoxy)butyrimidate; Maassen JA et al.; The structure of the dimeric form of the protein L7/L12 from ribosomes from Escherichia coli was studied by using the heterobifunctional cross-linker 4-(6-formyl-3-azidophenoxy)butyrimidate . The imidate group of the cross-linker reacts very specifically with Lys-51 of L7/L12 . Subsequent cross-linking of this modified L7/L12 by reductive alkylation of the aldehyde group of the cross-linker results in the formation of a covalent cross-link between both polypeptide chains of the L7/L12 dimer . This covalently cross-linked dimer is fully active in reconstitution of elongation factor G dependent GTP hydrolysis of 50S cores lacking L7/L12, suggesting a conformation of the cross-linked protein similar to the conformation of native L7/L12 . Analysis of the tryptic peptides of cross-linked L7/L12 shows the points of attachment of the cross-linker to be Lys-51 in one polypeptide chain and Lys-29 in the other . On the basis of a combination of this result with published data, a structure for the N-terminal region of L7/L12 dimers is proposed . The important feature of this model is a shifted parallel alignment of both polypeptide chains resulting in one free N-terminal stretch for each L7/L12 dimer which attaches the protein to the ribosome via protein L10. Biochem J, 1981 Feb 15, 194(2), 395 - 406 Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential . The cytochromes b-c segment; Papa S et al.; 1 . A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles . 2 . Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562 . Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562 . At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired . 3 . At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562 . Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566 . 4 . Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562 . The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values . 5 . Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566 . This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles . 6 . In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity . The transmembrane pH gradient (delta pH) on the other hand, decrease slightly . 7 . The results presented provide evidence that the delta psi component of the aerobic delta microH+ (the sum of the proton chemical and electrical activities) exerts a pH-dependent constraint on forward electron flow from cytochrome b566 to cytochrome b562 . This effect is explained as a consequence of anisotropic location of cytochromes b566 and b562 in the membrane and the pH-dependence of the redox function of these cytochromes . Transmembrane delta pH, on the other hand, exerts control on electron flow from cytochrome b562 to c cytochromes. Nature, 1981 Feb 12, 289(5798), 606 - 7 Assignment of the disulphide bonds of leukocyte interferon; Wetzel R; Nucleotide sequencing of cloned cDNA can lead to rapid and precise preduction of the primary amino acid sequence of gene products, but it cannot establish such post-translational modifications as the existence and arrangement of disulphide bonds . For example, the complete sequences of at least one fibroblast and seven leukocyte interferon genes are already known, but knowledge derived from analysis of the proteins is confined to information on the N-termini and some tryptic fragments . The presence of at least one disulphide bond in leukocyte interferon is suggested by that molecule's sensitivity to reducing agents . In addition, comparison of all leukocyte interferon gene sequences so far reported indicates four highly conserved cysteines . One of these genes has been engineered for efficient direct expression in Escherichia coli and we have purified the gene product, leukocyte interferon A (IFN-alpha A, Fig . 1) from bacterial extracts to a single species of molecular weight (MW) 19,400 . I report here the determination of the disulphide bonds of the purified protein by analysis of tryptic fragments . The results indicate that Cys 1 is bonded to Cys 98, and Cys 29 is bonded to Cys 138. Nucleic Acids Res, 1981 Feb 11, 9(3), 647 - 61 Escherichia coli ribosome unfolding in low Mg2+ solutions observed by laser Raman spectroscopy and electron microscopy; King TC et al.; Ribosomes unfolded by the removal of Mg2+ at 25 degrees C were studied by Raman spectroscopy and electron microscopy . Raman spectra showed a reduction in the 813 cm-1 phosphodiester signal of 30S and 50S ribosomes compared to intact ribosomes, suggesting that a fraction of the ribose moieties had shifted from the 3' endo (ordered) to the 3' exo (disordered) conformation . The maximum diameters of unfolded 30S and 50S ribosomes, judged by electron microscopy, were 1.8 and 2.5-fold greater, respectively, than those of intact ribosomes . Most unfolded 30S ribosomes had three distinct structural domains and appeared "Y-shaped"; whereas most unfolded 50S ribosomes had four distinct domains and appeared "X-shaped" . When ribosomes were partially unfolded (by brief exposure to 0.04 mM Mg2+ or EDTA), several possible intermediates in the unfolding process were observed . Both the shapes of particles and their Raman spectra reached the same final state in 0.04 mM Mg2+, where more than 50% of the rRNA phosphates are discharged by Mg2+, as in 10 mM EDTA, where less than 1% are discharged. Nucleic Acids Res, 1981 Feb 11, 9(3), 563 - 77 Rho-independent termination: dyad symmetry in DNA causes RNA polymerase to pause during transcription in vitro; Farnham PJ et al.; Termination of transcription by RNA polymerase at rho-independent sites appears to depend primarily upon two structural features, a region of GC-rich dyad symmetry in the DNA preceding the stop point and a stretch of uridines at the 3' end of the transcript . The possibility that the former might be responsible for slowing elongation prompted us to perform a kinetic analysis of transcription across the leader and terminator regions of the E . coli tryptophan (trp) operon . Regions where the elongation rate is dramatically slowed or stopped are identifiable because they generate discrete transcript hands on a gel . Species derived from pause sites, unlike those resulting from termination sites, are transient and detectable only within the first two minutes of transcription, since polymerase eventually resumes elongation . At two mutant trp attenuator sites (trp a135 and trp a1419), where termination is incomplete or absent in vitro, a substantial pause is nevertheless observed . Likewise, a significant pause occurs at trp t, the termination site at the end of the operon . Our experiments also reveal a major pause site at about position 90 in the trp leader sequence, just past a region of dyad symmetry . The RNA hairpin corresponding to this site is U-rich, and pausing is strongly enhanced by incorporation of BrUTP . In contrast, this analog does not affect pausing at the attenuator or terminator sites with hairpins that are GC-rich . These results strongly support the hypothesis that pausing of the polymerase is an obligatory prelude to rho-independent termination . Moreover, the termination event evidently results from consecutive but discrete responses to separate structural features of these sites. Nucleic Acids Res, 1981 Feb 11, 9(3), 489 - 501 The nucleotide sequence of the very low density lipoprotein II mRNA from chicken; Wieringa B et al.; The nucleotide sequence of an almost complete, double-stranded cDNA of chicken Very Low Density Lipoprotein II mRNA, carried in recombinant plasmid pVLDLII 3.33 (Wieringa et al., 1979, 7: 2147-2163) is presented . A stretch of 318 nucleotides codes for the pre-VLDLII polypeptide, which consists of a 24 amino acids signal and a 82 amino acids secreted protein . The coding stretch is flanked by 57 nucleotides in the 5'-leader sequence of the mRNA, and 258 nucleotides in the 3'-non-coding region . Hypothetical self-complementary structures of parts of the mRNA are presented. Nucleic Acids Res, 1981 Feb 11, 9(3), 529 - 43 Organization of the lexA gene of Escherichia coli and nucleotide sequence of the regulatory region; Miki T et al.; The product of the lexA gene of Escherichia coli has been shown to regulate expression of the several cellular functions (SOS functions) induced by treatments which abruptly inhibit DNA synthesis . We have cloned and mapped the lexA gene on a small segment of approximately 600 base pairs . The lexA promotor was located by transcription R-loop analysis, and the lexA product of 22,000 daltons was identified by protein synthesis in vitro . An unknown gene was found which directed the synthesis of a protein of 35,000 daltons in a region downstream from the lexA gene . Nucleotide sequence of the regulatory region of the lexA gene was determined . The sequence contained inverted repeats homologous to that of the recA regulatory region . These inverted repeats may be recognized by the lexA protein, because the protein is considered to repress both the genes as a common repressor. Nucleic Acids Res, 1981 Feb 11, 9(3), 503 - 17 Isolation, characterization and restriction endonuclease mapping of the Petunia hybrida chloroplast DNA; Bovenberg WA et al.; A procedure is developed for the isolation of intact chloroplast DNA (ctDNA) from Petunia hybrida . The molecular weight, calculated from contour length measurements, is 96.0 +/- 4.5 x 10(6) daltons . This value is in good agreement with the value of 101.2 x 10(6) daltons that was estimated from the electrophoretic mobilities of restriction endonuclease fragments of ctDNA . Analysis of petunia ctDNA in neutral CsCl gradients revealed the presence of only one type of DNA at a buoyant density of 1.6987 +/- 0.0005 gcm-3 . This corresponds with a GC-content of 39.3 +/- 0.5% . A physical map of petunia ctDNA was constructed by using the restriction endonucleases Sal I, Bgl I, Hpa I and Kpn I . The map indicates that petunia ctDNA contains two copies of a sequence in an inverted orientation . The inverted repeat regions have a minimum length of 10 x 10(6) daltons . Hybridization data indicate that part of the inverted repeat regions contain the genes for chloroplast ribosomal RNAs. Biochemistry, 1981 Feb 3, 20(3), 610 - 7 Diffusion-enhanced energy transfer shows accessibility of ribonucleic acid polymerase inhibitor binding sites; Meares CF et al.; Rifamycin and Cibacron Blue F3GA are powerful inhibitors of Escherichia coli deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase . In addition, both inhibitors strongly absorb visible light, making them suitable for use as acceptors in energy-transfer experiments . Transfer of energy to these acceptors from freely diffusing energy donors with long excited-state lifetimes (approximately 10(-3) s) depends strongly on whether donor and acceptor can make direct intermolecular contact . We observe that the rate constant for energy transfer from a small terbium chelate to enzyme-bound rifamycin is 1 X 10(7) M-1 s-1, which is about half as large as the rate constant observed for free rifamycin in solution . This relatively small change upon binding indicates that enzyme-bound rifamycin is highly accessible to small molecules in the solvent . In the case of Cibacron Blue, under conditions where approximately 90% of this inhibitor is bound to RNA polymerase, the small amount of unbound inhibitor accounts for practically all of the observed energy transfer . This implies that enzyme-bound Cibacron Blue is relatively inaccessible to energy donors in the solution . The dependence of energy transfer on the accessibility of the acceptor is illustrated by using simple geometric models . synthesis of a stable, electrically neutral terbium chelate which can be efficiently excited with UV radiation is also described. Biochemistry, 1981 Feb 3, 20(3), 506 - 11 Irreversible inhibition of glutamate decarboxylase by alpha-(fluoromethyl)glutamic acid; Kuo D et al.; alpha-(Fluoromethyl)glutamic acid (FMG) was synthesized and shown to be an active site directed irreversible inhibitor of glutamate decarboxylase (EC 4.1.1.15) from Escherichia coli . The KI for the active enantiomer is 1.4 microM, and the kinh = 5.9 X 10(-3) s-1 . Substrates for the enzyme, such as L-glutamate, and competitive inhibitors, such as citrate, decrease the rates of FMG-mediated inactivation of the enzyme . A profound change in the ultraviolet spectrum of the enzyme accompanies the inactivation process . When {3H}-FMG is used, it can be shown that the enzyme incorporates radioactivity at the same rate as that of inactivation . There is a 1:1 stoichiometry of {3H}FMG incorporated to pyridoxal phosphate binding subunits of the enzyme . From these and other studies it is concluded that FMG is a substrate for the enzyme and alkylates it as a consequence of this turnover. Am J Surg, 1981 Feb, 141(2), 194 - 8 Acute acalculous cholecystitis; Howard RJ; Sixty-three patients, 49 men and 14 women, developed acute cholecystitis without gallbladder stones . Only eight patients had a history suggestive of gallbladder disease . In 17 patients cholecystitis developed in the postoperative period, and cholecystitis occurred in 7 patients who had extensive trauma . The signs and symptoms did not differ markedly from those found when acute cholecystitis is associated with cholelithiasis . Pain and tenderness in the right upper abdominal quadrant, vomiting, abdominal distention, decreased bowel sounds, jaundice and fever were common . Thirty (47.6 percent) gallbladder specimens had gangrene, and perforation occurred in five instances . Bacteria were cultured from 28 of 43 bile specimens . E . coli was the most common organism . A high incidence of acalculous gallbladders is found when acute cholecystitis occurs in the postoperative period or after trauma and in children . Decreased blood flow to the gallbladder, cystic duct obstruction and concentrated bile are necessary to produce experimental cholecystitis . These factors are probably necessary in humans also . Decreased gallbladder perfusion caused by shock, congestive heart failure and arteriosclerosis probably contributed to the development of acute acalculous cholecystitis in these patients. J Cell Sci, 1981 Feb, 47, 295 - 309 Chromosomal basis of dosage compensation in Drosophila . X . Assessment of hyperactivity of the male X in situ; Chatterjee RN et al.; The results of examination of the template activity of the fixed polytene chromosomes of Drosophila hydei, monitored by 3H-UTP, under in situ assay conditions, upon the use of endogenous Drosophila polymerase, exogenous Escherichia coli RNA polymerase (holoenzyme) and exogenous Drosophila RNA polymerase II (or B) have been presented . Analysis of the data reveals that the transcription patterns with the 3 enzymes are not strictly comparable with the pattern obtained under in vivo conditions . Yet, with each of the 3 conditions of assay, there is a reasonable concordance between the template activity on the single X chromosome of the male and the paired Xs of the female, as observed under in vivo . There is also, in every case, a high positive correlation between the 3H-UMP incorporation into the X chromosome and that into a specific autosome . A site-wise analysis of 3H-UMP labelling under the 3 assay conditions also reveals that for most of the regions, the sites which are highly active in vivo also show high labelling in situ, and the proportionally is maintained in both sexes . These result have been interpreted to have suggested that the hyperactivity of the male X vis-a-vis dosage compensation in Drosophila is primarily a property of the inherent organization of the X chromosome itself and is achieved through modulation in the organization, rather than exclusively through autosomal factor(s), although a secondary level of autosomal regulation has not yet been ruled out. J Gen Microbiol, 1981 Feb, 122(Pt 2), 335 - 8 Importance of polymorphonuclear leucocytes in protection of mice against Escherichia coli; Tsuru S et al.; Bacterial growth and lethality of Escherichia coli infection of mice were enhanced by X-irradiation but not by treatment with carrageenan . Since carrageenan depletes macrophages but not polymorphonuclear leucocytes, it is concluded that protection against E . coli, at least in the early phases, depends mainly on polymorphonuclear leucocytes. J Gen Microbiol, 1981 Feb, 122(Pt 2), 255 - 61 Respiratory biogenesis during the cell cycle of aerobically grown Escherichia coli K 12 . The accumulation and ligand binding of cytochrome o; Scott RI et al.; A quantitative assay is described for the measurement of cytochrome o in intact cells of E . coli . The procedure involves flash photolysis of the CO-liganded, reduced enzyme in the absence of O2 at temperatures (approx . -100 degrees C) at which the rate of recombination of CO is immeasurably slow . Other CO-binding pigments known to be present, particularly cytochrome d, are excluded from the photodissociation spectrum under these conditions . Measurement of the content of cytochrome o in bacteria separated into size (and thus age) classes by zonal centrifugation shows that the cytochrome accumulates continuously, probably exponentially, throughout the cell cycle and thus constitutes a constant proportion of cell protein during the cycle . The velocity of recombination of CO with cytochrome o at -65 degrees C is invariant over the cell cycle. Biochem J, 1981 Feb 1, 193(2), 541 - 52 The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts; Packman LC et al.; 1 . Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits . Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes . The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3) . 2 . The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography . The alpha beta 3 variant was not recovered and may be unstable in vitro . 3 . The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers . 4 . Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers . Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent . Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid . The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried . 5 . A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described. Acta Pathol Microbiol Scand {C}, 1981 Feb, 89(1), 23 - 8 Experimental Escherichia coli 06 infection in mice . IV . On the relative importance of complement factors and antibodies for the host defence; Ahlstedt S; Treatment of CBA mice with cobra venom factor (CVF) decreased their C3 levels to less than 10 per cent of normal and resulted in a simultaneous increase of their susceptibility to intraperitoneal E . coli 06 infection . Animals immunized and treated with CVF had an infection resistance similar to untreated controls but considerably less than mice immunized only . The CVF treatment did not affect the antibody formation after immunization . Untreated or immunized AKR mice deficient in C5 did not show decreased resistance to the infection . Exposure of NMRI mice to preformed antigen-antibody complexes unrelated to the infection did not increase the susceptibility of the animals to infection, neither did it impair their C3 levels . Two out of five freshly frozen (-70 degrees C) normal rabbit sera gave significant, heat-labile protection of mice to the E . coli infection without containing any antibodies detectable with the ELISA . This protective capacity was not abolished with zymosan treatment affecting the levels of complement factors and could be "natural antibodies" stimulated from the environment . In testing whether similar protective factors as those found in normal rabbit serum could be raised in CBA mice, such animals were orally given killed E . coli 06 bacteria . This did not affect the resistance to the E . coli infection or to the rise of specific antibodies after immunization. Acta Pathol Microbiol Scand {C}, 1981 Feb, 89(1), 15 - 22 Experimental Escherichia coli 06 infection in mice . III . Effects of malnutrition, immunization and nutritional restoration; Ahlstedt S; Inbred mice of the CBA and A strains were from weaning malnourished on a low protein diet . During a three-week period their weight increased from about 8 to about 10 g, while mice on normal feeding increased to about 16 g . This malnutrition resulted in a 10-fold increase of the susceptibility to intraperitoneally injected E . coli 06:K2a,2c:H1 bacteria compared to conventionally fed controls . Immunization as well as transfer of fresh immune serum to malnourished or normally-fed mice resulted in an increase in resistance, 6 to 10 fold that of non-immunized controls, while transfer of normal serum did not have a great impact on the resistance . Antibody titers to El coli 06 antigen in mice on poor and normal feeding were similar, as were there non-specific cellular responsiveness measured as lymphoblast transformation by ConA, PHA and LPS . The effect of the malnutrition seemed to be an impairment of the clearance of live E . coli 06 bacteria 1 h after a sub-lethal infection compared with that of normally-fed controls . The noted increased susceptibility to injections in the malnourished mice was rapidly restored by normal feeding for one day. J Dairy Sci, 1981 Feb, 64(2), 227 - 35 Endotoxin induced migration of leukocytes from blood to milk; McKenzie WN Jr et al.; Guinea pigs were separated from young on days 5 to 7 of lactation . They were anesthetized with ether and were infused intramammary via the teat canal with either sterile saline (.5 ml) or Escherichia coli endotoxin (026:B6:500 microgram/.5 ml) . Each animal served as its own control by having sterile saline in one gland and endotoxin in the other . Animals were sacrificed 4, 6, 8, and 12 h later to determine the time of maximum migration of polymorphonuclear leukocytes from blood to milk . A control group of animals having had no intramammary infusion was sacrificed . Tissues were prepared for observation by light and electron microscopy . Five fields per slide, five slides per animal, and three animals per period were examined by light microscopy, and numbers of polymorphonuclear leukocytes per field were categorized for presence a) in the capillary, b) in interstitial space, c) touching the basal lamina, d) between epithelial cells of an alveolus, and e) in the lumen of an alveolus . Polymorphonuclear leukocytes were in small numbers in the lumen by 4 h and peaked at about 8 h after intramammary infusion of endotoxin . By 12 h numbers had decreased from 8 h . Polymorphonuclear leukocytes were observed by electron microscopy in the intercellular spaces of the alveolar epithelium . A time sequence model using the guinea pig to study mechanisms of leukocyte migration into milk based upon tissue studies may be used in research aimed at controlling mastitis. J Gen Physiol, 1981 Feb, 77(2), 121 - 35 Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli; Nikaido H et al.; Nutrients usually cross the outer membrane of Escherichia coli by diffusion through water-filled channels surrounded by a specific class of protein, porins . In this study, the rates of diffusion of hydrophilic nonelectrolytes, mostly sugars and sugar alcohols, through the porin channels were determined in two systems, (a) vesicles reconstituted from phospholipids and purified porin and (b) intact cells of mutant strains that produce many fewer porin molecules than wild-type strains . The diffusion rates were strongly affected by the size of the solute, even when the size was well within the "exclusion limit" of the channel . In both systems, hexoses and hexose disaccharides diffused through the channel at rates 50-80% and 2-4%, respectively, of that of a pentose, arabinose . Application of the Renkin equation to these data led to the estimate that the pore radius is approximately 0.6 nm, if the pore is assumed to be a hollow cylinder . The results of the study also show that the permeability of the outer membrane of the wild-type E . coli cell to glucose and lactose can be explained by the presence of porin channels, that a significant fraction of these channels must be functional or "open" under our conditions of growth, and that even 10(5) channels per cell could become limiting when E . coli tries to grow at a maximal rate on low concentrations of slowly penetrating solutes, such as disaccharides. Am J Vet Res, 1981 Feb, 42(2), 229 - 31 Experimental intramammary infection of the dairy cow with Escherichia coli during the nonlactating period; McDonald JS et al.; The nonlactating mammary gland was experimentally inoculated with Escherichia coli . During the first half of the nonlactating period, 32% of 34 inoculated glands were temporarily infected . All intramammary infections were eradicated by the cow without therapy and no signs of mastitis were observed . During the 30 days before parturition occurred, 88% of 42 inoculated glands in the cows became infected . Twenty-three intramammary infections were eradicated by the cow and infection in 14 glands persisted after parturition occurred . Peracute toxic mastitis occurred in those cows with infected glands. Acta Pathol Microbiol Scand {B}, 1981 Feb, 89(1), 37 - 9 A study of the elimination phase of phagocytosis of 32P-labelled Escherichia coli by human polymorphonuclear cells; Melby K et al.; A study of the elimination phase of phagocytosis of a radio-labelled strain of Escherichia coli by human polymorphonuclear cells is presented . In the presence of normal serum during the ingestion and post-ingestion phases, the elimination was 55% . Omission of normal serum during the post-ingestive phase resulted in an elimination of 35% . Without serum in both phases the elimination was approximately 50% . Microscopically the bacterial cell envelope appeared unchanged during the post-ingestive period (3h). Biokhimiia, 1981 Feb, 46(2), 346 - 81 {Kinetics of fumarate hydratase reaction catalyzed by free cells of Escherichia coli}; Gubnitskii LS et al.; The kinetics of the fumarate hydratase (fumarase) reaction catalyzed by the cells of E . coli strain 85 at high concentrations of the substrate (potassium fumarate) were studied . An automatic procedure for determination of the reaction product--malonic acid--including the use of commercial malate dehydrogenase from porcine heart was developed . The fumarate activity of bacterial cells was studied at different concentrations of the substrate and at different pH values with intact and disrupted cells of E . coli 85 used as the enzyme source . The rate of the fumarase reaction in the E . coli cells was shown to depend on the diffusion and transport processes of the reagent transfer across the cell wall and the cytoplasmic membrane of bacterial cells . The pH optimum of the reaction in free E . coli cells (8-9) and the rate of malonic acid synthesis from potassium fumarate under optimal conditions, which varies within the concentration range of (6--13) x 10(-5) mkmole per mg of protein depending on the quality of cell, were determined. J Biochem (Tokyo), 1981 Feb, 89(2), 411 - 20 An in vitro study of the methylation of methyl-accepting chemotaxis protein of Escherichia coli . Construction of the system and effect of mutant proteins on the system; Minoshima S et al.; An in vitro system for the methylation of methyl-accepting chemotaxis proteins (MCP's), which have been shown to be membrane integral proteins, was constructed . The system, consisting of the membrane, the cytoplasm, and labeled S-adenosyl methionine, showed the following characteristics . 1 . The methylation of MCP in the membrane required the cytoplasm . The rate of incorporation of the labeled methyl group into MCP was dependent on the amount of the cytoplasm . 2 . Incorporation of the labeled methyl moiety into MCP reached a steady state, and the level of the steady state incorporation was dependent on the concentration of the cytoplasm when the concentration of the membrane protein was constant . 3 . The methyl moiety which had been incorporated into MCP before the steady state could be exchanged . It was suggested that the amount of methyl group introduced into MCP was equal to that of taken from MCP . 4 . The methylated MCP was demethylated faster in the presence of a methyl donor than in its absence . 5 . The membranes obtained from cheX-, cheB-, and cheZ mutants were inactive in the present in vitro system even when they were mixed with the wild type cytoplasm. Proc Natl Acad Sci U S A, 1981 Feb, 78(2), 922 - 5 Molecular basis of valine resistance in Escherichia coli K-12; Lawther RP et al.; The relationship of valine resistance to the expression of the ilvGEDA operon of Escherichia coli K-12 has been determined . DNA sequence and in vivo protein analyses indicate that in wild-type E . coli K-12 there is a frameshift site within the gene (ilvG) for valine resistance . The ilvG+2096 (formerly designated ilv02096) mutation displaces this frameshift site, resulting in the expression of ilvG and the relief of transcriptional polarity on the distal genes of this operon . Thus, the "ilv0" mutation, which concomitantly confers valine resistance and increased expression of the ilvEDA genes, is, in fact, the "reversion" of a polar site within the first structural gene of the ilvGEDA operon. Proc Natl Acad Sci U S A, 1981 Feb, 78(2), 898 - 902 Metal cation influence on activity and regulation of aspartate carbamoyltransferase; Honzatko RB et al.; At saturating carbamoylphosphate and nonsaturating aspartate concentrations, Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Al3+, and Gd3+ inhibit aspartate carbamoyltransferase (carbamoylphosphate:L-asparate carbamoyltransferase, EC 2.1.3.2) from EScherichia coli . When nucleotide triphosphates are present, these inhibitory effects are displaced to higher concentrations of cation . At lower levels of cation and saturating carbamoylphosphate concentration, Mg2+, Mn2+, Al3+, and Gd3+ partially relieve allosteric inhibition by GTP but have little influence on activation by ATP and inhibition by CTP . At nonsaturating carbamoylphosphate concentrations, however, Mg2+, Mn2+, Al3+, and Gd3+ increase enzymatic activity to 170% over the level when GTP alone is present . In addition, Mg2+, Mn2+, and Al3+ show enhancement of ATP activation by 120-130% but only slight relief of CTP inhibition . We suggest that three modes of action by the metal can account for the observed kinetic behavior . (i) In the absence of nucleotide, metals inhibit catalytic activity either by a direct interaction with the enzyme or indirectly by complexing carbamoylphosphate . (ii) The metal-nucleotide complex interacts allosterically with the enzyme to enhance enzymatic activity relative to that produced by the free nucleotide, as noted above . (iii) By chelating to nucleotides, the metal diminishes their tendency to bind competitively at the carbamoylphosphate portion of the active site, as shown particularly by experiments on the catalytic subunit. Proc Natl Acad Sci U S A, 1981 Feb, 78(2), 861 - 5 Enzymatic release of 7-methylguanine from methylated DNA by rodent liver extracts; Margison GP et al.; Rat and hamster liver extracts were found to contain DNA glycosylases capable of removing 3-methyladenine and 7-methylguanine from methylated DNA . The activity of 7-methylguanine-DNA glycosylase was greater tin hamster than in rat liver extracts . This finding is consistent with previous reports that the half-life of 7-methylguanine in DNA after treatment with the carcinogen dimethylnitrosamine is longer in rats than in hamsters . These enzymes may, therefore, play an important role in the removal of abnormal alkylation products from mammalian cell DNA . Rodent liver extracts also contained a DNA glycosylase able to remove from alkylated DNA the imidazole-ring-opened form of 7-methylguanine which is produced by treatment with alkali . Although this product may occur in vivo after treatment with alkylating agents to only a very small extent, the enzyme may be needed to minimize its potentially harmful biological effects. Proc Natl Acad Sci U S A, 1981 Feb, 78(2), 834 - 7 Covalent modification and repressed transcription of a gene in hepatoma cells; Nakhasi HL et al.; When liver cells undergo malignant transformation, certain genes cease being expressed . We have studied the structure of one such gene, whose protein product we have designated hepatic protein 22 (hp22), which is not expressed in the two Morris hepatomas studied . We have prepared a chimeric clone of pBR322 containing cDNA sequences complementary to mRNA coding for this protein . By using this cloned cDNA, we have examined changes in expression of this gene and changes in the restriction pattern of the DNA isolated from normal liver and these hepatomas . In both hepatomas, studies using the isoschizomeric pair of restriction enzymes Msp I and Hpa II have indicated hypermethylation of a cytosine residue within or proximal to the hp22 gene . Other differences in the restriction pattern between normal liver and hepatoma DNA were also detected with EcoRI and Ava I . Thus, in the nontranscribed form of this gene, the DNA has undergone covalent modification, distinguishing these two hepatomas from each other and from normal liver. J Clin Pathol, 1981 Feb, 34(2), 194 - 8 Rapid screening for bacteriuria using a particle counter, pulse-height analyser, and computer; Alexander MK et al.; A system for the rapid screening of urines for the presence of bacteriuria has been devised using a Coulter Counter Model ZBI linked to a multichannel pulse-height analyser (Coulter "Channelyser") with computer analysis of the output . In a series of 215 urines containing growth of a single pathogen of more than 100 x 10(6)/l (greater than 100 000/ml) satisfactory level of sensitivity (99.1%) was obtained using only two different amplification settings by means of a brief treatment (5-10 seconds) of the undiluted specimen with low intensity ultrasound; 85-90% of mixed growths of 10 x 10(6)/l (greater than 10 000/ml) were detected . Sonication did not improve the results in this group . Specimens showing abnormal cyturia of more than 10 x 10(6)/l (greater than 10 000/ml) but no growth on culture were positive in 33% of cases without the use of ultrasound but in 72% after sonication. Can J Physiol Pharmacol, 1981 Feb, 59(2), 204 - 8 Circulatory effects of acute or chronic endotoxemia in rats; Keeler R et al.; A study was made of the effects of acute (4 h) infusion of Escherichia coli endotoxin on cardiovascular function in rats . Rats with acute endotoxemia had a reduced cardiac output but maintained their arterial blood pressure . Fractional distribution of the cardiac output was increased to the liver and reduced to the gastorintestinal tract and skin . No changes in fractional distribution to the kidneys, lungs, or heart were observed although absolute blood flow to these areas was reduced . Rats with chronic endotoxemia had a reduced cardiac output and hypotension with no change in peripheral resistance . Other changes resembled those seen in acute endotoxemia apart from a low renal fraction of the cardiac output . Calculation and interpretation of blood flow changes in these animals was difficult because of a large fall in hematocrit and changes in organ weight. J Infect Dis, 1981 Feb, 143(2), 286 - 90 The role of plasmids in adherence of invasive Escherichia coli to mammalian cells; Nandadasa HG et al.; Strains of Escherichia coli isolated from persons with dysentery-like diarrheal disease were demonstrated to adhere to the surface of cultured HEp-2 (human epithelial) cells under conditions that removed nonpathogenic control bacteria and to cause hemagglutination of human red blood cells . The plasmid content of 13 stains surveyed was found to be variable with respect to resistance to antibiotics and the presence of small cryptic plasmids . Conjugal transfer to resistance plasmids from two of the clinical isolates to a number of nonpathogenic laboratory and field isolates of E . coli was not accompanied by transfer of the capacity either for specific interaction with cultured HEp-2 cells or for hemagglutination of human red blood cells . Furthermore, cured derivatives of the enteroinvasive strains retained positive reactions in the assay systems. Infect Immun, 1981 Feb, 31(2), 712 - 5 Use of 51Cr-labeled mononuclear cells for measuring the cellular immune response in mouse lungs; Zarkower A et al.; Spleen cells labeled with 51Cr were used in sensitized syngeneic mice to measure the degree of mononuclear cell infiltration into antigen-challenged tissues . With this method, increased cellular infiltration was found after footpad challenge of mice sensitized with sheep erythrocyte, Escherichia coli, and BCG antigens . Cellular response also was determined by using this technique in the lungs of mice sensitized with sheep erythrocytes and BCG . This procedure offers the opportunity to measure cellular infiltration, whether due to cellular or humoral influences, in tissues not easily accessible to conventional immunological manipulation. Eur J Biochem, 1981 Feb, 114(2), 429 - 37 Factors modulating transcription and translation in vitro of ribosomal protein S20 and isoleucyl-tRNA synthetase from Escherichia coli; Wirth R et al.; The DNA-dependent protein-synthesizing system developed by Zubay {Zubay, G . (1973) Annu . Rev . Genet . 7, 267--287} was optimized for the transcription and translation of genes from the 0.5-min region of the Escherichia coli chromosome carried by transducing lambda phages . The E . coli gene products synthesized were isoleucyl tRNA synthetase, ribosomal protein S20, dihydrodipicolinic acid reductase and (possibly) the two subunits carbamoyl-phosphate synthetase . Formation of ribosomal protein S20 is specifically stimulated by the addition of 16-S rRNA and not by 5-S or 23-S rRNA . 16-S rRNA increases the rate of S20 synthesis, the final yield of product depends on the duration of persistence of the RNA added . Addition of 16-S rRNA to the separate transcription and translation systems showed that it is the translation of the S20 mRNA which is enhanced . Furthermore, S20 synthesis is stimulated more than fourfold when concomitant synthesis of rRNA occurs from a plasmid carrying an rrn transcriptional unit . The results described are explained in terms of a model which suggests that ribosomal protein S20 feedback inhibits its synthesis at the translational level and that removal of S20 into ribosomal assembly (i.e . binding to 16-S rRNA) releases inhibition . The model postulates a direct link between synthesis of ribosomal RNA and ribosomal protein and between the rates of ribosomal assembly and ribosomal protein synthesis . The stimulatory effect of guanosine 3'-diphosphate 5'-diphosphate on isoleucyl-tRNA synthetase formation and its inhibition of the synthesis of ribosomal protein S20 in vitro occurs at the level of transcription . Its relevance in vivo, however, remains to be demonstrated . Formation of isoleucyl-tRNA synthetase in vitro is not influenced either by the addition of a surplus of purified enzyme nor by the limitation of protein synthesis by the addition of anti-(isoleucyl-tRNA synthetase) serum . There is no evidence, therefore, that isoleucyl-tRNA synthetase is autogenously regulated. Eur J Biochem, 1981 Feb, 114(2), 413 - 20 Dihydrolipoamide dehydrogenase component of the pyruvate dehydrogenase complex from Escherichia coli K12 . Comparative characterization of the free and the complex-bound component; Schmincke-Ott E et al.; The regulation of the biosynthesis of dihydrolipoamide dehydrogenase is dependent on the biosynthesis of the pyruvate dehydrogenase complex . The gene coding for the dihydrolipoamide dehydrogenase appears to be included in the regulation of the pyruvate dehydrogenase operon . Possibly a secondary promoter is inserted . Dihydrolipoamide dehydrogenase was purified in its free form using a dihydrolipoamide-agarose affinity column and avoiding denaturating conditions . The enzyme shows complete cross-reactivity with antibodies against the pyruvate dehydrogenase complex and has a higher specific activity than any preparations described thus far . The transition state activation energy of the catalytic activity is smaller for the complex-bound enzyme than that found for the free enzyme . In its complexed form, the enzyme also proves to be somewhat more stable under alkaline conditions . The reaction catalyzed by the dihydrolipoamide dehydrogenase shows the behaviour of a ping-pong mechanism . The Michaelis constants for the substrates NAD and dihydrolipoamide found with the free enzyme are about four times those observed with the enzyme integrated into the native complex . The catalytic reaction of both forms of the dihydrolipoamide dehydrogenase is inhibited by NADH . The mechanism of this inhibition cannot simply be explained by a product inhibition . Rather the further reduction of the catalytically active, half-reduced enzyme form to the catalytically inactive, fully reduced form has to be considered as causing the inhibition. Eur J Biochem, 1981 Feb, 114(2), 407 - 11 Pyruvate dehydrogenase component of the pyruvate dehydrogenase complex from Escherichia coli K12 . Purification and characterization; Saumweber H et al.; Free pyruvate dehydrogenase component of the Escherichia coli K12 pyruvate dehydrogenase complex was isolated from a mutant lacking the dihydrolipoamide transacetylase component . The procedure, employing three chromatographic steps, yields a product that is electrophoretically pure . The purified enzyme reassociates with the residual complex lacking this component to a fully active enzyme complex . The kinetic characteristics of the free component were compared to that of the enzyme integrated into the native complex molecule . No essential differences could be detected regarding the behaviour of the catalytic reaction with variations in temperature, pH and substrate concentration . An inhibition, competitive to pyruvate, of the pyruvate dehydrogenase component (Ki = 18 microM) by fluoropyruvate was observed with both enzyme forms . Pyruvate exerts a cooperative effect both upon the partial enzyme reaction of the pyruvate dehydrogenase component as well as upon the overall reaction of the native enzyme complex . However, there is a clear difference in the shape of the saturation curves of both types of reaction . Experiments with the free enzyme, with the component integrated into the native complex molecule and with enzyme complexes which are partially deficient in the pyruvate dehydrogenase component demonstrate that the type of saturation curves obtained were characteristic for the reaction observed rather than for the interaction of a high number of subunits of the pyruvate dehydrogenase complex. Am J Clin Nutr, 1981 Feb, 34(2), 245 - 51 Vitamin E and aspirin depress prostaglandins in protection of chickens against Escherichia coli infection; Likoff RO et al.; The hypothesis was tested that vitamin E protects chickens from a lethal Escherichia coli infection by inhibiting the biosynthesis of prostaglandins, thereby activating humoral immunity and phagocytosis . When chickens were fed supplement vitamin E at the level of 300 mg/kg diet, which is six times the presently used dietary level, endogenous PGE1, PGE2, and PGF2 alpha levels decreased in the immunopoietic organs, bursa, and spleen . Antibody titers to E . coli lipopolysaccharide and phagocytosis increased at the same time . Infection slightly increased prostaglandin levels and vitamin E appeared to compensate for this increase . Aspirin, a known prostaglandin inhibitor acted synergistically with vitamin E in depressing endogenous PG levels in bursa and decreasing mortality from E . coli infection. Mutat Res, 1981 Feb, 80(2), 229 - 38 Mutagenic interactions between near-ultraviolet (365 nm) radiation and alkylating agents in Escherichia coli; de Moraes EC et al.; The mutagenic interaction between near-ultraviolet (365 nm) radiation and the alkylating agents ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS) was studied in a repair-competent and an excision-deficient strain of Escherichia coli . Near-UV radiation modified the metabolic response of exposure to these chemicals and either reduced or increased their mutagenic efficiency . Based on these results, an experimental model was formulated to explain the mutagenic interactions that occur between near-UV and various agents that induce prototrophic revertants via error-prone repair of DNA . According to this model, low doses of near-UV provoke conditions for mutation frequency decline (MFD) and lead to a mutagenic antagonism . With increasing near-UV doses, damage to constitutive error-free repair systems increases, favouring the error-prone system and inhibiting the MFD . Under these conditions there will be a progressive decrease in antagonism until at high doses an enhancement of mutation frequency (positive interaction) will occur. J Clin Microbiol, 1981 Feb, 13(2), 301 - 8 Analysis of parameters affecting the hemagglutination activity of Escherichia coli possessing colonization factor antigens: improved medium for observing erythrocyte agglutination; Sedlock DM et al.; The hemagglutination (HA) activity of two strains of Escherichia coli, each possessing different colonization factor antigens (CFA), was examined under different test conditions . The effects of ionic strength, temperature, pH, cations, and reaction surface on erythrocyte (RBC) agglutination were analyzed . Strain H-10407 (CFA/I) caused the agglutination of human, bovine, and chicken RBC, whereas strain CL-9699 (CFA/II) agglutinated only bovine and chicken RBC . The HA activity of both strains increased with decreasing ionic strength, pH, and temperature, the effects of temperature being negligible at low ionic strength . When accounting for ionic strength, the presence of Ca2+, Mg2+, Fe2+, or Fe3+ ions did not increase the HA activity of these bacteria . Optimum conditions for HA of reactive RBC by bacteria included low ionic strength (less than 50 mM) and slightly acidic pH (6.0 to 7.0) . Use of a low-ionic-strength medium permitted application of microtitration methods to visualize the HA reactions . Storage of RBC in low-ionic-strength medium did not change their HA properties, and the use of this medium proved superior to saline in overcoming HA variation observed with different preparations of RBC. J Pharmacol Exp Ther, 1981 Feb, 216(2), 410 - 4 Reduction in immunogenicity and clearance rate of Escherichia coli L-asparaginase by modification with monomethoxypolyethylene glycol; Kamisaki Y et al.; Escherichia coli L-asparaginase was modified with monomethoxypolyethylene glycol using cyanuric chloride as a coupler . The modified enzyme did not cross-react with anti-L-asparaginase antibody in precipitin reaction, but retained some catalytic activity (8% of the original activity) . It has the same Km value for L-asparagine and the same optimal pH as the native enzyme . The immunogenicity of the modified enzyme was substantially reduced because mouse antiserum to it showed no significant increase in hemagglutinin titer of L-asparaginase-coated sheep red blood cells . After a single i.p . injection of the modified enzyme (80 I.U./kg) into rats, enzyme activity was detected in the serum within 30 min and persisted for over 3 weeks . Concomitant depletion of serum L-asparagine persisted for more than 3 weeks . On the other hand, the active enzyme was rapidly cleared from the serum . The half-lives of the modified and native enzymes were calculated to be 56 and 2.9 hr, respectively . This modified L-asparaginase may be much more useful than the native enzyme for the clinical treatments of tumors because of its reduced immunogenicity. J Bacteriol, 1981 Feb, 145(2), 904 - 13 Isolation and characterization of amber mutations in gene dnaA of escherichia coli K-12; Schaus N et al.; Amber mutants with defects in the dnaA gene of Escherichia coli K-12 were isolated after localized mutagenesis of the tna-dnaA region of the chromosome . We isolated 36 mutants defective in the initiation of deoxyribonucleic acid replication as determined by their dependence upon integrative suppression by a P2 sig5 prophage . Three of the 36 mutants were shown to contain amber mutations through the use of a temperature-sensitive amber suppressor . These mutations, which mapped between gyrB and tna, were characterized genetically and biochemically as amber mutations in dnaA. J Bacteriol, 1981 Feb, 145(2), 845 - 9 Regulation of D-alanine carboxypeptidase and peptidoglycan cross-linkage in amino acid-deprived Escherichia coli; Harkness RE et al.; The effect of amino acid deprivation on the activities of D-alanine carboxypeptidase (CPase) and peptidoglycan transpeptidase in Escherichia coli was determined . Enzymes were assayed in ether-treated bacteria (ETB) which were permeable to peptidoglycan nucleotide precursors . ETB were prepared at intervals from cultures grown in the presence and absence of a required amino acid . The specific activity of CPase in ETB decreased 50 to 85% during amino acid deprivation . This was paralleled by a 60 to 70% decrease in the specific activity of peptidoglycan transpeptidase . Both enzymes reached their lowest level of activity about 40 min after the onset of amino acid deprivation . The decrease in CPase activity apparently was not due to degradation of the enzyme, since full activity was restored after disruption of ETB by sonication . A decrease in CPase activity was associated with an enhancement of transpeptidation . The peptidoglycan synthesized in vitro by amino acid-deprived ETB was 1.7 times more cross-linked than the peptidoglycan synthesized by control ETB These results support the proposal that CPase may be involved in regulating transpeptidation in E . coli. J Bacteriol, 1981 Feb, 145(2), 840 - 4 sfrA and sfrB products of Escherichia coli K-12 are transcriptional control factors; Beutin L et al.; The mechanisms whereby mutations in Escherichia coli K-12 genes sfrA and sfrB reduce expression of the transfer functions of sex factor F have been examined by assaying the levels of tra messenger ribonucleic acid and of tra proteins . The sfrA product was necessary for efficient transcription of the control gene traJ and, directly or indirectly, for transcription of the traY leads to Z operon . In the absence of sfrA, reduced levels of the traJ and traT proteins were observed in the outer membrane . The sfrB product was needed to prevent premature transcription at one or more rho-dependent termination sites . sfrB mutations also reduced synthesis of full-length lipopolysaccharide molecules, of several chromosomally determined outer membrane proteins, and of functional flagella . Thus, the sfrB product may act as an antiterminator in transcription of several operons encording cell envelope components. J Bacteriol, 1981 Feb, 145(2), 803 - 7 Effect of arsenate on chemotactic behavior of Escherichia coli; Arai T; Escherichia coli cells treated with arsenate cannot tumble . The relationship between cellular adenosine 5'-triphosphate (ATP) level and the ability to tumble has been studied . (i) Cells incubated with arsenate completely lost their tumbling ability, and the cellular ATP level was decreased to less than 0.3 nmol/mg of protein . (ii) Incubation with 10 mM arsenate-1 mM phosphate reduced the cellular ATP level to less than 0.25 nmol/mg of protein . However, the cells were still able to tumble . (iii) Tumbling of the arsenate-treated cells was completely recovered after addition of a slight amount of phosphate, although the ATP level was still as low as 0.2 nmol/mg of protein . (iv) The cellular ATP level of an arsenate-treated uncA mutant (Ca2+,Mg2+-adenosine triphosphatase defective) was lower than 0.1 nmol/mg of protein even after the addition of 5 5 mM phosphate . However, tumbling ability was almost completely restored upon addition of the phosphate. J Bacteriol, 1981 Feb, 145(2), 780 - 7 Transformation of Escherichia coli with plasmid deoxyribonucleic acid: calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions; Weston A et al.; Plasmid deoxyribonucleic acid (DNA) was tightly bound to cells of Escherichia coli at 0 degrees C in the presence of divalent cations . During incubation at 42 degrees C, 0.1 to 1% of this DNA became resistant to deoxyribonuclease . Deoxyribonuclease-resistant DNA binding and the ability to produce transformants became saturated when transformation mixtures contained 1 to 2 micrograms of plasmid NTP16 DNA and about 5 X 10(8) viable cells . Under optimum conditions, between 1 and 2 molecule equivalents of 3H-labeled NTP16 DNA per viable cell became deoxyribonuclease resistant . Despite this, only 0.1 to 1% of viable cells became transformed by saturating amounts of the plasmid . The results suggest that transport of DNA across the inner membrane is a limiting step in transformation . After transformation the bulk of labeled plasmid DNA remained associated with outer membranes . However, in vitro assays indicated that plasmid DNA would bind equally well to preparations of inner or outer membranes provided divalent cations were present to preparations of inner or outer membranes provided divalent cations were present . Divalent cations promoted differing levels of binding to isolated inner and outer membranes in the order Ca2+ much greater than Ba2+ greater than Sr2+ greater than Mg2+ . This parallels their relative efficiencies in promoting transformation . Binding of plasmid DNA was greatly reduced when outer membranes were treated with trypsin; this suggests that protein components may be required for the binding or transport of DNA (or both) during transformation. J Bacteriol, 1981 Feb, 145(2), 722 - 8 Catabolite repression of Escherichia coli heat-stable enterotoxin activity; Martinez-Cadena MG et al.; The Escherichia coli heat-stable enterotoxin (ST) coded for by plasmid pYK007 (Apr ST+) showed a dependence for cyclic adenosine 3',5'-monophosphate (cAMP) to express ST activity in an adenyl cyclase (cya) deletion mutant; no ST activity was detected in the presence of cAMP in a cAMP receptor protein (crp) deletion mutant or in a double deletion mutant (delta cya delta crp) . The cya-crp effect on ST activity could not be accounted for by a modification of the copy number of plasmid deoxyribonucleic acid per chromosome equivalent or by an alteration in the secretion of an active intracellular enterotoxin. J Bacteriol, 1981 Feb, 145(2), 1110 - 1 Selection for loss of tetracycline resistance by Escherichia coli; Maloy SR et al.; An improved medium for the direct, positive selection of tetracycline-sensitive clones from a population of tetracycline-resistant strains of Escherichia coli is described. J Bacteriol, 1981 Feb, 145(2), 1091 - 4 Escherichia coli growth studied by dual-parameter flow cytophotometry; Steen HB et