Escherichia coli

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 al.; The growth of Escherichia coli cells has been analyzed for the first time by dual-parameter flow cytophotometry, in which the deoxyribonucleic acid and protein contents of single bacteria have been measured simultaneously with an accuracy of a few percent and at a rate of 3,000 cells/s.

J Bacteriol, 1981 Feb, 145(2), 1036 - 41
Incomplete flagellar structures in Escherichia coli mutants; Suzuki T et al.; Escherichia coli mutants with defects in 29 flagellar genes identified so far were examined by electron microscopy for possession of incomplete flagellar structures in membrane-associated fractions . The results are discussed in consideration of the known transcriptional interaction of flagellar genes . Hook-basal body structures were detected in flaD, flaS, flaT, flbC, and hag mutants . The flaE mutant had a polyhook-basal body structure . An intact basal body appeared in flaK mutants . Putative precursors of the basal body were detected in mutants with defects in flaM, flaU, flaV, and flaY . No structures homologous to the flagellar basal body or its parts were detected in mutants with defects in flaA, flaB, flaC, flaG, flaH, flaI, flaL, flaN, flaO, flaP, flaQ, flaR, flaW, flaX, flbA, flbB, and flbD . One flaZ mutant had an incomplete flagellar basal body structure and another formed no significant structure, suggesting that flaZ is responsible for both basal body assembly and the transcription of the hag gene.

J Bacteriol, 1981 Feb, 145(2), 1031 - 5
Cysteine and growth inhibition of Escherichia coli: threonine deaminase as the target enzyme; Harris CL; Cysteine has been shown to inhibit growth in Escherichia coli strains C6 and HfrH 72, but not M108A . Growth inhibition was overcome by inclusion of isoleucine, leucine, and valine in the medium . Isoleucine biosynthesis was apparently affected, since addition of this amino acid alone could alter the inhibitory effects of cysteine . Homocysteine, mercaptoethylamine, and mercaptoethanol inhibited growth to varying degrees in some strains, these effects also being prevented by addition of branched-chain amino acids . Cysteine, mercaptoethylamine, and homocysteine were inhibitors of threonine deaminase but not transaminase B, two enzymes of the ilvEDA operon . Cysteine inhibition of threonine deaminase was reversed by threonine, although the pattern of inhibition was mixed . These results suggest a relationship between the growth-inhibitory effects of cysteine and other sulfur compounds and the inhibition of isoleucine synthesis at the level of threonine deaminase.

Surgery, 1981 Feb, 89(2), 257 - 63
The effect of prostacyclin infusion on endotoxin-induced lung injury; Demling RH et al.; Lung injury produced by endotoxin is characterized by both pulmonary hypertension and increased pulmonary vascular permeability . Prostacyclin (PGI2) has been found to be a vasodilator and a membrane stabilizer . The purpose of this study was to determine the effectiveness of PGI2 in preventing endotoxin injury . Eight sheep with chronic lung lymph fistula were given both Escherichia coli endotoxin (2 microgram/kg) alone and endotoxin plus an immediate infusion of PGI2 (0.1 to 0.2 microgram/kg/min) for a 5-hour period; the studies were performed 4 days apart . The endotoxin injury was characterized by early severe pulmonary hypertension, with pulmonary artery pressure increasing from 18 +/- 0.6 to 40 +/- 3.1 mm Hg and lung lymph flow (QL) increasing threefold . This was followed in about 3 hours by an increase in permeability characterized by an increasing lymph to plasma protein ratio (0.63 to 0.74) and a threefold increase in QL . In seven of eight animals infused with PGI2 the pulmonary hypertension and alteration in QL in the early and later phases were significantly decreased . In four paired studies, prostaglandins PGE, PGF2 alpha, and PGI2 as 6-keto PGF1 alpha were measured in lymph and plasma . PGF2 alpha and PGI2 were significantly increased in lung lymph during the early hypertensive phase immediately after endotoxin injection, but returned to baseline during the later phase . In the PGI2 infusion studies, PGF2 alpha showed the same pattern of response, but PGI2 was increased to much higher levels in lymph and plasma, as compared to values of endotoxin alone . The higher plasma values corresponded with less severe lung injury . The one animal not protected by PGI2 had the lowest plasma PGI2 level . We therefore found PGI2 to protect the lung against injury from endotoxin.

J Gen Microbiol, 1981 Feb, 122(Pt 2), 323 - 33
Kinetics of growth of the ciliate Tetrahymena pyriformis on Escherichia coli; Watson PJ et al.; The growth of the ciliate Tetrahymena pyriformis on non-growing Escherichia coli has been studied by following the time courses of population densities and protozoan mean cell volume in batch cultures . Viable, non-encysted protozoa always stopped feeding before the bacterial density was reduced to zero and non-feeding ciliates tended to swim faster than feeding ciliates . In addition, the number of bacteria and other particles of bacterial size consumed in the formation of one new ciliate, when averaged over the lag and reproductive phases of a culture, declined toward a limiting value of about 1.6 x 10(4) particles per ciliate as the initial density of such particles was increased.

Can J Biochem, 1981 Feb, 59(2), 100 - 5
The anomeric specificity of beta-galactosidase and lac permease from Escherichia coli; Huber RE et al.; Beta-Galactosidase was found to act on alpha-lactose slightly more than twice as rapidly as on beta-lactose for both the hydrolysis and transgalactosylis reactions . The effect was shown to be on the Vmax values; the Km values for the different anomeric forms were the same . The step of the reaction for which the enzyme has anomeric specificity was shown to be glycosidic bond breakage . The steps in glucose release or in the glucose acceptor reaction were not affected by anomeric composition . Neither allolactose hydrolysis nor transport of lactose into the cells by lac permease was sensitive to the anomeric composition of the substrate . The implications of these results for lac operon induction and for lactose metabolism are discussed.

J Biochem Biophys Methods, 1981 Feb, 4(2), 91 - 100
A simple method for the determination of DNA molecular weight distribution; Surowiec A; A method is described for the molecular weight distribution of DNA which is determined from sedimentation-velocity analysis . Knowing the distribution of sedimentation coefficients for a single DNA concentration it is possible to extrapolate such a distribution to infinite dilution of the solute in a simple way . Two versions (using two or three terms of a series) of extrapolating equations are considered and discussed in detail . The sedimentation coefficients distribution calculated from these equations differs only insignificantly with that obtained in a conventional way.

Res Commun Chem Pathol Pharmacol, 1981 Feb, 31(2), 217 - 22
Effect of endotoxin on thiourea induced pulmonary edema and pleural effusion; Hollinger MA et al.; Administration of endotoxin prior to an LD50 dose of thiourea protected rats against pulmonary edema and pleural effusion . These results are similar to those seen with endotoxin pretreatment and pulmonary O2 toxicity.

Surg Gynecol Obstet, 1981 Feb, 152(2), 159 - 62
Effects of naloxone therapy on hemodynamics and metabolism following a superlethal dosage of Escherichia coli endotoxin in dogs; Raymond RM et al.; Experiments were done upon anesthetized and unanesthetized dogs given endotoxin only, endotoxin plus naloxone or naloxone only . Dogs given endotoxin and treated with naloxone showed marked hemodynamic and metabolic improvements compared with the dogs given endotoxin only . Beneficial effects of naloxone treatment following the administration of endotoxin are attentuated hypotension, hemoconcentration and acidosis and prevention of hypoglycemia . Results of mortality studies in unanesthetized dogs given endotoxin suggest that naloxone treatment increases the survival time.

Proc Natl Acad Sci U S A, 1981 Feb, 78(2), 1115 - 8
Evidence that ribosomal protein S10 participates in control of transcription termination; Friedman DI et al.; We report the isolation of an Escherichia coli K-12 strain with a mutation, nusE71, that results in a change in ribosomal protein S10 . Phage lambda fails to grow in hosts carrying the nusE71 mutation because the lambda N gene product is not active . The N product regulates phage gene expression by altering transcription complexes so that they can overcome termination barriers . This suggests that a ribosomal protein is involved in antitermination of transcription.

Arch Microbiol, 1981 Feb, 128(4), 360 - 4
Iron uptake in pseudorevertants of Escherichia coli K-12 mutants with multiple defects in the enterochelin system; Pickett CL et al.; Iron uptake in pseudorevertants of Escherichia coli K-12 strains which lack the ability to synthesize enterochelin, 2,3 dihydroxybenzoate, and the ferrienterochelin receptor protein was characterized . In four independent pseudorevertants, the suppressor mutations which permitted growth in iron-poor environments appeared to be located in omp B, the regulatory locus for the porin proteins . Unlike wild-type cells, the pseudorevertants were unable to utilize ferrienterochelin and could acquire iron from citrate without induction by prior growth in citrate . The energy requirements of the pseudorevertant system appeared to be identical to those of the enterochelin system . Evidence that loss of the porin proteins results in the secretion by the pseudorevertants of a molecule with siderophore activity is presented; this siderophore is able to remove iron from the non-biological iron chelators nitrilotriacetic acid and alpha, alpha'-dipyridyl but not fom the siderophores ferrichrome and enterochelin.

J Virol, 1981 Feb, 37(2), 738 - 47
In vitro transcription of the inverted terminal repetition of the vaccinia virus genome: correspondence of initiation and cap sites; Venkatesan S et al.; Specific RNAs synthesized in vitro by vaccinia virus cores were analyzed with the aid of DNA from the terminal 9,000 base pairs of the genome that was cloned in phage lambda, pBR322, and the single-stranded phage fl . Three mRNA's coding for polypeptides with molecular weights of 7,500 (7.5K), 19K, and 42K were shown to have sizes and map positions similar to those described for mRNA's made early in infection . A previously undescribed transcript made in vivo and in vitro, with a 5' end at about 8.7 kilobase pairs from the end of the genome, was also detected . After chemical removal of the terminal 7-methylguanosine residue, the 5' ends of the RNAs were specifically labeled by enzymatic capping and the mapped by gel electrophoresis of nuclease-resistant RNA.DNA hybrids, as well as by hybridization of the end-labeled RNA to immobilized DNA restriction fragments . Analysis of the purified cap structures demonstrated that three of the mRNA's have both m7G(5')pppAm and, m7G(5')pppGm ends, indicating some degree of terminal heterogeneity . The fourth transcript has exclusively m7G(5')pppAm ends . By synthesizing RNA in the presence of {beta-32P}GTP, it could be shown that cap sites correspond to sites of initiation of RNA synthesis.

Infect Immun, 1981 Feb, 31(2), 631 - 5
Specific inhibition of Escherichia coli ferrienterochelin uptake by a normal human serum immunoglobulin; Moore DG et al.; Normal human serum contains an enterochelin-specific antibody which presumably acts with transferrin to hinder iron assimilation by enterochelin-producing pathogens . This antibody can be isolated from serum by sodium sulfate fractionation or affinity chromatography by employing an enterochelin-derived ligand (2,3-dihydroxy-N-benzoyl-L-serine) attached to aminohexyl Sepharose 4B . In assays of iron uptake by whole cells, the antibody inhibited enterochelin-directed uptake but not that mediated by citrate or ferrichrome . Also, the growth stimulatory effect of enterochelin on an Ent- strain of Escherichia coli was blocked by the immunoglobulin . This antibody has a high affinity for enterochelin; various elution procedures employing high salt concentrations and low pH failed to remove it from affinity columns . Elution with 3 M sodium thiocyanate or 13 mM 2,3-dihydroxybenzoic acid proved successful . Two pieces of evidence indicate the enterochelin-specific antibody is primarily of the immunoglobulin A (IgA) isotype . It could be removed from serum with goat antihuman IgA and was present only in sodium sulfate fractions of serum known to contain IgA.

J Immunol, 1981 Feb, 126(2), 709 - 14
T11: a new protein marker on activated murine T lymphocytes; Gately MK et al.; Murine lymphocytes were activated in vitro in mixed lymphocyte cultures (MLC) or by the addition of the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), or E . coli lipopolysaccharide (LPS) . Activated lymphocytes were internally labeled with 35S-methionine and then disrupted by hypotonic lysis . A plasma membrane-enriched fraction was isolated from each cell population, and the 35S-labeled proteins in this fraction were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) . An intensely labeled band, the position of which indicated an apparent m.w . of 11,000, was observed when plasma membrane-enriched fractions from MLC- and Con A-activated cells were subjected to SDS-PAGE . In contrast, plasma membrane-enriched fractions from normal spleen cells, LPS-activated cells, PHA-activated cells, and EL4, RDM4, and P815 tumor cells possessed little or none of this protein, which we have designated T11 . T11 was not found in the soluble cytoplasmic protein from MLC-activated cells . Hence the presence of T11 in the plasma membrane-enriched fraction from these cells cannot be attributed to contamination by cytoplasmic protein . Removal of T cells from populations of MLC-activated cells by treatment with monoclonal anti-Thy 1 and complement removed T11 . These results suggest that T11 may represent a new protein marker on a subclass of activated T lymphocytes.

J Gen Microbiol, 1981 Feb, 122(Pt 2), 171 - 9
Partial replacement of succinate dehydrogenase function by phage- and plasmid-specified fumarate reductase in Escherichia coli; Guest JR; Phages capable of transducing succinate dehydrogenase mutants (sdh) of Escherichia coli were isolated from pools of artificially constructed recombinant lambda phages using a selective casein digest medium . These phages produced characteristically dense turbid plaques, and as prophages they increased the aerobic growth efficiencies of sdh mutants on complex media but were unable to promote growth with succinate as sole carbon and energy source (an essential feature of sdh + strains) . The phages were identified as fumarate reductase transducing phages (lambda frdA) by the presence of a characteristic 4.9 kilobase pairs R.HindIII fragment of bacterial DNA, the expression of a polypeptide with a relative molecular mass of 72,000 (the frdA gene product) and by comparing their transducing activities with authentic lambda frdA phages . In parallel studies a strain containing a ColE1-frd hybrid plasmid (pGS1 = pLC16.43) was characterized . Transfer of pGS1 to sdh mutants was accompanied by increased aerobic growth efficiencies on complex media and the ability to utilize succinate as sole carbon and energy source . It was concluded that fumarate reductase can replace succinate dehydrogenase but the extent of the reversal of the sdh lesion depends on frd gene dosage and the titration of the repressor which normally prevents aerobic synthesis of the reductase.

Acta Med Okayama, 1981 Feb, 35(1), 1 - 17
Reconstitution of chromatin at physiological ionic strength and regulation of transcription of the reconstituted chromatin; Hidaka H; Rat liver and simian virus 40 (SV40) chromatin were reconstituted in vitro under physiological conditions of ionic strength and temperature . The nucleosome assembly under the conditions was mediated in the presence of chromatin extracts, rich in nicking-closing activity, from rat liver or cultured CV-1 cells . The frequency of nucleosome assembly on DNA was dependent on both the incubation time and the weight ratio of histone to DNA . The regulatory effects of host cellular histones on the transcription of SV40 DNA were investigated by using reconstituted SV40 chromatin containing or lacking histone H1 . The cellular histones composing the chromatin were prepared from permissive CV-1 cells . Transcription of chromatin was analyzed in vitro using Escherichia coli DNA-dependent RNA polymerase . The rate of incorporation of ribonucleoside triphosphates into RNA synthesized on SV40 chromatin containing Hl as the template was 5 to 10% of the rate for RNA synthesized on supercoiled SV40 DNA . The rate of incorporation for SV40 chromatin lacking Hl was approximately 40 to 50% of that for SV40 DNA . RNA products transcribed from both these chromatin and SV40 DNA were fairly homogeneous 16 to 28S species with several identical peaks.

Proc Natl Acad Sci U S A, 1981 Feb, 78(2), 843 - 7
Catenation and knotting of duplex DNA by type 1 topoisomerases: a mechanistic parallel with type 2 topoisomerases; Brown PO et al.; Escherichia coli omega protein, a type 1 topoisomerase, can catenate and knot duplex DNA circles . Previously, these activities were thought to be limited to type 2 topoisomerases . Catenation by omega requires a nick in one of the participating molecules, but it is not necessary that both be nicked . Catenation does not depend on sequence homology and is greatly stimulated by DNA-condensing agents such as spermidine . A eukaryotic type 1 topoisomerase can also interlock duplex DNA circles . These activities cannot easily be explained by the widely held topoisomerase model in which a reversible nick in DNA allows free rotation about the unbroken strand . We suggest instead passage of a DNA segment though a transient enzyme-bridged break in a single DNA strand . This is analogous to the sign inversion mechanism of the type 2 topoisomerases, and thus expresses an essential mechanistic unity among topoisomerases . As expected for relaxation by a single-strand passage, omega changes the linking number of DNA in steps of 1.

Proc Natl Acad Sci U S A, 1981 Feb, 78(2), 815 - 9
Expression of herpes simplex virus type I thymidine kinase gene in Escherichia coli; Garapin AC et al.; A herpes simplex virus type I DNA fragment containing the sequence coding for thymidine kinase was fused to the very beginning of the Escherichia coli lac Z gene in the three possible reading frames . When the thymidine kinase sequence was in the orientation fit to be transcribed from the lac promoter, functional thymidine kinase was made under lac control in all three cases . Sequences data indicate that translation reinitiation occurs at the 5' end of the thymidine kinase gene after stop signals . Two T+A-rich sequences, which may be part of eukaryotic promoters, are found in the same region.

Proc Natl Acad Sci U S A, 1981 Feb, 78(2), 752 - 6
Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli; Lee NL et al.; The DNA-protein contact sites in the ara regulatory region, which contains the promoters for araBAD and araC, have been determined for araC protein, the cyclic AMP-binding protein, and RNA polymerase, by using the methylation protection and DNase I protection methods . The functional significance of binding was assessed by correlating the state of occupancy of these sites with promoter activity in transcription initiation . Our results suggest that the basis for araC autoregulation is that araC protein, in either its activator (P2) or repressor (P1) form, acts as a repressor for araC, by binding to the RNA polymerase attachment site at the araC promoter . We also found that the araC and araBAD promoters share a common site of positive control by the cyclic AMP-binding protein, located 90 bases from the araBAD and 60 bases from the araC transcriptional start points . A model for the mechanism of regulation of araBAD and araC expression by the catabolite gene-activator protein, P1, and Pe is proposed . An earlier model proposed by Ogden et al . {Ogden S., Haggerty, D., Stoner, C . M., Kolodrubetz, D . & Schleif, R . (1980) Proc . Natl . Acad . Sci, USA 77, 3346-3350} is discussed in the light of the data presented in this paper.

Proc Natl Acad Sci U S A, 1981 Feb, 78(2), 707 - 11
Movement and site selection for priming by the primosome in phage phi X174 DNA replication; Arai K et al.; The Escherichia coli priming system used for initiation of DNA chains on phage phi X174 single-stranded DNA is a multiprotein unit called the primosome {Arai, K . & Kornberg . A . (1981) Proc . Natl . Acad . Sci . USA 78, 69-73} . Assembled with participation of seven prepriming proteins and primase at a unique place on the phi X174 DNA template, the primosome is bound tightly to the DNA, yet moves rapidly and unidirectionally opposite to primer and DNA chain synthesis . Contributions of protein n' and dnaB protein, two components of the primosome, to movement and site selection for priming are considered in this report . Figuratively, the primosome can be likened to a locomotive that depends on protein n' as its engine and dnaB protein as the engineer . Protein n', a DNA-dependent ATPase (dATPase) appears to use the energy of hydrolysis of the nucleoside triphosphate for processive translocation of the primosome . dnaB protein, A DNA-dependent ribonucleosidetriphosphatase, depends on allosteric effects of a nucleoside triphosphate to induce changes in the structure of the single-stranded DNA at preferred sequences that enable primase to synthesize a short primer for initiation of DNA synthesis (unpublished data) . These primosome properties have important implications for the progress of the replication fork of the E . coli chromosome.

J Bacteriol, 1981 Feb, 145(2), 914 - 9
Transductional mapping of ksgB and a new Tn5-induced kasugamycin resistance gene, ksgD, in Escherichia coli K-12; Fouts KE et al.; We have mapped the Escherichia coli ksgB gene to min 36.5, 0.8 min from man and 0.7 min from aroD . A new kasugamycin resistance (Ksgr) gene, ksgD, has been isolated, using a transposon, Tn5 . ksgD::TN5 is 44% cotransducible with sbcA, unlinked to trp, and unlinked to man (by P1 transduction) . The ksgD::Tn5 has a late time of entry from HfrB7 (PO43) . These data place ksgD clockwise from sbcA (which enters early from HfrB7) at min 30.4 . The reistance of ksgB ksgD single and double mutant strains has been quantitated . Single mutations, ksgB or ksgD, gave resistance to 600 micrograms of kasugamycin per ml, whereas a ksgB ksgD strain was able to grow in the presence of kasugamycin levels in excess of 3,000 micrograms/ml . This indicates that the mechanisms of resistance coded for by the two genes are independent and synergistic.

J Bacteriol, 1981 Feb, 145(2), 861 - 6
Isolation and analysis of multicopy extragenic suppressors of dnaA mutations; Projan SJ et al.; Recombinant plasmids were constructed from restriction enzyme digests of Escherichia coli chromosomal deoxyribonucleic acid and pMB9 plasmid deoxyribonucleic acid and selected for correction of the dnaA phenotype . The three plasmids isolated, all retransformed dnaA cells, both recA+ and recA, such that all tetracycline-resistant transformants selected at permissive temperature simultaneously became temperature resistant . Restriction enzyme mapping of the plasmids showed all three to be different, and it was subsequently shown that none contained the dnaA+ gene . Though each of the three plasmids suppressed three different temperature-sensitive dnaA alleles, none corrected the phenotype of an unsuppressed dnaA amber allele . It was concluded, therefore, that each plasmid contained a unique extragenic suppressor of dnaA and that the suppression was observed because of the elevated gene dosage of the cloned material . The plasmids were unstable in the absence of selection.

Gastroenterology, 1981 Feb, 80(2), 356 - 65
Antisecretory effects of indomethacin on rabbit ileal mucosa in vitro; Smith PL et al.; Prior in vivo studies have shown that indomethacin, which inhibits prostaglandin (PG) synthesis, affects fluid transport in the small bowel, enhancing spontaneous fluid absorption and reducing the amount of fluid that accumulates in response to cholera toxin and other secretory stimuli . To further explore the mechanisms involved, we determined the effects of indomethacin on ion transport, cAMP concentration, and PGE2 production in rabbit ileal mucosa in vitro . Indomethacin (1 mM), when added alone, had no significant effect on short-circuit current (either basal or glucose-stimulated), Cl fluxes, or cAMP concentration . Indomethacin did, however, inhibit the ion transport changes caused by several secretagogues: Effects of theophylline, Ca-ionophore A23187, and arachidonate were reversibly inhibited by at least 65%, whereas effects of dibutyryl cAMP, 16,16-dimethyl PGE2, cholera toxin, and heat-stable Escherichia coli enterotoxin were inhibited by about 30% . Indomethacin also inhibited the theophylline-evoked increase in cAMP concentration . Indomethacin decreased PGE2 production under basal conditions and in the presence of theophylline and A23187, which may partly explain the antisecretory action of the drug . Since arachidonate increased PGE2 release from the mucosa more than 10-fold and indomethacin did not inhibit this effect, indomethacin at high concentration (0.5-1 mM) appears to also inhibit the action of intestinal secretagogues by a prostaglandin-independent mechanism . This study also demonstrates that the antisecretory effect of indomethacin is not simply due to stimulation of an unrelated absorptive process.

Biull Eksp Biol Med, 1981 Feb, 91(2), 188 - 90
{Effect of mitogens on suppression of antibody formation and proliferation in dense cultures}; Korukova AA et al.; A study was made of the effect of mitogens on general proliferation and primary immune response to sheep red blood cells in density-inhibited cultures of mouse spleen cells . The mitogens applied included fetal calf serum and both B cell- and T cell-specific mitogens (dextran sulfate, LPS and ConA) . Experiments with 3H-thymidine incorporation demonstrated that the proliferation was equally enhanced by any mitogen in both optimal and density-inhibited cultures . The mitogens did not remove the density inhibition of antibody formation.

J Virol, 1981 Feb, 37(2), 748 - 54
Mechanism of action of Moloney murine leukemia virus RNase H III; Gerard GF; The mechanism of action of Moloney murine leukemia virus RNase H III was studied, utilizing the model substrate (A)n . (dT)n and polyacrylamide gel electrophoresis to assay enzyme activity . Examination by electrophoresis on 15% polyacrylamide gels in 7 M urea and on DEAE-cellulose paper in 7 M urea revealed that, early in a reaction with {3H}(A)n . (dT)n as substrate, RNase H III generated products ranging in length from 80 to 90 nucleotides to less than 10 nucleotides and that after extended incubation the limit digest products generated were 3 to 15 nucleotides long . Product oligomers were of the following configuration: {5'-P, 3'-OH}(A)n . RNase H III was shown to be an exonuclease requiring free ends in its substrate for activity by the inability to degrade RNA inserted in Escherichia coli ColE1 plasmid DNA . The enzyme was capable of attacking RNA in RNA-DNA hybrids in the 5' to 3' and 3' to 5' directions as demonstrated by the use of {3H, 5'-32P}(A)600 . (dT)n and cellulose-{3H}(A)n . (dT)n . Rnase H III was random in its mode of action because addition of excess unlabeled (A)n . (dT)n to an ongoing reaction with {3H}(A)n . (dT)n as substrate resulted in immediate inhibition of enzyme activity.

Am J Vet Res, 1981 Feb, 42(2), 173 - 7
Protection against enteric colibacillosis in pigs suckling orally vaccinated dams: evidence for pili as protective antigens; Moon HW; Pregnant gilts were vaccinated orally with Escherichia coli that produced pilus antigens K99 or 987P . The vaccines were live or dead enterotoxigenic E coli (ETEC) or a liver rough non-ETEC strain which has little ability to colonize pig intestine . Pigs born to the gilts were challenge exposed orally with K99+ or 987P+ ETEC, which did not produce heat-labile enterotoxin or flagella and which produced somatic and capsular antigens different from those of the vaccine strains . Control gilts had low titers of serum and colostral antibodies against pilus antigens, and their suckling pigs frequently had fatal diarrhea after challenge exposure . Serum antibody titers against pilus antigens of the vaccine strains increased in the gilts after vaccination with liver ETEC, and the colostral antibody titers of these gilts were higher than those of controls . Pigs suckling such vaccinated gilts were more resistant than controls to challenge strains were of different pilus types, and it could not be attributed to enterotoxin neutralization by colostrum . In contrast to the live ETEC vaccines given to the pregnant gilts, the liver rough non-ETEC and dead ETEC vaccines stimulated little or no production of antibody against pilu, and the pigs born of these vaccinated gilts remained highly susceptible to challenge exposure . The results support the hypothesis that pilu can be protective antigens in oral ETEC vaccines . It was indicated that in the system reported, protection depended on living bacteria for the production of pilus antigens in vivo or for the transport of pilus antigens across intestinal epithelium.

J Pharm Pharmacol, 1981 Feb, 33(2), 65 - 8
The measurement of phenol coefficients by flow microcalorimetry; Beezer AE et al.; Microcalorimetric bioassay procedures have been applied to the determination of phenol coefficients and dilution coefficients . The derived values are compared with those from a standard AOAC test procedure . This microcalorimetric results indicate close agreement with AOAC values, good reproducibility (+/- 2%), rapidity (30 min per test), potential for automation and the determination of in-use dilutions of disinfectants.

J Bacteriol, 1981 Feb, 145(2), 687 - 95
Synthesis and metabolism of uracil-containing deoxyribonucleic acid in Escherichia coli; Warner HR et al.; Significant amounts of uracil were found in the deoxyribonucleic acids (DNAs) of Escherichia coli mutants deficient in both uracil-DNA glycosylase (ung) and deoxyuridine 5'-triphosphate nucleotidohydrolase (dut) activities, whereas little uracil was found in the DNAs of wild-type cells and cells deficient in only one of these two activities . The amounts of uracil found in the DNAs of dut ung mutants were directly related to the growth temperature of the cultures, apparently because the deoxyuridine 5'-triphosphate nucleotidohydrolase synthesized by dut mutants was temperature sensitive . The dut mutant used failed to grow exponentially, became filamentous at temperatures above 25 degrees C, and exhibited a hyperrec phenotype; however, the ung mutation suppressed all of these effects . Although the dut ung mutants grew exponentially at all temperatures, their growth rates were always slower than the growth rate of the wild type . Since pool size measurements indicated that both deoxyuridine triphosphate and deoxythymidine triphosphate pools were markedly elevated in dut mutants, the reduced growth rate of dut ung cells apparently was due to the actual presence of uracil in the DNA, rather than to a deficiency of deoxyuridine triphosphate and deoxyribosylthymine triphosphate for DNA synthesis . The presence of uracil in E . coli donor DNA also markedly reduced the recombination frequency when the recipient cells were ung+, indicating that DNA repair commenced before the entering DNA could be replicated.

Gastroenterology, 1981 Feb, 80(2), 321 - 6
Effect of chlorpromazine on intestinal secretion mediated by Escherichia coli heat-stable enterotoxin and 8-Br-cyclic GMP in infant mice; Robins-Browne RM et al.; Subcutaneously administered chlorpromazine reduced intestinal fluid accumulation induced by Escherichia coli heat-stable enterotoxin in infant mice . The antisecretory effect of chlorpromazine, although dose related, was, even with high doses, less than that observed with respect to cholera toxin . Whereas 100 micrograms chlorpromazine abolished cholera toxin-induced intestinal secretion almost completely, 500 microgram chlorpromazine (equivalent to 200 microgram/g body wt) lowered secretion induced by heat-stable enterotoxin by only 41% . The effect of chlorpromazine on intestinal secretion was quantitatively similar regardless of whether heat-stable enterotoxin or the cyclic GMP analogue, 8-Br-cyclic GMP, was the secretagogue . This finding, which suggested that the inhibitory effect of chlorpromazine on heat-stable enterotoxin was independent of guanylate cyclase, was confirmed by assaying this enzyme in intestinal homogenates from mice that had been inoculated with chlorpromazine, and also in experiments in which chlorpromazine was added to guanylate cyclase assay mixtures in vitro . Caution is advised before chlorpromazine is routinely adopted for the treatment of all syndromes of watery diarrhea.

Nature, 1981 Jan 29, 289(5796), 417 - 20
Removal of O6-methylguanine from DNA of normal and xeroderma pigmentosum-derived lymphoblastoid lines; Sklar R et al.; The ability to excise (repair) UV-induced pyrimidine dimers in Escherichia coli is not related to its ability to remove N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced O6-methylguanine (O6-MeG) from DNA . It was therefore surprising that certain xeroderma pigmentosum cell lines, deficient in dimer excision, were also unable to remove O6-MeG . We find that removal of O6-MeG occurs rapidly with a half life of less than 1 h . Two cell types can be distinguished: mex+, which remove O6-MeG residues produced by incubation with 0.5 microgram ml-1 MNNG, and mex- cells, which are unable to remove the adduct . Xeroderma pigmentosum-derived lymphoblastoid lines of complementation groups A, C or D may be either mex+ or mex- . The biochemical mechanism for the removal of O6-MeG in human cells is distinct from the excision of adducts produced by compounds such as N-acetoxy-N-2-acetylaminofluorene (AAAF) or by UV irradiation but it is not clear whether the distinction between mex+ and mex- lines is genetic or epigenetic.

Biochim Biophys Acta, 1981 Jan 29, 652(1), 139 - 50
Hydrophobic interactions of Escherichia coli ribosomes; Fabry M et al.; We have demonstrated the presence of hydrophobic sites on the surface of Escherichia coli ribosomes by means of hydrophobic chromatography on Octyl-Sepharose . Both 30-S and 50-S ribosomal subunits adsorb to Octyl-Sepharose at a low salt concentration, and can be eluted from it with a nonionic detergent without substantial changes in structure or activity . By testing a series of LiCl-derived ribosomal cores for their ability to adsorb to Octyl-Sepharose we have shown that the interaction of ribosomal particles with Octyl-Sepharose is dependent on the presence of certain ribosomal proteins; the core particles which lack these proteins do not bind to Octyl-Sepharose . The binding of a series of different ribosomal cores to nitrocellulose filters (Millipore) yielded the same pattern as was observed with Octyl-Sepharose, i.e . the more protein-depleted the particles, the less they were adsorbed . Thus, the adsorption of ribosomes to Millipore filters and to Octyl-Sepharose is presumably of the same hydrophobic nature.

Biochim Biophys Acta, 1981 Jan 26, 663(1), 1 - 13
Incorporation and metabolism of 2-acyl lysophospholipids by Escherichia coli; Homma H et al.; The incorporation of 2-acyl lysophospholipids into Escherichia coli, and their metabolism were studied . 2-{14C}Acyl lysophosphatidylethanolamine could penetrate into E . coli cells and was mainly incorporated into phosphatidylethanolamine . 2-Acyl lysophosphatidylethanolamine was partially degraded, but some of it was incorporated into membrane phospholipids by acylation . 2-Acyl lysophosphatidylcholine also entered cells and was acylated to phosphatidylcholine . The acylation of 2-acyl lysophospholipid by the envelope fraction was also studied . Fatty acids were incorporated into 2-acyl lysophospholipids by the envelope fraction in the presence of ATP and Mg2+, and the incorporation was stimulated by acyl carrier protein, but not by coenzyme A . No acylation was observed with acyl coenzyme A as acyl donor . The acylation activities of the inner and outer membranes were examined . Pathways for degradation and modification of membrane phospholipids in E . coli are proposed.

Biochim Biophys Acta, 1981 Jan 26, 663(1), 46 - 57
Activity of N-acetylmuramoyl-L-alanine amidase in phospholipidic environments; Vanderwinkel E et al.; A purified preparation of N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), a murein hydrolase from Escherichia coli, was found to lose its activity during incubation in the presence of bacterial phospholipid suspensions . Whether it was co-dispersed with the phospholipids or added to sonicated phospholipid suspension, the enzyme was inhibited (or inactivated) from the first minutes of incubation at 37 degree C . As phosphatidylglycerol/cardiolipin ratio of the phospholipid suspension as increased (all other things being equal), a further decrease of amidase activity was observed . The highest losses of activity were found after co-dispersion of the enzyme and the substrate together with the phospholipids, the resulting suspension being formed of larger multilayered vesicles, as revealed by electron microscopy . In these conditions, the effect on enzyme activity was only partially accounted for by the proportion of the enzyme that was entrapped in the vesicles . The entrapment capacity of the enzyme (using a 35S-labelled enzyme preparation) and of the substrate (3H-labelled) by the multilamellar phospholipidic vesicles did not significantly change as a function of their relative content of phosphatidylglycerol and cardiolipin . The possible physiological meaning of the results is discussed is connection with our previous data and with other works related to membranous phospholipid distribution and to septum formation control in bacteria.

J Biol Chem, 1981 Jan 25, 256(2), 905 - 11
A histone-like protein (HTa) from Thermoplasma acidophilum . II . Complete amino acid sequence; DeLange RJ et al.; The complete amino acid sequence of the DNA-binding histone-like protein (Hta) from Thermoplasma acidophilum has been established by sequence studies directly on the protein and on tryptic, chymotryptic, and thermolysin peptides derived from the protein . The sequence of the 89-residue form of HTa is: H2N-Val-Gly-Ile-Ser-Glu-Leu-Ser-Lys-Glu-Val-Ala-Lys-Lys-Ala-Asn-Thr-Thr-Gln-Lys -Val-Ala-Arg-Thr-Val-Ile-Lys-Ser-Phe-Leu-Asp-Glu-Ile-Val-Ser-Glu-Ala-Asn-Gly-Gl y-Gln-Lys-Ile-Asn-Leu-Ala-Gly-Phe-Gly-Ile-Phe-Glu-Arg-Arg-Thr-Gln-Gly-Pro-Arg-L ys-Ala-Arg-Asn-Pro-Gln-Thr-Lys-Lys-Val-Ile-Glu-Val-Pro-Ser-Lys-Lys-Lys-Phe-Val- Phe-Arg-Ala-Ser-Ser-Lys-Ile-Lys-Tyr-Gln-Gln-COOH The molecular weight calculated from the sequence is 9,934 . Another form of HTa probably differs only by the presence of an additional residue (methionine) at the NH2 terminus (the calculated molecular weight of this form is 10,065) . HTa resembles eukaryotic histones in several ways, including some sequence homology, HTa also shows sequence homology with the Escherichia coli DNA-binding proteins NS1 (or HU-1) and NS2 (or HU-2).

J Biol Chem, 1981 Jan 25, 256(2), 815 - 22
Substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli; Bisswanger H; The investigation of the substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli allows a description of the binding region of pyruvate . Substrate analogs with electronegative substitutions in the methyl group show a strong competitive inhibition of the overall reaction of the pyruvate dehydrogenase complex . The most efficient inhibitor is fluoropyruvate which has a more than 100-fold higher affinity for the enzyme than pyruvate (Ki = 1.4 x 10(-6) M) does . The affinity of alpha-keto acids decreases with increasing chain length . Branched chain alpha-keto acids are even less effective inhibitors (Ki = approximately 0.02 M) . alpha-Ketobutyrate is the only alpha-keto acid which is able to substitute for pyruvate as a substrate in the overall reaction of the enzyme complex . The Km value (3 mM) is 10-fold greater than that for pyruvate . The steady state kinetics of the overall reaction of alpha-ketobutyrate exhibits the same cooperativity (nh = 1.9) as seen with pyruvate . Small modifications of the carbonyl or the carboxyl group of pyruvate prevent binding completely . Binding of pyruvate to the pyruvate dehydrogenase complex may thus require interaction with two independent electrophilic centers . The acceptance of the methyl group seems not so much due to lipophilic interactions as to a steric effect . The experiments were carried out with an enzyme which was purified by a modified procedure which is faster and more convenient than previous methods . The procedure is applicable up to 0.5 liter of crude extract.

J Biol Chem, 1981 Jan 25, 256(2), 736 - 42
Phospholipid synthesis in Escherichia coli . Characteristics of fatty acid transfer from acyl-acyl carrier protein to sn-glycerol 3-phosphate; Rock CO et al.; Two kinetically distinguishable sn-glycerol 3-phosphate (glycerol-P) acryltransferase activities were detected in Escherichia coli inner membranes using acyl-acyl carrier protein (ACP) substrates . The first system was characterized as having a Michaelis constant (Km) for glycerol-P of 90 microM and utilized palmitoyl-ACP to form primarily 1-acylglycerol-P . Palmitoyl-CoA and cis-vaccenoyl-ACP were also utilized by this system but, with these substrates, significantly more phosphatidic acid was formed as compared to palmitoyl-ACP . Although palmitoyl-ACP and palmitoyl-CoA had kinetically indistinguishable glycerol-P sites, distinct acyl donor binding sites were inferred from kinetic experiments using acyl carrier protein as an acyltransferase inhibitor . A second enzyme system, characterized as having a Km for glycerol-P of 700 microM, was found using palmitoyl-ACP as a substrate . This acyltransferase had a slightly higher pH optimum than the low Km acyltransferase activity, and phosphatidic acid was the major product . Two degradative reactions were identified in this system . One reaction yielded diacylglycerol when palmitoyl-ACP was the substrate . The other degradative reaction produced glycerol . Glycerol was formed in all incubations but was most pronounced when palmitoyl-ACP was the substrate.

J Biol Chem, 1981 Jan 25, 256(2), 767 - 72
Kinetic alpha-deuterium isotope effects for Escherichia coli purine nucleoside phosphorylase-catalyzed phosphorolysis of adenosine and inosine; Stein RL et al.; Kinetic alpha-deuterium isotope effects have been measured for the purine nucleoside phosphorylase-catalyzed phosphorolysis of adenosine and inosine by a competitive double label technique at saturating concentrations of the second substrate, phosphate . Under these conditions the observed isotope effect, kH/kD, is on the second order rate constant, Vmax/Km, for reaction of nucleoside with the Michaelis complex of enzyme and phosphate . For adenosine, at neutral pH, the isotope effect is unity . For inosine, kH/kD was determined as a function of pH (the numbers in parentheses are the ratios of Vmax at that pH to Vmax at pH 7.3): 1.10 at pH 5.0 (0.19); 1.10 at pH 6.1 (0.72); 1.01 at pH 7.3 (1.00); 1.16 at pH 8.4 (0.22); and 1.18 at pH 9.4 (0.04) . These values suggest a mechanism for purine nucleoside phosphorylase involving a change in rate-limiting step from one at pH values near neutrality for which cleavage of the nucleoside C-N bond is not rate limiting to a step at extremes of pH with a transition state having considerable oxocarbonium ion-like character.

J Biol Chem, 1981 Jan 25, 256(2), 560 - 2
Identification of the malK gene product . A peripheral membrane component of the Escherichia coli maltose transport system; Shuman HA et al.; The malK gene product of Escherichia coli has been identified through the use of a previously described technique that employs gene fusions (Shuman, H . A., Silhavy, T . J., and Beckwith, J . R . (1980) J . Biol . Chem . 255, 168-174) . This protein, along with the four other products of the malB locus, comprise the complete maltose transport system . The malK protein has a molecular weight of approximately 40,000 and is located in the cell envelope . In mutant strains which lack another component of the transport system, the malG protein, the malK protein is located in the cytoplasm . This alteration in location suggests that the malK protein is associated with the inner surface of the cytoplasmic membrane via an interaction with the malG protein.

J Biol Chem, 1981 Jan 25, 256(2), 882 - 6
Affinity labeling of the active site of Escherichia coli glutamine synthetase by 5'-p-fluorosulfonylbenzoyladenosine; Foster WB et al.; The interaction of Escherichia coli glutamine synthetase with the adenosine 5'-triphosphate analogue, 5'-p-fluorosulfonylbenzoyladenosine (5'-FSO2BzAdo), has been studied . This interaction results in the covalent attachment of the 5'-FSO2BzAdo to the enzyme with concomitant loss of catalytic activity . Although adenine nucleotides interact with glutamine synthetase at three distinct sites--a noncovalent AMP effector site, a regulatory site of covalent adenylylation, and the catalytic ATP/ADP binding site--our studies suggest that reaction with 5'-FSO2BzAdo occurs only at the active center . When glutamine synthetase was incubated with 5'-FSO2BzAdo, the decrease in catalytic activity obeyed pseudo-first order kinetics . The plot of the observed rate constant of inactivation versus the concentration of 5'-FSO2BzAdo was hyperbolic, consistent with reversible binding of the analogue to the enzyme prior to covalent attachment . Protection against inactivation was afforded by ATP and ADP; L-glutamate did not protect the enzyme against inactivation, but rather enhanced the rate of inactivation, consistent with the observations of others (Timmons, R . B., Rhee, S . G., Luterman, D . L., and Chock, P . B . (1974) Biochemistry 13, 4479-4485) that there is synergism in the binding of the two substrates to the enzyme . The incorporation of approximately 1.09 mol of the 5'-FSO2BzAdo/mol of glutamine synthetase subunit resulted in the total loss of enzymatic activity . The results suggest that 5'-FSO2BzAdo occupies the ATP binding site at the active center of glutamine synthetase and binds covalently to an amino acid residue nearby.

J Biol Chem, 1981 Jan 25, 256(2), 644 - 8
Subunit interaction in unadenylylated glutamine synthetase from Escherichia coli . Evidence from methionine sulfoximine inhibition studies; Rhee SG et al.; Although glutamine synthetase from Escherichia coli is composed of 12 identical subunits, there is no evidence that homologous subunit interactions occur in fully unadenylylated or fully adenylylated enzyme . Meister and co-workers (Manning, J . M., Moore, S., Rowe, W . B., and Meister, A . (1969) Biochemistry 8, 2681-2685) have shown that L-methionine-S-sulfoximine, one of the four diastereomers of methionine sulfoximine, preferentially inhibits glutamine synthetase irreversibly in the presence of ATP, due to the formation of tightly bound products, ADP, and methionine sulfoximine phosphate . Using highly purified unadenylylated glutamine synthetase and the two resolved diastereomers of L-methionine-S,R-sulfoximine, we have studied both the kinetics of glutamine synthetase inactivation in the presence of excess methionine sulfoximine and ATP, and the binding of methionine sulfoximine to the enzyme . The results reveal that (a) the apparent first order rate constant of irreversible inactivation by the S isomer decreases progressively from the expected first order rate, indicating that an inactivated subunit retards the reactivity of its neighboring subunits toward methionine sulfoximine and ATP; (b) the R isomer does not inactivate glutamine synthetase irreversibly in the presence of ATP; however, the R isomer is capable of protecting the enzyme temporarily from the irreversible inhibition by the S isomer; and (c) the binding of the S isomer monitored by changes in protein fluorescence exhibits an apparent negative cooperative binding isotherm, whereas the R isomer yields an apparent positive cooperative pattern.

Nucleic Acids Res, 1981 Jan 24, 9(2), 401 - 14
Regulation of aromatic amino acid transport by tRNA: role of 2-methylthio-N6-(delta2-isopentenyl)-adenosine; Buck M et al.; E . coli growing rapidly in media where ferric iron is not freely available contain a population of specifically undermodified tRNAs . These tRNAs contain isopentenyl adenosine instead of the usual methylthioisopentenyl adenosine adjacent to the 3' end of the anticodon . Iron restricted E . coli also show an enhanced capacity to transport aromatic amino acids into the cell . Our work shows that undermodified tRNAs for phe, tyr and trp can function as positive regulatory elements of the aromatic amino acid transport system in E . coli . This iron related metabolic control, mediated through a specific post-transcriptional modification of the tRNAs, may be an important mechanism for adapting E . coli for growth in an iron restricted environment.

Nucleic Acids Res, 1981 Jan 24, 9(2), 293 - 307
The secondary structure of the protein L1 binding region of ribosomal 23S RNA . Homologies with putative secondary structures of the L11 mRNA and of a region of mitochondrial 16S rRNA; Branlant C et al.; An heterologous complex was formed between E . coli protein L1 and P . vulgaris 23S RNA . We determined the primary structure of the RNA region which remained associated with protein L1 after RNase digestion of this complex . We also identified the loci of this RNA region which are highly susceptible to T1, S1 and Naja oxiana nuclease digestions respectively . By comparison of these results with those previously obtained with the homologous regions of E . coli and B . stearothermophilus 23S RNAs, we postulate a general structure for the protein L1 binding region of bacterial 23S RNA . Both mouse and human mit 16S rRNAs and Xenopus laevis and Tetrahymena 28S rRNAs contain a sequence similar to the E . coli 23s RNS region preceding the L1 binding site . The region of mit 16S rRNA which follows this sequence has a potential secondary structure bearing common features with the L1-associated region of bacterial 23S rRNA . The 5'-end region of the L11 mRNA also has several sequence potential secondary structures displaying striking homologies with the protein L1 binding region of 23S rRNA and this probably explains how protein L1 functions as a translational repressor . One of the L11 mRNA putative structures bears the features common to both the L1-associated region of bacterial 23S rRNA and the corresponding region of mit 16S rRNA.

Nucleic Acids Res, 1981 Jan 24, 9(2), 267 - 75
Adenosine dimethylation of 16S ribosomal RNA: effect of the methylgroups on local conformational stability as deduced from electrophoretic mobility of RNA fragments in denaturing polyacrylamide gels; Van Charldorp R et al.; The electrophoretic mobility of RNA fragments derived from the 3'-end of 16S rRNA on slabs of polyacrylamide gel in the presence of urea is strongly influenced by dimethylation of the N6-aminogroup of two adjacent adenosines . This is not due to the presence of the methylgroups per se, but must be ascribed to an effect of methylation on long range intramolecular interactions at these denaturing conditions . When it is assumed that the electrophoretic mobilities of the RNA fragments in the polyacrylamide matrix are determined by the conformational state(s) of the fragments, dimethylation of the adenosines leads in the smaller fragments to a less compact average conformation and in the larger fragments to a more compact average conformation . An effort is made to comprehend the effects of adenosine dimethylation in terms of secondary structure based on nucleotide sequence.

Nucleic Acids Res, 1981 Jan 24, 9(2), 415 - 36
Salt dependence and thermodynamic interpretation of the thermal denaturation of small DNA restriction fragments; Hillen W et al.; The influence of cation concentration on the thermal denaturation of DNA restriction fragments from the E . coli lac regulatory region and from pVH51, ranging in size from 43- to 880- bp, is described . Upon increasing the ionic strength, the melting transitions broaden in a cooperative manner at salt concentrations characteristic for the specific fragment . For three fragments studied in detail, the salt concentration dependence at the midpoint varied between 0.03 and 0.19 M Na+ . Along with the broadening, the melting transitions become more symmetrical . This result is discussed with respect to the irreversibility of melting transitions at low ionic strength . After a cooperative broadening, the shape of the melting curves remains constant up to salt concentrations of 0.5 M Na+ . The dTM/dlog{Na+} values for three fragments fall between 15.7 and 16.7 . An easily applicable approximation of the van't Hoff equation is used to evaluate the enthalpies of 13 transitions arising from the denaturation of 43 to 600 bp . The results of this analysis are compared to calculations of the expected enthalpies based on calorimetric measurements . The TMs of most transitions were directly related to the base composition, but several deviations from the predicted behavior were observed . The possible influences of fragment length and sequence on the thermal stability are discussed . The experimental and mathematical procedure to resolve a thermal denaturation transition with a width f 0.17 +/- 0.01 degrees and its distinction from another preceeding transition only approximately 0.15 degrees away in an 880-bp Hae III fragment from pVH51 is described . This transition is about half as wide as the smallest one reported to date.

Nucleic Acids Res, 1981 Jan 24, 9(2), 309 - 21
A system for shotgun DNA sequencing; Messing J et al.; A multipurpose cloning site has been introduced into the gene for beta-galactosidase (beta-D-galactosidegalactohydrolase, EC 3.21.23) on the single-stranded DNA phage M13mp2 (Gronenborn, B . and Messing, J., (1978) Nature 272, 375-377) with the use of synthetic DNA . The site contributes 14 additional codons and does not affect the ability of the lac gene product to undergo intracistronic complementation . Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations . Using the new phage M13mp7, DNA fragments generated by cleavage with a variety of different restriction endonucleases can be cloned directly . The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding the new array of restriction sites.

Nucleic Acids Res, 1981 Jan 24, 9(2), 277 - 92
Cyclic AMP receptor proteins interacts with lactose operator DNA; Schmitz A; The wildtype lac promoter-operator region of E . coli was reinvestigated for cyclic AMP receptor protein (CRP) binding sites using the 'footprinting' method . Two distinct interaction sites were found, one in the -60 base pair region, which has previously been described, and a second one covering the lac operator . These two sites are similar in extent, orientation and the characteristic pattern of CRP-induced DNaseI enhanced cutting sites.

Biochim Biophys Acta, 1981 Jan 21, 672(2), 207 - 13
Production of an aromatic amino acid auxotroph of a trimethoprim-resistant Escherichia coli and deuteration of the aromatic amino acids in its dihydrofolate reductase; Florance J et al.; Interpretation of the 1H-NMR spectra of Escherichia coli dihydrofolate reductase is complicated by the large number of overlapping resonances due to protonated aromatic amino acids . Deuteration of the aromatic protons of aromatic amino acid residues is one technique useful for simplifying the 1H-NMR spectra . Previous attempts to label the dihydrofolate reductase from overproducing strains of Escherichia coli were not completely successful . This labeling problem was solved by transducing via P1 phage a genetic block into the de novo biosynthetic pathway of aromatic amino acids in a trimethoprim resistant strain of E . coli, MB 3746 . A new strain, MB 4065, is a very high level producer of dihydrofolate reductase and requires exogenous aromatic amino acids for growth, therefore allowing efficient labeling of its dihydrofolate reductase with exogenous deuterated aromatic amino acid.

Biochemistry, 1981 Jan 20, 20(2), 449 - 52
Interaction of elongation factor Tu with the aminoacyl transfer ribonucleic acid dimer Phe-tRNA-Glu-tRNA; Yamane T et al.; The effect of EF-Tu.GTP on the codon-anticodon interaction of AA-tRNA was studied by using as a model system the interaction of AA-tRNAs with complementary anticodons, namely, dimerization between yeast or Escherichia coli Phe-tRNAPhe (anticodon GmAA) and E . coli Glu-tRNAGlu (anticodon s2UUC) or nonacylated tRNAGlu in the presence or absence of EF-Tu.GTP . The present data indicate that the ternary complexes Phe-tRNA-EF-Tu.GTP and Glu-tRNA-EF-Tu.GTP can form dimers with a binding constant of (0.9 +/- 0.2) X 10(6) M-1, which is identical in magnitude with that of the dimer of the nonacylated tRNAs tRNAPhe-tRNAGlu and also with that of the complex Phe-tRNA-EF-Tu.GTP with nonacylated tRNAGlu . These results show that the anticodon region is not affected by complexation with EF-Tu.GTP; however, this conclusion does not preclude the possibility of structural changes in the anticodon loop that have no effect in energetic terms . In addition, this model codon-anticodon interaction does not stimulate hydrolysis of the GTP bound in the ternary complex.

Biochemistry, 1981 Jan 20, 20(2), 372 - 8
Ribonucleic acid-protein cross-linking in Escherichia coli ribosomes: (4-azidophenyl)glyoxal, a novel heterobifunctional reagent; Politz SM et al.; We have used the heterobifunctional reagent (4-azidophenyl)glyoxal (APG) to cross-link RNA to protein in Escherichia coli 30S ribosomal subunits . Synthesis and characterization of the reagent are described . Like other dicarbonyl reagents (e.g., kethoxal), APG reacts specifically with guanosine among the four ribonucleosides . The azido group in APG can be photolyzed with UV light (lambda greater than 300 nm), yielding an unstable nitrene which is potentially reactive with many groups in proteins and nucleic acids . Conditions for APG modification of guanylic acid residues in 30S subunits are described; photolysis of bound APG results in cross-linking of approximately 5% of the total 30S proteins to 16S RNA . A specific subset of the 30S proteins is cross-linked to 16S RNA by APG.

Biochemistry, 1981 Jan 20, 20(2), 290 - 7
Hydrogen-1 and carbon-13 nuclear magnetic resonance of the aromatic residues of fd coat protein; Cross TA et al.; The aromatic residues of fd coat protein in sodium dodecyl sulfate micelles are characterized by 1H and 13C NMR . Resonances from both types of nuclei show structure-induced chemical shift dispersion and line widths indicative of a folded native structure for the protein . The two tyrosines were found to have pKas of 12.3 and 12.5 by 1H NMR and spectrophotometric titrations . 13C relaxation measurements show that two of the three Phe rings have significant internal mobility, the two Tyr rings have moderate internal mobility, and the Trp side chain is completely immobilized . Qualitative comparisons are made between the intact virus and the isolated coat protein.

Biochem J, 1981 Jan 15, 194(1), 91 - 8
Dependence on androgens of the specific DNA-binding activity of rat ventral-prostate non-histone chromosomal proteins; Lesser BH et al.; Interactions between rat prostate non-histone chromosomal proteins and DNA were studied by using a nitrocellulose-filter-binding technique to monitor the formation of DNA--protein complexes . The total binding activity of the non-histones, as measured by binding of proteins to a trace quantity of labelled DNA, displays no preference for rat DNA relative to Escherichia coli DNA . Sequestration of non-specific binding proteins by preincubation with unlabelled bacterial DNA enables detection of a fraction of rat prostate non-histones that binds preferentially to labelled rat DNA relative to labelled E . coli DNA . After castration of adult male rats, both total and specific binding activities decrease . Administration of 5 alpha-dihydrotestosterone to castrated rats stimulates both total and specific DNA-binding activities of prostate non-histones; specific binding is stimulated to a greater extent than total DNA, indicating that the specific binding proteins constitute a larger fraction of the non-histone proteins in the presence of androgens . The specific DNa-binding activity is dependent on the dose of steroid administered.

Nucleic Acids Res, 1981 Jan 10, 9(1), 183 - 8
The nucleotide sequence of spinach chloroplast methionine elongator tRNA; Pirtle R et al.; The nucleotide sequence of spinach chloroplast methionine elongator tRNA (sp . chl . tRNAm Met) has been determined . This tRNA is considerably more homologous to E . coli tRNAm Met (67% homology) than to the three known eukaryotic tRNAm Met (50-55% homology) . Sp . chl . tRNAm Met, like the eight other chloroplast tRNAs sequenced, contains a methylated GG sequence in the dihydrouridine loop and lacks unusual structural features which have been found in several mitochondrial tRNAs.

J Biol Chem, 1981 Jan 10, 256(1), 526 - 32
Construction and preliminary characterization of the rat casein and alpha-lactalbumin cDNA clones; Richards DA et al.; We have constructed a double-stranded cDNA library using total poly(A)-containing RNA extracted from 8-day lactating rat mammary gland and have utilized this library to isolate clones for each of the four major milk proteins . These four cDNA clones, representing the three major rat caseins and alpha-lactalbumin, were initially identified by colony hybridization with labeled cDNA probes synthesized from individual mRNA fractions purified by preparative gel electrophoresis . Additional characterization was accomplished by hybridizing individual clones labeled with 32P by nick translation to a Northern gel blot of an enriched fraction of the four major milk protein mRNAs . The individual mRNAs were clearly resolved by electrophoresis on fully denaturing methylmercury hydroxide agarose gels . The identity of each milk protein clone was further established by the location of unique restriction enzyme sites within each clone . Final identification of each clone was performed by hybrid-arrested cell-free translation . The sizes of the milk protein cDNA clones ranged frm 70% for the alpha-lactalbumin gene to essentially full length for the gamma-casein gene, in comparison to their respective mRNAs . This represents the first isolation of a family of peptide hormone-responsive genes.

J Biol Chem, 1981 Jan 10, 256(1), 49 - 53
Fluorescence studies of the location of proteins L7 and L12 on the Escherichia coli ribosome; Lee CC et al.; The location of ribosomal protein L7/L12 has been examined by fluorescence spectroscopy . Protein L7 modified mainly at the COOH terminus by 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonic acid, and yeast tRNAPhe-containing fluorescent dyes located specifically near the anticodon or at the 3'-end were used in these studies . Fluorescence-quenching measurements indicate that the COOH terminus of protein L7/L12 is very accessible to the solvent . Steady state anisotropy measurements indicate that this region has a high degree of rotational mobility . Singlet-singlet energy transfer measurements show that the COOH-terminal region of L7/L12 is far away from the peptidyltransferase center, and the main body of the ribosome in general . These results are most easily explained if all four copies of L7/L12 are in a parallel arrangement . Further energy transfer experiments show that the COOH terminus of L7/L12 must be greater than 70 A away from both the 3'-end and the anticodon region of ribosome-bound tRNAs . This indicates that all four copies of L7/L12 must be oriented so that their COOH termini are far away from the tRNAs . However, changes in both the spectrum and anisotropy of labeled L7/L12 accompany tRNA binding . The most simple explanation for these results is a structural change in the 70 S ribosome which somehow alters the environment or location of L7/L12 when tRNA is present . One simple possibility is an alteration in the quaternary arrangement of the multiple copies of L7/L12.

J Biol Chem, 1981 Jan 10, 256(1), 244 - 52
Membrane biogenesis . Evidence that a soluble chimeric polypeptide can serve as a precursor of a mutant lac permease in Escherichia coli; Fried VA; A mutant in the Escherichia coli lac permease, called Yf, appears to be defective in the biogenesis and proper assembly of this membrane protein . It was proposed that this defect led to the accumulation of a precursor of the mutant permease (Fried, V . A . (1977) J . Mol Biol . 114, 477-490) . In this communication, evidence is presented that the lacYf mutant accumulates a novel lac-specific soluble polypeptide with a molecular weight of 87,000 . Detected by double-label analysis on sodium dodecyl sulfate gels, and identified as a lac-specific polypeptide on a two-dimensional gel system, this polypeptide is immunoprecipitated by anti-transacetylase antibody . Pulse-chase experiments are consistent with the hypothesis that it is converted in vivo into a lac-specific membrane protein with an apparent molecular weight of 28,000, which appears to be the mutant lac permease . The results suggest that the 87,000-dalton soluble protein is a precursor of the mutant lac permease . It is proposed that this precursor is a polyprotein chimera containing both the lacY and lacA gene products.

J Biol Chem, 1981 Jan 10, 256(1), 198 - 204
Purification and properties of alanine tRNA synthetase from Escherichia coli A tetramer of identical subunits; Putney SD et al.; Escherichia coli alanine tRNA synthetase has been purified from a strain which carries the gene on a recombinant pBR322 plasmid . Several per cent of the soluble protein can be obtained as alanine tRNA synthetase in the plasmid containing host cell . The enzyme was proven to be an alpha 4 tetramer with a Mr = 380,000 by the following criteria: i) a single band of Mr = 95,000 was found in sodium dodecyl sulfate gel electrophoresis; ii) gel filtration chromatography gives a single peak at a Mr = 360,000 for the native enzyme; iii) dimethyl suberimidate cross-linking indicates a tetrameric structure; iv) a single undecapeptide N-terminal sequence was found which is Ser-Lys-Ser-Thr-Ala-Glu-Ile-Arg-Gln-Ala-Phe . Limited proteolysis generates a fragment of the native enzyme . The fragment has a Mr = 48,000 (one half that of the native subunit) and, in contrast to the native enzyme, oligomerization of the fragment could be not detected either by gel filtration chromatography or by dimethyl suberimidate cross-linking . The fragment is derived from the NH2-terminal half of the native subunit as shown by their common decapeptide NH2-terminal amino acid sequences . The ATP-PPi exchange activity of 1 mol of fragment is identical with that of 1 mol of native subunit, but aminoacylation activity is absent from the fragment . Therefore, in the fragment, the aminoacyl adenylate formation activity is cleanly separated from tRNA aminoacylation and subunit association properties . These results also mean that, with respect to aminoacyl adenylate formation activity, each subunit in the native tetramer acts independently.

Nucleic Acids Res, 1981 Jan 10, 9(1), 47 - 53
The nucleotide sequence of spinach chloroplast tryptophan transfer RNA; Canaday J et al.; Spinach chloroplast tRNATrp, purified by column chromatography and two-dimensional gel electrophoresis, has been sequenced using in vitro labeling techniques . The sequence is : pG-C-G-C-U-C-U-U-A-G-U-U-C-A-G-U-U-C-Gm-G-D-A-G-A-A-C-m2G-psi-G-G-G-psi-C-U-C-A-A*-A-A-C-C-C-G-A-U-G-N-C-G-U-A-G-G-T-psi-C-A-A-G-U-C-C-U-A-C-A-G-A-G-C-G-U-G -C-C-AOH . Like the E . coli suppressor tRNA psu+UGA which translates both the opal terminator codon U-G-A and the tryptophan codon U-G-G, spinach chloroplast tRNATrp has C-C-A as an anticodon and contains an A-U pair in the D-stem.

J Biol Chem, 1981 Jan 10, 256(1), 494 - 8
Immunological reagents for comparisons of DNA polymerase-alpha and DNA polymerase-beta; Chang LM et al.; Antibodies to homogeneous calf thymus DNA polymerase-beta and calf thymus DNA polymerase-alpha preparations were raised in rabbits . The antiserum against calf thymus DNA polymerase-beta cross-reacts with all vertebrate DNA polymerase-beta preparations tested, but does not cross-react with trypanosome DNA polymerase-beta, DNA polymerase-gamma, terminal transferase, yeast DNA polymerases, and Escherichia coli DNA polymerase I . The antibodies against calf thymus DNA polymerase-alpha cross-react with DNA polymerase-alpha from mouse, human, and chicken, but do not cross-react with DNA polymerase-alpha from sea urchin embryos and Drosophila embryos, DNA polymerase-beta, DNA polymerase-gamma, terminal transferase, yeast DNA polymerases, and E . coli DNA polymerase I.

Nucleic Acids Res, 1981 Jan 10, 9(1), 95 - 119
Polyethylene glycol derivatives of base and sequence specific DNA ligands: DNA interaction and application for base specific separation of DNA fragments by gel electrophoresis; Muller W et al.; Various base pair specific DNA ligands comprising a phenyl phenazinium dye, a triphenylmethan dye and Hoechst 33258 were covalently bound to polyethylene glycol (PEG) via ester or ether bonds . The DNA interactions of the PEG derivatives formed were shown to exhibit the same base pair specificity as the parent compounds . Since the PEG chains thus bound to the DNA could be expected to increase drastically the frictional coefficient of the DNA, the PEG derivatives were used for base specific DNA separations in agarose and polyacrylamide gel electrophoresis . The procedures, which do not require any special techniques, are described in detail . The resolution observed in agarose gels allows one to separate equally sized DNA fragments differing as little as 1% in base composition at mean travel distances of about 10 cm . Examples of gels showing the base compositional heterogeneity of restriction fragments obtained from lambda DNA, E . coli DNA and calf thymus DNA are given.

Nucleic Acids Res, 1981 Jan 10, 9(1), 189 - 201
Some substrate properties of analogues of oligothymidylates with p-s-C5' bonds; Rybakov VN et al.; The action of T4 polynucleotide kinase, T4 DNA polymerase, E . coli DNA polymerase I, snake venom phosphodiesterase (VPDE) and S1 nuclease on analogues of oligothymidilates with p-s-C5' bonds and the ability of these analogues to prime the replication of poly (dA) by T4 DNA polymerase were studied . These analogues were shown to be substrates for all these enzymes . Substitution of these analogues for corresponding oligothymidilates in the reaction mixtures resulted in drop in rates of enzymic reactions . This drop in reactions rates was not significant when these oligonucleotides were phosphorylated with T4 polynucleotide kinase or used as a primers, however in comparison with oligothymidilates these analogues were found to be considerably more resistant to nucleolytic hydrolysis . Some possible applications of these analogues are discussed.

J Biol Chem, 1981 Jan 10, 256(1), 533 - 8
Restriction enzyme mapping and heteroduplex analysis of the rat milk protein cDNA clones; Richards DA et al.; Detailed restriction enzyme maps have been determined for the three major rat casein and the fourth principal milk protein, alpha-lactalbumin, cDNA clones . Each of the milk protein genes exhibited unique and characteristic restriction enzyme sites . A comparison of the restriction enzyme maps of the three rat caseins revealed no apparent sequence homology among their gene sequences . The orientation of each cDNA gene sequence within the parent plasmid, pBR322, was determined by hybridization with a 3' specific cDNA probe synthesized from a partially hydrolyzed total poly(A) mRNA preparation following isolation by chromatography on oligo(dT)-cellulose . This technique provided a rapid procedure for determining the 5'-3' orientation of the cloned DNA sequences . Three casein clones were selected, which were in the same orientation, and were employed for a heteroduplex analysis to determine whether minor regions of homology existed within the alpha-, beta-, and gamma-casein genes . No heteroduplex formation was observed among these genes even under the low stringency conditions of hybridization employed, suggesting that considerable sequence divergence has occurred within the rat casein gene family.

Nucleic Acids Res, 1981 Jan 10, 9(1), 65 - 84
The construction, identification and characterisation of plasmids containing human alpha-lactalbumin cDNA sequences; Hall L et al.; We describe the cloning of double-stranded cDNA synthesized from lactating human mammary gland total poly(A)-containing RNA, into the EcoRI site of the plasmid pAT153 . Nine recombinants were shown to contain alpha-lactalbumin cDNA sequences as determined by positive hybridisation translation of complementary RNA . Restriction enzyme maps were determined for six of these . Alignment of the restriction map with the known amino acid sequence of human alpha-lactalbumin provided evidence that two plasmids, designated phO-53 and phB-35, contained the complete coding sequence of the primary translation product (pre-alpha-lactalbumin) . Hybridisation studies using purified human, monkey and guinea-pig alpha-lactalbumin cDNA demonstrated that greater nucleotide sequence divergence has occurred within the rodents than the primates, and that rodent alpha-lactalbumin mRNAs retain regions of homology with primate alpha-lactalbumin mRNAs.

Nucleic Acids Res, 1981 Jan 10, 9(1), 133 - 48
Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information; Zuker M et al.; This paper presents a new computer method for folding an RNA molecule that finds a conformation of minimum free energy using published values of stacking and destabilizing energies . It is based on a dynamic programming algorithm from applied mathematics, and is much more efficient, faster, and can fold larger molecules than procedures which have appeared up to now in the biological literature . Its power is demonstrated in the folding of a 459 nucleotide immunoglobulin gamma 1 heavy chain messenger RNA fragment . We go beyond the basic method to show how to incorporate additional information into the algorithm . This includes data on chemical reactivity and enzyme susceptibility . We illustrate this with the folding of two large fragments from the 16S ribosomal RNA of Escherichia coli.

J Biol Chem, 1981 Jan 10, 256(1), 81 - 6
A GTPase reaction accompanying the rejection of Leu-tRNA2 by UUU-programmed ribosomes . Proofreading of the codon-anticodon interaction by ribosomes; Thompson RC et al.; The characteristics of a GTPase reaction between poly(U)-programmed ribosomes, EFTu . GTP, and the near-cognate aminoacyl (aa)-tRNA, Leu-tRNA Leu 2, have been studied to assess the role of this reaction in proofreading of the codon-anticodon interaction . The reaction resembles the GTPase reaction with cognate aa-tRNAs and EFTu . GTP in its substrate requirements, in its involving EFTu . GTP . aa-tRNA ternary complexes, and in its requiring a free ribosomal A-site . The noncognate reaction differs from the cognate one in that aa-tRNA becomes stably bound to the ribosomes only 5% of the time; it therefore seems best characterized as an abortive enzymatic binding reaction . The rate of reaction is a significant fraction (4%) of that of the cognate aa-tRNA, indicating that recognition of ternary complexes by ribosomes involves a level of error greater than that of translation as a whole . The rejection of the noncognate aa-tRNA following GTP hydrolysis is therefore a vital step in the translation process and fulfills the criteria set for a proofreading reaction . Leu-tRNA Leu 2 which escapes rejection through proofreading, forms a stable complex with the ribosomal A-site, so it appears that the Leu-tRNA2 which was rejected never reached the A-site and that proofreading precedes full A-site binding.

J Biol Chem, 1981 Jan 10, 256(1), 41 - 8
The number of copies of ribosome-bound proteins L7 and L12 required for protein synthesis activity; Lee CC et al.; Poly(U)-dependent poly(Phe) synthesis and elongation factor G (EF-G)-dependent GTPase activity were used to study the partial reconstitution of L7/L12-deficient ribosomes with proteins L7/L12 and fluorescent conjugates . Seventy-five per cent of these activities are restored when unmodified L7/L12 dimer is added to L7/L12-deficient cores at a ratio of 1:1 . Various covalent fluorescent conjugates of L7/L12 bind to these cores about as well as unmodified protein . A fluorescein-5-isothiocyanate derivative of L12 shows almost no functional activity when bound . However, mixed reconstitutes of this conjugate and unmodified L12 have 75% functional activity when half the protein is unmodified . These results can be explained by a model in which there are two independent binding sites on the ribosome for two dimers of L7/L12 . The binding of dimers to ribosomes is totally random and complete; the particle is 100% active so long as it has one active dimer bound to either one of the two sites . However, more complex models cannot be ruled out . An 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonic acid (IAEDANS) derivative of L7 is labeled semispecifically at the COOH terminus . This conjugate shows partial functional activity . When assay results are analyzed using the above model, it appears that the specific COOH-terminal modification has no effect on activity . However, all but a small fraction of the nonspecific IAEDANS modifications lead to inactivation.

Biochemistry, 1981 Jan 6, 20(1), 87 - 93
Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: equilibrium kinetics and mechanism of enzyme i phosphorylation; Hoving H et al.; The phosphorylation of enzyme I from the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system was studied by means of isotope exchange between phosphoenolpyruvate and pyruvate . Experiments monitoring 1H--2H exchange showed that enzyme I phosphorylation is accompanied by the transfer of a proton from the enzyme to the C-3 atom of the substrate . 14C--12C-exchange experiments with both deuterated and protonated pyruvate exhibited a kinetic isotope effect (nu V/nu D = 1.9), showing that the proton transfer is (partly) rate determining and is an essential step in the mechanism of phosphoryl group transfer . Under certain reaction conditions, a more than proportional increase of the 14C exchange rate with increasing total enzyme concentration was observed, indicating that only the dimeric form of enzyme I is phosphorylated . From the dependence of the 14C exchange rate on the phosphoenolpyruvate and pyruvate concentrations, the forward and reverse second-order rate constants of the reaction were determined to be 3 X 10(7) and 8 X 10(5) M-1 min-1, respectively, yielding an equilibrium constant of approximately 40 and a delta G degree for enzyme I phosphorylation of --2.3 kcal/mol . The significance of the values of these rate constants for the thermodynamics of the phosphotransferase system is discussed.

Biochemistry, 1981 Jan 6, 20(1), 8 - 15
Stereochemical control of ribosomal peptidyltransferase reaction . Role of amino acid side-chain orientation of acceptor substrate; Bhuta A et al.; The substrate specificity of the acceptor site of peptidyltransferase of Escherichia coli 70S ribosomes was investigated in the fMet-tRNA.A-U-G.70S ribosome and AcPhe-tRNA.poly(U).70S ribosome systems by using a series of 2'- and 3'-aminoacyldinucleoside phosphates as acceptors . These chemically synthesized compounds are analogues of the 3' termini of either 2'(3')-, 2'-, or 3'-aminoacyl transfer ribonucleic acids (AA-tRNAs) of the types C-A-aa, C-2'-dA-aa, C-3'-dA-aa, C-3'-dA-3'-NH-aa, and C-2'-dA-2'-NH-aa (aa = Phe, D-Phe, Lys, Leu, Ala, Glu, Pro, Gly, Asp, Met, and alpha-aminoisobutyryl) . It was found that the 3'-aminoacyl derivatives of optically active amino acids are much better acceptors of N-formyl-L-methionine (fMet) or N-acetyl-L-phenylalanine (AcPhe) residues than the isomeric 2'-aminoacyl derivatives with affinity constant ratios (KM 2'/3') greater than 100 . Likewise, C-A(D-Phe) is a weaker acceptor than the corresponding L derivative C-A-Phe . In contrast, all glycyl derivatives (C-2'-dA-Gly, C-3'-dA-Gly, C-3'-dA-3'-NH-Gly and C-2'-dA-2'-NH-Gly) are good acceptors of the fMet residue, with ratios (KM 2'/3') of approximately 2 . On the basis of these results, a model for the stereochemical control of the peptidyl-transferase reaction is proposed . It assigns a major role to the orientation of the amino acid side chain in 2'- or 3'-AA-tRNA . A detailed model of the interaction of the acceptor terminus of 3'-AA-tRNA with the acceptor site of peptidyltransferase is also proposed . The model is strikingly similar to those for the active sites of proteolytic enzymes.

C R Seances Acad Sci III, 1981 Jan 5, 292(1), 5 - 8
{Analysis of the stability and local cooperativity of DNA : elements permitting the recognition of promoters by DNA-dependent RNA polymerase}; Ehrlich R et al.; Analysis of the local stability of promoters processed by E . Coli RNA polymerase shows that they bear a characteristic stability profile: two segments of low stability are located around --35 and --8 bases ahead of the transcription start and are generally bordered by more stable segments . This key profile may act as a recognition signal for both attracting and positioning the RNA polymerase in the promoter site.

Int Arch Allergy Appl Immunol, 1981, 64(1), 11 - 8
Immunological studies on modified enzymes . I . soluble L-asparaginase/mouse albumin copolymer with enzyme activity and substantial loss of immunogenicity; Yagura T et al.; For the successful administration of exogenous proteins and enzymes for therapeutic purposes, abrogation or modifications of antigenicity and immunogenicity of the protein must be achieved without substantial loss of the biological activity necessary for therapeutic purposes . In order to try this, a model experiment was carried out in the present study . Escherichia coli L-asparaginase, whose chemical properties have been well defined and which is used for the treatment of acute lymphocytic leukemia and malignant lymphomas, was modified by copolymerization with isologous albumin, and alterations of its immunogenicity were examined in mice . The results demonstrated that the immunogenicity of the modified L-asparaginase was remarkably reduced and satisfactory enzyme activity was preserved.

Adv Shock Res, 1981, 6, 107 - 20
Hemodynamics, hypoglycemia, and hepato-pancreatic pathology during the course of endotoxin shock; Manson NH et al.; The interrelationships between hemodynamics and hypoglycemia during the course of endotoxin shock (ES) has not been fully defined . In the following study, ES (E . coli, 1 mg/kg; n = 7) was induced in a canine model and systemic hemodynamics, glucose metabolism, and hepatic and pancreatic function monitored for 5 hr and compared to time-matched controls (TMC, n = 7) . Total peripheral resistance (TPR, dynes-sec-cm-5) increased from 3227 +/- 430 to 4050 +/- 750 at 30 min and then declined to 3050 +/- 1100 at 90 minutes . TPR progressively increased to 6225 +/- 749 by 5 hours . Plasma glucose did not significantly differ from control values (105 +/- 4 mg%) for the first 90 min but then declined to 68 +/- 6 mg% at 4.5 hours . (TMC = 103 +/- 17, P less than 0.05) . Serum amylase during the 5 hr protocol was not elevated (TMC = 110.9 +/- 2.4; ES = 100 +/- 1.97%; P greater than 0.1), and light microscopy of the exocrine pancreas demonstrated normal acinar structure . Islet cell structure from the ES group is not significantly different from the TMC . Hepatic histology in the ES group demonstrated periportal and perilobular degranulation and hepatocyte disruption not seen in the TMC . It is hypothesized that ES results in a circle of positive feedback initiated by an increase in TPR and subsequent decrease in flow resulting in hepato-pancreatic ischemia . Ischemic damage is most apparent at the liver and leads to changes in hepatic metabolic activities which contribute to the developing hypoglycemia of the late phase of ES.

Pol J Pharmacol Pharm, 1981, 33(6), 593 - 6
The effect of sodium salicylate on body temperature in normothermic and febrile rabbits with enhanced level of Cu2+; Hac EE; Increased level of Cu2+, induced by intravenous injection of CuCl2 at a dose of 2 mg Cu2+/kg in normothermic and Escherichia coli lipopolysaccharide treated rabbits, enhanced the antipyretic effect of sodium salicylate . Furthermore, in the presence of CuCl2, sodium salicylate caused a fall of body temperature in normothermic rabbits.

Acta Chir Acad Sci Hung, 1981, 22(1-2), 23 - 9
{Effect of pretreatment with radiation-detoxified endotoxin on endotoxin shock in dogs}; Balogh A et al.; The protective action of the radiation detoxified endotoxin preparation (Tolerin) on the endotoxin shock of dogs was studied . Endotoxin shock was induced by the intravenous administration of 2 mg/kg of Escherichia coli endotoxin . It was found that the intraperitoneal administration of 4 mg of radiation-detoxified endotoxin (Tolerin) 48 hours prior to the inducement of shock prevents the drop in blood pressure and the decrease of cardiac output which promptly appear in the acute phase of endotoxin shock.

Prog Clin Biol Res, 1981, 64, 467 - 74
Effects of metabolic inhibitors on the assembly of fd phage; Ng YC et al.; Treatment of E . coli cells that are actively producing fd phage with KCN, arsenate, DNP or CCCP inhibits production of infectious phage . These results are consistent with (but do not prove) an energy dependence for the assembly of fd phage . The major difficulty is to distinguish between a direct dependence on the proton motive force and an indirect one . Very preliminary experiments with valinomycin, which can be used to manipulate the voltage gradient of the cell membrane, and nigericin, which can be used to alter the pH gradient, suggest that a voltage gradient is not required for phage assembly but that pH gradient is required.

Carcinogenesis, 1981, 2(12), 1293 - 8
Limited capacity for the removal of O6-methylguanine and its regeneration in a human lymphoma line; Sklar R et al.; The Raji human lymphoma line is able to remove O6-methylguanine (O6MeG) lesions introduced by treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . The reaction has a rapid phase in which approximately 40% of the O6MeG is removed in the first 10 min . The capacity of cells for rapid O6MeG removal is limited and is saturated at concentrations of MNNG which do not saturate the systems removing 3-methyladenine . Pretreatment of cells with MNNG inhibits their ability to remove O6MeG produced by a subsequent dose given after 2 h . Treatment with N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) is effective in diminishing cellular capacity for O6MeG removal, and cells unable to remove O6MeG and sensitive to the cytotoxic effects of MNNG are also more sensitive to ENNG than their removal competent counterparts . Regeneration of the ability to remove O6MeG requires incubation of cells for periods greater than 24 h . The O6MeG removal system is similar to that found in adapted Escherichia coli although the capacity of the Raji lymphoma line much lower than that of the induced bacteria per unit of DNA.

Acta Biochim Pol, 1981, 28(2), 135 - 46
Stimulatory effect of hnRNA on template activity of isolated rat liver chromatin; Grabowska M et al.; Transcription of chromatin isolated from rat liver with E . coli RNA polymerase was stimulated up to five-fold with heterogeneous nuclear RNA (hnRNA) from rat liver . The transcription product had features of DNA-primed RNA . Neither cytoplasmic poly(A)-containing RNA nor high or low molecular weight cytoplasmic poly(A)-lacking RNA from rat liver exhibited stimulation of transcription . The stimulatory effect seems to be confined to middle repetitive sequences of hnRNA . With purified DNA templates, addition of hnRNA resulted in complete inhibition of transcription . The stimulatory effect of hnRNA on chromatin transcription was also observed with endogenous RNA polymerase . This effect was dependent ionic strength and was most pronounced under conditions optimal for RNA-polymerase B activity.

Adv Shock Res, 1981, 5, 163 - 8
Effects of endotoxin in murine burns; Spillert CR et al.; Previous studies in our laboratory have demonstrated the protective effect of the spleen in murine burns . Since the spleen is a reservoir of the reticuloendothelial system (RES) and since small doses of endotoxin stimulate RES activity, the purpose of this investigation was to perform studies to determine whether endotoxin administration will further augment burn protection in splenectomized and nonsplenectomized animals . Swiss white mice were randomly assigned to one of two groups . One group was splenectomized and the other received a sham splenectomy . Twelve weeks later, under pentobarbital anesthesia, all animals were burned uniformly on the lower back . Log doses of Escherichia coli endotoxin or an equal volume of saline were administered intraperitoneally immediately after thermal injury . Severity of burns at 24 hours was assessed from gross and microscopic appearances using a grading scale of 0 (normal) to 4 (severe) . Splenectomized controls receiving saline had more severe burns than the sham-splenectomized controls . Endotoxin decreased burn severity in both splenectomized and non-splenectomized mice . However, endotoxin afforded greater burn protection in the splenectomized mice when compared with the non-splenectomized animals.

Adv Shock Res, 1981, 5, 103 - 11
Glucocorticoid effect on glycolytic intermediates in septic rat heart; Kuttner RE et al.; Glycolytic intermediates in rat liver show similar changes in both endotoxic and peritonitis shock models suggesting a common etiology . To determine if this parallelism exists for other organs, adult rat hearts were analyzed 5 hours after E . coli endotoxin injection (2 mg/100 gm rat weight) or after sepsis produced by cecal incision . Tissue obtained by freeze-clamp biopsy was deproteinized and the metabolites were enzymatically assayed . Glucocorticoids were injected IV: 1 mg dexamethasone sodium phosphate (DMS) and 3 mg methylprednisolone sodium succinate (MPS) per 100 gm rat weight at time of operation . Glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) in septic hearts (N = 10) declined 36% and 37% from sham-operated control values (N = 11) of 640 nmole and 136 nmole per gm wet tissue . This finding is consistent with accelerated glycolysis . No changes of other metabolites indicative of severe hypoxic conditions were noted . A 2.5-fold increase in lactate was observed . This may reflect a shift to anaerobiosis in peripheral muscle and greater extraction by heart . Confirming earlier endotoxin rat heart data, glucocorticoids did not prevent decreases in G6P and F6P in septic hearts which declined 37% and 36% below sham controls (N = 10 for pooled glucocorticoid data) . Hexose monophosphates in sham animals treated with GC alone were also found to be lowered . Glucocorticoids were synergistic with endotoxin in lowering beta-hydroxybutyrate in heart samples . It is concluded that endotoxin and sepsis produce identical responses on myocardial carbohydrate metabolites and that glucocorticoids do not counter these effects, which reflects the lack of gluconeogenic potential in this organ.

Toxicology, 1981, 21(3), 251 - 60
In vitro screening of potential anti-cancer chemicals: effect of purine pyrimidine analogues on seed germination; Dwivedi CM et al.; Germinating Cicer arietinum seeds were used to simulate embryonic tissue for evaluating toxicity of purine and pyrimidine analogues . 6-Mercaptopurine stimulated germination, whereas 8-azaadenine, 8-azaguanine, 2,6-diaminopurine, 6-amino, 2-hydroxypurine and 5-fluorouracil inhibited germination . 6-Methyluracil, 5-aminouracil and methylxanthines, viz . caffeine, theophylline, theobromine and uric acid exhibited little inhibitory effects . RNA synthesis ensued immediately after water imbibition and reached maximum at 12 h of germination . 2,6-Diaminopurine, 8-azaadenine, 8-azaguanine and 5-fluorouracil inhibited RNA synthesis . Cyclic-AMP, adenosine, indole-3-acetic acid (IAA) and folic acid partially reversed the inhibitory effects produced by purine pyrimidine analogues . Several hitherto untested pyrimidine analogues inhibited germination as well as RNA synthesis . Inhibition of Escherichia coli dihydrofolate reductase was used for purposes of comparison.

Circ Shock, 1981, 8(5), 551 - 61
Hemodynamic consequences of endotoxemia in sheep; Traber DL et al.; Sheep which have been previously prepared for cardiopulmonary studies and collection of lung lymph were given 0.75 micrograms/kg of E coli endotoxin iv . This induced sepsis produced a triphasic response . Phase 1 occurs during the first hour after endotoxin administration and is characterized by a decreased cardiac output, lymph to plasma protein ratio, neutrophil count, and an increased hematocrit, total plasma protein concentration, lymph flow, and pulmonary artery pressure . During phase 2 these variables tend to return toward their baseline values . Phase 3 begins 2.5 hr after the administration of endotoxin and shows many of the same changes that were observed in phase 1 . Also during the late phase, the plasma protein concentration and lymphocyte count were reduced and the lymph to plasa protein ratio was increased . It is concluded that (1) An early fall in cardiac output occurs as a consequence of hypovolemia . The decreased volume is the result of fluid movement from the vascular compartment to the interstitial space consequent to a microvascular pressure increase . Since the changes occur coincidentally with a drop in neutrophil count, these cells may bear some causal relationship to the response . (2) The late fall in cardiac output in phase 3, also the result of a diminished vascular volume, occurs secondarily to extravasular fluid movement as a consequence of both an elevated microvascular pressure and an increased permeability to protein . The latter may be causally related to a fall in lymphocytes.

Carcinogenesis, 1981, 2(7), 637 - 43
Mass spectral characterisation of the major DNA--carcinogen adduct formed from the metabolically activated carcinogen 15,16-dihydro-11-methylcyclopenta{a}phenanthren-17-one; Wiebers JL et al.; E . coli DNA, labelled with {14C}adenine and {14C}-guanine, was allowed to react with the {3H}-labelled carcinogen 15,16-dihydro-11-methylcyclopenta{a}phenanthren-17-one in the presence of a microsomal metabolising system . Enzymatic hydrolysis of the DNA followed by Sephadex LH20 chromatography of its constituent nucleosides established that the major DNA - carcinogen adduct involved guanine, and not adenine . This was confirmed by submitting calf thymus DNA, which had been allowed to react with the unlabelled carcinogen, to pyrolysis electron impact mass spectrometry without further derivatisation . Analysis of a selected ion product (m/z 368) by means of mass-analysed kinetic energy spectrometry, a technique which allows study of the further fragmentation of the single, selected ion, revealed that the guanine moiety was attached via the nitrogen atom of its exocyclic amino group to C-1 of a 1,2,3,4-tetrahydro-2,3,4-trihydroxy derivative of the original carcinogen.

J Mol Evol, 1981, 17(2), 94 - 102
Periodic correlations in DNA sequences and evidence suggesting their evolutionary origin in a comma-less genetic code; Shepherd JC; Strong rhythms with a period of three bases have been seen while correlating the relative positions of purines and pyrimidines and of the four individual bases in the complete DNA sequence of the viruses phi X174, G4 and fd . Generally weaker variations of the same type have been found in the DNA virus SV40, the plasmid pBR322, the RNA virus MS2, and elsewhere in procaryotes and eucaryotes (e.g . in a ribosomal protein gene cluster of E . coli and the sea urchin histone genes) . From the interrelation of four-base with purine-pyrimidine rhythms it seems that the purine-pyrimidine relationships have a basic significance . An explanation is proposed in terms of the former use of a comma-less genetic code (i.e . readable only in one frame) of the general form RNY (R = purine, Y = pyrimidine and N = purine or pyrimidine) . In spite of subsequent mutation, there appears to be still enough of the primitive messages remaining to produce these periodic variations with their characteristic properties in phase and amplitude . Particularly good evidence for this hypothesis is provided by the fact that the phases for the stronger rhythms are the same in all the genomes tested and can be successfully predicted by a simple consideration of the original RNY pattern . With regard to amplitude it can be similarly foreseen which variations will be more clearly marked than others . The observed behaviour of the amplitude as the separation between correlated bases increases is also explained by the insertions, deletions and point mutations which have occurred . Additionally it is possible to account for some notable features of the non-random use of codons for the same amino acid by this theory.

Circ Shock, 1981, 8(3), 351 - 60
Endotoxin-induced lung injury in unanesthetized sheep: effect of methylprednisolone; Demling RH et al.; Our purpose was to study the effect of methylprednisolone (MPS) on endotoxin-induced lung injury . We used lung lymph flow and lymph protein content at sensitive indicators of pulmonary microvascular integrity in unanesthetized sheep . Seven animals were given E coli endotoxin (2 micrograms/kg) alone and also endotoxin with MPS pretreatment (30 mg/kg) in paired separate studies . The endotoxin injury was divided into an early hypertensive phase (30-90 minutes) characterized by a significant increase in pulmonary artery pressure (Ppa) and lymph flow, QL . This was followed later (3-5 hours) by a significant increase in protein-rich QL with normal pressures . MPS pretreatment significantly increased both injury phases in all animals . White cell count decreased in both groups; however, lymph beta-glucuronidase increased only in the nontreated group . Platelet count decreased in the nontreated group . Seven animals were then compared using endotoxin alone or with MPS treatment one hour after endotoxin . Only four of seven animals had a decrease in the lung injury after MPS treatment . We conclude that methylprednisolone is very effective in preventing lung injury, but only minimally effective in reversing the injury from endotoxin.

Circ Shock, 1981, 8(3), 301 - 12
Evidence for an early lipid soluble cardiodepressant factor in rat serum after a sublethal dose of endotoxin; Carli A et al.; The administration of a sublethal dose of E coli endotoxin (2 mg/kg) has been found to induce in the intact rat a slight decrease in mean carotid arterial pressure only 16 hours later . No death occurred whereas the same dose injected in the adrenalectomized rat caused lethality, and so did the higher dose (3 mg/kg) in the intact rat . Sera sampled two, four and 16 hours after administration of endotoxin (2 mg/kg) depressed contractile activity and the chronotropic response of isoproterenol in cultured rat heart cells . Such properties were not noted with sera sampled one or 24 hours after endotoxin administration . These cardiodepressant effects were localized in the lipid soluble fraction of endotoxin serum and absent in the fat-free portion . And yet neither endotoxin nor nonesterified fatty acid (NEFA) serum fraction was involved, since they did not alter cell culture activity . Moreover, though total or individual NEFA levels in endotoxic serum were higher than those of control rat serum four hours after endotoxin injection, they were not significantly different from control 16 hours later . It is concluded that the administration of a sublethal dose of endotoxin to the intact rat induces early and prolonged (up to 14-16 hours) release of one or more humoral lipid soluble cardiodepressant factor(s), even in the absence of systemic hypotension.

Acta Chir Scand, 1981, 147(3), 161 - 2
Effects of acetylsalicylic acid and prostaglandin on endotoxin-induced pulmonary platelet sequestration; Thornee LJ et al.; Previous experimental studies have shown that soft tissue trauma and endotoxin shock cause pulmonary platelet trapping . In the soft tissue trauma model it has been found that this event can be obviated by pretreatment with aspirin and prostaglandin E1 in combination . This study was made to determine the effect of these two drugs on endotoxin-induced pulmonary platelet trapping . Dogs with 51Cr-tagged platelets were injected with 20 mg endotoxin E . coli . The dogs were kept in shock for 2 hours and then sacrificed . The dogs were kept in shock for 2 hours and then sacrified . The amount of platelet sequestration in the lungs was determined by measuring the 51Cr activity in the lungs . The results of the study showed that prostaglandin alone decreases pulmonary platelet trapping induced by endotoxin, but aspirin, either alone or together with prostaglandin E1, has no positive effect . This could be explained by the fact that acetylsalicylic acid depresses endogenous prostaglandin, and thus decreases the blood concentration of available prostaglandin.

Curr Eye Res, 1981-82, 1(11), 683 - 5
A method for the intraocular injection of micro-volumes in small animal species; Hammond BR et al.; A method has been developed for the intraocular administration of small volumes (0.5-1.5 microliters) in small animal species such as guniea pigs and rats . The method was found to yield reproducible injection volumes and resulted in minimal ocular trauma . The aqueous humor of eyes injected with saline contained normal levels of protein and was free of white blood cells . Responses to Prostaglandin E2 and E.Coli endotoxin were dose-dependent and as expected.

Cancer Treat Rep, 1981, 65 Suppl 4, 123 - 30
Asparaginase and amino acids in cancer therapeutics . Cancer and Leukemia Group B Investigators; Holland JF et al.; Asparaginase was found ineffective in improving induction frequency of acute lymphocytic leukemia of children from vincristine and prednisone, but prolonged remission duration . Prolongation of remission duration was accomplished more effectively and at lower toxic cost by administration of asparaginase after the vincristine and prednisone treatment had been completed . Asparaginase is markedly more effective against human leukemia T-cells in vitro than against several types of B-lymphocytes . This is consistent with in vivo experience . Leukemic T-cells resistant to asparaginase have been induced in vitro . Deficiency of other amino acids causes absolute cessation of RNA biosynthesis, constituting evidence for other amino acid targets critical to leukemic cell survival.

Anticancer Res, 1981, 1(6), 373 - 6
Pharmacology of Escherichia coli-L-asparaginase polyethylene glycol adduct; Park YK et al.; The polyethylene glycol (PEG) adduct of Escherichia coli L-asparaginase was administered intravenously to 4 patients with chemotherapy refractory cancers . The PEG-enzyme in plasma exhibited a half-life of 16-25 days . Doses of 250IU/m2 or greater reduced plasma asparagine to undetectable levels for as long as enzyme was detectable in plasma . All doses of enzyme administered (250-1000 IU/m2) caused similar increases in plasma aspartate, i.e . no dose-response relationship . Pleural fluid and ascites contained detectable enzyme but at a value 10-15% of simultaneously drawn plasma levels . Toxicity in this small group of patients was minimal; nausea and transient fever predominated . There were no clinical signs of PEG-asparaginase-induced pancreatitis, renal dysfunction, hypocalcemia and hyperglycemia . No patient developed evidence of a PEG-asparaginase allergic reaction; no patient formed antibodies to asparaginase or PEG-asparaginase . Two patients with large cell lymphoma showed a partial response to treatment.

Ann Rech Vet, 1981, 12(3), 253 - 7
{Frequence of K99 antigen and antibioresistance in Escherichia coli from calves in France (author's transl)}; Martel JL et al.; During the winter of 1979-1980, an epizootiological study of diarrhoeic calves revealed the presence of K99+ E . coli among 8.2 p . cent of clinically healthy calves and in 18.9 p . cent of diseases calves . Some calves which seemed healthy on the day of sampling possibly became diarrhoeic on the following days . In diarrhoeic calves, K99+ E . coli were mainly found during the early life, i.e . in 33.7 p . cent of calves less than 4 days old . These results were obtained with 147 healthy calves and 1053 diarrhoeic calves . They confirmed previous results obtained with more limited numbers of animals . Moreover, K99+ E . coli were found in all breeding systems . Antibiotics resistance among the isolated E . coli were very high, especially in K99+ strains . The results proved the interest of E . coli K99 diagnosis in liquid diarrhoea of very young calves.

Antonie Van Leeuwenhoek, 1981, 47(4), 325 - 37
Genetic and biochemical characterization of an Escherichia coli K-12 mutant with an altered outer membrane protein; Tommassen J et al.; The properties of an Escherichia coli K-12 mutant are described which seemingly produces a "new" major outer membrane protein with an apparent molecular weight of 40 000 . This 40K protein was purified and its cyanogen bromide (CNBr) fragments were compared with those of several known major outer membrane proteins . A similarity was found between the CNBr fragments of the 40K protein and those of the OmpF protein (molecular weight 37 000) . In addition, the 40K protein was found to be regulated exactly like the OmpF protein, and the mutation which causes the production of the 40K protein has been localized in (or very close to) the ompF gene . It is concluded that the 40K protein is a mutant form of the OmpF protein . The results provide additional evidence that the ompF gene at minute 21 is the structural gene for the OmpF protein.

Ann Rech Vet, 1981, 12(2), 137 - 41
Chlorpromazine and propranolol extend survival of infant mice inoculated with enterotoxigenic Escherichia coli; Bertin A; Effects of chlorpromazine and propranolol were tested in infant mice model of Enterotoxigenic Escherichia coli diarrhea . Reference strain B41 inoculated orally from 18 h to 48 h after birth causes death of almost all animals in less than 48 h . Both drugs, which are known to reverse submaximal induced secretion by heat-stable toxin in the suckling mouse assay, used as single treatment, were able to extend the survival of mice.

Natl Inst Anim Health Q (Tokyo), 1981 Summer, 21(2), 68 - 72
Vero cytotoxin produced by Escherichia coli strains of animal origin; Kashiwazaki M et al.; A heat-labile Vero cytotoxin (VT) was detected by the Vero cell assay in the culture supernatants of ten Escherichia coli strains isolated from a variety of clinical sources . The VT titer varied with strains, ranging from 1:8 to 1:2,048 . No VT was found to be cytotoxic for Y1 mouse adrenal cells or to induce fluid accumulation in the rabbit or porcine ileal loops . No strains produced a heat-labile enterotoxin as defined by either tissue culture or the standard ileal loop reaction . Three strains originated from piglets with diarrhea were confirmed by the infant mouse reaction to produce a heat-stable enterotoxin . Neutralization studies revealed antigenically the presence of two different forms of VT produced by E . coli strains of animal origin.

Dev Biol Stand, 1981, 50, 287 - 92
The eucaryotic cell-lipochromosome as a new host-vector system in gene amplification and expression; Miller AC et al.; Although procaryotes such as E . Coli are generally considered to be ideal hosts, able to amplify eucaryotic gene sequences contained within hybrid DNA plasmid molecules (3, 14), recent experimental evidence such as the decreased bacterial viability observed during the cloning of mouse mitochondrial DNA, clearly shows the limitations of this type of approach (6) . In addition, major technical difficulties are encountered during the isolation and purification of the specific messenger RNA (mRNA) needed to synthetize the complementary DNA (cDNA) molecule (1, 4) . Last but not least, after having screened many different bacterial clones -- provided that the foreign gene product is not toxic for its host -- it is still a question of good luck to get one clone producing adequate quantities of the biologically active protein of interest, free of contaminants . On the other hand, the low but significant frequency with which eucaryotic cells exposed to fragmented DNA molecules or metaphasic chromosomes phenotypically express a particular marker suggests that this approach might offer an alternative to the bacteria-plasmid system used in "classic" genetic engineering (10, 17, 18, 19) . It is the purpose of this work to briefly discuss some of the difficulties involved in this approach and to propose solutions.

Vet Med Nauki, 1981, 18(8), 12 - 8
{Drug resistance of Escherichia coli strains isolated from poultry}; Giurov B et al.; Studied was the sensitivity of a total of 143 strains of Escherichia coli, isolated from young birds and broilers died from coli septicaemia, to antibiotics and chemotherapeutics . The following descending order was established: gentamycin, carbenicillin, ampicillin, furazolidon, borgal, kanamycin, strep tomycin, chloramphenicol, neomycin sulphathiazole, and tetracycline . Markers of resistance were established with all strains with regard to the therapeutic agents in current and prospective use in industrial poultry farming . It is stated that a preliminary antibiogram is indispensable in order to obtain dependable results in the treatment of animals affected with colibacteriosis . An alternative is to apply directly those drugs to which the strains have shown highest sensitivity.

Mol Biol (Mosk), 1981 Jan-Feb, 15(1), 62 - 71
{Role of the carboxylic groups in interaction of phenylalanyl-tRNA synthetase with substrates}; Gorshkova II et al.; The effect of modification of carboxylic groups of phenylalanyl-tRNA synthetase by p-toluene sulfonate N-cyclohexyl-N'-beta-(4-methylmorpholine) ethylcarbodiimide (CMEC) on the activity of the enzyme was investigated . It was shown that modification of two moles of carboxylic groups per mole of the enzymes leads to the diminution of negative charge of the enzyme and to inactivation in ATP-{32P}PPi-exchange and aminoacylation reactions . The inactivation is completely reversed by mild alkaline hydrolysis . ATP in concentration 2 X 10(-4) M partially protects the enzyme against inactivation, protective effect being stimulated by Mg2+ and 0.4-0.7 moles of carboxylic groups per mole of the enzyme are protected against inactivation is observed although the depth of modification is increased . Other substrates do not have protective effect . Modification of the enzyme by CMEC increases Kdiss value of {14C}-Phe-tRNA enzyme complex and Km value for tRNAPhe in aminoacylation by factor of three . Vmax for all substrates in both aminoacylation and leads to 40% increase of Hill's coefficient for ATP in ATP-{32P}PPi-exchange reaction but not in aminoacylation . The carboxylic groups modified by CMEC are assumed to take part in ATP recognition and in catalysis of the ATP conversion and in catalysis of transfer of activated amino acid residues on tRNA.

Mol Biol (Mosk), 1981 Jan-Feb, 15(1), 5 - 26
{Processing of tRNA}; Venkstern TV; The article reviews various aspects of tRNA biosynthesis in pro- and eukaryotes . Some data on the tRNA gene localization are presented . Structures of tRNA precursors are given; their conversion into mature tRNA molecules and the corresponding enzymatic systems are discussed in detail . Special emphasis is given to the transcription of tRNA genes and processing of tRNA precursors in vitro . Similarities and diversities of tRNA processing in pro- and eukaryotes are discussed.

Mol Biol (Mosk), 1981 Jan-Feb, 15(1), 45 - 53
{Alkylation of tRNA-Phe free and bound to phenylalanyl-tRNA synthetase with 4-(N-2-chloroethyl-N-methylamino)benzylamine}; Vlasov VV et al.; Reactivities toward the alkylating reagent 4-(N-2-chloroethyl-N-methylamino)benzylamine of guanosine residues in the tRNAPhe free and bound to the phenylalanyl-tRNA synthetase were determined . This highly efficient reagent modifies mainly guanosine in single-stranded as well as in helical regions of tRNAs . The reaction is sensitive to the elements of the macrostructure of tRNAs . It is found the phenylalanyl-tRNA synthetase protects the D-stem of the tRNAPhe (guanosines G10, G22, G24) from alkylation.

Mol Gen Genet, 1981, 184(3), 560 - 1
{DNA degradation and survival of ozone-resistant and -sensitive mutants isolated in Escherichia coli B (author's transl)}; Hamelin C et al.; Cell survival and DNA degradation were measured in mutants of Escherichia coli B either more sensitive or resistant to ozone than wild-type . Results indicate that the ozrA and ozrB gene products intervene differently in the resistance of E . coli to ozone and that lesions other than DNA strand breaks are induced by this agent.

Mol Gen Genet, 1981, 184(3), 526 - 30
Complementation studies with the repair-deficient uvrD3, uvrE156, and recL152 mutations in Escherichia coli; Siegel EC; Previous work showed that the mutations uvrD3, uvrE156, and recL152 were closely linked and increased UV-sensitivity . They were phenotypically distinguishable in that only the uvrD3 mutation significantly decreases host cell reactivation of UV-irradiated phage (Hcr-) and repair of methylmethane sulfonate (MMS)-induced damage, and only the uvrE156 mutation increased mutation rates (Mut-) . MMS-resistant revertants of a uvrD3 mutant were still UV-sensitive and fell into two phenotypic classes . Hcr- Mut+ (non-mutator) and Hcr+ Mut- . In this work complementation tests were done by examining UV- and MMS-sensitivity and host cell reactivation in heterogenotes containing combinations of uvrD3, uvrE156, recL152, and the MMS-resistant mutations derived from uvrD3 . The mutations could not complement each other in the repair of UV-damage, the one trait all had in common, indicating that they were in one gene . For the most part, the different mutations were able to complement each other in respect to traits in which one was deficient and the other had wild type activity.

Mol Gen Genet, 1981, 184(3), 504 - 7
Escherichia coli merodiploids with altered ratios of episomal to chromosomal RNA polymerase activity; Tittawella IP; Starting with an E . coli merodiploid strain (rpoB+ (rifS)/rpoB- (rifR)) containing equal amounts of rifampicin-sensitive and resistant RNA polymerase activities, mutants were isolated that had a disproportionately high ratio of the rifampicin-resistant enzyme activity . With one strain it is shown that the mutation responsible for this phenotype is closely linked to the rifR (rpoB-) allele.

Mol Gen Genet, 1981, 184(3), 421 - 9
Induced radioresistance: an aspect of induced repair; Pollard EC et al.; Irradiation of Escherichia coli cells with UV or X-rays followed by incubation under conditions in which protein synthesis can occur results in a population of cells that is resistant to X-rays; however, this resistance develops only if the cells are recA+ and lexA+, a fact that associates the phenomenon with induced (S.O.S.) repair . By observing separately the component of a culture that is resistant and the component that retains its normal growth, the fraction of induced and uninduced cells for a dose of UV or X-rays can be estimated . Such estimates show that the dose-response for UV induction of resistant cells agrees with that of the recA gene product . Thus induced radioresistance is considered to be due to the changes in the cell occasioned by the derepression of recA and lexA . These changes are expected to be involved with the synapsis of homologous genomes that is necessary for the use of a second genome to repair damage occurring in both strands of a duplex at the same base, as exemplified by a double-strand break or an interstrand crosslink . This consideration is additionally supported by the increased resistance of cells grown to contain multiple genomes in the same envelope, an increased resistance not found in recA- or lexA- cells . The condition of a completed chromosome is also resistant, again not in recA- or lexA- cells . We suggest that cell killing by X-rays is due to the double-strand breaks which are not repaired by molecular synapsis before the arrival of the replication polymerase at the break.

Mol Gen Genet, 1981, 184(1), 40 - 5
The target of the negative regulator of pMB1 replication overlaps with part of the repressor coding sequence; Cesareni G; Plasmid mutants (svir), insensitive to inhibition by the repressor of initiation of pMB1 replication, have been selected by exploiting their ability to support lambda growth in the presence of lambda repressor and inhibitor of plasmid replication . The alteration in the mechanism that controls plasmid replication causes a change in the plasmid copy number, svir mutants are dominant, as expected for mutants in the target of a repressor, but at the same time they are unable to synthesise a repressor active on the wild-type target . This lack of cross interaction between svir mutants and a co-resident wild-type plasmid results in their compatibility . These findings are explained by postulating that the target of the inhibitor of pMB1 replication coincides with part of the DNA segment that codes for the inhibitor itself . As a consequence single base pair changes in the target result in altered repressor molecules.

Mol Gen Genet, 1981, 183(3), 557 - 8
On the role of the Escherichia coli RNA polymerase sigma factor in T4 phage development; Zograff YN; The rpoD800 mutation causing the temperature sensitivity of Escherichia coli RNA polymerase sigma factor has been used to demonstrate that the bacterial sigma factor is necessary throughout T4 phage development . In T4-infected RpoD800 mutant cells RNA synthesis is equally depressed at restrictive temperature at early and late stages of infection . The results show the bacterial sigma factor to be necessary for the synthesis of all RNA types in infected cells.

Mol Gen Genet, 1981, 183(3), 490 - 6
Plasmid replication functions . VII . Electron microscopic localization of RNA polymerase binding sites in the replication control region of plasmid R6-5; Lurz R et al.; RNA polymerase binding sites on the R6-5 miniplasmid derivative, plasmid pKT401, were mapped by electron microscopy of DNA:RNA polymerase complexes formed with both circular-supercoiled and restriction endonuclease-linearized plasmid DNA molecules . Of eight specific binding sites on pKT401 that were identified, three were found to be in the P-6 fragment of the plasmid replication region, three in the Tn3 element, and two in other parts of the plasmid molecule . Binding sites 1 and 3 in the P-6 fragment are most probably the promoters of the copB and copA/incA plasmid replication control genes, respectively, whereas site 2 in this fragment appears to be the promoter of the essential replication gene, repA . The location of these promoters in relation to the site of action of the plasmid replication control elements, copT, and the origin of replication, oriV, suggests that replication control may be effected by regulation of transcription events initiated at site 2, or of the activity of transcripts initiated from this site, i.e., by regulation of the expression of the repA gene or another function dependent upon these events.

Mol Gen Genet, 1981, 183(3), 473 - 7
Ribosomal protein modification in Escherichia coli . III . Studies of mutants lacking an acetylase activity specific for protein L12; Isono S et al.; Among mutants of E . coli selected for temperature-sensitive growth, four were found to possess alterations in ribosomal proteins L7/L12 . Of these, three apparently lack protein L7, the acetylated form of protein L12 . Genetic analyses have revealed that the mutation responsible for this alteration maps at a locus around 34 min of the current E . coli genetic map, which is clearly different from the location for the structural gene for protein L7/L12 which is situated at 89 min . Hence, the gene affected in these mutants was termed rimL . Tryptic and thermolysin fingerprints of the protein L12 purified from the rimL mutants showed a profile indistinguishable from that of wild-type protein . It was found that the assayed in vitro, in the high-speed supernatant prepared from mutant cells . These results indicated that the three mutants contain mutations in the gene rimL codes for an acetylating enzyme specific for ribosomal protein L12.

Mol Gen Genet, 1981, 183(3), 418 - 21
Methylation of ribosomal proteins during ribosome assembly in Escherichia coli; Chang FN; In Escherichia coli, a number of ribosomal proteins are methylated . The time of methylation of L7 and L11 during ribosome assembly was studied . It was observed that the methylation of L7 could occur in the free protein stage . Both the 32S and 40S ribonucleoprotein intermediates also contained methylated L7 although the extent of methylation in these particles was not as high as in the free L7, the 45S or the 50S particles . Free L11 could also be partially methylated but the bulk of methylation of this protein was found in the 45S and the 50S particles . It was previously reported that the methylation of L7 is inversely proportional to the growth temperature (Chang 1978), we now show that once L7 is methylated at 25 degree, the methyl group is stable when the culture is shifted to 37 degree C . However, a partial turnover of the methyl group of L7 is observed when the methylated ribosome is chased at 25 degree C . On the other hand, the methyl groups of L11 appear to be stable at either 25 degree C or 37 degree C . We also observe that the extent of methylation of both L7 and L11 stays nearly constant during the cell growth cycle from early log to stationary phase.

Adv Exp Med Biol, 1981, 137, 603 - 22
Passive immunity of the G . I . tract; Wilson RA; The need for continued pursuit to identify all of the components necessary for effective passive immunity is obvious . With a focus upon natural phenomenon, we need to expand our understanding of antigenic modulation while determining the distribution of specific antibodies among the various isotypes available in lacteal secretions and capable of transfer to the intestinal tract of the neonate and broaden the scope of investigations to include the significance of cell mediated immune mechanisms present in lacteal secretions . Passively acquired immunocytes, plasma cells, macrophages and polymorphonuclear leucocytes may contribute to the immune status of suckling ruminants.

Mol Gen Genet, 1981, 184(2), 200 - 7
Interplasmidic and intraplasmidic recombination in Escherichia coli K-12; Laban A et al.; The construction of plasmids which facilitate the study of interplasmidic and intraplasmidic recombination is described . In this system, a single recombination event between two mutated Tcr genes on separate plasmids or on one plasmid leads to a change in the host phenotype from sensitivity to resistance to tetracycline . Recombination proficiencies have been determined for different E . coli K-12 strains: both interplasmidic and intraplasmidic recombination are independent of the recBC gene product . RecA mutations decrease the proficiency of plasmidic recombination 40 - 100 fold . Intraplasmidic and interplasmidic recombination via the recE pathway are more efficient than via the recBC pathway . Intraplasmidic recombination, but not interplasmidic recombination via the recE pathway is independent of a functional recA product.

Mol Gen Genet, 1981, 184(2), 166 - 73
Effects of reduced amount of RNA polymerase sigma factor on gene expression and growth of Escherichia coli: studies of the rpoD450 (amber) mutation; Osawa T et al.; A mutant of Escherichia coli K-12 carrying an amber mutation (rpoD40) in the structural gene for RNA polymerase sigma factor and a temperature-sensitive amber suppressor (supF-Ts6) grows virtually normally at 30 degrees C, but does not grow at 42 degrees C due to the inability to synthesize sigma polypeptides (Osawa, T . and Yura, T., Mol Gen Genet 180, 293 - 300, 1980) . When the mutant cells are transferred from 30 to 42 degrees C, the cellular amount of sigma relative to total protein is found to decrease from 50% (at 30 degrees C) to 10% of the wild-type level after about 2 h . The decrease of sigma is accompanied by a gradual decrease in RNA and protein syntheses and a sudden loss of viability . At the highest temperature (36 degrees C) that permits steady growth of this mutant, the amount of sigma and the growth rate become 6% and 50 to 60% of the wild type, respectively . These results suggest that the minimum level of sigma required for growth is 0.02 to 0.04 in terms of molar ratio of sigma to core enzyme, that is 6 to 10% of the wild type . Two-dimensional gel electrophoresis of proteins synthesized under the reduced sigma level reveals either markedly increased or decreased syntheses of several polypeptides, while no detectable effect is observed in the majority of polypeptides . Notably, the synthesis of a set of major heat-shock polypeptides is greatly enhances . Hence, the decrease of RNA polymerase holoenzyme relative to the core enzyme seems to affect the synthesis of individual proteins differentially, primarily at the level of transcription . The expression of the groE operon, one of the major heat-inducible operons in E . coli is also studied in some detail.

Mol Gen Genet, 1981, 183(2), 277 - 82
In vivo synthesis of a polycistronic messenger RNA for the ribosomal proteins L11, L1, L10 and L7/12 in Escherichia coli; Bruckner R et al.; Sucrose density gradient centrifugation and DNA/RNA hybridization have been used to analyse the mRNA synthesized from the ribosomal protein - RNA polymerase subunits gene cluster rplKAJL-rpoBC in Escherichia coli . DNA/RNA hybrids obtained from total E . coli RNA and specific DNA restriction fragments from this chromosomal area were further subjected to endonuclease S1 digestion . This analysis permits the mapping of the ends of mRNA molecules for specific genes or operons by sizing the S1 resistant hybrids . Our results show that the predominant mRNA synthesized under conditions of balanced growth from the rplKAJL-rpoBC region codes for the four ribosomal proteins L11, L1, L10 and L7/12 . This tetracistronic mRNA puts the transcription of the following rpoBC genes under the main control of the L11 promoter . Smaller distinct mRNA species could also be detected by this technique . They originate from intercistronic transcription termination and re-initiation as well as from processing of the larger polycistronic mRNA.

Mol Gen Genet, 1981, 183(1), 78 - 81
RNA polymerase is required for DNA initiation in vitro; Projan SJ et al.; We have previously reported in vitro complementation assays for chromosome initiation that enable dnaA and dnaC mutant extracts to synthesize DNA . To examine the role of RNA polymerase in chromosome initiation, inhibitors of the enzyme and anti-RNA polymerase antibody were used . Though rifampicin failed to efficiently inhibit ribonucleoside triphosphate polymerization under the assay conditions, both streptolydigin and anti-RNA polymerase antibody abolished ribonucleic acid synthesis completely . Antibody effectively inhibited chromosome initiation in the dnaA mutant based reaction but streptolydigin did not . Neither streptolydigin nor antibody affected the dnaC-dependent assay . It was concluded that RNA polymerase is required for initiation but not necessarily to polymerize a polyribonucleotide . A scheme for the sequence of initiation events is presented.

Mol Gen Genet, 1981, 183(1), 37 - 44
Effect of recB21, uvrD3, lexA101 and recF143 mutations on ultraviolet radiation sensitivity and genetic recombination in delta uvrB strains of Escherichia coli K-12; Wang TV et al.; The interaction of the recB21, uvrD3, lexA101, and recF143 mutations on UV radiation sensitization and genetic recombination was studied in isogenic strains containing all possible combinations of these mutations in a delta uvrB genetic background . The relative UV radiation sensitivities of the multiply mutant strains in the delta uvrB background were: recF recB lexA greater than recF recB uvrD lexA, recF recB uvrD greater than recA greater than recF uvrD lexA greater than recF recB, recF uvrD greater than recF lexA greater than recB uvrD lexA greater than recB uvrD greater than recB lexA, lexA uvrD greater than recB greater than lexA, uvrD greater than recF; three of these strains were more UV radiation sensitive than the uvrB recA strain . There was no correlation between the degree of radiation sensitivity and the degree of deficiency in genetic recombination . An analysis of the survival curves revealed that the recF mutation interacts synergistically with the recB, uvrD, and lexA mutations in UV radiation sensitization, while the recB, uvrD, and lexA mutations appear to interact additively with each other . We interpret these data to suggest that there are two major independent pathways for postreplication repair; one is dependent on the recF gene, and the other is dependent on the recB, uvrD, and lexA genes.

Mol Gen Genet, 1981, 183(1), 25 - 31
Constriction of a fused operon consisting of the recA and kan (kanamycin resistance) genes and regulation of its expression by the lexA gene; Miki T et al.; The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322 . A DNA fragment containing the promoter-operator region of the recA gene was inserted into the promoter region of the cloned kan gene to produce a fused operon, recA-kan . Plasmid pMCR685 carrying recA-kan expressed a low level of activity of the kan gene product (kanamycin phosphotransferase; KPT) in the wild-type cells of Escherichia coli, while the plasmid showed an increased level of the activity in the SPr- mutant cells which produce the inactive lexA protein . The KPT activity in the wild-type cells harboring the plasmid increased 6- to 11-fold upon treatment of the cells with mitomycin C or nalidixic acid, both of which are known to induce synthesis of recA protein . Expression of the recA-kan operon fusion was remarkably repressed by the lexA gene cloned into a plasmid carrying the operon fusion . Higher concentrations of mitomycin C were required for maximal induction of KPT activity in the cells harboring the resulting plasmid pMCR687 . These results strongly suggest that the lexA gene product can be itself repress the recA gene, and that pMCR687 is a useful vector to clone genes whose expression is harmful to the host cell growth.

Mol Gen Genet, 1981, 183(1), 163 - 70
The phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system enzymes II as chemoreceptors in chemotaxis of Escherichia coli K 12; Lengeler J et al.; In Escherichia coli K12, eight substrate-specific, membrane-bound enzymes II of the PEP-dependent carbohydrate: phosphotransferase system (PTS), specific for hexoses, hexosamines and hexitols, have been characterised in a series of isogenic and constitutive strains . In such mutants, lacking all but one enzyme II, the transport and vectorial phosphorylation activities as well as the chemotactical response in capillary tube assays have been compared . According to the data obtained, all enzymes II not only are directly involved in the transport and vectorial phosphorylation of their substrates, but they have also a primary role as the chemoreceptors for these substrates: (1) Metabolism of the attractant beyond the phosphorylation step is not a pre-requisite to eliciting positive chemotaxis . (2) Mutants, having only one enzyme II react in the capillary tube assay only to substrates of this enzyme II, but not to substrates of the missing enzymes II . This holds for enzymes II consisting of one membrane-bound protein as well as for systems containing a soluble factor III (FIII) . (3) The substrate specificities or affinities, whether tested by transport and chemotaxis assays in vivo or by phosphorylation tests in vitro, are in correspondence . (4) The activities of enzymes II, regulated in a complex way at the level of enzyme synthesis and activity and tested as above, are also in agreement, (5) Mutants lacking the soluble proteins enzyme I or HPr of the PTS no longer respond chemotactically to any substrate taken up and phosphorylated by enzymes II . It is concluded that in PTS enzymes II some functions required for transport and chemotaxis are identical . It is suggested furthermore, that the alternation of intrinsic membrane-bound proteins between a phosphorylated and a dephosphorylated state, rather than binding of the substrate to the enzyme II, is the decisive stimulus in the chemotaxis toward carbohydrates taken up by these transport systems.

Mol Gen Genet, 1981, 183(1), 139 - 43
The process of general recombination in Escherichia coli K-12: structure of intermediate products; Bresler SE et al.; A review of the data on the genetic determination of general recombination in Escherichia coli introduces three alternative pathways of recombination, RecBC, RecF, and RecBCF . One recBC-dependent pathway is functional in recF- cells . An initiating endonuclease is involved, acting on the chi-sites of DNA . The second is recF-dependent, acting in the double mutant recBC sbcB . The corresponding endonuclease uses the fre-sites as a substrate . A third pathway acting in wild-type cells is mixed . Both enzymatic systems participate in the overall process . We shall call it RecBCF . Using the thermosensitive recA44 mutant it became possible to study the kinetics of integration of donor DNA into the recipient chromosome via the RecF and RecBCF pathways of recombination . The RecF pathway is characterized by delayed recombination; not less than 14 h being needed to complete the process at 35 degrees C . By the RecBCF pathway (wild-type recipient) the reaction is fast, as described by Lloyd and Johnson (1979) . The two stage nature of the RecF pathway is important . First an intermediate product is formed during a short time interval . This product is resistant to the degrading exonuclease V . Afterward the intermediate product is slowly integrated into the recipient chromosome . Autoradiography of this intermediate product, extracted from exconjugants, shows that it consists of closed DNA circles . Their length is within the limits 2--15 min on the E . coli map . Their average value is in fair agreement with genetic estimations of the integrated DNA fragments . Taking into consideration the similarity between genetic determinations of the fre-effects and the heterogeneity of the progeny, we conclude that the intermediate structures formed contribute to this heterogeneity.

Gastroenterol Jpn, 1981, 16(5), 465 - 71
Pathophysiological aspect of the liver in acute obstructive suppurative cholangitis; Sakoda K et al.; This study is prompted in order to clarify the pathophysiological aspect of the liver in acute obstructive suppurative cholangitis (AOSC) . An experimental model of AOSC, was prepared by intracholedochal infusion of endotoxin in dogs with and without obstructive jaundice . In the patients with AOSC, liver function was aggravated remarkably compared with that in the stage of non suppurative cholangitis . A rapid fall of platelet count occurred . Experimentally, after intracholedochal infusion of endotoxin, liver function revealed significant hepatic cellular damage . A considerable increase in the S-OCT level was accompanied by a marked rise in blood ammonia concentration . Liver damage due to obstructive jaundice was further aggravated by endotoxin infusion . The level of serotonin in the liver tissue increased markedly after endotoxin infusion . This was accompanied by a rapid fall of platelet counts . Serotonin is considered to be a factor which may cause an impairment of hepatic microcirculation and then hepatic cellular damages . It may be concluded that AOSC is induced by cholangio-venous reflux of endotoxin . Liver function is impaired remarkably due to increased bile canalicular pressure, to the direct affect of endotoxin on liver cells during the process of cholangio-venous reflux, and to impairment of hepatic microcirculation in endotoxin shock . Liver dysfunction contributes to develop this clinical entity and play an important role to make its outcome fatal.

Acta Chir Scand, 1981, 147(4), 225 - 30
Gentamicin treatment in severe surgical infections--serum levels, interactions, toxicity and efficacy; Athlin L et al.; A retrospective and a prospective group of 38 and 20 patients respectively were studied with regard to bacterial strains, clinical effect, toxic side effects and serum concentration of gentamicin in severe surgical infections . A high degree of therapeutic efficacy evaluated as cure or improvement was noted (68 and 75% in the two groups) . The serum levels were especially closely checked in the prospective study to keep the gentamicin concentration within the therapeutic range but below toxic levels . This aim was achieved in most cases probably explaining the relatively low incidence of side effects (nephrotoxicity 4 and fungal infection 11 patients) . The hazards of gentamicin interactions with drugs like furosemide, penicillins or cephalosporins are discussed.

Zentralbl Bakteriol Naturwiss, 1981, 136(6), 505 - 8
Effect of anilazine on growth, respiration and enzyme activity of Escherichia coli; Garg FC et al.; The effect of anilazine on growth, respiration and enzyme activity of E . coli has been studied . Anilazine delays the growth of E . coli by prolonging the lag period . It inhibits glucose oxidation by 60% and succinate oxidation by 100% . It also inhibits in vitro succinic dehydrogenase activity . It seems that the inhibition of E . coli by anilazine is because of inhibition of respiratory enzymes.

Vet Med Nauki, 1981, 18(5), 39 - 47
{Pathomorphological changes in colibacteriosis in pigs}; Veselinova A et al.; A total of 46 suckling piglets spontaneously affected with colibacteriosis and 20 pigs aged from 1 to 10 days, experimentally infected with two strains of Escherichia coli (0149 and 0138) were investigated morphologically . Both in the animals with spontaneous infection and in those with experimentally induced disease there were severe morphologic lesions not only in the small intestines, but also in the other viscera and the brain . Catarrhal to hemorrhagic enteritis was observed in 56.5 per cent of the spontaneous cases and 57.1 per cent of the test animals . Parenchymal dystrophy with hemorrhages were seen in the liver in 89.1 per cent of the pigs of the first group and 57.1 per cent of those of the experimental group . Kidney lesions were noted in 65.2 per cent and 28.6 per cent, respectively . Spontaneously affected piglets showed bacterial thrombi in the lumen of some small blood vessels, and in the adrenals (23.9 per cent) and the thymus (13.00 per cent) . In the brain there was meningoencephalitis in 54.3 per cent of the spontaneously affected animals and in 85.9 per cent of the experimentally infected ones . In the test animals infected with serotype 0149 there were more serious morphologic changes than in those infected with serotype 0138 . It was demonstrated that occasionally pathogenic E . coli organisms had a broader spectrum of action in the body of host sucklings . So colibacteriosis is considered a severe generalised disease which runs its course in sucking pigs as septicaemia and toxaemia.

Radiat Environ Biophys, 1981, 20(1), 37 - 51
Studies on possible genetic effects of microwaves in procaryotic and eucaryotic cells; Dardalhon M et al.; The biological effects of microwaves in the hyperfrequency range, 9,4 GHz, and 70-75 GHz were investigated in bacteria and yeast . At power densities below 60 mW/cm2 and SAR values not exceeding 28 mW/g no significant effects on survival of repair competent an deficient strains were observed in Escherichia coli and Saccharomyces cerevisiae . In addition, microwaves of 17 GHz did not induce mutations in E . coli B/r WP2 trp- uvr- above the spontaneous level, and the induction of nuclear reversions, cytoplasmic "petite" mutations and mitotic recombination as well as the efficiency of sporulation was not affected in yeast.

Genetika, 1981, 17(11), 1945 - 51
{Repair and mutagenesis in Escherichia coli cells on induction in the DNA of monoadducts and cross-links by light-activated 8-methoxypsoralen . Dependence on the uvrA and polA genes}; Peshekhonov VT et al.; Relative efficiency of (psoralen + near UV)-induced monoadducts and inter-strand cross-links of Escherichia coli cells has been studied, as well as the role of uvrA and polA genes in these processes . Inter-strand cross-links have been shown to be more effective, as compared with monoadducts both in cell inactivation and in mutagenesis . At least 3 ways for repair of DNA carrying monoadducts have been found, and only 2 for DNA carrying inter-strand cross-links, each of these being controlled by genes uvrA and polA in different modes.

Genetika, 1981, 17(11), 1904 - 8
{Plasmid pKM101 sensitization of Escherichia coli strains to the action of ionizing radiation: the effect of the plasmid on survivability and induced mutagenesis}; Aleshkin GI et al.; The wild type Escherichia coli K-12 has been shown to be sensitized to inactivation by gamma-irradiation by the plasmid pKM101 . The dnaA strains of E . coli are more sensitive to gamma-rays killing effect, as compared with the wild type E . coli, pKM101 plasmid showing only slight sensitizing effect . "Cis" or "trans" position of the plasmid in relation to the chromosome plays no role in sensitization, while the plasmid effect on UV-induced killing and mutability depends on "trans" position of the plasmid before irradiation . gamma-Rays induced mutability to prototrophy is completely dependent on the presence of pKM101 in "trans" in wild type and dnaA strains before irradiation.

Acta Microbiol Acad Sci Hung, 1981, 28(4), 413 - 7
Selection of mouse virulent non-motile strains of Escherichia coli by the soft agar technique; Takahashi M et al.; Using strain SME-12 of Escherichia coli and its variants A and B showing large round, diffuse and compact-type colonial morphology, respectively, in soft-agar medium, their capsule showed that although these strains were similar in toxicity, the parent strain exhibited a large capsule, a large cell volume and a high mouse virulence . Variant A had no capsule, its cell volume was remarkably lower than that of the original strain, and was avirulent; variant B had no capsule and displayed the lowest cell volume and mouse virulence . With 193 fresh isolates of E . coli, the majority of colonies of 16, 152 and 25 strains were of large round, diffuse and compact types, respectively . Fifteen strains of pure growth type from each of the three groups were tested for mouse virulence . The majority of strains showing large round-type growth was virulent, diffuse-type strains displayed a low virulence, while no mouse was killed by compact-type strains.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1981, 250(1-2), 52 - 62
The lipopolysaccharide of escherichia coli C- studies on the anomeric configurations of the hexoses in the R1 core; Feige U; Lipopolysaccharide from E, coli C as well as lipopolysaccharides from submutants of E . coli with incomplete core structures in their lipopolysaccharides were isolated and quantitatively analyzed . Core oligosaccharides were isolated from lipopolysaccharides by acetic acid degradation and were purified by gel chromatography . The difference in molecular rotations of the core oligosaccharides from E . coli C and 6 submutants thereof with incomplete core structure were correlated to the differences in sugar compositions . The anomeric configurations have been deducted from the high or low contribution of each individual sugar to the molecular rotation of the core oligosaccharide from E . coli C . The primary structure of the hexose region of the lipopolysaccharide from E . coli C is primary structure of the hexose region of the lipopolysaccharide from E . coli C is, see formula in text . The anomeric configurations of glucoses I, II, and III were confirmed by precipitation reactions of alkali treated lipopolysaccharides from E . coli C, C23 . 1, and C21 with Concanavalin A . The alpha-anomeric configurations of both the galactoses were confirmed by degradation studies with alpha-galactosidase (E.C.3.2.1.22) from green coffee beans with the isolated and purified core oligosaccharide from E . coli C71.

Vet Med Nauki, 1981, 18(4), 25 - 32
{Pathomorphology of gastroenteritis in newborn calves}; Dzhurov A et al.; The morphologic changes were studied in the parenchymal and digestive organs of a total of 39 calves with gastroenteritis from which pathogenic Escherichia coli organisms were isolated . A correlation was established between the age of calves and the forms of gastroenteritis manifestation . In 1 to 3-day-old calves 63.6 per cent of the cases presented histologic changes of enteritis, and 36.3 per cent presented the septic form of the disease, while in 4 to 7 day-old calves these forms were presented in equal percent . In 8 to 12-day-old animals septic changes were seen in 71.4 per cent of the cases, and toxic changes-in 28.5 per cent . The same age group showed initial changes typical of catarrhal (28.2 per cent) and interstitial (37.7 per cent) pneumonia . With the advance in age the histologic changes of septicaemia showed a rising trend as against the enterotoxaemic and enterotoxic form . The coli infections in 90 per cent of the investigated cases were accompanied by degenerative changes in the kidneys . In calves with nervous symptoms there were hyperemia, perivascular and pericellular edema and status cribrosus of the brain, and occasionally - lymphocytic and leukocytic infiltrations in the leptomeninges.

Urol Radiol, 1981, 3(2), 113 - 5
Perinephric abscess with renal cell carcinoma; Chintapalli K et al.; A case of perinephric abscess with renal cell carcinoma is presented . Hematuria is uncommon in cases of perinephric abscess . When hematuria is present in a patient with perinephric abscess further evaluation is necessary to rule out an associated malignant process.

Scand J Infect Dis, 1981, 13(2), 129 - 32
A new principle for prevention of diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) possessing colonization factor antigen (CFA/I); Wadstrom T et al.; The infant rabbit model, developed to study human ETEC with CFA/I (Evans et al., 1975), was used to evaluate agarose (Sepharose) gel substituted with a hydrophobic ligand for prevention of diarrhoeal symptoms . In a group of rabbits given 0.02 g of palmitoyl Sepharose 1 h after an oral infection with 10(9) ETEC organisms with CFA/I antigen, only 2/11 animals developed diarrhoea, while in control groups given the unsubstituted Sepharose (hydrophilic) gel or ETEC organisms alone, all animals developed diarrhoea . In groups of rabbit given the hydrophobic gel 6 h after the ETEC organisms, only 2/6 animals showed a short period of diarrhoea . The "hydrophobic principle" to prevent ETEC infections will be studied in other animal models and in human volunteers.

Prog Clin Biol Res, 1981, 63, 371 - 81
Multiple sites of methylation in the methyl accepting chemotaxis proteins of Escherichia coli; Chelsky D et al.; The methyl-accepting chemotaxis proteins (MCP) of E coli show at least five bands when subjected to SDS-gel electrophoresis . The intensity of the individual bands varies depending on the environment of the cells before solubilization . The faster migrating bands are enhanced following attractant stimulation, whereas the slower migrating bands are enhanced following attractant dilution or repellent increase . The time scale of these intensity changes is similar to that for adaptation of the behavioral response in these cells suggesting that methylation of the MCP is involved in producing these bands . Peptide mapping experiments show three methylated peptides in both MCP I and MCP II . These results suggest multiple sites of methylation, which are responsible, at least in part, for the observed multiple bands of the MCPs.

Microbiol Immunol, 1981, 25(10), 1059 - 66
Chemotaxis of Kupffer cells isolated from rodent liver; Katano M et al.; Kupffer cells (KC) were isolated from the liver of guinea pigs, rats, and mice using enzymatic digestion with collagenase, followed by differential centrifugation and plastic adherence . Purity of the isolated KC was 96.0 +/- 2.2, 97.2 +/- 2.1, and 96.0 +/- 2.3 per cent in guinea pigs, rats, and mice respectively . These isolated KC were tested for migratory response to bacterial factor, which is one of the representative chemotactic factors for inflammatory macrophages, using a modified Boyden chamber technique . KC from the three animal species similarly migrated to the bacterial factor . The migratory response of the KC to the bacterial factor is due to chemotaxis but not chemokinesis . These results show the possibility that KC may recognize a chemoattractant and directionally migrate to it.

Microbiol Immunol, 1981, 25(10), 1047 - 58
Stimulation of nonspecific host resistance to infection induced by muramyldipeptides; Matsumoto K et al.; The effect of muramyldipeptide (MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine {MDP(Ala)}, and its analogs on bacterial infection was studied using the experimental model of sepsis infection in mice . Injection of MDP(Ala) gave mice definitive protection against E . coli infection, but only partial protection against P . aeruginosa or K . pneumoniae infection . Several factors influencing the protective activity of MDP(Ala) on E . coli infection were studied, and it was demonstrated that the activity was induced by various routes of administration of MDP(Ala), including the oral route, and was markedly influenced by the bacterial inoculum size . It was also shown that the effective dose of MDP(Ala) was 100 micrograms per mouse for intraperitoneal, intravenous or subcutaneous injections and 1,000 microgram per mouse when administered orally . Furthermore, the optimal interval between MDP-treatment and infection was 24 hr when the treatment was carried out before infection . Clearance of bacterial cells in blood was observed after E . coli infection in mice treated with MDP(Ala) . The efficacy of MDP(Ala) and two analogs, N-acetylmuramyl-L-valyl-D-isoglutamine {MDP(Val)} and N-acetylmuramyl-L-seryl-D-isoglutamine {MDP (Ser)}, was evaluated for the E . coli infection; MDP(Val) was proven to be slightly less active than MDP(Ala), and MDP(Ser) to be the least effective, although MDP(Val) or MDP(Ser) was reported to have higher adjuvanticity than MDP (Ala) for the development of delayed-type hypersensitivity.

Genetika, 1981, 17(10), 1730 - 6
{Regulation of the activity of the Escherichia coli K-12 uridine phosphorylase gene . I . Mapping of the structural gene mutations and a determination of the direction of transcription}; Alkhimova RA et al.; A fine genetic mapping of some point and deletion mutations for uridine phosphorylase (udp) gene, including deletions covering simultaneously the udp and the adjacent metE gene was carried out . Deletions of the latter type do not recombine with the udp promoter mutations isolated previously (Mironov, Sukhodolets 1979) thus suggesting that the udp promoter is situated at the site adjoining metE.

Genetika, 1981, 17(10), 1719 - 29
{Disturbance of the tandem promotor functioning of the Escherichia coli K-12 deo-operon in the genome of the rho15(ts) mutant for the transcription termination factor}; Sukhodolets VV et al.; The effect of the rho15(ts) mutation on the expression of Escherichia coli deo operon's genes is studied . In relation to the regulatory deoR and cytR genes, the rho15 mutation causes in wild type genome 2,5-fold increase in both thymidine phosphorylase (deoA gene) and purine nucleoside phosphorylase (deoD gene) activity, while the deoxyriboaldolase activity controlled by the proximal deoC gene almost does not differ in the rho+ and rho15 strains . The effect of rho15 for the expression of the deo genes in constitutive deoR genome depends on the allele of crp gene: in the crp+ bacteria rho15 leads to a decrease, while in the crp bacteria - to an essential increase in the activity of deo enzymes . These data suggest a possible role of CRP protein as an inhibitor of transcription initiated from deoP promoter . The presence of rho15 in a bacterial genome leads to the complete block of the cytP promoter activity under conditions of both induction of deo enzymes by cytidine and their depression in cytR genome . Based on these data, it is proposed that proximal to cytP promoter, i . e . between deoP and ctyP a Rho-dependent attenuator is located which is usually responsible for termination of the deoP-initiated transcription . An activity of the inner deo operon OP3 promoter is possibly also inhibited in the rho15 genome as shown by the data on the absence of induction of purine nucleoside phosphorylase by inosine in the rho15 bacteria.

Bioelectromagnetics, 1981, 2(3), 285 - 9
Inhibition of growth rate of Escherichia coli induced by extremely low-frequency weak magnetic fields; Ramon C et al.; Cultures of Escherichia coli kept at 0 degree C in a phosphate buffer solution were exposed to a sinusoidal weak 60- or 600-Hz magnetic field of strength 2 X 10(-3) Tesla . A decrease of more than 40% in bacterial count was observed after a 60-h exposure to the magnetic field . Electron micrographs of exposed bacteria show ruptured cell walls, possibly due to the breaking away of flagella under the influence of the sinusoidally varying electromotive force.

Adv Shock Res, 1981, 5, 143 - 8
Thromboxane, prostacyclin, and hemodynamic events in primate endotoxin shock; Fletcher JR et al.; Prostaglandins participate in the pathophysiology of septic shock; however, their exact role is unclear . In this study we investigated the possibility that thromboxane and prostacyclin, the most recently discovered prostaglandins, may be related to the pulmonary arterial hypertension (thromboxane) and systemic arterial hypotension (prostacyclin) during endotoxin shock in the baboon . There are no previously reported studies in the subhuman primate . In this study ten male baboons received an LD70 dose of E . coli endotoxin . Cardiac output, mean systemic arterial pressure, pulmonary arterial pressure, blood gases, WBC and platelet counts, and prostaglandins were determined at 0, 15, 60, 120, 180, and 240 minutes . Thromboxane and prostacyclin levels were significantly (P less than 0.05) increased after the endotoxin injection . Systemic arterial PGI values increased within 15 minutes, peaked at two hours, and was directly related to the fall in systemic arterial pressure (r = 0.93) . In contrast, thromboxane values peaked at 15 minutes and directly related (r = 0.90) to the rise in pulmonary artery pressure . Thromboxane and prostacyclin are significantly increased in subhuman primate endotoxin shock . The temporal relationship of thromboxane and pulmonary arterial pressure suggests that thromboxane may mediate the effects of endotoxin on the pulmonary vasculature.

Mol Gen Genet, 1981, 182(3), 511 - 3
Are pyrimidine dimers non-instructive lesions?
Lawrence CW.
Published data from yeast and E . coli show that base substitution induced by UV in pyrimidine-pyrimidine sequences is not random, and suggest that fidelity of DNA replication is not entirely lost during transdimer synthesis . These observations question whether cyclobutane pyrimidine dimers are truly non-instructive lesions.

Mol Gen Genet, 1981, 182(3), 508 - 10
Colicin Ib does not cause plasmid-promoted abortive phage infection of Escherichia coli K-12; Boulnois GJ; A 2.2 kilobase (kb) fragment of ColIdrd-1 cloned in pBR325 causes phage T5 and BF23 infections of Escherichia coli K-12 to abort . This abortive infection is associated with leakage of beta-galactosidase from the cell . A recombinant plasmid containing a 2.8 kb fragment of ColIdrd-1 encodes colicin Ib but fails to cause abortive infection . Tn5 and Tn10 insertions into ColIdrd-1 that abolish colicin Ib production have no effect on the abortive infection phenotype . These findings are inconsistent with a previously proposed role for colicin Ib in causing phage infections to abort.

Mol Gen Genet, 1981, 182(3), 369 - 76
Localization of the 3' end of 16S rRNA in Escherichia coli 30S ribosomal subunits by immuno electron microscopy; Luhrmann R et al.; The location of the 3' end of 16S rRNA in E . coli 30S ribosomal subunits has been determined by immuno electron microscopy . The 3' terminal adenosine of isolated 16S rRNA was oxidized with sodium periodate and reacted with N-gamma-(2,4-dinitrophenyl) aminobutyric acid hydrazide . Functionally active 30S subunits were reconstituted from DNP-16S rRNA and total 30S ribosomal proteins . DNP-30S subunits were complexed with DNP-specific IgG-antibody and examined in the electron microscope . The 3' end of the 16S rRNA was mapped at a single region located at the inner side of the large lobe of the 30S subunit . The location of the 3' end also provides information as to the topography of the binding domain of natural mRNA on 30S subunits, since a pyrimidine-rich sequence at the 3' terminal region of 16S rRNA participates in the correct alignment of natural mRNAs during initiation complex formation.

Genetika, 1981, 17(9), 1644 - 8
{Effect of alpha-tocopherol on the mutagenic repair pathways in Escherichia coli cells}; Kalinina LM et al.; It is shown in the present work that the antimutagenic effect of alpha-tocopherol is related to suppression of mutagenic repair controlled by the lexA gene . During this process mutations occur in chromosomal replication forks, provided cells are in the logarithmic phase of growth . Lesions in nonreplicating DNA resulting from the direct alkylation of nitrosoguanidine are apparently realized in the course of repair errors formation, the process being unaffected in any significant way by alpha-tocopherol.

Genetika, 1981, 17(9), 1606 - 17
{Escherichia coli K-12 mutants for the thymidine phosphorylase structural gene that retain the anabolic function of the enzyme}; Moskaleva ND et al.; From the Escherichia coli thymine auxotroph carrying a constitutive mutation for deo-enzymes (thy deoR) mutants (tpp38, tpp39 and tpp40) for thymidine phosphorylase (catalyzing a conversion of thymine to thymidine) were isolated via selection for a low thymine requirement . In the thy deoR+ genome these mutations led to inability of bacteria to use thymidine as the sole carbon source for growth, though the ability to utilize thymine was retained at the level comparable to that of thy deoR+ tpp+ bacteria . In the thy deoR genome mutations obtained led to a more efficient utilization of thymine in comparison with the thy deoR tpp++ strain . At the same time, the thymidine phosphorylase activity, as determined by a degradation of thymidine in bacterial extracts was lower in thy deoR strains carrying tpp38, tpp39 and tpp40 by factors 5,25 and 22, respectively, in comparison with the thy deoR tpp+ strain . The mutations tpp38, 39, 40 were localized in the distal part of the tpp structural gene (tpp39 and tpp40 being in the extreme distal position), whereas the earlier described tpp-leaky mutants incapable of using exogenous thymine for growth were mapped in the extreme proximal part of the tpp gene (Sukhodolets et al., 1971) . It is proposed that the tpp-leaky mutations obtained enhance the thymidine phosphorylase affinity to deoxyribose-1-phosphate, a product in a reversible reaction of the thymidine phosphorolysis.

Eur Surg Res, 1981, 13(2), 152 - 68
Clotting and other plasma factors in experimental endotoxemia: inhibition of degradation by exogenous proteinase inhibitors; Jochum M et al.; Endotoxemia in dogs was induced by a slow intravenous infusion of E . coli endotoxin for 2 h . Thereby, a significant decrease was observed in the plasma levels of several clotting, fibrinolysis and complement factors . The changes were studied over an experimental period of 14 h and checked for statistical significance by three-way analysis of variance . Application of the broad-spectrum proteinase inhibitor aprotinin (Trasylol) from bovine organs clearly lowered the endotoxin-induced decline of the plasma proteins studied . By intravenous application of a specific granulocytic proteinase inhibitor (Bowman-Birk inhibitor from soybeans), the endotoxin-induced reduction of the plasma proteins was prevented in a similar manner . It can be concluded that at least some of the pathobiochemical mechanisms observed in clotting, fibrinolysis and complement systems during endotoxemia are not only caused by a severe consumption reaction but also by unspecific proteolytic degradation due to neutral granulocytic proteinases.

Carcinogenesis, 1981, 2(9), 909 - 14
Comparison of the mutagenic activity of dialkylnitrosamines in animal-mediated and in vitro assays using an Escherichia coli indicator; Kerklaan P et al.; An intrasanguineous host-mediated assay was used to establish the mutagenic potential of a series of dialkylnitrosamines to E . coli K12/343/113 in the liver and spleen of mice . For calibrating purposes, dose- and time-dependent kinetics of mutation induction by the model compound diethylnitrosamine in this assay was determined . Comparison with the results of the same compound in vitro reveals that activation in the intact liver of living mice is more efficient and proceeds for a longer period of time than during incubation in the presence of a liver homogenate . The mutagenicity of five other dialkylnitrosamines (dimethyl-, diethanol-, diisopropyl-, methylethyl-, and methyl-n-propylnitrosamine) was also determined . The results of host-mediated assays in livers and spleens of mice indicate a good correlation with the carcinogenic properties of these compounds as far as the effect on the liver is concerned . The mutagenic activity in vitro shows, however, a poor correlation with carcinogenicity data, mainly because some of the carcinogenicity data, mainly because some of the carcinogenic nitrosamines are not detectable in those tests . It is concluded that, under the present experimental conditions, intrasanguineous host-mediated assays are more sensitive and more representative of the carcinogenic activity of dialkylnitrosamines than in vitro assays using S-9 liver fractions.

Ann Sclavo, 1981 Jan-Feb, 23(1), 106 - 15
{Properties of 5 H2-S-positive strains of "Escherichia coli" (author's transl)}; Quarto M et al.; Some properties of 5 H2S+ strains of E . coli (labeled EC1-EC5, of which 4 isolated from the feces of subjects with gastroenteritis and 1 from a patients with cystopyelitis) has been studied . Of these, two resulted to belong to the O140 and one to the O127 serological groups; other 2 strains were found to react respectively with two (O84 and O108) and three (O65, O70 and O71) E . coli antisera . H2S- segregants were spontaneously obtained from 3 of these strans, but with different frequency: it was very low in EC5 (1.4%) and especially in EC3 (0.2%) strains, but appeared higher in EC4 (39.3%) strain . Clones from EC4 strain were also raffinose-negative; 11 of 12 obtained from EC5 strain (resistant to tetracycline) appeared sensible to tetracycline . Only one strain (EC4) has been able to transfer the H2S+ character with 4.7% frequency; another strain (EC5) transferred tetracycline-resistance character, but with lower frequency . Raffinose-positive character has never been transferred.

Z Allg Mikrobiol, 1981, 21(6), 439 - 45
Further studies on a temperature-sensitive mutant of Escherichia coli with defective repair capacity; Morfiadakis I et al.; A temperature-sensitive mutant of E . coli, WG24, was studied with respect to its sensitivity to photodynamic action, its capacity to perform host controlled reactivation, and its sensitivity to transduction at elevated temperatures . Mutant cells are much more sensitive than wild type cells to photodynamic action by thiopyronine and visible light at elevated temperatures . As well defined rec mutants, WG24 cells are less able to reactivate UV irradiated lambda c phages at elevated temperatures, while their ability to repair T1 phages is less impaired . Mutant cells cannot be transduced to T6 resistance at a detectable rate at elevated temperature . It is concluded, therefore, that some rec gene carries a ts mutation in this mutant.

Mol Gen Genet, 1981, 182(2), 364 - 6
The initiation of chromosome replication in a dnaAts46 and a dnaA+ strain at various temperatures; Frey J et al.; The regulation of chromosome replication initiation was studied at various temperatures with an E . coli dnaA46 strain and its dnaA+ parent . We find that, in both strains, the "initiation mass" varies depending upon growth temperature while the replication time remains constant relative to the cell doubling time . In the permissive temperature range, the initiation mass of the dnaA46 mutant strain is larger by a constant factor than that for a dnaA+ strain . We conclude that, even at temperatures permissive for growth of the dnaA46 strain, the activity of the dnaA46 product is lower than that of the wild-type protein . The dnaA gene product, therefore, plays an important role in regulating initiation.

Mol Gen Genet, 1981, 182(2), 358 - 60
Ribosomal mutation in Escherichia coli affecting membrane stability; Bosl A et al.; Mutations in ribosomal protein L6 cause (i) loss of viability of cells at 0 degrees C, which can be prevented by the presence of sodium chloride or 20% sucrose in the medium, (ii) influx of compounds at low temperature that normally cannot penetrate, and (iii) a defective assembly and maturation of 30S and 50S subunits at low temperature . It is proposed that abnormal interaction of immature subunits (or mutant 70S ribosomes) with the cytoplasmic membrane is responsible for triggering breakdown of membrane stability during cold shock.

Mol Gen Genet, 1981, 182(2), 279 - 87
Isolation of fusions between the lac genes and several genes of the exu regulon: analysis of their regulation, determination of the transcription direction of the uxaC-uxaA operon, in Escherichia coli K-12; Hugouvieux-Cotte-Pattat N et al.; Gene fusions between the lac structural genes and different genes of the hexuronate system were isolated by the two methods described by Casadaban (1976, 1979) . Mud (Apr lac) mutants which have the lac genes fused to the regulatory region of exuT, uxaC, uxaA and uxaB genes were constructed . Separately, the lac genes carried by a lambda plac-Mu hybrid phage were placed into a Mucts prophage inserted into uxaC gene and fusions were obtained, by selection of deletions putting the lac genes under the control of the uxaC regulatory elements . Induction of the lysogen led to the isolation of a lambda transducing phage bearing this uxaC-lac fusion . In all the fusion strains, beta-galactosidase expression was shown to be inducible by the natural inducers of hexuronate system enzymes, sensitive to catabolite repression and under the negative control of the exuR regulatory gene . The mutants with Mu insertion were used to establish the direction of transcription of the uxaC-uxaA operon, which is counter-clockwise on the circular genetic map of Escherichia coli (from uxaC to uxaA).

Mol Gen Genet, 1981, 182(2), 263 - 7
Initiation of chromosomal DNA synthesis in vitro; Projan SJ et al.; An in vitro complementation assay for initiation of chromosomal DNA replication is described . The initiation reaction is dependent upon extract from either of two hybrid-plasmid containing strains . Each hybrid plasmid carries a suppressor of dna A-ts mutations . The in vitro DNA synthesis is heavily biased toward the origin region, and the origin of replication (oriC) is replicated as determined by DNA-DNA hybridizations.

J Mol Evol, 1981, 17(6), 354 - 60
The density distribution of gene loci over the genetic map of Escherichia coli: its structural, functional and evolutionary implications; De Martelaere DA et al.; A quantitative analysis was carried out on the dispersion of gene loci over the E . coli genetic map . Therefore, the map was divided into regions characterized by an homogeneous gene density . This created a distribution pattern of gene loci that contained a symmetry axis located near to the origin of DNA replication . The pattern could be subdivided into a set of 22 functional domains containing gene loci whose products revealed a biochemical or functional relatedness . A correlation was found between the boundary positions of these domains and the distribution of F plasmid- and DNA insertion sites over the E . coli chromosome . The structural, functional and evolutionary implications of these findings are discussed.

Folia Psychiatr Neurol Jpn, 1981, 35(2), 175 - 9
The diagnostic value of cerebrospinal fluid lactic acid levels in meningitis; Murata R et al.; To assess the reliability of cerebrospinal fluid (CSF) lactate levels in children for differentiating between bacterial and viral meningitis, we measured the CSF lactate levels using the carboxylic acid analyzer, enzymatic method and colorimetric determination . Lactate determination by these methods is a highly reliable indicator of the presence or absence of bacterial meningitis . Moreover, the duration of elevated CSF lactate levels coincided with the clinical response to therapy.

Folia Microbiol (Praha), 1981, 26(4), 270 - 6
Intracellular location of NR1-plasmid DNA in Escherichia coli minicells; Hochmannova J et al.; Intracellular location of plasmid NRI (M = 58 Mg/mol, stringent control of replication 1-2 copies per Escherichia coli chromosomal equivalent) was compared with that of plasmid R6D delta 1 (M = 21 Mg/mol, relaxed control of replication, 10-15 copies per E . coli chromosomal equivalent), both in E . coli minicells . Considerable difference in relative distribution of molecules of these two plasmid DNA's between the cytoplasm and the membrane fraction was found when components of the corresponding minicell lysates were fractionated by sedimentation in a double-linear gradient of caesium chloride and sucrose . Also the differences in relative numbers of NRI DNA and R6K delta 1 DNA molecules stably associated with the membrane of minicells, determined by electron-microscopic examination of the fractions containing plasmid DNA-membrane complexes, was evaluated as statistically significant . The association of NRI DNA molecules with E . coli minicell membrane was found to be a much more frequent event than such association of R6K delta 1 molecules . The absolute amount of plasmid DNA associated with membrane in a single minicell corresponds to one molecule for both NRI and R6K delta 1.

Experientia, 1981, 37(8), 846 - 7
High magnesium content of Escherichia coli B; Moncany ML et al.; We have found that the intracellular concentration of magnesium in exponentially growing Escherichia coli B is much higher than has been previously assumed; it is about 100 mM . Results of equilibrium dialysis suggest that nearly all of this Mg is bound, probably most of it to nucleic acids . These findings could have important consequences for the study of protein-DNA interactions and the in vitro simulation of protein biosynthesis.

Cytobios, 1981, 30(119), 153 - 66
A quantitative study of the phagocytosis of Escherichia coli by myxamoebae of the slime mould Dictyostelium discoideum; Glynn PJ; A method is described for the rapid and sensitive assay of phagocytosis which utilizes radioactively labelled bacteria . Ingestion is proportional to the concentration of amoebae in the incubation mixture and a double reciprocal plot of the initial rate of ingestion versus bacterial concentration yields a straight line . In the presence of excess bacteria, the kinetics of ingestion are approximately linear during the early stages of the time course, and then continue at a steadily decreasing rate, eventually levelling off after about 60 min incubation . At this stage, the average number of bacteria per amoebae is between 65 and 70 . Phagocytosis is sensitive to low temperature and inhibitors of aerobic respiration but is unaffected by substances which block glycolysis . Digestion of the engulfed bacteria begins rapidly and can apparently proceed to completion since very little radioactivity is lost to the medium for a period of 8 h following ingestion.

Arch Exp Veterinarmed, 1981, 35(3), 337 - 48
{Diarrhea in young calves . 4 . Redox potential of the gastrointestinal tract of clinically healthy calves, and calves with spontaneous diarrhea, experimental Escherichia coli infection, and cyclophosphamide treatment}; Schulze F et al.; The redox potential (ROP) dropped strongly in a calf, from two hours after birth to 48 hours of age . The drop in the colon was more strongly pronounced than that in the small intestine . -The Eh values recorded throughout the small intestine of clinically intact calves, aged between four and ten days, were more negative than those in the stomach . The most negative ROP values, which differed with significance from those of all proximal gastro-intestinal sections, were recorded from the caecum . -Intracolonic negativation of ROP in calves, during the first hours of life, must be attributed to progressing germ population . The same relationship is borne out clearly by a pronounced rise in anaerobic germ count accompanied by significant ROP negativation in the colon . -Growing age was accompanied by drastic change in ROP values throughout the gastro-intestinal tract . Calves aged between six and nine weeks were compared to animals aged between four and ten days . ROP values in stomach, proximal and distal jejunum as well as in the ileum of the former group were negative with significance, whereas ROP values in the caecum and colon of the same group were more positive . -Animals with diarrhoea exhibited significant ROP deviations only in the ileum . They were more negative, as compared to clinically intact calves aged between four and ten days . Pathophysiological intestinal disorders which could be measured via ROP were found to play no role at all in the genesis and course of diarrhoea of calf . -No pronounced gastro-intestinal ROP deviations were exhibited by calves with experimentally induced Escherichia coli infection and manifest coli dysentery nor by animals which received cyclophosphamide treatment.

Prog Clin Biol Res, 1981, 62, 115 - 21
Influence of Escherichia coli endotoxin on palmitate, glucose, and lactate utilization by isolated dog heart myocytes; Liu MS et al.; The in vitro effects of E . coli 0127:B8 endotoxin on the oxidation of palmitate, glucose, and lactate by isolated, beating, adult dog heart myocytes were studied . The result were correlated with the in vitro effect of endotoxin on the transport of palmitate and glucose by using their respective analog, hexadecanol and 3-O-methyl-D-glucose . Endotoxin decreased palmitate but increased glucose and lactate oxidation in a dose-dependent manner . The changes in palmitate and lactate oxidation induced by endotoxin could not be corrected by addition of L-carnitine to the reaction mixture . The transport of hexadecanol or of 3-O-methyl-D-glucose was not affected by endotoxin . These results suggest that the endotoxin-induced alteration in palmitate and glucose oxidation by the heart is not the result of an impairment in metabolite transport, but rather a defect in the subsequent intracellular oxidative processes.

Mol Gen Genet, 1981, 181(2), 192 - 200
ExpA: a conditional mutation affecting the expression of a group of exported proteins in Escherichia coli K-12; Dassa E et al.; A mutant of Escherichia coli K-12 was isolated as conditionally deficient in the expression of two exported proteins simultaneously (i.e . two acid phosphatases) . The mutant was found to be thermosensitive on minimal medium at 37 degrees C and above, but grew normally on rich media at these temperatures . The mutation, named expA and located at 22 min on the recalibrated linkage map, depressed the levels of six periplasmic enzymatic activities in bacteria grown at 37 degrees C . At least ten proteins were greatly reduced in the periplasm under these conditions . The mutation also affected some outer membrane proteins, among which were the ompF protein and a protein which may be protein III, but had little effect on cytoplasmic membrane proteins . The gel patterns of the soluble cytoplasmic proteins were not modified except for one major protein of MW 47,000 . The activities of beta-galactosidase and of aspartate transcarbamylase were unmodified . After growth at 30 degrees C no difference was observed between expA and expA+ isogenic strains . The results are discussed with respect to the mechanism of protein export.

Mol Gen Genet, 1981, 181(2), 164 - 8
Expression of the L11-L1 operon in mutants of Escherichia coli lacking the ribosomal proteins L1 or L11; Stoffler G et al.; Using a variety of immunological techniques, the supernatant levels of ribosomal proteins were measured in mutants lacking the ribosomal proteins L1 or L11, and in wild-type strains . There was a 2.5--5-fold elevation of protein L11 level in the supernatant of strains lacking protein L1, compared to wild-type . In contrast, there was no elevation, but rather a diminution, in the corresponding L1 level in strains lacking protein L11, compared to wild-type . These results are consistent with a model for the control of expression of the L11-L1 operon in which protein L1 is an inhibitor of expression of that operon, but protein L11 is not . The supernatant concentrations of other proteins were indistinguishable in all strains.

Circ Shock, 1981, 8(4), 451 - 64
A comparison of the cardiovascular, renal, and coronary effects of dopamine and monensin in endotoxic shock; Somani P et al.; The present investigation was carried out in anesthetized dogs to compare the cardiovascular effects of dopamine and monensin, a carboxylic ionophore, in normal and E coli endotoxin-induced shock conditions . In control animals, monensin increased cardiac contractility, cardiac output, systemic blood pressure, and coronary blood flow in a dose-dependent manner, but had little or no effect on heart rate . However, unlike dopamine, which selectively increased renal blood flow, monensin did not affect renal blood flow in doses that produced a maximal increase in coronary blood flow or other hemodynamic effects . The duration of action of monensin was longer than 2 hr . Both dopamine and monensin reversed the cardiac depression and hypotension produced by E coli endotoxin, and in these experiments also, the duration of action of monensin was longer than 2 hr . Regional blood flow measurements with the radioactive microsphere (15 mu) technique demonstrated a marked decrease in organ blood flows at 60 min postendotoxin, but some recovery was observed at 90 min . In the left ventricle, reduction of flow to the endocardial region was greater than to the epicardium . In dogs with endotoxic shock, monensin produced a significant increase in organ blood flows towards or even above control values.

Circ Shock, 1981, 8(4), 435 - 50
Augmented venous return: a model of left ventricular afterload reduction during the course of endotoxin shock; Hess ML et al.; Utilizing a canine model of endotoxin shock (E coli, 4 mg/kg, B6:026) the major determinants of cardiac output (preload, afterload, contractility, and heart rate) were simultaneously followed for 5 hr in four study groups: Group I: time-matched controls, Group II: endotoxin shock, Group III: endotoxin shock and femoral-femoral A-V shunt, and Group IV: A-V shunt control . Groups II and III demonstrated an initial, abrupt increase in total peripheral resistance (TPR), coronary vascular resistance (CVR), and pulmonary vascular resistance (PVR), and a decrease in cardiac output (CO), coronary flow (CF) and heart rate (HR) and stroke work (P less than 0.05) . Group II then demonstrated a decrease in TPR, CVR, PVR with an increase in CO and CF but systemic arterial pressure did not return to control values . At approximately 3 hr, Group II developed a progressive increase in TPR, CVR, and PVR, and a decrease in CO, CF, and SW . Heart rate did not change . In contrast, at 3 hr Group III demonstrated no significant increase in TPR, CVR, or PVR, a progressive increase of CO and CF, and preservation of SW . It is hypothesized that endotoxin shock is characterized by an initial phase characterized by an increase in resistance and decrease in flow that is not affected by an augmented venous return . However, in the intermediate and latter stages of shock, there is a progressive increase in resistance and decrease in flow, increasing impedance to left ventricular ejection that results in an imbalance between myocardial oxygen supply and demand, contributing to the observed myocardial failure . An augmented venous return, by decreasing resistance (afterload) and increasing venous return (preload) preserves cardiac output and myocardial function and thus serves as a model of left ventricular afterload reduction during the course of endotoxin shock.

Carcinogenesis, 1981, 2(4), 261 - 4
Mutagenicity of nitrosated cimetidines; Ichinotsubo D et al.; Dinitrosocimetidine and mononitrosocimetidine were tested in a series of short term assay systems . Both compounds were mutagenic in the absence of rat liver microsomes . The dinitrosocimetidine produced higher mutagenic or clastogenic effects at concentrations that were 50 to 500 times lower than the concentrations at which the mononitrosocimetidine produces its maximum effects . The most sensitive short term assay system was the Chinese hamster ovary cell culture system . Dinitrosocimetidine caused sister chromatid exchanges at a concentration of 10(-8) M and chromosome aberrations at 10(-7) M in this system . Dinitrosocimetidine had moderate activity in the bacterial short term assay systems . In the Ames test, strain TA 100 was the most sensitive . The compound was of lower activity in the E . coli WP-2 and the E . coli rec- systems.

Carcinogenesis, 1981, 2(3), 189 - 94
A differential killing assay for mutagens and carcinogens based on an improved repair-deficient strain of Escherichia coli; Tweats DJ et al.; The lexA gene suppresses the spontaneous inviability of recA strains of Escherichia coli without affecting their repair deficiency . We have taken advantage of this to construct a uvrA recA lexA triple mutant CM871 that combines extreme repair deficiency with near wild-type growth . We have used this strain in conjunction with an isogenic strain WP67 uvrA polA and isogenic wild-type strain WP2 in a differential killing test . Dilute suspensions in buffer of the repair-deficient strains and repair-proficient control are incubated for 2 h at 37 degrees C with test compound in the presence or absence of a S9 metabolising system . Survival is determined by Miles Misra plating . For each strain three 10 microliter spots of each of three concentrations of test compound and of the untreated control are placed on a nutrient agar plate . Following overnight incubation survivors are counted . Results with reference compounds are given and the advantages and disadvantages of the test are discussed.

Carcinogenesis, 1981, 2(2), 141 - 6
The induction of errors during in vitro DNA synthesis following chloroacetaldehyde-treatment of poly(dA-dT) and poly(dC-dG) templates; Hall JA et al.; Chloroacetaldehyde, a rearranged metabolic product of the human carcinogen vinyl chloride, reacts with the DNA-like polymers poly(dA-dT) and poly(dC-dG) to form etheno-adducts of the adenine and cytosine bases . These treated polymers, when used as templates for E . coli DNA polymerase I in an in vitro assay, show a decreased ability to direct DNA synthesis . At the same time, increased relative levels of non-complementary nucleotides are incorporated . With the poly(dA-dT) templates 1 dGMP residue is incorporated for every approx 60 ethenoadenine residues present whilst no increased misincorporation of dCMP was detected . With the poly(dC-dG) templates 1 misincorporation of dAMP or dTMP occurred in the presence of approx 30 and 80 ethenocytosine residues respectively . A nearest neighbour analysis shows that with the modified poly(dC-dG) templates the majority of the errors were incorporated opposite cytosine (or modified cytosine) bases.

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