Escherichia coli

Nucleic Acids Res, 1984 May 25, 12(10), 4207 - 28
Isolation of cDNA and genomic DNA clones encoding type II collagen; Young MF et al.; A cDNA library constructed from total chick embryo RNA was screened with an enriched fraction of type II collagen mRNA . Two overlapping cDNA clones were characterized and shown to encode the COOH propeptide of type II collagen . In addition, a type II collagen clone was isolated from a Charon 4A library of chick genomic fragments . Definitive identification of the clones was based on DNA sequence analysis . The 3' end of the type II collagen gene appears to be similar to that of other interstitial collagen genes . Northern hybridization data indicates that there is a marked decrease in type II collagen mRNA levels in chondrocytes treated with the dedifferentiating agent 5-bromodeoxyuridine . The major type II collagen mRNA species is 5300 bases long, similar to that of other interstitial collagen RNAs.

Nucleic Acids Res, 1984 May 25, 12(10), 4139 - 52
Targeted mutagenesis in vitro: lac repressor mutations generated using AMV reverse transcriptase and dBrUTP; Mott JE et al.; We have cloned the gene for the lac operon repressor (lacI) of Escherichia coli into the M13 related phage f1 . Mutagenesis of the lacI gene was performed in vitro by filling dsDNA molecules gapped over the lacI gene with Avian Myeloblastosis Virus (AMV) reverse transcriptase . LacI mutants are found at a frequency of 1 in 10(4) using a genetic screen in vivo . For two-thirds of the 60 mutants, lesions were identified within the first 400 bases of lacI, by dideoxy sequencing . An unexpectedly wide range of different lesions were observed, including transitions, transversions, and deletions (of which the most common were the removal of single base pairs) . The replacement of dTTP by dBrUTP in the filling reaction resulted in a doubling of deletions in the sample population as well as the anticipated T to C and C to T transitions . Although the lacI gene has been extensively studied in vivo, the power of this technique for mutagenesis in vitro is demonstrated by the generation of three previously undescribed lacI mutations.

Biochemistry, 1984 May 22, 23(11), 2367 - 72
Molecular mechanisms of chemical mutagenesis: 9-aminoacridine inhibits DNA replication in vitro by destabilizing the DNA growing point and interacting with the DNA polymerase; Topal MD; 9-Aminoacridine was found to inhibit dNTP incorporation into DNA homopolymer duplexes by phage T4 DNA polymerase in vitro . Systematic variation of the molar ratio of 9-aminoacridine to DNA, to DNA polymerase, and to DNA precursors demonstrated that this inhibition at 9-aminoacridine concentrations below 10 microM was mainly due to interaction of 9-aminoacridine with the DNA and suggested that the basis for the preferential inhibition of incorrect precursor incorporation was destabilization of the DNA growing point . Consistent with destabilization, 9-aminoacridine stimulated the hydrolysis of correctly base paired DNA by the 3'-5' exonuclease activity of phage T4 DNA polymerase . This is the first indication to my knowledge that an intercalating dye destabilizes the DNA growing point, whereas it raises the overall Tm of the DNA . At 9-aminoacridine concentrations above 10 microM overall incorporation of dNTPs was inhibited by 9-aminoacridine interaction with the DNA polymerase . A possible explanation for the induction of both deletion and addition frameshift mutations by 9-aminoacridine during DNA biosynthesis is discussed in light of growing-point destabilization.

Biochemistry, 1984 May 22, 23(11), 2363 - 7
Cysteinyl residues of Escherichia coli recA protein; Kuramitsu S et al.; The Escherichia coli recA protein has three cysteinyl residues at positions 90, 116, and 129 . All of them are reactive with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) . In the presence of ATP or ADP, only one cysteinyl residue reacts with DTNB . The residue was also reactive with N-{7-(dimethylamino)-4-methylcoumarinyl}maleimide (DACM) in the presence of ATP . The results on an analysis of the DACM-modified protein cleaved at the nonmodified cysteinyl residues after cyanation with 2-nitro-5-(thiocyano)benzoic acid show that two cysteinyl residues protected in the presence of ATP or ADP are identified as Cys-90 and Cys-129 . When the ionic strength is higher than 1, one cysteinyl residue does not react with DTNB . This residue is Cys-90 or Cys-129, because one of the two cysteinyl residues, which are not modified with DACM in the presence of ATP, does not react with DTNB at high ionic strength . The binding of single-stranded DNA to the recA protein does not change the reactivity of the cysteinyl residues with DTNB.

Biochim Biophys Acta, 1984 May 22, 804(1), 118 - 24
In vivo 19F-NMR of 5-fluorouracil incorporation into RNA and metabolites in Escherichia coli cells; Gochin M et al.; 19F resonances from RNA with 5-fluorouracil incorporated could be observed in intact Escherichia coli cells, as well as in tRNA isolated from the cells . 19F-NMR signals from the metabolic breakdown products of the fluorinated RNA were also detected in vivo . By observing the 19F-NMR spectrum, variations in the metabolic disposition of administered 5-fluorouracil could be monitored as a function of time and be compared when the cells were deprived of oxygen and other nutrients, subjected to ethidium bromide treatment, or grown in the presence of mitomycin C.

FEBS Lett, 1984 May 21, 170(2), 290 - 4
The effect of aminoacyl- or peptidyl-tRNA at the A-site on the arrangement of deacylated tRNA at the ribosomal P-site; Babkina GT et al.; Photoreactive derivatives of E . coli tRNAPhe bearing arylazido groups on guanine residues (azido-tRNA) were used for affinity labelling of E . coli ribosomes in the region of the P-site when the A-site was either free or occupied by aminoacyl- or peptidyl-tRNA . Corresponding complexes of azido-tRNA with ribosomes and poly(U) were obtained both nonenzymatically and with the use of elongation factors . UV-irradiation of the complexes resulted in labelling of ribosomal proteins (preferentially of 30 S subunit) . Proteins S9 and S21 were labelled only when the A-site was free; S14 - only when it was occupied; S11, S13, S19 - in both cases; S5, S7, S12, S20 - in some states.

Nature, 1984 May 17-23, 309(5965), 215 - 9
Role of RecA protein spiral filaments in genetic recombination; Howard-Flanders P et al.; Physical and enzymatic studies on RecA protein from Escherichia coli provide the basis for a molecular model of general genetic recombination, a novel feature of which is the role attributed to spiral filaments of RecA protein.

Biochem Biophys Res Commun, 1984 May 16, 120(3), 946 - 52
Selective inhibition of eukaryotic RNA polymerase: a possible new mechanism of antitumor drug action; Chuang LF et al.; N-Trifluoroacetyladriamycin-14-O- Hemiadipate (AD 143), a new derivative of adriamycin with greater antitumor activity and lower cardiotoxicity, was shown not to interact with DNA and yet inhibit the activities of both RNA polymerases I and II of chicken myeloblastosis cells in vitro with ID50 values equal to 6.5 microM and 7 microM, respectively . On the other hand, an approximately 35-fold higher concentration of AD 143 was required to cause a similar inhibition of the activity of DNA polymerase alpha from chicken myeloblastosis cells . Under the same assay conditions, AD 143 had even less effect on either RNA polymerase or DNA polymerase I of E . coli cells (ID50 greater than 265 microM for both enzymes) . These studies suggest that AD 143, in contrast to its parental drug adriamycin, may have a selective inhibitory effect against eukaryotic RNA polymerases.

Biochem Biophys Res Commun, 1984 May 16, 120(3), 939 - 45
Mn-Mn interaction in adenylylated and unadenylylated glutamine synthetase; Gibbs EJ et al.; The distance between the two catalytically important metal ions of glutamine synthetase was determined by electron paramagnetic resonance (EPR) . Mn(II) binds more tightly to the n1 site of this enzyme in the presence of methionine sulfoximine and the influence of Mn(II) bound at the n2 site on the EPR spectrum of Mn(II) at n1 was studied . A monotonic increase in the EPR spectrum of Mn(II) was observed at Mn:E (subunit) ratios of 0 to 0.8 . After this point as Mn(II) was added to about 1.8 Mn:E, a decrease in the EPR signal was observed . This phenomenon was found for both adenylylated and unadenylylated forms of glutamine synthetase . The data were analyzed using a theory for dipolar electron-electron relaxation and a distance of 10-12 A was computed for the Mn(II)-Mn(II) separation . These data demonstrate that both modified and unmodified forms of glutamine synthetase which have different catalytic activities have a similar spatial relationship between the two catalytic metal ion sites.

Eur J Biochem, 1984 May 15, 141(1), 109 - 14
Replication of M13mp7 single-stranded DNA in vitro by the 9-S DNA polymerase alpha from calf thymus; Grosse F et al.; The replication of M13 single-stranded DNA by the 9S DNA polymerase alpha from calf thymus has been studied in vitro . Priming conditions, the nature of the replication products and conditions for optimal elongation have been investigated . Oligonucleotides comprising only four nucleotides can serve as primers . Both ribo and deoxy oligonucleotides can be elongated . Priming by the short oligonucleotides occurs at multiple sites on the M13 genome . If replication is primed at single sites with a specific pentadecamer or with RNA in the origin of replication, specific pausing sites are observed . These pausing sites can partly be correlated with secondary structures in the template DNA . Addition of Escherichia coli single-stranded DNA binding protein leads to a weakening of pausing sites and to the synthesis of longer products . The 9S enzyme is able to proceed through most of the pausing sites resulting in the synthesis of product molecules as long as 6600 nucleotides . The 9S DNA polymerase alpha contains a potent DNA primase activity which enables it to initiate replication on a single-stranded template in the presence of the four NTPs . However, priming is also possible in the presence of ATP alone . The priming sites are not randomly distributed over the M13 DNA.

Toxicology, 1984 May 14, 31(2), 151 - 63
Ferritin: protection of enzymatic activity against the inhibition by divalent metal ions in vitro; Price DJ et al.; Ferritin binds a large quantity of Be2+ (Price D.J . and Joshi, J.G . J . Biol . Chem . 258 (1983) 10873) as well as other divalent metal ions . Therefore the ability of this protein to protect enzymes against or reverse the inhibition by metal ions was studied . Evidence presented here shows that the inhibition by Be2+ of the enzymes Na+K+ATPase, alkaline phosphatase and phosphoglucomutase is reversed by ferritin . Be2+ can be transferred reversibly between phosphoglucomutase and ferritin depending upon the relative concentrations of the 2 proteins . Ferritin also reactivated phosphoglucomutase inhibited by Zn2+, Cu2+, or Cd2+ . Incubation of ferritin containing Be2+ with 4-10 fold molar excess of phosphoglucomutase (with respect to Be2+) removed 90% of the Be2+ from ferritin . The rates of inactivation of phosphoglucomutase by Be2+ donated by apoferritin or ferritin were identical . Based upon these observations it is suggested that Be2+ bound to the protein shell and to the iron core are in equilibrium with each other with the equilibrium favoring ferritin-Be2+ complex.

Biochim Biophys Acta, 1984 May 11, 793(3), 379 - 86
pH-dependent modulation of phospholipase A2 activity by alkaline cations and catecholamines in a granule-enriched fraction of adrenal medulla; Bartolf M et al.; Phospholipase A activity was measured in the soluble fractions from bovine adrenal medullary granules and rat liver lysosomes . The adrenal medulla preparation, enriched 2.5-fold in chromaffin granules and lysosomes, hydrolyzes the phospholipids of {1-14C}oleate-labelled autoclaved Escherichia coli in the pH range 3.5-7.0 in an alkaline cation (Na+, K+, Ca2+, Mg2+)-dependent fashion . At low alkaline cation concentrations the apparent pH optimum is near 6.5 but decreases to about 4.5 with increasing cation concentrations . When measured at high alkaline cation concentrations phospholipase activity in the adrenal fraction has a pH profile and optimum similar to those of rat liver lysosomes . Amine-containing buffers, millimolar concentrations of catecholamines and micromolar concentrations of the amine-containing drug, trifluoperazine, modulate the adrenal medulla phospholipase activity in a pH- and alkaline cation-dependent manner . Studies with specifically labelled phosphatidylethanolamines confirm previous conclusions that activity at pH 6.4 is almost exclusively phospholipase A2; but in contrast to previous conclusions (Smith, A.D . and Winkler , H . (1968) Biochem . J . 108, 867-874) we find significant phospholipase A2 activity at pH 4.2.

Nucleic Acids Res, 1984 May 11, 12(9), 3727 - 46
Differential expression of human interferon genes; Hiscott J et al.; We developed a method for quantitating closely related mRNAs by S1 mapping and used it to determine the levels of mRNAs for IFN-beta, IFN-gamma and various alpha IFNs (IFN-alpha 1, -alpha 2, -alpha 4, -alpha 5, -alpha 6, -alpha 7, -alpha 8 and -alpha 14) in human peripheral blood leukocytes, lymphoblastoid (Namalwa), HeLa and human fibroblastic cells, induced in different fashions . The ratio of alpha to beta IFN transcripts varied greatly, depending on the cell type . The levels of the individual IFN-alpha RNAs were very different: IFN-alpha 1, -alpha 2 and -alpha 4 RNAs constituted the major fraction of the IFN-alpha transcripts measured . Moreover, there was a striking difference in the proportion of individual IFN-alpha mRNA species in different cell types . Use of different induction protocols did not significantly affect the proportion of IFN mRNAs . IFN production was not proportional to mRNA level in all cases, as lymphoblastoid cells induced by incubation at high density and virus-induced HeLa cells contained high levels of IFN-beta but produced little antiviral activity.

Nucleic Acids Res, 1984 May 11, 12(9), 4011 - 7
Oligonucleotide mutagenesis of the lacPUV5 promoter; Munson LM et al.; Synthetic oligonucleotides were used to introduce mutations into the lacPUV5 promoter . Four mutations were obtained at positions -13, -14, and -15, with respect to the transcriptional start site . The effects of these mutations were measured in vivo and the results are discussed with respect to the consensus sequence and other promoter mutations located in this region.

Science, 1984 May 11, 224(4649), 605 - 7
Suppression of prolactin in pigs by Escherichia coli endotoxin; Smith BB et al.; An endotoxin produced by Escherichia coli caused a decrease in prolactin concentrations in the plasma of sows when given at low dosages 2 days postpartum . Five to tenfold increases occurred in the plasma cortisol concentrations . Piglet growth, used as an indicator of milk secretion by the sows, was significantly depressed after the endotoxin administration . Some cases of lactation failure in the periparturient sow may thus be due to endotoxins suppressing prolactin concentrations . This appears to be the first report of a bacterial endotoxin having an effect on prolactin in any species.

Nucleic Acids Res, 1984 May 11, 12(9), 3937 - 50
The influence of messenger RNA secondary structure on expression of an immunoglobulin heavy chain in Escherichia coli; Wood CR et al.; A gene for murine mu heavy chain immunoglobulin has been inserted into a bacterial expression plasmid containing the Escherichia coli trp promoter and ribosome binding site . A low level expression of mu protein was detected . Secondary structure analysis showed the presence of a hairpin loop burying the mu initiation codon . Alteration of secondary structure at this site by oligonucleotide replacement mutagenesis revealed a correlation between mu expression levels and accessibility of the ribosome binding site . Abolition of secondary structure increased mu protein expression over ninety-fold, to a level approximately equal to that of a trpE -mu fusion protein using the native trpE ribosome binding site.

Nucleic Acids Res, 1984 May 11, 12(9), 3873 - 93
Selection of initiation sites by eucaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence for preproinsulin; Kozak M; Recombinant plasmids that direct synthesis of rat preproinsulin under the direction of the SV40 early promoter have been used to probe the mechanism of initiation of translation . Insertion of an upstream AUG triplet that was out-of-frame with respect to the coding sequence for preproinsulin reduced the yield of proinsulin, in keeping with the predictions of the scanning model . The extent to which an upstream AUG codon interfered depended on sequences surrounding the AUG triplet; with two constructs ( p255 /20 and C2) the 5'-proximal AUG codon constituted an absolute barrier: there was no initiation at the downstream start site for preproinsulin . With two other constructs ( p255 /9, p255 /21), however, proinsulin was made despite the presence of an upstream, out-of-frame AUG codon in a favorable context for initiation . In those cases the reading frame set by the first AUG triplet was short, terminating before the start of the preproinsulin coding sequence . The interpretation that ribosomes initiate at the first AUG, terminate, and then reinitiate at the AUG that directly precedes the preproinsulin coding sequence was tested by introducing a point mutation that eliminated the terminator codon: the resulting mutant made no proinsulin.

Nucleic Acids Res, 1984 May 11, 12(9), 3857 - 72
Cloning and delimiting one chloroplast DNA replicative origin of Chlamydomonas; Wang XM et al.; The EcoR1 restriction fragments containing D-loops which marked the replication origin of chloroplast DNA were identified in two different species of Chlamydomonas . Each fragment was cloned in the E . coli plasmid pBR325 . The cloned fragments were compared by restriction endonuclease analyses and by heteroduplex analyses in the electron microscope . The relative position of the D-loop regions and the homologous regions between the 2 fragments was determined . The D-loops were located within one short homologous region of 0 . 42kb in length between the 2 cloned restriction fragments . The homologous region was subcloned in pBR322 . Closed circular plasmid DNAs containing the short homologous region showed preferred denaturation in the D-loop region under physiological salt concentration which suggested that D-loop region was AT rich . Sequence divergence was detected at both ends of the D-loop region . Southern blot analyses indicated the presence of species-specific repetitive sequences within the divergent regions.

J Biol Chem, 1984 May 10, 259(9), 5632 - 6
Decoding at the ribosomal A site . The effect of a defined codon-anticodon mismatch upon the behavior of bound aminoacyl transfer RNA; Hornig H et al.; Ribosomes from Escherichia coli were programmed by being allowed to bind a molecule of tRNAMetf or fMet-tRNAMetf and the hexanucleotide messenger AUGN1N2N3 . The interaction of the ternary complex {EF-Tu X GTP X Phe-tRNAPhe} with the A site (containing the codon N1N2N3) was then studied by measuring the extent of (i) the binding of Phe-tRNAPhe to the ribosome, (ii) the hydrolysis of GTP, and (iii) the formation of the dipeptide fMet-Phe . By variation of N1,N2, and N3, a defined degree and position of mismatch could be obtained; the correct A-site codon UUU was compared with the incorrect codons CUU, UCU, GUU, and UUG . Each single-point alteration led to catalytic hydrolysis of GTP and to a strong reduction in the amounts of Phe-tRNAPhe binding and of dipeptide formation . The observations were explicable qualitatively by a hypothesis according to which the behavior of the bound aa-tRNA, after hydrolysis of GTP and before peptidyl transfer, is determined principally by the energy of binding of the aminoacyl-tRNA to the A site . This binding in turn was found to depend upon both the nature and the position of the mismatch . The results further suggest a steric interplay between the 3' (acceptor) end of the A-site tRNA and the second and third positions of the anticodon, so that a mismatch at one of these positions can impair directly the interaction between the aminoacylated 3' end and the ribosome and can thus reduce the rate of peptide bond formation and contribute to the overall fidelity of the elongation cycle.

J Biol Chem, 1984 May 10, 259(9), 5601 - 5
Characterization of a novel lipoprotein mutant in Escherichia coli; Giam CZ et al.; Mutants altered in the structural gene for murein lipoprotein in Escherichia coli can be isolated by globomycin selection . We have isolated a unique globomycin-resistant mutant, strain 6-23, which synthesizes a structurally altered, albeit modified and processed, lipoprotein . DNA sequence analysis of the mutant lpp allele and determination of the amino acid composition of the mutant lipoprotein revealed a single amino acid substitution of cysteine for arginine at the 68th amino acid residue of prolipoprotein . Pulse-chase experiments revealed that the kinetics of lipoprotein maturation was affected by this alteration in the structure of lipoprotein.

J Biol Chem, 1984 May 10, 259(9), 5543 - 8
DNA glycosylase activities for thymine residues damaged by ring saturation, fragmentation, or ring contraction are functions of endonuclease III in Escherichia coli; Breimer LH et al.; A DNA glycosylase activity that excises oxidized, fragmented thymine residues from a polydeoxyribonucleotide has been purified 9,500-fold to apparent homogeneity from Escherichia coli . The purified enzyme also excises thymine glycol and cleaves DNA at apurinic sites, and appears to be identical with E . coli DNA endonuclease III . The enzyme catalyzes the release of several different forms of oxidized thymine, including urea, methyltartronylurea and 5-hydroxy-5-methylhydantoin . The molecular weight of the native protein is 25,000, and the same value is obtained for the denatured homogeneous protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

J Biol Chem, 1984 May 10, 259(9), 5430 - 9
Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli; Marasco WA et al.; Chemotactic factor-enriched butanol extracts from Escherichia coli culture filtrates were fractionated and purified by high pressure liquid chromatography . The yield from individual fractions of biological activity (lysosomal enzyme secretion) and antigenic activity (competition with {3H}fMet-Leu-Phe for binding to rabbit anti-fMet-Leu-Phe) revealed an average 50% recovery of original material . Five peaks of biological activity were separated as demonstrated by enzyme-releasing activity . Three of these peaks coincided exactly with peaks of antigenic activity, suggesting that at least 3 and as many as 5 distinct formyl-methionyl peptides had been separated . The majority of recovered activity appeared in peak 3 and represented 70% of the total biological and antigenic activities recovered . The five peak fractions were subsequently analyzed by dipeptidyl carboxypeptidase gas chromatography-mass spectrometry (DCP/GC-MS) to determine amino acid sequences . After digestion, the formyl-Met peptide was demonstrated in only one of the five peak fractions (peak 3) . Furthermore, both the GC retention times and mass spectra indicated that peak 3 contained formyl-methionyl-leucyl-phenylalanine . The DCP/GC and MS data were confirmed with tests made on authentic fMet-Leu-Phe . Butanol extracts from E . coli filtrates to which were added synthetic fMet-Leu-Phe resulted in increased biological and antigenic activity in the precise high pressure liquid chromatography fractions of peak 3 where the fMet-Leu-Phe produced by E . coli was found . Finally, the analysis of recovered biological and antigenic activities indicated that the formyl peptides were found in nanomolar concentrations in culture filtrates . These results demonstrate that the NH2-terminal formyl peptides produced by E . coli, of which formyl-methionyl-leucyl-phenylalanine appears to be the major component, are the peptide mediators responsible for leukocyte chemotactic activity in the bacterial culture extracts.

J Biol Chem, 1984 May 10, 259(9), 6033 - 8
Sequences of the Escherichia coli photolyase gene and protein; Sancar GB et al.; We have determined the nucleotide sequence of a 2039-base pair segment of Escherichia coli chromosomal DNA containing the phr gene, which encodes deoxyribopyrimidine photolyase . The coding region of phr is 1416 base pairs and is preceded by regions homologous to consensus sequences for E . coli promoters and ribosome binding sites . The phr gene is preceeded by an open reading frame of 169 codons (orf169) which is transcribed in the same direction . The proximity of orf169 to phr suggests that both are members of a single operon containing one or more internal promoters allowing differential expression of phr . An unusually large number of rare or infrequently used codons are utilized in phr, which may contribute to the low copy number of photolyase . The sequence at the NH2 and COOH termini and the overall amino acid composition of mature photolyase, determined using purified protein, agrees with predictions based upon the nucleotide sequence . Photolyase consists of 471 amino acids and has a calculated molecular weight of 53,994.

J Biol Chem, 1984 May 10, 259(9), 6028 - 32
Purification of Escherichia coli DNA photolyase; Sancar A et al.; Escherichia coli photolyase is a DNA repair enzyme which monomerizes pyrimidine dimers, the major UV photoproducts in DNA, to pyrimidines in a light-dependent reaction . We recently described the construction of a tac-phr plasmid that greatly overproduces the enzyme (Sancar, G . B., Smith, F . W., and Sancar, A . (1983) Nucleic Acids Res . 11, 6667-6678) . Using a strain carrying the overproducing plasmid as the starting material, we have developed a purification procedure that yields several milligrams of apparently homogeneous enzyme . The purified protein is a single polypeptide that has an apparent Mr of 49,000 under both denaturing and nondenaturing conditions . The enzyme has no requirement for divalent cations and it restores the biological activity of irradiated DNA only in the presence of photoreactivating light . The purified photolyase has a turnover number of 2.4 dimers/molecule/min; this value agrees well with the in vivo rate of photoreactivation in E . coli.

J Biol Chem, 1984 May 10, 259(9), 6013 - 8
Mechanism of localization of major outer membrane lipoprotein in Escherichia coli . Studies with the OmpF-lipoprotein hybrid protein; Yu F et al.; A chimera gene consisting of the ompF promoter, the coding regions for the signal peptide and the NH2-terminal 11 amino acid residues of outer membrane OmpF protein, and the coding region for the major outer membrane lipoprotein devoid of the NH2-terminal 7 amino acid residues was constructed . Escherichia coli carrying the cloned chimera gene produced a hybrid protein with the predicted chemical structure . The protein was localized in the periplasmic space with an interaction with the peptidoglycan layer . These results indicate that the hybrid protein was expressed, secreted across the cytoplasmic membrane, and processed for the signal peptide normally . The hybrid protein, however, was not incorporated into the outer membrane, suggesting the importance of the lipid domain in the assembly of the lipoprotein into the outer membrane . Although a larger part of the protein was extractable with sodium dodecyl sulfate, a part of the hybrid protein was covalently bound to the peptidoglycan layer as the lipoprotein is . Upon treatment with lysozyme of the envelope the hybrid protein became water soluble . The solubilized protein most probably existed as a trimer . These results most likely suggest that the major lipoprotein exists as a trimer in the periplasmic space with interactions with the peptidoglycan layer through the protein domain on one side and with the outer membrane through the lipid domain on the other side.

J Biol Chem, 1984 May 10, 259(9), 5567 - 73
The interaction of DNA polymerase III and the product of the Escherichia coli mutator gene, mutD; DiFrancesco R et al.; A comparison of DNA polymerase III core enzyme (McHenry, C . S., and Crow, W . (1979) J . Biol . Chem . 254, 1748-1753) prepared from wild type Escherichia coli and a strain harboring the mutator gene, mutD5 (Degnen, G . E., and Cox, E . C . (1974) J . Bacteriol . 17, 477-487) has revealed several differences in their properties . Among these are alterations in the heat stability, divalent cation requirement, pH optimum, 3'----5'-single strand exonuclease activity, and DNA-dependent conversion of a deoxynucleoside triphosphate to its corresponding monophosphate ("turnover") . The decrease in the 3'-single strand exonuclease and turnover indicate a defect in the editing function of the mutD strain, which is at least in part responsible for the high spontaneous mutation rate in mutD . Transformation of mutD by a hybrid plasmid, pRD3, constructed from an EcoRI restriction fragment of E . coli and pBR322, cures mutD of its abnormally high mutation rate, and simultaneously restores its 3'-exonuclease activity . These observations are consistent with the notion that the mutD gene product is a subunit of DNA polymerase III, and it either contains the catalytic site for the 3'-exonuclease or modulates its activity . From a consideration of the known molecular weights of the subunits in DNA polymerase III core (McHenry C . S., and Crow, W . (1979) J . Biol . Chem . 254, 1748-1753) the molecular weights of the two proteins translated in maxicells transformed with pRD3, and from a comparison of our results with those obtained with the mutator dnaQ (Horiuchi, T., Maki, H., Maruyama, M., and Sekiguchi, M . (1981) Proc . Natl . Acad . Sci . U . S . A . 78, 3770-3774) and the work of Cox and Horner (Cox, E . C., and Horner, D . L . (1983) Proc . Natl . Acad . Sci . U . S . A . 80, 2295-2299) as well as Echols et al . (Echols, H., Lu, C., and Burgers, P . M . J . (1983) Proc . Natl . Acad . Sci . U . S . A . 80, 2189-2192) we tentatively assign the mutD gene product to the epsilon subunit of DNA polymerase III.

Biochemistry, 1984 May 8, 23(10), 2221 - 6
lac Repressor cysteine-140 reacts selectively with a fluorescent probe bound to the core-headpiece interface; Schneider JM et al.; The fluorescent probe N-{(iodoacetyl)amino}-ethyl}-5-naphthylamine-1-sulfonate (I-AEDANS) reacts selectively with Cys-140 of the lac repressor . The reasons for this selectivity were investigated . The ability of 8-anilino-1-naphthalenesulfonate and 5,5'-bis(8-anilino-1-naphthalene-sulfonate) to bind noncovalently to the interface between the core and headpiece regions of the repressor suggested that I-AEDANS might also bind to this interface and then react intramolecularly with Cys-140 nearby . Two observations strongly support this model . (1) The selectivity for Cys-140 was lost when the headpiece regions were removed from the repressor . The rate of reaction with Cys-140 relative to Cys-107 in the repressor was 13.5 +/- 1.4, from trypsin digestions of labeled repressor . This ratio decreased to 2.1 +/- 1.0 for the core protein . (2) Iodoacetamide, which lacks the naphthylaminesulfonate portion of I-AEDANS, showed little selectivity for Cys-140 in either the repressor or the core . Nonreactive analogues of I-AEDANS did not alter the reaction of I-AEDANS with the repressor, presumably because they bound too weakly . Decreasing the ionic strength from 0.61 M to 56 mM decreased the selectivity of I-AEDANS for Cys-140 in the repressor, suggesting that I-AEDANS is not bound to the repressor by ionic interactions . Decreasing the pH from 8.5 to 7.5 increased the selectivity for Cys-140 only slightly . Fluorescent probes attached to Cys-140 appear to be ideally located to report motions of the headpieces , relative to the core, that attend DNA binding.

J Mol Biol, 1984 May 5, 175(1), 39 - 55
Synthesis of the isoleucyl- and valyl-tRNA synthetases and the isoleucine-valine biosynthetic enzymes in a threonine deaminase regulatory mutant of Escherichia coli K-12; Singer PA et al.; A mutation in the structural gene for threonine deaminase, ilvA538 , results in lower than normal levels of the isoleucyl, valyl- and leucyl-tRNA synthetases . Moreover, this regulatory mutation decreases the level of expression of the ilv biosynthetic operons and renders their expression non-responsive to limitations of the branched-chain amino acids . In this paper, we present in vitro evidence for the inhibition of isoleucyl- and valyl-tRNA synthetase activity by threonine deaminase and 2-ketobutyrate, the product of the threonine deaminase reaction, through the formation of a high molecular weight complex of the three molecules . Based on these results, we propose a model to explain the regulation of the isoleucyl- and valyt -tRNA synthetases in which transient inhibition of the synthetase enzyme activities by threonine deaminase and 2-ketobutyrate increases the expression of ileS and valS , the structural genes for isoleucyl- and valyt -tRNA synthetase, respectively . Further, the results suggest that the hyperattenuated expression of the ilv biosynthetic operons is due to an increased rate of complex formation of valyl and isoleucyl-tRNA synthetases and the altered form of threonine deaminase of the ilvA538 mutant strain.

J Mol Biol, 1984 May 5, 175(1), 29 - 38
An effect of codon context on the mistranslation of UGU codons in vitro; Carrier MJ et al.; Effects of codon context on nonsense codon suppression may act either through release factor recognition of termination codons or aminoacyl-tRNA selection by the ribosome . The latter hypothesis has been studied by comparing misreading by Escherichia coli UGA suppressor tryptophan tRNA of UGU (cysteine) codons in two synthetic polymers, poly(U-G) and poly( U5 , G), which differ in sequence around the UGU codons . In vitro translation of these polymers in a cell-free system from E . coli yielded selection errors of 4 X 10(-3) and 1.75 X 10(-2) for UGU codons in poly(U-G) and poly( U5 , G), respectively . This difference suggests that codon context may significantly affect misincorporation of amino acids into protein.

J Mol Biol, 1984 May 5, 175(1), 19 - 27
Codon context effects in missense suppression; Murgola EJ et al.; After our first observation of codon context effects in missense suppression ( Murgola & Pagel , 1983), we measured the suppression of missense mutations at two positions in trpA in Escherichia coli . The suppressible codons in the trpA messenger RNA were the lysine codons, AAA and AAG, and the glutamic acid codons, GAA and GAG . The mRNA sites of the codons correspond to amino acids 211 and 234 of the trpA polypeptide, positions at which glycine is the wild-type amino acid . Our data demonstrated codon context effects with both pairs of codons . The results indicate that suppression of AAA and AAG by mutant lysine transfer RNAs was more efficient at 211 than at 234, whereas suppression of GAA and GAG by two different mutant glycine tRNAs was more efficient at 234 than at 211 . In general, the context effects were more pronounced with GAG and AAG than with GAA and AAA . (In some instances it appeared that suppression of GAA or AAA at a given position was more effective than suppression of GAG or AAG.) By contrast, no context effects were observed with a glyT suppressor of AAA and AAG, a glyT GAA/G-suppressor, and a glyU suppressor of GAG . Our observation of this phenomenon in missense suppression demonstrates that codon context can affect polypeptide elongation and that the effects can be different depending on the codons and tRNAs examined . It is suggested that tRNA-tRNA interaction on the ribosome is involved in the observed context effects.

Eur J Biochem, 1984 May 2, 140(3), 553 - 6
Biosynthesis of the 09 antigen of Escherichia coli . Core structure of rfe mutant as indication of assembly mechanism; Weisgerber C et al.; The chemical structure of the outer (hexose) regions of the core oligosaccharide from Escherichia coli 09 with the complete R1 core, and from a R1-derived rfe mutant were analyzed using compositional analysis, methylation and gas chromatography/mass spectrometry . It was found that, in contrast to the branched outer region of the R1 core, the outer region of the core from the rfe mutant lacked terminal glucose and was linear . These results are in agreement with recent findings on the biosynthesis of the 09 antigen . They suggest a cotransfer of glucose with the 09-specific mannan to a 'pre-core' lacking terminal glucose, as the assembly (translocation) step in the 09 antigen synthesis . Thus it is suggested that the initiation of O-chain synthesis (by the formation of an acceptor glucolipid ) and the termination of core synthesis are closely correlated . In conjunction with previous biochemical data, the analytical results presented here indicate a novel core synthesis.

Acta Paediatr Scand, 1984 May, 73(3), 296 - 301
Daily ingestion of immunologic components in human milk during the first four months of life; Butte NF et al.; The amounts of lactoferrin, lysozyme, SIgA and SIgA antibodies to E . coli somatic antigens in human milk ingested per day and per kg per day by breast-fed infants were determined during the first four months of life . A gradual decline in the amounts of lactoferrin, SIgA, and SIgA antibodies ingested per day or per kg per day was found, whereas the quantities of lysozyme ingested by the infants rose during that period . These data suggest that the production and secretion of these immunologic factors by the mammary gland may be linked to the ontogeny of the production or catabolism of those components at mucosal tissues of the recipient infant.

Radiat Res, 1984 May, 98(2), 227 - 33
Action spectra for inactivation of dry phage T1 after monochromatic (150-254 nm) synchrotron irradiation in the presence and absence of photoreactivation and dark repair; Maezawa H et al.; Dry phage T1 was irradiated with monochromatic uv radiation (150-254 nm) in vacuo . The inactivation sensitivity was highest at 150 nm . On Escherichia coli Bs-1, the phage inactivation sensitivity was two and four times higher at 150 nm than at 254 and 230 nm, respectively . Action spectra for phage inactivation on both E . coli B and Bs-1 fit the absorption spectrum of phage DNA, except around 190 nm . The host-cell- reactivable fraction for vacuum-uv radiation (below 190 nm) was smaller than that with far-uv radiation . There was almost no photoreactivation at 150 nm, in contrast to a photoreactivation sector of about 0.3 at 254 nm.

Eur J Cell Biol, 1984 May, 34(1), 193 - 205
Nuclear bodies in mouse lymphoid cells stimulated by lipopolysaccharide; Radoux D et al.; C57 BL/6J mouse spleen lymphocytes have been stimulated by a polyclonal mitogene, the lipopolysaccharide of E . coli (LPS) . Depending on the LPS concentration, two pathways of B lymphocyte differentiation can be obtained . At low dose, the population is mainly composed of blast cells (85%) and at a high dose, the latter transforms into plasma cells (80%) . Four types of nuclear bodies have been distinguished and quantitatively studied at several stages of cell differentiation . Only the simple nuclear bodies type A, which could be related to the nuclear matrix, show quantitative modifications in small lymphocytes . Connections between granular nuclear bodies (type D) and nucleolar material have been observed . Some granular nuclear bodies exhibit a morphological zone similar to the nucleolar fibrillar centre as well as fibrillar and granular components . Autoradiographic studies indicate that the granular nuclear bodies contain RNA synthesized elsewhere in the nucleus and that this RNA subsequently migrates to the cytoplasm . Furthermore connections between granular nuclear bodies and chromatin have also been observed.

Appl Environ Microbiol, 1984 May, 47(5), 910 - 4
Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1; Mallison SM 3rd et al.; Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides . Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus . Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU . P . boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ) . One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium . Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-{2-( ethylsulfinyl )ethyl}O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ) . Two pesticide-resistant strains of P . boryanum were isolated against DCMU and Atrazine . These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals . The results indicate that P . boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.

Mutat Res, 1984 May, 140(1), 13 - 9
The effect of pretreatment of Escherichia coli CM891 with ethylenediaminetetraacetate on sensitivity to a variety of standard mutagens; Mitchell ID et al.; The effect of EDTA pretreatment on the sensitivity of E . coli CM891 to 10 standard mutagens was assayed for cytotoxicity, trp- reversion and mutation to A2Cr . There was no obvious correlation of effect with molecular weight but good correlation with water solubility . EDTA pretreatment did not improve the sensitivity of E . coli to agents which were very soluble in water viz . MMS, CP, ACR, DR and GM . However, with very water-insoluble agents, EDTA pretreatment usually gave a significant, 2-5-fold increase in mutation, viz . with 2NF , BP and CAP (trp locus) but not with 9AA . 2AP , which was moderately soluble in distilled water, only showed a small significant increase at the A2C locus after EDTA pretreatment . Increases in mutation were not always paralleled by increases in cytotoxicity.

J Bacteriol, 1984 May, 158(2), 460 - 3
Proline transport and metabolism in Rickettsia prowazekii; Winkler HH et al.; Purified Rickettsia prowazekii cells were able to transport L-proline . The influx of this amino acid had a Kt of 14 microM and a Vmax of about 64 pmol/min per mg of protein . Proline could not be transported by heat-killed or metabolically poisoned rickettsiae or at 0 degrees C . The uptake of proline was linear for almost 2 h . More than 90% of the accumulated intracellular radioactivity was proline . This intracellular pool could not be chased out of the cell by excess non-radioactive proline and did not exit into a proline-free medium . These results indicate that intracellular proline was bound or that the cell had a very limited efflux component for proline transport . The influx of proline was specific: among various analogs tested, only 3,4-dehydro-D,L-proline was effective in inhibiting proline uptake . R . prowazekii cells were unable to utilize proline as an energy source to drive hemolysis, and no measurable evolution from the rickettsiae of CO2 derived from proline occurred . The activities of the enzymes pyrroline-5-carboxylate-reductase and pyrroline-5-carboxylate dehydrogenase were not detectable . These enzymes are important in anabolism and catabolism of proline, respectively, and, if present in R . prowazekii have activities less than 1% of those in Escherichia coli.

Infect Immun, 1984 May, 44(2), 268 - 73
Properties of cross-linked toxoid vaccines made with hyperantigenic forms of synthetic Escherichia coli heat-stable toxin; Klipstein FA et al.; The ability of hyperantigenic preparations of synthetically produced Escherichia coli heat-stable toxin (ST) to provide an immunogenically more potent vaccine when cross-linked by the glutaraldehyde reaction to the heat-labile toxin B subunit was assessed . Three synthetic ST preparations were evaluated: ST(S) had the same antigenicity and toxicity (secretory potency in the suckling mouse assay) as native ST, ST 1056 had 3.5-fold more antigenicity and 1% toxicity, and ST(C) had 15-fold greater antigenicity and 31% toxicity . Vaccines that contained equal antigenic proportions of ST and B subunit, as determined by enzyme-linked immunosorbent assays, consisted by weight of 52% ST(S), 25% ST 1056, and 9% ST(C) . The initially lower toxicity and smaller proportions by weight of hyperantigenic ST preparations yielded vaccines that had nearly 10-fold less residual ST toxicity than the ST(S) vaccine . Immunization of rats with graded dosages of vaccines containing 9% ST(C) and 51% ST(S) by weight, but equal amounts of ST(S) antigenicity, raised to the same degree dose-dependent increases in mucosal immunoglobulin A antitoxin titers to ST(S) which correlated with the amount of protection against challenge with a viable LT-/ST+ strain . These observations indicate that hyperantigenic synthetic ST preparations provide immunologically more potent vaccines than those obtained with the previously used synthetic ST(S) preparation, which has the same biological properties as native ST.

Cancer Res, 1984 May, 44(5), 1867 - 70
Arrest of DNA elongation by DNA polymerases at guanine adducts on 4-hydroxyaminoquinoline 1-oxide-modified DNA template; Yoshida S et al.; In vitro modification of M13 phage single-stranded DNA with 4-hydroxyaminoquinoline 1-oxide (4HAQO) resulted in four kinds of adducts: three guanine adducts, QGI, QGII, and QGIII; and one adenine adduct, QA, at ratios of 16.4 47.3, 13.7, and 22.6, respectively . The carcinogen-modified DNA, initiated with a sequence-defined oligodeoxynucleotide primer, was replicated in vitro with Escherichia coli DNA polymerase I (Klenow fragment) and calf thymus DNA polymerases alpha and beta . The reaction products were analyzed on a DNA-sequencing gel . DNA elongation by DNA polymerase I was arrested at putative guanine adducts on the template in three ways: at one base prior to guanine; at positions opposite to guanine; and at one base beyond guanine . Similar patterns of elongation arrest were also obtained with the mammalian DNA polymerases alpha and beta . In contrast to guanine adducts, the adenine adduct, QA, might lack the capacity to arrest DNA chain elongation by DNA polymerases.

Bioorg Khim, 1984 May, 10(5), 641 - 7
{Physico-chemical properties of DNA-dependent RNA-polymerase from Escherichia coli and its subunits}; Deshko TN et al.; CD and UV spectroscopy were employed to study at different temperatures the conformational states of the DNA-dependent RNA polymerase core- and holo-enzymes, as well as of its alpha and beta subunits . Both core- and holo-enzyme were shown to have a higher percentage of regular structures than the separate subunits . CD and fluorescence methods were used to monitor the complex formation between rifamycin SV or its derivative, rifampicin, with the RNA polymerase from the E . coli wild and mutant (Rpo B255) types, the former enzyme being sensitive and the latter being resistant to these antibiotics . Complexation led to concomitant changes in the conformation of antibiotics and local structural rearrangements of the protein in vicinity of the binding site which comprises at least one tryptophan residue in a hydrophobic microenvironment.

Tsitologiia, 1984 May, 26(5), 520 - 4
{Changes in the orientation of the fracture plane of cell membranes during freeze-fracturing as an index of change in membrane structure}; Kirillov VA et al.; Using freeze-fracture method it is shown that the direction of the fracture plane is a sensitive indicator of the membrane structural organization . A decrease in the proportion of longitudinal fractures is noted upon the membrane rearrangement presumably associated with changes in the lipid phase, and, on the contrary, an increase in the proportion of these fractures is observed upon some actions resulting, presumably, in changes of the protein phase of membranes.

Mol Biol (Mosk), 1984 May-Jun, 18(3), 751 - 8
{Comparison of the physical properties of ribosomal proteins from Escherichia coli 50S subparticles isolated by different methods}; Tumanova LG et al.; Physical properties of ribosomal proteins obtained with or without denaturing agents were compared . CD measurements and PMR studies have shown that proteins L2, L19, L24 and L30 isolated under denaturing conditions have the same properties as those prepared avoiding denaturing agents . CD and PMR data of L1, L6, L11, L23, L25 and L29 obtained by us under denaturing conditions practically coincide with the data for these proteins obtained in "mild" conditions and published in the literature . These findings indicate that the differences of physical properties reported in the literature can be due to different procedures of protein renaturation rather than to the methods of their isolation.

J Gen Microbiol, 1984 May, 130 ( Pt 5), 1279 - 84
Pyruvate and ethanol as electron donors for nitrite reduction by Escherichia coli K12; Pope NR et al.; Pyruvate and ethanol were both effective electron donors for nitrite reduction by Escherichia coli K12 . The pyruvate-dependent rate decreased by approximately 50% when either a cysG mutation, which results in loss of NADH-dependent nitrite reductase activity (EC 1.6.6.4), or a chl mutation, which results in loss of the formate-nitrite oxidoreductase activity, was introduced into the prototrophic parental strain CGSC4315 . A double mutant deficient in both of these previously described activities retained only 2% of the rate of nitrite reduction of the parental strain after growth on glucose or 5% after growth on pyruvate . We conclude that any third pathway for nitrite reduction contributes little to the in vivo rate of nitrite reduction by wild-type strains.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1984 May, 257(1), 93 - 107
Phagocytosis of non-opsonized Escherichia coli by mouse peritoneal macrophages . An electron microscopic study; Rollag H et al.; Phagocytosis of non-opsonized Escherichia coli by mouse peritoneal macrophages (MPM) was studied by means of light microscopy, scanning electron microscopy and transmission electron microscopy . During a phagocytosis period of 90 min the surface morphology changed . Early in the phagocytosis period the MPM were polar with many ridges and villi, and little ruffling . At the end of the period the cells appeared well spread with a smooth surface and extensive ruffling . Two modes for ingestion of bacteria seemed to exist . The bacteria were ingested either by membrane folds rising from the macrophage surface, fitting tightly to the bacteria or by bacteria sinking into the cytoplasm of the MPM . Early in the period of phagocytosis most bacteria were attached to the surface . Ten per cent of the bacteria attached were never ingested . Bacteria ingested were located in phagolysosomes that were either of a tight or a loose type . After a phagocytosis period of 90 min the phagolysosomes contained bacteria at different stages of degradation . During the degradation the bacteria showed several morphological changes including a decrease in the density of the endoplasm, detachment of the bacterial membrane from the cell wall and deformities in the bacterial cell wall.

Pathol Res Pract, 1984 May, 178(5), 491 - 8
A study on renal papillary necrosis experimentally produced by the Shwartzman mechanism in rabbits; Nakano F et al.; To produce renal papillary necrosis experimentally by means of the Shwartzman mechanism in rabbits, E . coli endotoxin was injected into the renal pelvis unilaterally through the ureter as a preparative procedure after pretreatment by local administration of alcohol, and the same endotoxin was given again 24 hours later, but intravenously this time via the ear vein, as a provocation . Marked necrosis was produced in the renal papillae, where many intravascular fibrin thrombi were found histologically . Such papillary necrosis was largely prevented by heparin administration, and this lesion was considered to be the univisceral Shwartzman reaction occurring in the renal papillae . The lesion produced in the new experimental system of renal papillary necrosis described here has a good similarity to that of human cases in etiology, pathogenesis and morphology . The present system may therefore be a good model of human renal papillary necrosis, and should be useful for future studies.

Mikrobiologiia, 1984 May-Jun, 53(3), 495 - 9
{Introduction of a radioactive label into Escherichia coli cells}; Shevchenko VP et al.; A labeled cell culture was obtained by treating Escherichia coli cells with diluted tritium in the presence of rhodium catalysts and by incubating them with sodium {14C}acetate . Most of the cells retained their ability for division . The rate of cell growth declined with increasing the molar radioactivity of sodium acetate.

Mikrobiologiia, 1984 May-Jun, 53(3), 419 - 22
{Participation of the chemotactic system in regulating cell division in Escherichia coli}; Sherman MIu et al.; The possible role of the chemotaxis system in regulating cell division of Escherichia coli was studied . Attractants increased the rate of division whereas repellents reduced it . Non-metabolisable attractants analogues were also effective in stimulating cell division . Fucose, a non-metabolisable analogue of galactose, increased the rate of division by 20-25% . Co2+ at concentrations which had no effect on the tar-mutant division suppressed the division of the wild type . Likewise, indole at concentrations which did not influence the division of the tsr-mutant, suppressed the division of the wild type.

J Biochem (Tokyo), 1984 May, 95(5), 1315 - 21
Studies on the metabolism of unsaturated fatty acids . XIV . Purification and properties of NADPH-dependent trans-2-enoyl-CoA reductase of Escherichia coli K-12; Nishimaki T et al.; NADPH-dependent trans-2-enoyl-CoA reductase was purified to homogeneity from Escherichia coli . The molecular weight of the native enzyme was estimated to be 80,000 by gel filtration and that of the subunit was estimated to be 40,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis . The enzyme was most active toward trans-2-hexenoyl-CoA and trans-2-octenoyl-CoA but was less active toward longer chain substrates, whereas the Km values decreased progressively with increasing carbon chain length of substrates . The reductase appears to have a functional thiol group . The enzyme activity was rapidly decreased by p-hydroxymercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid), and slowly by N-ethylmaleimide . The enzyme was protected from inhibition by these SH-reagents by the addition of NADPH . The enzyme was also inhibited by saturated acyl-CoA esters.

Atherosclerosis, 1984 May-Jun, 51(2-3), 269 - 80
In vitro immune aggression against rabbit aortic smooth muscle cells; Scebat L et al.; Rabbit aortic smooth muscle cells cultivated with certain antisera underwent growth changes and necrosis . These cytotoxic antisera were obtained by immunizing rabbits against rat aorta, human or pig aortic glycoproteins, human serum glycoproteins and E . coli lipopolysaccharide . These different antigens share some biochemical characteristics, and contain four main amino acid residues (Glu, Ala, Asp, Gly) and four sugars (mannose, galactose, glucose, N-acetyl glucosamine) . The cytolytic properties of these antisera, however, probably correspond to structural analogies, since although ovalbumin is a glycoprotein, anti-ovalbumin antiserum was not cytotoxic . Antibody cytotoxicity against rabbit arterial smooth muscle cells may depend on the biochemical structure of the antigen used to produce antiserum.

Anal Biochem, 1984 May 1, 138(2), 291 - 7
Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities; Lee MY et al.; The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated . The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease . The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined . The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted . Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates . Magnesium was found to reinforce the binding of the enzyme to these affinity supports . DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns . These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.

Zh Mikrobiol Epidemiol Immunobiol, 1984 May, (5), 44 - 8
{Properties of Escherichia coli alpha-hemolysin}; Favorov VV et al.; SH-reagents (alpha-ethyl maleimide, n-chlor mercuribenzoate, dithioglycolic acid) and hydrogen peroxide induce insignificant changes in the activity of hemolysin . Reducing reagents (2-mercaptoethanol and ascorbate) inhibit hemolytic activity . Cholesterol at a concentration of 4.3 X 10(-5) M reduces this activity by 50% . Hemolysin has no phospholipase A activity . The energy necessary for activating the interaction of hemolysin with the membranes of erythrocytes (lag period) is 13800 cal/mol and for inducing the lysis of erythrocytes, 10 600 cal/mol . The above values are much less than those of O-labile hemolysins.

Zh Mikrobiol Epidemiol Immunobiol, 1984 May, (5), 40 - 4
{Determination of the viability of Escherichia coli M17 by its respiratory activity}; Shimchuk LF et al.; The possibility of the determination of the number of viable E . coli M17 in Colibacterin by the rate of oxygen consumption, based on measuring the slow fluorescence of these bacteria, has been studied . The direct correlation between the number of live E . coli and their respiratory activity in the standard sample of Colibacterin has been established . The method of measuring the oxygen consumption rate is recommended as an additional rapid method for the control of the content of live E . coli in the process of the manufacture and storage of the preparation.

Gene, 1984 May, 28(2), 201 - 9
The selection and characterisation of two novel mutations in the overlapping promoters of the Escherichia coli galactose operon; Busby S et al.; Mutations that result in small decreases or increases in expression from the Escherichia coli galactose operon promoter region can be detected by using a plasmid in which the gal promoters were fused to the lac operon . We describe how the level of lac expression was adjusted so that the Lac phenotype of host cells was optimally sensitive to changes in the gal promoter sequence . We have investigated the properties of two new gal promoter mutations both in vivo and in vitro, and have determined their effects on the two overlapping gal promoters, P1 and P2 . Although one mutation causes only a small reduction in overall expression in vivo, it completely suppresses transcription initiation at the P1 promoter . However, it also increases expression from the P2 promoter, which compensates for the change at P1 . This mutation, a GC to AT transition, falls in a zone just upstream of the P1 Pribnow box, which is essential for P1 activity, whilst improving the homology between the P2 Pribnow box and the consensus sequence . The second mutation causes a small increase in P1 activity . This change, a GC to AT transition at -23, falls in the spacer region between the Pribnow box and the -35 region, a zone containing no known promoter consensus sequences . We suggest that this mutation, which creates a stretch of five AT base pairs, acts by increasing the twist angle of the sequences in the spacer region . We argue that the increase in promoter activity is due to this twist changing the relative orientation of the Pribnow box and -35 regions.

Gene, 1984 May, 28(2), 147 - 52
Cloning of a gene required for tryptophan biosynthesis from Leptospira biflexa serovar patoc into Escherichia coli; Yelton DB et al.; A clone bank, consisting of approx . 8100 colonies, has been created for the spirochete Leptospira biflexa serovar patoc in Escherichia coli using pBR322 as the vector . One of these clones contains the genetic information needed to complement a defect in the trpE gene of E . coli . The information resides on a 20.5-kb plasmid designated pYC1, which carries a 16-kb insert consisting of three HindIII fragments . It does not complement defects in other genes needed for the biosynthesis of tryptophan in E . coli.

Genetika, 1984 May, 20(5), 756 - 9
{Escherichia coli K-12 mutants with enhanced resistance to ionizing radiation . IV . Recombination characteristics of Gamr mutants}; Bresler SE et al.; In the radiation-resistant Gamr444 mutant the inheritance frequency of long F' episomes ORF1 (purE+ tsx+ procC+ lac+) and F'14 (ilv+--argE+) is lower, and the frequencies of chromosome mobilization and integrative suppression of temperature-sensitive dnaA46 mutation by the sex factor F are much higher than those in the wild-type strain AB1157 and another radiation-resistant mutant Gamr445 . In this respect, the mutant Gamr444 is very similar to the recRC sbcB mutant (RecF-pathway of recombination).

Genetika, 1984 May, 20(5), 746 - 55
{Escherichia coli K-12 mutants with enhanced resistance to ionizing radiation . III . The effect of rec and lexA mutations on radioresistance}; Bresler SE et al.; Lethal action of gamma-rays on derivatives of the wild-type strain AB1157 and of two radiation-resistant mutants (Gamr444 and Gamr445) containing additional mutations dnaA46, recB21, recF143, recA56, recA430, lexA3, lexA102 or lexA3 recAo98, was studied . When the mean number of genomes per cell was reduced by means of pre-incubation at 43 degrees C, radioresistance of the strains AB1157 dnaA46 and Gamr445 dnaA46 was not changed, and that of the strain Gamr444 dnaA46 was reduced to the level of the Gamr445 dnaA46 strain . Introduction of additional mutations recB21, recA56 or lexA3 (lexA102) into the genome of the strains Gamr444 or Gamr445 made them as radiosensitive as the corresponding variants of AB1157 . Additional mutations recF143 or recA430 (lexB30) significantly decreased the radioresistance of Gamr444 and Gamr445 mutants, although did not level them to corresponding derivatives of AB1157 . Operator-constitutive mutation recAo98 enhanced radioresistance of all lexA3 derivatives tested but not to the level of the corresponding lexA+ strains . The role of recombinational repair and the inducible SOS system in enhanced radioresistance of Gamr mutants is discussed . The data of post-irradiation DNA degradation in various derivatives of the strains AB1157 and Gamr suggest that Gamr mutants have a constitutive inhibitor of degradation which does coincide with RecA protein.

Br J Pharmacol, 1984 May, 82(1), 289 - 94
Failure of drugs that selectively inhibit thromboxane synthesis to modify endotoxin shock in conscious rats; Furman BL et al.; The effects of two thromboxane synthetase inhibitors ( dazoxiben and UK 38485) were investigated on the cardiovascular and metabolic effects of Escherichia coli endotoxin infusion in the conscious, unrestrained rat . Infusion of E . coli endotoxin (41.7 ng kg-1 min-1) for 4 h produced a fall in mean arterial pressure, an increase in heart rate, a transient hyperglycaemia (at 1 h) followed by hypoglycaemia (evident at 6 h), an elevation in plasma lactate and a profound thrombocytopenia . The above changes were accompanied by a marked elevation in plasma thromboxane B2 concentrations (e.g . endotoxin-treated 935 +/- 150 pg ml-1 at 1 h compared with pre-endotoxin values of 125 +/- 30 pg ml-1) . The administration of either dazoxiben (30 mg kg-1 i.v., given 30 min before starting the endotoxin infusion) or UK 38485 (15 mg kg-1 given 30 min before, and again 4 h after, starting the endotoxin infusion) prevented the rise in plasma thromboxane B2 concentrations . Neither dazoxiben nor UK 38485 prevented the metabolic, cardiovascular or thrombocytopenic effects of endotoxin and did not modify mortality . These results suggest that, although large amounts of thromboxane are generated in response to endotoxin, they do not play an important role in the major pathophysiological consequences of acute endotoxaemia.

Am J Vet Res, 1984 May, 45(5), 963 - 6
Combined effects of deoxycorticosterone and furaltadone on Escherichia coli infection in chickens; Gross WB; After low antibody-response line chickens were challenge exposed with Escherichia coli, 10% of the controls, 35% of those fed furaltadone , 37% of those fed deoxycorticosterone (DOC), and 92% of those fed furaltadone and DOC gained weight . Concentrations of DOC above or below the optimal concentration (40 mg/kg of feed) were less effective, eg, when 100 mg of DOC/kg of feed was fed with furaltadone , 45% of low antibody-response line birds gained weight as compared with 85% of the birds when furaltadone was fed alone . However, the optimal amount of DOC did vary among genetic stocks and with environment . Stocks of birds also differed in their response to the concentration of furaltadone and DOC . Socialization increased the effectiveness of furaltadone .

Virology, 1984 May, 135(1), 200 - 6
Formation of phage T1 concatemers by the RecE recombination pathway of Escherichia coli; Pugh JC et al.; Infections of nonpermissive ( sup0 ) Escherichia coli by T1 phage with amber mutations in either gene 3.5 or gene 4 exhibit a variety of defective phenotypes, including premature arrest of T1 DNA synthesis, failure to make concatemeric DNA, formation of an abnormal DNA replication intermediate, failure to package phage DNA, and reduced genetic recombination . The lethal effect of gene 3.5 or 4 mutations is suppressed when the sup0 bacteria express the RecE recombination pathway . This RecE suppression occurs by partial restoration of the capacity to make concatemeric molecules and partial reversal of the DNA arrest defect which, in turn, leads to the formation of viable progeny . Infection by T1+ or by mutants defective in any of the four DNA synthesis genes (genes 1, 2, 3.5, and 4) inhibited the ATP-dependent exonuclease present in uninfected cells (presumably the RecBC enzyme, exonuclease V) . Extracts from T1+ infections also showed increased levels of an ATP-independent exonuclease activity which was absent from gene 4 mutant extracts . It is concluded that gene 4, together with gene 3.5, specifies an activity related to that of the RecE exonuclease VIII and essential for T1 concatemer formation and recombination.

Proc Natl Acad Sci U S A, 1984 May, 81(10), 3019 - 23
Internal homologies in the two aspartokinase-homoserine dehydrogenases of Escherichia coli K-12; Ferrara P et al.; In Escherichia coli, AK I- HDH I and AK II- HDH II are two bifunctional proteins, derived from a common ancestor, that catalyze the first and third reactions of the common pathway leading to threonine and methionine . An extensive amino acid sequence comparison of both molecules reveals two main features on each of them: (i) two segments, each of about 130 amino acids, covering the first one-third of the polypeptide chain, are similar to each other and (ii) two segments, each of about 250 amino acids and covering the COOH-terminal 500 amino acids also present a significant homology . These findings suggest that these two regions may have evolved independently of each other by a process of gene duplication and fusion previous to the appearance of an ancestral aspartokinase-homoserine dehydrogenase molecule.

J Surg Res, 1984 May, 36(5), 516 - 25
Effects of nonsteroidal anti-inflammatory drugs on renal function in septic dogs; Fink MP et al.; The effects of nonsteroidal anti-inflammatory drugs on renal function were studied in 15 chronically instrumented, unanesthetized beagles . In 9 dogs, bacterial peritonitis was induced by implanting in the peritoneal cavity a fibrin clot containing viable Escherichia coli . Six (control) dogs were subjected to laparotomy but were not implanted with an infected clot . Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were estimated using standard clearance methods . All measurements were performed after resuscitation with Ringer's lactate to a pulmonary capillary wedge pressure of 6 Torr . When measured 24 hr after laparotomy, there were no significant changes (relative to baseline) in GFR or ERPF in either the septic or control groups . In septic dogs, 60 min after the administration of either indomethacin (2 mg/kg) or ibuprofen (25 mg/kg), GFR decreased an average of 26 +/- 11 ml/min (P = 0.043) and ERPF decreased an average of 100 +/- 27 ml/min (P = 0.02) . In controls, administration of indomethacin (2 mg/kg) did not significantly affect either GFR or ERPF . These results suggest that renal function should be carefully monitored in clinical trials of nonsteroidal anti-inflammatory drugs in septic patients.

J Surg Res, 1984 May, 36(5), 420 - 7
Complement and endotoxin-induced lung injury in sheep; Horn JK et al.; Intravenous infusions of endotoxin in sheep cause lung injury characterized by edema due to increased microvascular permeability . Similar increases in pulmonary microvascular permeability are seen in septic patients with the adult respiratory distress syndrome . Since endotoxin-induced lung injury may be mediated by interactions between products of complement activation and polymorphonuclear leukocytes, plasma and lung lymph from six unanesthetized sheep infused with Escherichia coli endotoxin (1.0 micrograms/kg over 30 min) were examined for complement-derived chemotactic activity . By 2-3 hr following infusion of endotoxin, all animals had the increased lung lymph fluid and protein flows characteristic of permeability edema . Preinfusion samples of plasma and lung lymph did not contain chemotactic activity for polymorphonuclear leukocytes . Following infusion of endotoxin, however, significant chemotactic activity was detected in plasma at 0.5-3.5 hr (P less than 0.05) and in lymph at 1.5-6.5 hr (P less than 0.025) . The chemotactic activity was heat stable (56 degrees C for 30 min) but was abolished by treatment with antibodies to C5 . These data indicate that infusions of endotoxin lead to the generation in plasma, and the appearance in lung lymph, of C5-derived peptides with chemotactic activity for polymorphonuclear leukocytes . C5-derived peptides may account for the pulmonary microvascular leukostasis and endothelial injury that lead to increased permeability edema after infusions of endotoxin.

J Bacteriol, 1984 May, 158(2), 762 - 3
Genetic mapping of pheU, an Escherichia coli gene for phenylalanine tRNA; Gallagher PJ et al.; We report the genetic mapping of pheU , an Escherichia coli gene for phenylalanine tRNA . This gene was located near 94.5 min on the E . coli map . There are no other known tRNA or ribosomal genes in its immediate vicinity.

J Bacteriol, 1984 May, 158(2), 760 - 1
Transformability of galE variants derived from uropathogenic Escherichia coli strains; van Die IM et al.; Transformation of uropathogenic Escherichia coli strains with plasmid DNA was in general unsuccessful or very inefficient . Transformation was much more efficient when galE mutants of such strains, in which the lipopolysaccharide chains appeared shorter, were used as recipients.

J Bacteriol, 1984 May, 158(2), 757 - 9
Colicin V-treated Escherichia coli does not generate membrane potential; Yang CC et al.; Colicin V-treated Escherichia coli was inhibited in its capacity to carry out active transport of proline and was unable to generate a membrane potential . Colicin V also prevented membrane potential formation by isolated cytoplasmic membrane vesicles . We conclude that a primary effect of this colicin involves the cytoplasmic membrane as a target.

J Bacteriol, 1984 May, 158(2), 749 - 53
Long repair replication patches are produced by the short-patch pathway in a uvrD252 (recL152) mutant of Escherichia coli K-12; Rothman RH et al.; The uvrD252 mutation leads to increased UV sensitivity, diminished dimer excision and host cell reactivation capacity, and an increase in the average patch size after repair replication . A recA56 uvrD252 double mutant was far more resistant to UV than was a recA56 uvrB5 double mutant . Its host cell reactivation capacity was identical to that of uvrD252 single mutant and was far greater than that of the uvrB5 single mutant . The strain showed no Weigle reactivation . From these results, we concluded that the double mutant has no inducible DNA repair (including long-patch excision repair) but retains dimer excision capabilities comparable to the uvrD252 single mutant . It appears, therefore, that the long patches detected in the uvrD mutant were not identical to the recA-dependent patches seen in wild-type cells.

J Bacteriol, 1984 May, 158(2), 737 - 8
Expression of a Thiobacillus ferrooxidans origin of replication in Escherichia coli; Rawlings DE et al.; A cryptic plasmid from an autotrophically grown arsenic-resistant strain of Thiobacillus ferrooxidans was isolated and cloned into pBR325 . The origin of replication of pBR325 was deleted, and the recombinant plasmid was shown to replicate in Escherichia coli, using an origin of replication located on the Thiobacillus plasmid.

J Bacteriol, 1984 May, 158(2), 727 - 9
recF-dependent and recF recB-independent DNA gap-filling repair processes transfer dimer-containing parental strands to daughter strands in Escherichia coli K-12 uvrB; Wang TV et al.; The processes for repairing DNA daughter-strand gaps were studied in UV-irradiated uvrB, uvrB recB, uvrB recF, and uvrB recB recF cells of Escherichia coli K-12 . The dimer-containing parental DNA was found to be joined to daughter strands during postreplication repair in all four strains examined . Therefore, both the major (recF-dependent) and the minor (recF recB-independent) gap-filling processes repair DNA daughter-strand gaps by transferring parental strands into daughter strands.

J Bacteriol, 1984 May, 158(2), 632 - 5
Mapping of the lipoprotein signal peptidase gene (lsp); Regue M et al.; A pBR322 plasmid which contains a fragment of Escherichia coli DNA encoding the lipoprotein signal peptidase gene was used to transform Hfr polA1 strains . Ampr transformants were used as donors in conjugation experiments, and the location of the plasmid amp gene adjacent to the chromosomal lsp gene was determined to be near the thr ara loci of the E . coli chromosome . P1 transduction experiments established that the location of the lsp gene is closely linked to that of dapB , at 0.5 to 0.6 min on the E . coli genetic map . The position of the lsp gene was further determined to be between ileS and dapB by complementation analysis of an E . coli mutant showing temperature-sensitive prolipoprotein signal peptidase activity.

J Bacteriol, 1984 May, 158(2), 615 - 20
Escherichia coli K-12 lysyl-tRNA synthetase mutant with a novel reversion pattern; Hirshfield IN et al.; Fast-growing revertants have been selected from a slow-growing lysyl-tRNA synthetase mutant . All of the revertants had increased lysyl-tRNA synthetase activity compared with the mutant (5- to 85-fold), and in some revertants this amounted to two to three times the wild-type synthetase activity . Two-dimensional gel electrophoresis of a whole-cell extract of revertant IH2018 (1.5- to 2-fold wild-type synthetase activity) showed that the increase in synthetase activity is due to the induction of cryptic lysyl-tRNA synthetase forms and not to a change in the constitutive lysyl-tRNA synthetase . Genetic studies have shown that a locus termed rlu (for regulation of lysU ) which is cotransducible with purF at 49.5 min influences the amount of the cryptic lysyl-tRNA synthetase.

J Bacteriol, 1984 May, 158(2), 590 - 6
Overproduction of fumarate reductase in Escherichia coli induces a novel intracellular lipid-protein organelle; Weiner JH et al.; The expression of fumarate reductase in Escherichia coli has been amplified over 30-fold by utilizing a recombinant plasmid, pFR63 , carrying the fumarate reductase operon . More than 50% of the inner-membrane protein could be accounted for by the enzyme, whereas the total amount of protein associated with the membrane fraction doubled . The membrane accommodated this excess fumarate reductase without reducing the levels of other membrane-associated enzymes . At the same time, the amount of membrane lipid increased such that the lipid/protein ratio remained constant, indicating that the total amount of membrane had doubled . Small alterations in fatty acid composition as well as a large increase in cardiolipin were detected in the fumarate reductase-enriched membranes . The excess membrane was localized in novel tubular structures which were observed in thin-section and negatively stained electron-microscopic preparations . The tubules only appeared after the cytoplasmic membrane became highly enriched in fumarate reductase . They branched from the cytoplasmic membrane and were fumarate reductase . They branched from the cytoplasmic membrane and were composed of an aggregate of fumarate reductase and lipid.

Carcinogenesis, 1984 May, 5(5), 691 - 3
Differences in the promutagenic nature of 3-methylcytosine as revealed by DNA and RNA polymerising enzymes; Saffhill R; Poly(dC,3- MedC ) has been synthesised and used as a template to compare the miscoding properties of 3-methylcytosine (3-MeC) during DNA and RNA synthesis . Although 3-MeC was promutagenic with the RNA polymerase incorporating both AMP and UMP in the ratio of approximately 5:1 (agreeing with results reported by earlier workers) no non-complementary nucleotide incorporation was observed with DNA polymerase I . The results show that 3-MeC, which is a strong inhibitor of DNA synthesis, is only promutagenic with the less accurate RNA polymerase and that the reported differences in promutagenicity for this modified base with the two nucleotide polymerising enzymes arise from different specificities for the two enzymes.

AJR Am J Roentgenol, 1984 May, 142(5), 941 - 6
The variable CT appearance of hepatic abscesses; Halvorsen RA et al.; Fifty computed tomographic (CT) scans in 33 patients with 37 separate episodes of hepatic abscess were reviewed retrospectively . Abnormalities were detected in all but one case (97% sensitivity) . However, a characteristic CT appearance of hepatic abscess was not evident . Most intrahepatic foci were solitary, but multiple abscesses were present in 34% of cases . The CT appearance of the lesions varied from well defined, rounded cavities with contents near water density, resembling poorly defined hepatic cysts, to higher-density foci indistinguishable from hepatic neoplasms . In only 19% was gas present within the abscess . Rim enhancement was seen in only 6% . This study suggests that CT is a sensitive test for detecting hepatic abscess but is often nonspecific.

Proc Natl Acad Sci U S A, 1984 May, 81(9), 2831 - 5
Stimulation of recombination between homologous sequences on plasmid DNA and chromosomal DNA in Escherichia coli by N-acetoxy-2-acetylaminofluorene; Luisi-DeLuca C et al.; A plasmid containing a wild-type lac operon and a tetracycline-resistance gene was covalently modified by N-acetoxy-2-acetylaminofluorene and used to transform two series of Lac- Escherichia coli cell types . Each set contained wild-type and repair-deficient mutants . One set of cells contained a lacY mutation and the other a deletion of the entire lac operon . Survival and mutagenesis of the plasmid were measured as a function of the N-acetoxy-2-acetylaminofluorene concentration . The results indicate that when no homologous sequences are present in the chromosomal DNA, mutations occur at a low frequency: at 10% survival the frequency was 1-2 X 10(-4) mutants per transformant . When homologous sequences, the lacY allele, are present in the chromosomal DNA, Lac- plasmids are found at a high frequency in a recA-dependent, lexA-independent fashion: at 10% survival the frequency was 5-10 X 10(-2) mutants per transformant . Southern blot analysis of the restriction enzyme profiles of the resulting plasmid and host-cell DNA sequences showed recombinational transfer of host sequences to the N-acetoxy-2-acetylamino-fluorene-treated plasmid had occurred . When the host chromosomes contained Lac+ homologous sequences no mutants were found, indicating that the results were not caused by error-prone recombination.

J Dermatol Surg Oncol, 1984 May, 10(5), 380 - 1
Meshed skin grafts versus sheet skin grafts on a contaminated bed; Nappi JF et al.; An experimental model was designed to test the premise that meshed skin grafts survive better than sheet grafts on contaminated wounds . In this rat model, meshing of grafts significantly improved graft take, and expansion of the mesh led to an even greater improvement . The results of this study indicate a need for further investigation of this technique in the management of contaminated wounds.

Infect Immun, 1984 May, 44(2), 514 - 8
In vitro adhesion of enterotoxigenic Escherichia coli to human intestinal epithelial cells from mucosal biopsies; Knutton S et al.; An adhesion assay with isolated human enterocytes prepared from duodenal biopsies has been developed and tested by using human enterotoxigenic Escherichia coli expressing colonization factor antigens I and II (CFA/I and CFA/II) and type 1 fimbriae . Enterotoxigenic E . coli strains H10407 (CFA/I) and B2C (CFA/II) bound to duodenal enterocytes to a much greater extent (mean of 4.6 and 4.0 bacteria per brush border) than did strain H10407P, a CFA/I- mutant of H10407 (mean of 0.1 bacteria per brush border) . Type 1 fimbriae also promoted adhesion of strain H10407P to duodenal enterocytes but attachment was to basolateral rather than brush border surfaces . CFA/I and CFA/II, on the other hand, promoted adhesion only to human enterocyte brush borders.

Infect Immun, 1984 May, 44(2), 409 - 20
Coli surface antigens 1 and 3 of colonization factor antigen II-positive enterotoxigenic Escherichia coli: morphology, purification, and immune responses in humans; Levine MM et al.; Enterotoxigenic Escherichia coli (ETEC) of serotype O6:H16, biotype A, bearing colonization factor antigen II (CFA/II) possesses two distinct coli surface antigens, CS1 and CS3, whereas CFA/II-positive ETEC of serotype O8:H9 manifests only CS3 . CS1 has been shown to be fimbrial in nature, but heretofore the morphology of CS3 has not been described . Accordingly, by immune electron microscopy we investigated the morphological characteristics of CS3 on bacterial cells and after purification . CS3 was found to consist of thin (2-nm), flexible, wiry, "fibrillar" fimbriae, visible both on bacteria (O6:H16, biotype A, and O8:H9 strains) and in the pure state . In contrast, CS1 exists as wider (6-nm), rigid fimbriae on the surface of O6:H16, biotype A, strains . By the use of antisera to CS1 and CS3 in immune electron microscopy, immunodiffusion in gel, and immunoblotting techniques, CS1 and CS3 were found to be immunologically as well as morphologically distinct . Six of nine volunteers who developed diarrhea after challenge with an O139:H28 ETEC strain bearing CS1 and CS3 had significant serological rises to purified CS1 and CS3 antigens, suggesting that both antigens are elaborated in vivo, play a role in pathogenesis, and stimulate an immune response.

Arch Biochem Biophys, 1984 May 1, 230(2), 430 - 9
The tryptophan synthase alpha 2 beta 2 complex: a comparison of the reactivity of amino groups in the alpha and beta 2 subunits and in the complex by differential labeling studies; Miles EW et al.; The interaction of the alpha and beta 2 subunits of tryptophan synthase of Escherichia coli to form an alpha 2 beta 2 complex has been probed by differential labeling studies . In the first step the separate alpha or beta 2 subunit or the alpha 2 beta 2 complex was labeled by reductive methylation with trace amounts of {3H}HCHO in the presence of NaCNBH3 . In the second step the 3H-labeled preparation was fully labeled under denaturing conditions with {14C}HCHO and NaCNBH3 . Peptides containing labeled monomethyl or dimethyl amino groups were isolated after thermolytic digestion or after cyanogen bromide treatment . The 3H/14C ratio of each peptide is a measure of the relative reactivity of the amino group or groups in each peptide . The most reactive amino group in the alpha subunit, lysine-109, is strongly shielded from modification in the alpha 2 beta 2 complex . The most reactive amino group in the beta 2 subunit, the amino-terminal threonine, is not shielded from modification in the alpha 2 beta 2 complex.

Surgery, 1984 May, 95(5), 553 - 61
A conscious septic dog model with hemodynamic and metabolic responses similar to responses of humans; Shaw JH et al.; We have developed a conscious septic dog model suitable for in vivo tracer studies . Dogs weighing 10 to 20 kg underwent general anesthesia followed by the insertion of long-term arterial, venous, and portal cannulas and the formation of a long-term tracheostomy . After 7 to 10 days of convalescence, the animals were fed in the morning and 4 hours later 10(10) live Escherichia coli organisms were infused intra-arterially over approximately 30 minutes . One hour later a second dose of 5 X 10(9) bacteria was given, again over 30 minutes . Resuscitation was provided by infusion of 1000 ml of lactated Ringer solution over 3 hours . Twenty-four hours after the induction of sepsis the animals were hemodynamically stable and suitable for study . Cardiac output was increased from the control value of 185 +/- 35 ml/kg X min to 308 +/- 44 ml/kg X min in the septic animals . Heart rate was increased from 98 +/- 10 to 125 +/- 5 beats/min, and arterial pressure was not significantly altered . We employed indirect calorimetry and primed constant infusions of both radioactive and stable isotopes to assess a variety of metabolic parameters . The metabolic rate was increased approximately 25%, and the energy for this increase was primarily provided by the increased oxidation of both free fatty acids and triglyceride . The release of free fatty acids was approximately three times greater than the control value, and triglyceride synthesis increased 500% . The oxidation rate of free fatty acids and the fatty acids contained in very low density lipoproteins-triglyceride increased 40% and 900%, respectively . Glucose production was maintained at approximately the control value, and the rate of glucose oxidation (as measured with 14C-glucose) was also not significantly altered . The plasma insulin concentration was moderately elevated, and plasma glucagon concentration was five to six times greater than the control value . Plasma catecholamine levels were increased significantly . This model is suitable for the performance of metabolic studies in sepsis . The induction of a hyperdynamic septic state in less than 24 hours avoids the complications of starvation and dehydration frequently seen in the various peritonitis and abscess models . Most importantly, the model is predictable in its time course and reproducibly creates a situation that hormonally, hemodynamically, and metabolically resembles what is commonly seen in humans with sepsis.

Surg Gynecol Obstet, 1984 May, 158(5), 427 - 30
Subphrenic abscess; van der Sluis RF; A series of 55 patients with subphrenic abscess is presented . In 50 patients, the abscess occurred postoperatively, most frequently after biliary tract and gastric operations . The surgical approach to drainage was selected on the basis of clinical signs and symptoms and previous operation . Among 34 patients in whom the abscesses were drained extraserously initially, nine subsequently underwent a transperitoneal approach because of inadequate drainage . Of all patients, 17 (30.9 per cent) had multiple space abscesses . These figures support the use of midline exploration . Twenty-six patients (47.3 per cent) had complications develop after surgical drainage; ten (18.2 per cent) of them died . The value of early diagnosis is stressed.

Rev Infect Dis, 1984 May-Jun, 6 Suppl 2, S487 - 93
Nucleotide sequence from neurovirulent and attenuated strains of type 3 poliovirus; Almond JW et al.; As part of an investigation into the molecular basis of attenuation in the Sabin poliovirus vaccines, the genomes of three strains of type 3 poliovirus differing in neurovirulence were cloned in Escherichia coli . The nucleotide sequence of the region of the genome encoding the virion protein 1 (VP1) polypeptide from each of these strains is presented . Very few changes were observed, a finding suggesting that the mutation(s) involved in the attenuation of type 3 poliovirus probably lies elsewhere in the genome . The complete nucleotide sequence of one of the strains, P3/Sabin, has been obtained and is compared with the published sequences of type 1 poliovirus . Between serotypes 1 and 3, there is 77% homology in nucleotide sequence and 90% homology in the amino acid sequence predicted for viral proteins.

Gene, 1984 May, 28(2), 133 - 46
Analysis of the cya locus of Escherichia coli; Koop AH et al.; A 9500-bp DNA segment containing the adenylate cyclase gene (cya) of Escherichia coli has been isolated and analyzed . Four large proteins are encoded within this fragment - the adenylate cyclase protein (92 kDal), two proteins of unknown function (37 and 32 kDal), and a part of the uvrD-coded protein . Various truncated adenylate cyclase proteins, made from cya genes having as much as 60% of their carboxy-terminal end deleted, are sufficient to complement cya- hosts . When these truncated cya genes are present on a multicopy plasmid in a cya- host, the synthesis of beta-galactosidase is still regulated by glucose . The "maxicell" technique was used to visualize the four proteins encoded by this region and some of the truncated adenylate cyclase proteins.

EMBO J, 1984 May, 3(5), 1187 - 92
The supercoil-stabilised cruciform of ColE1 is hyper-reactive to osmium tetroxide; Lilley DM et al.; Supercoiled pColIR215 contains a site of pronounced hyper-reactivity towards modification by osmium tetroxide, a reagent known to be single-strand-selective . The site of hypersensitivity has been mapped to the ColE1 inverted repeat, believed to extrude a cruciform in supercoiled DNA . Linear or relaxed plasmids are not modified by the reagent . We conclude that cruciform formation is responsible for the site-selective modification . Fine mapping of the modification site as a function of time has revealed that the initial reaction occurs at the centre of the inverted repeat, i.e., the unpaired loop of the cruciform, but that the modification region rapidly expands outwards from this point.

EMBO J, 1984 May, 3(5), 1175 - 80
The F plasmid origin of transfer: DNA sequence of wild-type and mutant origins and location of origin-specific nicks; Thompson R et al.; The DNA sequence of the F plasmid origin of conjugal DNA transfer, oriT , has been determined . The origin lies in an intercistronic region which contains several inverted repeat sequences and a long AT-rich tract . Introduction of a nick into one of the DNA strands in the oriT region precedes the initiation of conjugal DNA replication, and the position of the strand-specific nicks acquired by a lambda oriT genome upon propagation in Flac-carrying cells has been determined . The nicks were not uniquely positioned, rather there was a cluster of three major and up to 20 minor sites: the biological significance of this observation is not yet fully clear . Nine independent point mutations which inactivate oriT function have been sequenced and found to alter one or other of two nucleotide positions which lie 14 and 19 bp to one side of the rightmost (as drawn) major nick site . These key nucleotides may lie in a recognition sequence for the oriT endonuclease, since mutations at these sites prevent nicking at oriT .

EMBO J, 1984 May, 3(5), 1115 - 9
The identification and high level expression of a protein encoded by the yeast Ty element; Dobson MJ et al.; Transcription of the yeast Ty element, Ty1 -15, was placed under the control of the efficient PGK promoter on the yeast expression vector, pMA91 . In extracts of yeast transformants containing these constructions a new 52 K basic polypeptide was detected by one- and two-dimensional electrophoresis and was shown, by hybrid arrested translation, to be specifically encoded by the Ty element . The protein coding region was mapped to the first 1.45 kb of the transcribed region of Ty1 -15 . These data show for the first time that Ty elements encode proteins and illustrate the general usefulness of high efficiency expression vectors for the detection of rare products.

Proc Natl Acad Sci U S A, 1984 May, 81(10), 2955 - 9
Properties of a human immunoglobulin epsilon-chain fragment synthesized in Escherichia coli; Kenten J et al.; A fragment of the cloned gene for the human myeloma ND epsilon chain, coding for the second, third, and fourth domains of the immunoglobulin, has been coupled to the tryptophan control region of an expression plasmid and subcloned in Escherichia coli . Induction of gene expression results in the synthesis of the expected, antigenically active polypeptide of Mr 40,000, which constitutes 18% of total bacterial protein and yields 55 mg/liter of culture . The immunoglobulin, which is aggregated and packed into large inclusion bodies within the bacterial cell, can be dissolved by denaturing solvents and purified by affinity chromatography using anti-IgE Sepharose . Reduced monomeric chains assemble spontaneously into dimers . On assay to measure the inhibition of binding of 125I-labeled human E myeloma protein to Fc epsilon receptors on cultured human basophils, the cloned gene product exhibited 20% of the activity of the native protein.

J Bacteriol, 1984 May, 158(2), 674 - 82
Molecular cloning and characterization of genes required for ribose transport and utilization in Escherichia coli K-12; Iida A et al.; We isolated spontaneous and transposon insertion mutants of Escherichia coli K-12 that were specifically defective in utilization or in high-affinity transport of D-ribose (or in both) . Cotransduction studies located all of the mutations near ilv, at the same position as previously identified mutations causing defects in ribokinase ( rbsK ) or ribose transport ( rbsP ) . Plasmids that complemented the rbs mutations were isolated from the collection of ColE1 hybrid plasmids constructed by Clarke and Carbon . Analysis of those plasmids as well as of fragments cloned into pBR322 and pACYC184 allowed definition of the rbs region . Products of rbs genes were identified by examination of the proteins produced in minicells containing various rbs plasmids . We identified four rbs genes: rbsB , which codes for the 29-kilodalton ribose-binding protein; rbsK , which codes for the 34-kilodalton ribokinase ; rbsA , which codes for a 50-kilodalton protein required for high-affinity transport; and rbsC , which codes for a 27-kilodalton protein likely to be a transport system component . Our studies showed that these genes are transcribed from a common promoter in the order rbsA rbsC rbsB rbsK . It appears that the high-affinity transport system for ribose consists of the three components, ribose-binding protein, the 50-kilodalton RbsA protein, and the 27-kilodalton RbsC protein, although a fourth, unidentified component could exist . Mutants defective in this transport system, but normal for ribokinase , are able to grow normally on high concentrations of the sugar, indicating that there is at least a second, low-affinity transport system for ribose in E . coli K-12.

J Bacteriol, 1984 May, 158(2), 665 - 73
D-ribose metabolism in Escherichia coli K-12: genetics, regulation, and transport; Lopilato JE et al.; We have isolated mutants defective in high-affinity D-ribose transport . The mutations map in rbsT or rbsB , the structural gene for ribose binding protein . rbsT consists of at least one gene coding for a protein required for high-affinity transport . The high-affinity transport-defective mutants were able to utilize D-ribose, indicating that at least a second, low-affinity transport system for D-ribose is present in Escherichia coli K-12 . rbsT and rbsB are located at min 84 on the E . coli genetic map and, together with rbsK , the gene coding for ribokinase , constitute an rbs operon . The order of genes is rbsP /O rbsT rbsB rbsK . The rbs operon is subject to negative control by the product of the rbsR gene . rbsR is located distal to the rbs operon and appears to form a separate transcriptional unit.

J Bacteriol, 1984 May, 158(2), 551 - 61
Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death; Schoemaker JM et al.; Mutations in sulA (sfiA) block the filamentation and death of capR (lon) mutants that occur after treatments that either damage DNA or inhibit DNA replication and thereby induce the SOS response . Previous sulA-lacZ gene fusion studies showed that sulA is transcriptionally regulated by the SOS response system (lexA/recA) . SulA protein has been hypothesized to be additionally regulated proteolytically through the capR (lon) protease, i.e., in lon mutants lacking a functional ATP-dependent protease there would be more SulA protein . A hypothesized function for SulA protein is an inhibitor of cell septation . To investigate aspects of this model, we attempted to construct lon, lon sulA, and lon sulB strains containing multicopy plasmids specifying the sulA+ gene . Multicopy sulA+ plasmids could not be established in lon strains because more SulA protein accumulates than in a lon+ strain . When the sulA gene was mutated by a mini Mu transposon the plasmid could be established in the lon strains . In contrast, sulA+ plasmids could be established in lon+, lon sulA, and lon sulB strains . The sulA+ plasmids caused lon sulA and lon sulB cells to exist as filaments without SOS induction and to be sensitive to UV light and nitrofurantoin . Evidence implicated higher basal levels of SulA protein in these lon plasmid sulA+ strains as the cause of filamentation . We confirmed that the SulA protein is an 18-kilodalton polypeptide and demonstrated that it was induced by treatment with nalidixic acid . The SulA protein was rapidly degraded in a lon+ strain, but was comparatively more stable in vivo in a lon sulB mutant . Furthermore, the SulA protein was localized to the membrane by several techniques.

J Bacteriol, 1984 May, 158(2), 523 - 9
Genetic analysis of ColN plasmid determinants for colicin production, release, and immunity; Pugsley AP; Colicin N was identified as the 39,000-molecular-weight protein encoded by the 4,900-base-pair, multiple copy number, amplifiable plasmid ColN -284 . Its production was controlled by the SOS regulatory circuit and by catabolite repression . Colicin accumulated intracellularly to ca . 10(6) molecules per cell after growth for 2 to 3 h in medium containing 0.5 microgram of mitomycin C per ml and was then released as the cells underwent partial lysis . Strains carrying pColN -284 and its derivatives exhibited low-level immunity to colicin N and were fully sensitive to all other colicins tested . Regions of the plasmid responsible for colicin N activity (cna), for mitomycin-induced lysis ( cnl ), and for colicin N immunity ( cni ) were localized and characterized by cloning, transposon Tn5 and hydroxylamine mutagenesis, and restriction endonuclease deletion and mapping analysis . The results are discussed in terms of both the organization of the cna, cnl , and cni genes and the respective role of cnl expression and colicin N production in mitomycin sensitivity, colicin export, and induced partial lysis of ColN + cells.

J Bacteriol, 1984 May, 158(2), 455 - 9
Determination of the precise location and orientation of the Escherichia coli dnaE gene; Shepard D et al.; The minimal region required for expression of the dnaE gene of Escherichia coli has been determined relative to a detailed restriction endonuclease map . This has been accomplished by analysis of Bal 31 exonuclease-generated deletions from the termini of the E . coli DNA contained in plasmid pMWE303 , a plasmid that we have previously demonstrated to contain the dnaE gene (M . M . Welch and C . S . McHenry , J . Bacteriol . 152:351-356, 1982) . The competence of these deletion-containing plasmids in expressing the alpha subunit of DNA polymerase III holoenzyme has been determined by their ability both to complement a dnaE mutant and to direct the synthesis of a complete alpha subunit . The carboxyl-terminal coding region of dnaE has been identified through the detection of partial alpha polypeptides encoded by plasmids containing deletions from one end of the gene . This approach has permitted the precise determination of both termini of the dnaE gene and the determination of the orientation of the gene within the E . coli chromosome.

J Bacteriol, 1984 May, 158(2), 397 - 403
DNA supercoiling in gyrase mutants; Steck TR et al.; Nucleoids isolated from Escherichia coli strains carrying temperature-sensitive gyrA or gyrB mutations were examined by sedimentation in ethidium bromide-containing sucrose density gradients . A shift to restrictive temperature resulted in nucleoid DNA relaxation in all of the mutant strains . Three of these mutants exhibited reversible nucleoid relaxation: when cultures incubated at restrictive temperature were cooled to 0 degree C over a 4- to 5-min period, supercoiling returned to levels observed with cells grown at permissive temperature . Incubation of these three mutants at restrictive temperature also caused nucleoid sedimentation rates to increase by about 50%.

Genetics, 1984 May, 107(1), 9 - 18
Copy number control of Tn5 transposition; Johnson RC et al.; Transposition of Tn5 in Escherichia coli strains containing one or multiple copies of the transposable element was investigated . It was found that the overall frequency of transposition within a cell remained constant regardless of the number of copies of Tn5 present in that cell . Experiments measuring the transposition frequency of differentially marked Tn5s confirmed that the frequency of transposition of an individual Tn5 decreased proportionally with the total number of copies of the element present in a cell . The IS50R -encoded function, protein 2, which has previously been shown to be an inhibitor of transposition, is sufficient to mediate this inhibitory effect . The concentration of protein 2 in a cell appears to modulate the transposition of individual Tn5 elements in such a way that the overall transposition of Tn5 in a cell remains constant.

Cell, 1984 May, 37(1), 299 - 307
Structural requirements for the function of a yeast chromosomal replicator; Kearsey S; A sequence closely linked to the Saccharomyces cerevisiae HO gene confers autonomous replication in yeast . I have subjected this putative replication origin to deletion and point mutagenesis in order to identify structural features that are important requirements for autonomous replication in vivo . This analysis identifies a 14 bp core region, which is crucial for function and shows partial sequence conservation between a number of autonomously replicating sequences . Point mutations within the core region can abolish autonomous replication . The core region is flanked on one side by a sequence of about 20 bp, which is important for efficient autonomous replication . Deletion of this flanking sequence reduces, but does not necessarily eliminate, autonomous replication.

Cell, 1984 May, 37(1), 243 - 52
Intragenic suppressor mutations that restore export of maltose binding protein with a truncated signal peptide; Bankaitis VA et al.; A deletion mutation, malE delta 12-18, removes seven residues from the hydrophobic core of the maltose binding protein (MBP) signal peptide and thus prevents secretion of this protein to the periplasm of E . coli . Intragenic suppressor mutations of malE delta 12-18 have been obtained, some highly efficient in their ability to restore proper MBP export . Twelve independently isolated suppressors represent six unique mutational events . Five result in alterations within the MBP signal peptide; one changes the amino acid at residue 19 of the mature MBP . Analysis of these suppressors indicates that the length of the hydrophobic core is a major determinant of signal peptide function . The experiments further suggest that the hydrophobic core region serves primarily a structural role in mediating protein secretion, and that other sequences outside of this region may be responsible for providing the initial recognition of the MBP nascent chain as a secreted protein.

Proc Natl Acad Sci U S A, 1984 May, 81(9), 2757 - 61
Mechanism of the concerted action of recA protein and helix-destabilizing proteins in homologous recombination; Muniyappa K et al.; Secondary structure in single-stranded DNA impedes the presynaptic association of recA protein and consequently blocks the formation of joint molecules as evidenced by effects of temperature, nucleotide sequence, and ionic conditions . Escherichia coli single-strand-binding protein eliminates sequence-specific "cold spots" by removing folds even from sites of strong secondary structure . Thus, destabilization of secondary structure in single-stranded DNA is critical for the action of recA protein, whereas specific interactions directly between helix-destabilizing proteins and recA protein are unimportant.

Infect Immun, 1984 May, 44(2), 222 - 7
Cloning and expression of Legionella pneumophila antigens in Escherichia coli; Engleberg NC et al.; To isolate and characterize Legionella pneumophila antigens, we constructed a genomic library of L . pneumophila serogroup 1 (strain 130b) . L, pneumophila DNA fragments (2.5 to 7.5 megadaltons) obtained by partial digestion with Sau 3A endonuclease and size fractionation on a sucrose density gradient were inserted into the dephosphorylated BamHI site of vector pBR322; CaCl2-treated Escherichia coli cells of strain HB101 were transformed with hybrid plasmids . To detect expression of antigens, 2,559 ampicillin-resistant transformants were transferred to nitrocellulose paper, lysed in situ, and screened by enzyme immunoassay (EIA) with E . coli-absorbed rabbit anti-L . pneumophila sera . A total of 77 (3%) of the colonies were reactive by EIA; 31 (1.2%) were strongly reactive, and 6 were strongly reactive by EIA without colony lysis . Analysis of 29 stable, strongly reactive clones by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting showed antigenic bands in 18 clones by EIA with E . coli-absorbed antisera . Absorption of antisera with heat- and Formalin-killed L . pneumophila antigen eliminated or diminished the reactivity of the antigenic bands in representative clones . These studies confirm that several L . pneumophila antigens can be cloned and expressed in E . coli.

J Virol, 1984 May, 50(2), 343 - 51
Photoaffinity labeling with GTP of viral p21 ras protein expressed in Escherichia coli; Stein RB et al.; The v-ras oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton protein, p21, which mediates transformation produced by that virus . Previous work has shown that both p21v-rasH and the cellular homolog p21c-rasH appear to bind guanine nucleotides . We report here the expression in Escherichia coli of v-rasH to produce a biochemically active p21 fusion protein which retains both guanine nucleotide binding and autophosphorylating activity . Furthermore, direct interaction of this protein with GTP is unequivocally demonstrated by photoaffinity labeling it with {alpha-32P}GTP.

Mikrobiologiia, 1984 May-Jun, 53(3), 432 - 6
{Effect on the orientation of the membrane vesicles of Escherichia coli of various methods of cell disintegration}; Mikhaleva NI et al.; As was demonstrated using the Con-A polymer, membranous fractions prepared by various cell disintegration procedures are a heterogeneous population . The population includes right side out vesicles and inside out vesicles whose proportion depends on the procedure of disintegration . The orientation of these vesicles was studied by electron microscopy, their ATPase activity was assayed by cytochemical techniques, and the morphology of the vesicles was also investigated . The authors discuss the possible effect of Con-A on the reorganisation of membranes and the activity of ATPase.

J Biochem (Tokyo), 1984 May, 95(5), 1349 - 53
Solubilization and reconstitution of membrane proteins of Escherichia coli using alkanoyl-N-methylglucamides; Hanatani M et al.; Alkanoyl-N-methylglucamides, nonionic detergents, were utilized to solubilize membrane proteins of Escherichia coli and were used to reconstitute them into liposomes . First, critical micelle concentrations (CMC) of nonanoyl-N-methylglucamide and decanoyl-N-methylglucamide were determined to be 25 mM and 7 mM, respectively, by photometric assay . Then solubilization and reconstitution of the melibiose transport carrier were performed using these detergents at concentrations above the CMC . Melibiose counterflow activity was observed with the proteoliposomes reconstituted from the extracted proteins and phospholipids . The proton-translocating ATPase complex (F1-F0) was also solubilized with these detergents . These results indicate that nonanoyl- and decanoyl-N-methylglucamide are useful detergents for solubilization and reconstitution of membrane proteins.

Gene, 1984 May, 28(2), 159 - 70
Structural analysis of the dnaA and dnaN genes of Escherichia coli; Ohmori H et al.; The nucleotide sequence of the entire region containing the Escherichia coli dnaA and dnaN genes was determined . Base substitutions by such mutations as dnaA46, dnaA167, dnaN59, and dnaN806 were also identified . Analyses of coding frames, the mutational base substitutions, and other data indicate that dnaN follows dnaA, both have the same orientation, and are separated by only 4 bp . The deduced amino acid sequence specifies Mrs and isoelectric points consistent with those of the previously identified gene products . The transcriptional initiation site of the dnaA gene was assigned by analysis of in vitro RNA products . Examination of the intercistronic sequence and analysis of in vitro transcription supported the notion that the dnaA and dnaN genes constitute a single operon.

Proc Natl Acad Sci U S A, 1984 May, 81(9), 2689 - 92
A genetic switch in vitro: DNA inversion by Gin protein of phage Mu; Plasterk RH et al.; Inversion of the G segment in the DNA of Escherichia coli phage Mu depends on the Mu Gin protein and alters the host range of the phage . The frequency of the inversion reaction is low both in the lysogenic state and during lytic growth . A sensitive assay was developed to detect low levels of G inversion: the E . coli lac operon was inserted within the invertible G segment in such a way that the lac operon was expressed only by G(-) clones . As a result Gin-catalyzed inversion from G(+) to G(-) can be monitored as a lactose-negative to lactose-utilizing switch . Using a crude extract from a Gin-overproducing strain and this assay plasmid, we could detect a low level of G inversion in vitro (1% in 30 min) . The reaction depends on Mg2+ and a supercoiled substrate . Under optimized reaction conditions over 15% of the plasmids had the G segment inverted after incubation with Gin in vitro . The inversion was then visualized by agarose gel analysis of plasmid DNA digested by restriction endonucleases . The Gin protein retains its catalytic properties upon partial purification . The mechanism of this genetic switch can now be studied in vitro.

Mol Biol (Mosk), 1984 May-Jun, 18(3), 691 - 703
{Interaction of substrates and substrate-like inhibitors with the peptidyltransferase center from Escherichia coli ribosomes}; Kukhanova MK et al.; The binding isotherms of CACCA(3'NHPhe----Ac) and CACCA(3'NHPhe) to E . coli ribosomes and 50S subunits were measured . A theoretical model of adsorption for the case of cooperative interaction between two ligands adsorbed on a ribosome was designated . The analysis of the experimental binding isoterms leads to the following conclusions . A ribosome (or subunit) binds one CACCA (3'NHPhe----Ac) molecule to donor site of the peptidyl transferase center, but two CACCA (3'NHPhe) molecules to both donor and acceptor sites . The binding of CACCA (3'NHPhe) to ribosomes (or subunits) is a cooperative process, characterized by the cooperativity coefficient tau = 40 +/- 5 or more . When model substrates CACCA-Phe, CACCA-Leu and CACCA-Val were taken instead of CACCA (3'NHPhe) in the incubation mixture with ribosomes, dipeptides were obtained even in the case, when ratio {model substrate}: {ribosome} (in moles) was much lower than 1 . Puromycin binding to acceptor site with constant (1-2) X 10(4) M-1 also stimulates CACCA(3'NHPhe----Ac) adsorption to the donor site of ribosomes with cooperativity coefficient being equal to 1.5-2.5 . It is also shown that cytidine 5'-phosphate binding to the donor site increases kappa cat of the reaction of minimal donors with CACCA-Phe by 1.5 orders of magnitude but has no effect on Km of this reaction . These facts point out that cytidine 5'-phosphate being adsorbed on the corresponding area of the donor site leads to the conversion of low-productive complex {ribosome + minimal donor substrate + acceptor substrate} into high-productive complex {ribosome + minimal donor substrate + acceptor substrate + cytidine 5'-phosphate}.

Mol Biol (Mosk), 1984 May-Jun, 18(3), 637 - 42
{Selective binding of oligodeoxyribonucleotides with Escherichia coli RNA-polymerase and their effect on DNA-dependent RNA synthesis}; Savinkova LK et al.; It was shown previously, that E . coli RNA polymerase selectively binds certain fractions of oligoribonucleotides with the length greater than or equal to 5 nucleotides from the mixtures of random oligonucleotides of definite length . The data presented demonstrate, that E . coli RNA polymerase from the mixtures of random oligodeoxynucleotides of various length selectively binds oligodeoxynucleotides with the length greater than or equal to 9 . The activity of the enzyme correlates with its ability to bind oligodeoxynucleotides . The enzyme which has selectively bound oligodeoxynucleotides, manifests sedimentation position characteristic for E . coli RNA polymerase engaged in transcription . The oligodeoxynucleotides with high affinity to the enzyme act as competitive inhibitors of transcription catalyzed by E . coli RNA polymerase . The data suggest that E . coli RNA polymerase bound oligodeoxynucleotides mimic the nucleotide sequences of the promoter responsible for the binding of the enzyme . It was found that selectively bound oligoribo - and oligodeoxynucleotides do not compete for the site on the enzyme . This property of E . coli RNA polymerase is assumed to play a certain role in the regulation of transcription.

Plasmid, 1984 May, 11(3), 221 - 33
Transcriptional analysis of the leading region in F plasmid DNA transfer; Cram D et al.; Transcriptional activity associated with the leading region (53.8-66.7F) in F DNA transfer has been shown by RNA-DNA hybridization studies to occur on the anterior segment extending from 59.4 to 66.7F . Promoter-probe analysis of cloned leading region segments detected two promoters within the transcribed portion of the leading region . The promoter active across the 64.7F EcoRI site on the transferred F strand was associated with the expression of two polypeptides, 6d and 13.5p, located between 64.7-66.6F . However, no definite role could be ascribed to the second promoter operative through the 66.6F Bg/II site located in close proximity to oriT, the origin of transfer.

Prikl Biokhim Mikrobiol, 1984 May-Jun, 20(3), 428 - 31
{Use of a TLC method for the simultaneous determination of an enzyme complex of nucleic acid metabolism during the isolation of polynucleotide phosphorylase}; Pupkova VI et al.; For the simultaneous determining of several enzymes of nucleic acid metabolism during polynucleotide phosphorylase isolation TLC was used . It was found that using TLC one can simultaneously detect six and more enzymes, e . g . polynucleotide phosphorylase, 5'-nucleotidase, exoribonuclease together with nucleosidediphosphatase, desaminase etc . The method is simple and accessible.

Appl Environ Microbiol, 1984 May, 47(5), 1012 - 6
Protein synthesis in Escherichia coli during recovery from exposure to low levels of Cd2+; Mitra RS; Exposure of Escherichia coli to 3 microM Cd2+ results in 84 to 95% of the cells losing their ability to form colonies on plates of nutrient agar . Transfer of the cells to Cd2+-free liquid medium results in a recovery of colony-forming ability without significant synthesis of DNA . As an early event in recovery, the cells exhibit a rapid uptake of {3H}leucine . Recovery and this incorporation are inhibited by chloramphenicol or rifampin . Sodium dodecyl sulfate-gel electrophoresis of proteins from recovering cells labeled with {3H}leucine for 1 min indicated the synthesis of at least two classes of proteins with apparent molecular weights of 55,000 to 65,000 . One class bound Cd2+ and was absent in untreated cultures . The other class of proteins, which did not bind Cd2+, was synthesized at a rapid rate in recovering cells and may be a normal cellular protein.

J Biochem Biophys Methods, 1984 May, 9(2), 171 - 9
Yet another improved silver staining method for the detection of proteins in polyacrylamide gels; Tunon P et al.; Silver staining is very sensitive for detection of proteins in polyacrylamide gels and different procedures have been published . By combining and modifying some of the recipes, a very reproducible method, which is based upon staining with diamine complexes of silver, has been developed . The background staining is negligible and reduced silver does not precipitate on the gel surface . The technique works very well for sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both homogeneous and in gradient gels as well as for two-dimensional (2-D) PAGE . It was possible to detect 1-10 ng of protein corresponding to approximately 50 pg/mm2, provided that a discontinuous buffer system was used, which gives sharp bands.

Antimicrob Agents Chemother, 1984 May, 25(5), 622 - 5
Improved genetically modified Escherichia coli strain for prescreening antineoplastic agents; Bartus HR et al.; Clinical experience suggests that drugs that interact with and damage DNA are useful in cancer chemotherapy (H . Umezawa , p . 43-72, in V . T . DeVita , Jr., and H . Busch {ed.}, Methods in Cancer Research; Cancer Drug Development, vol . XVI, 1979) . Prescreening systems for antitumor agents in natural products require assays that are exquisitely sensitive, since the active components are often produced in quantities of micrograms per milliliter or less . One assay used to identify agents that interact with DNA is the biochemical induction assay, utilizing Escherichia coli BR 513 (R . K . Elespuru and R . J . White, Cancer Res . 43:2819-2830, 1983) . In this paper we describe a genetic modification of strain BR 513 that displays an expanded spectrum of activity . This strain may provide an improved prescreen for detecting natural products that interact with DNA.

Am J Vet Res, 1984 May, 45(5), 967 - 71
Monoclonal antibodies to Brucella surface antigens associated with the smooth lipopolysaccharide complex; Schurig GG et al.; Hybridomas producing antibodies to determinants associated with the lipopolysaccharide (LPS) of Brucella abortus and B melitensis were obtained by polyethylene glycol fusion of the SP2/0 myeloma cell line with B lymphocytes harvested from a Sprague-Dawley-derived rat previously immunized with whole B abortus strain 1119 organisms . Two clones, BRU38 and BRU28 , were selected for their ability to react with whole B abortus organisms and purified smooth-LPS ( f5p ) . The BRU38 monoclonal antibodies were absorbed with live, rough strain 45/20 and smooth strains of B abortus and B melitensis organisms, whereas only smooth strains absorbed the antibody activity from BRU28 . Complete inhibition of the monoclonal's activity could be achieved with crude smooth-LPS, a purified f5p fraction, and a water-soluble acid degraded polysaccharide . Absorption of BRU38 and BRU28 with rough Brucella LPS, polysaccharide-B antigen, keto- deoxyoctanoic acid, or with several sugars and fatty acids known to be components of the Brucella LPS complex had no effect on the monoclonals . The data indicate that antigenic determinants are associated with the smooth LPS complex, probably with the O-side chain, and are expressed patchwise and in different quantities on several strains of B abortus and B melitensis . The B abortus rough strain 45/20 contains surface determinants which lead to the agglutination of smooth strain 1119 organisms . The potential use of monoclonals in competitive enzyme-linked immunosorbent assays for diagnostic purposes is discussed.

J Bacteriol, 1984 May, 158(2), 535 - 42
Cloning, mapping, and expression of genes involved in the fatty acid-degradative multienzyme complex of Escherichia coli; Spratt SK et al.; Two protein subunits (42,000 and 78,000 daltons) encoded by the fadAB genes form a multifunctional enzyme complex containing thiolase, 3-hydroxyacyl-coenzyme A dehydrogenase, crotonase , epimerase, and isomerase activities (S . Pawar and H . Schulz, J . Biol . Chem . 256:3894-3899, 1981) . In an attempt to characterize the structural organization and regulatory properties of these genes, a 5.2-kilobase pair fragment containing the fadAB genes has been isolated . Plasmids containing this fragment (i) complement mutations in the fadAB genes; (ii) overproduce by 10- to 50-fold thiolase, 3-hydroxyacyl-coenzyme A dehydrogenase and crotonase ; and (iii) specify a 42,000- and a 78,000-dalton protein . The fadA gene, which encodes the 42,000-dalton protein, has been localized within the original clone to a 3.3-kilobase pair fragment . Thiolase activity, which is encoded by the 42,000-dalton protein, was not observed in the absence of the 78,000-dalton protein, suggesting that an intact complex is required for function . Transposon Tn5 insertional mutagenesis of the cloned fadAB genes has demonstrated that both fadA and fadB are transcribed as a single transcriptional unit with the direction of transcription from fadA to fadB . The molecular cloning and characterization of the fadAB region confirm the original genetic contention that the genes encoding the proteins for the multifunctional complex form an operon.

Biull Eksp Biol Med, 1984 May, 97(5), 567 - 9
{Analysis of the cyclase enzyme activity of the cell membrane following lymphocyte stimulation with a mitogenic polyanion}; Ataullakhanov RI; Stimulant action of the mitogenic polyanion, polyacrylic acid (PAA) was investigated in mouse lymphocyte culture in vitro . B cell division was induced by "impulsive" PAA treatment . Shortly after PAA treatment the activity of the membrane enzymes, adenylate and guanylate cyclases, was assayed according to the changes in the concentration of cAMP and cGMP . The effect of PAA on the time course of cAMP and cGMP in lymphocytes was compared to the effect of B cell mitogen of other chemical nature--bacterial lipopolysaccharide (LPS) . PAA was demonstrated to produce no effect on the activity of membrane cyclase enzymes . On the contrary, following LPS addition guanylate cyclase in the lymphocyte membrane was activated within the first 5-10 minutes . Later on (after 2h) the cells activated with LPS showed an increase in adenylate cyclase activity . By the 12th-24th hour the concentration of cAMP in the LPS-stimulated cells reached 250% of the control level . The differences are discussed between the mitogenic polyanion (PAA) and the lipid-modifying mitogen (LPS) in the molecular mechanisms by which the lymphocyte responses are activated.

Infect Immun, 1984 May, 44(2), 519 - 27
Ultrastructural study of adhesion of enterotoxigenic Escherichia coli to erythrocytes and human intestinal epithelial cells; Knutton S et al.; The adhesion to erythrocytes and human intestinal epithelial cells of enterotoxigenic Escherichia coli strains H10407, B2C, and H10407P, expressing colonization factor antigen I (CFA/I), CFA/II, and type 1 fimbriae, respectively, was examined by electron microscopy . CFA and type 1 fimbriae were visualized by negative staining in thin sections after en bloc staining with ruthenium red and by immune labeling with antisera raised against purified fimbriae . By negative and ruthenium red staining, CFA/I, CFA/II, and type 1 fimbriae were indistinguishable and appeared as approximately 7-nm-diameter hollow cylindrical structures up to 1.5 micron in length; strain B2C also produced 2- to 3-nm-diameter flexible fibrillar fimbriae . Bacteria producing CFA/I, CFA/II, and type 1 fimbriae adhered to and agglutinated human, bovine, and guinea pig erythrocytes, respectively; CFA/I and CFA/II also mediated attachment of bacteria to the brush border of isolated human duodenal enterocytes . Electron microscopy of agglutinated erythrocytes and enterocytes with adherent bacteria showed, in each case, that bacterial adhesion involved the formation of many interactions between the tips of fimbriae and receptors on the erythrocyte or enterocyte brush border membrane . Immune labeling allowed different fimbrial antigens mediating bacterial attachment to human enterocytes to be identified.

Infect Immun, 1984 May, 44(2), 493 - 501
Characterization of the mechanism of action of Escherichia coli heat-stable enterotoxin; Dreyfus LA et al.; The mechanism of activation of intestinal guanylate cyclase by Escherichia coli heat-stable enterotoxin (STa) has been studied by using isolated rat intestinal epithelial cells and purified brush border membrane (BBM) preparations . Inhibitors of prostaglandin biosynthesis, quinacrine and 5,8,11,14-eicosatetraynoic acid (ETYA), significantly reduced intracellular levels of cyclic guanosine 3', 5'-monophosphate in isolated cells treated with STa . Although these data suggested that activation of phospholipase A2 and metabolism of arachidonic acid are involved in the mechanism of action of STa, other data ruled out such a mechanism . (i) The rate of release of {3H}arachidonic acid by prelabeled intestinal cells incubated with STa was the same as control cells not treated with STa . (ii) Thin-layer chromatography of lipid extracts of intestinal cells treated with STa and untreated cells did not reveal any quantitative or qualitative differences in free fatty acids, neutral lipids, and phospholipids . (iii) Amounts of prostaglandin PGE2, prostaglandin PGF2 alpha, and thromboxane B2 in intestinal cells and BBM incubated with STa did not increase compared with controls not incubated with STa . When purified BBM preparations were incubated with phospholipase A2 inhibitors (p-bromophenacyl bromide and quinacrine) or cyclooxygenase inhibitors (ETYA and indomethacin), basal and STa-induced guanylate cyclase activities were significantly reduced . Inhibitors of calcium-calmodulin-mediated reactions (EGTA {ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid}, trifluoperazine, and chlorpromazine) and calcium channel blockers (verapamil and nifedipine) also nonspecifically inhibited both basal and STa-stimulated guanylate cyclase in BBM preparations . Lanthanum, a competitive inhibitor of membrane-bound calcium, did not affect either basal or STa-stimulated guanylate cyclase of BBM preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Int, 1984 May, 8(5), 715 - 23
Nucleosidediphosphate kinase in Escherichia coli: its polypeptide structure and reaction intermediate; Ohtsuki K et al.; A nucleosidediphosphate kinase (NDP-kinase) has been highly purified from E . coli . The purified enzyme was found to have a molecular weight of approximately 64,000 daltons and be a 16,000 dalton polypeptide tetramer . The enzyme rapidly formed a phosphoenzyme when incubated with ATP in the presence of divalent cations such as Mg2+ and Ca2+ (1 mM) even at low temperatures (below 4 degrees C) . The available evidence suggests that the phosphoenzyme is an enzyme-bound high energy phosphate intermediate, which functions as an intermediary in NDP-kinase action.

Mol Biol (Mosk), 1984 May-Jun, 18(3), 599 - 606
{Termination of DNA transcription by a poly(dA).poly(dT) fragment inserted into pBR325 plasmid}; Denisova LIa et al.; A recombinant DNA was constructed by inserting polynucleotide (dA).(dT) of 80-100 base pairs long into EcoRI site of the pBR325 plasmid DNA . Transcription of this DNA was studied in E . coli RNA polymerase system in vitro . Some transcripts obtained with the recombinant plasmid were shown to have poly(U) clusters at the 3'-ends . Obtained data indicate that poly(dA).poly(dT) sequence acts as a terminator of RNA synthesis . Orientation of this sequence in recombinant DNA was also established.

J Gen Microbiol, 1984 May, 130 ( Pt 5), 1271 - 8
Cloning of the aspartase gene (aspA) of Escherichia coli; Guest JR et al.; The aspartase gene (aspA) of Escherichia coli has been isolated in two plasmids, pGS73 and pGS94, which contain segments of bacterial DNA (12.5 and 2.8 kb, respectively) inserted into the tet gene of the vector pBR322 . The plasmids were constructed by sequential sub-cloning from a larger ColE1-frd+ hybrid plasmid . The location of the aspA gene confirmed predictions based on a correlation between the genetic and restriction maps of the corresponding region . The aspartase activities of plasmid-containing aspA mutants were amplified four- to sixfold relative to aspA+ parental strains . The aspA gene product was tentatively identified as a polypeptide of Mr 55 000, which is somewhat larger than previous estimates (Mr 45000 to 48000) for aspartase.

Plasmid, 1984 May, 11(3), 272 - 5
Stabilization of the cloning vector pACYC184 by insertion of F plasmid leading region sequences; Ray A et al.; The leading region of the F plasmid is, by definition, the first part of the plasmid DNA to be transferred to the recipient cell during conjugation . Restriction fragments of the leading region, when cloned into the plasmid vector pACYC184, extended the maintenance of the normally unstable p15A-derived vector replicon in rec+ Escherichia coli K-12 cells . Mutations in the host's general recombination systems were found to influence the maintenance of these hybrid plasmids.

Plasmid, 1984 May, 11(3), 253 - 9
Construction of a plasmid containing functional Escherichia coli uvrA, B, and C genes in a configuration potentially suitable for mammalian expression; DeLuca JG et al.; A plasmid, pUVABC-2, was constructed that encodes functional uvrA, B, and C genes of Escherichia coli . This plasmid also contains the gpt and ampr genes for positive selection in either bacterial or mammalian systems . Each of the uvrA, B, C, and gpt genes is located between SV40 initiation and termination signals and retains the original bacterial promoters . This recombinant vector conferred a wild-type UV resistance phenotype to uvrA-, B-, and C- strains of E . coli . The results indicate that each of the uvr genes contained in pUVABC-2 function in E . coli . The plasmid is a potential biological probe for DNA repair in mammalian cells.

Plasmid, 1984 May, 11(3), 243 - 7
Cloning and expression in minicells of the determinant of resistance to fosfomycin from the transposon Tn2921; Garcia-Lobo JM et al.; The fosfomycin resistance determinant of the transposon Tn2921 has been localized and cloned into the plasmid pBR322 . A DNA fragment of 1 kb contains all the information required for full expression of the resistance . The level of resistance was unaffected by the plasmid copy number and by the orientation in which the fragment was cloned . Studies in minicells showed that this 1-kb fragment directed the synthesis of a single polypeptide with a molecular mass of 16 kDa.

Thromb Haemost, 1984 Apr 30, 51(2), 232 - 5
Effects of human antithrombin III on mortality and blood coagulation induced in rabbits by endotoxin; Triantaphyllopoulos DC; Twenty-one rabbits were infused with 20 micrograms/kg/hr of E . coli endotoxin for 6 hr . Eight of the animals were preinjected immediately before the infusion of endotoxin, with a bolus dose of human AT III calculated to increase the antithrombin content of the plasma by about 4 units/ml . All eight animals which were preinjected with AT III survived, while 5 of the 13 control rabbits infused with endotoxin alone died . The changes in coagulation parameters from the baseline values, between the 8 control rabbits which survived and the 8 animals which were preinjected with AT III were compared . The concentration of the preinjected human AT III declined significantly faster (P: less than 0.01) than that of the native rabbit AT III . AT III prevented the decline of F.XII throughout the infusion of the endotoxin . However, the decline in F.V, fibrinogen, prothrombin and platelets was not affected (P: greater than 0.5) by the injection of AT III.

Biochem Biophys Res Commun, 1984 Apr 30, 120(2), 527 - 33
The DCCD-reactive aspartyl-residue of subunit C from the Escherichia coli ATP-synthase is important for the conformation of F0; Friedl P et al.; The effect of various point mutations in subunits a and and c of the E . coli ATP-synthase was characterized . In each of the mutants there was no F0-dependent H+-conduction, but still an ATPase-activity comparable to wildtype activities . In addition, the subunit b could be extracted from the mutant's F0 but not from the F0 of wildtype . The effects are interpreted as a change in the conformation of F0 caused by the different mutations.

Biochim Biophys Acta, 1984 Apr 27, 786(1-2), 79 - 87
Acidic ribosomal proteins of Neurospora crassa; Kim CH et al.; Neurospora crassa acidic ribosomal proteins from the high salt-ethanol extract of 80 S ribosomes have been fractionated by DEAE-cellulose chromatography . Six acidic ribosomal proteins were purified . All resemble Escherichia coli L7 and L12 in amino acid composition and molecular weight but each has a slightly different net charge at pH 3.2 . Four have an apparent molecular weight of approx . 14 000, and two have a molecular weight of approx . 14 800 . The amino acid compositions and circular dichroism (CD) spectra of the purified Neuropsora proteins are identical for the four 14 kDa proteins, but clearly distinguishable from the two 14.8 kDa proteins . The latter are also identical in amino acid composition and CD spectra . This suggests that there are two Neurospora acidic, or 'A', proteins, one of which exists in four microheterogeneous forms and the other exists in two forms.

J Biol Chem, 1984 Apr 25, 259(8), 5173 - 81
Paramecium mitochondrial genes . II . Large subunit rRNA gene sequence and microevolution; Seilhamer JJ et al.; Mature Paramecium mitochondrial large subunit rRNA consists of two stable segments: a 20 S segment described previously and a unique 283-base segment similar to 5.8 S rRNAs typically found in eucaryotic cytoplasmic RNA . pBR325 clones of both gene regions from both Paramecium primaurelia and Paramecium tetraurelia were sequenced and aligned . The gene segments lie adjacent to each other very near the replicative terminal end of the linear Paramecium mitochondrial genome and are transcribed from a common 23 S precursor . The precise gene ends were determined using nuclease S1 protection; the large subunit rRNA gene complex (consisting of "5.8 S-like" rRNA, a 19-26-base excised region, and 20 S rRNA) spans about 2654 base pairs . The gene complex is preceded by a 15-base poly(T) tract and terminates randomly within a 20-base A + T-rich segment immediately preceding the tRNATyr gene . The sequences from the two species were 4% divergent, the changes consisting of 59% transitions, 38% transversions, and 3% insertions or deletions . The sequences were aligned with Escherichia coli 23 S rRNA, and a secondary structure model is presented for the entire molecule based on structures proposed for E . coli 23 S rRNA.

J Biol Chem, 1984 Apr 25, 259(8), 4987 - 90
113Cd NMR . Arsenate binding to Cd(II) alkaline phosphatase; Gettins P et al.; Alkaline phosphatase from Escherichia coli contains three metal binding sites (A, B, and C) located at sites forming a triangle with sides of 4, 5, and 7 A (Wyckoff, H.W., Handschumacher, M., Murthy, K., and Sowadski, J.M . (1983) Adv . Enzymol . 55, 453) . When all three sites are occupied by Cd(II) the enzyme has a very low turnover; at least 10(3) slower than the native Zn(II) enzyme . The slow turnover number has made the Cd(II) enzyme useful in NMR studies of the mechanism of alkaline phosphatase . The binding of arsenate to two forms of Cd(II) alkaline phosphatase (Cd(II)2alkaline phosphatase and Cd(II)6alkaline phosphatase) has been studied by 113Cd NMR . Cd(II)2alkaline phosphatase, pH 6.3, binds arsenate at only one monomer of the dimeric enzyme and causes migration of Cd(II) from the A site of one monomer to the B site of the arsenylated monomer . This same migration has previously been observed to accompany metal ion-dependent phosphate binding, but is much more rapid in the case of arsenate . The acceleration of migration induced by arsenate supports the conclusion based on the phosphate data that the substrate anion binds to the A site metal ion of one monomer prior to migration and that only the metal ion at A site is required for phosphorylation (arsenylation) of serine 102 . The 113Cd chemical shifts of A and B site metal ions are very sensitive to the form of the bound arsenate, i.e . covalent (E-As) or noncovalent (E X As) complex . Like the analogous phosphate derivatives, the change of chemical shift of A site (to which phosphate is coordinated in the E X P complex) is much greater than that of the B site metal ion, when the arsenate shifts between the two intermediates, suggesting that arsenate is also coordinated to A site in the E X As intermediate . The chemical shifts of A and B site 113Cd(II) ions are considerably different in the arsenate and phosphate derivatives, while the C site 113Cd(II) ions have nearly identical chemical shifts . Thus the substrate appears to interact closely with both A and B sites, while C site appears relatively unimportant in phosphomonoester hydrolysis . The analogous behavior of arsenate and phosphate at the active center as evaluated by 113Cd NMR supports the validity of using the heavier arsenate derivative in x-ray diffraction studies.

J Biol Chem, 1984 Apr 25, 259(8), 5316 - 20
Crystallization of succinyl-CoA synthetase from Escherichia coli; Wolodko WT et al.; Well formed, tetragonal prisms of succinyl-CoA synthetase from Escherichia coli have been crystallized at room temperature from ammonium sulfate and mixtures of sodium and potassium phosphates . A systematic survey of the conditions for crystallization of the enzyme has been carried out . This has shown the addition of a small amount of an organic solvent (acetone, 2-methyl-2,4-pentanediol, tert-butyl alcohol, or tertamyl alcohol) to the phosphate media and of CoA to the sulfate media to be beneficial in producing large, single crystals suitable for analysis by x-ray diffraction methods . Preliminary examination of precession photographs reveals that the crystals from phosphate media have a unit cell of symmetry P4222 with dimensions a = b = 94 A and c = 248 A . Evidence suggests that there may be only half of the (alpha beta)2 tetramer/asymmetric unit in these crystals . The crystals from ammonium sulfate media have unit cell dimensions of a = b = 99 A and c = 399 A, a space group of P4122 (P4322), and one tetramer/asymmetric unit . They diffract to a resolution of 3.4 A . Both crystal types have large solvent contents of about 65% of the unit cell volumes . A parameter called "quality index" is introduced to facilitate comparison of crystals grown under a variety of conditions with respect to their quality of x-ray diffraction.

J Biol Chem, 1984 Apr 25, 259(8), 5167 - 72
Paramecium mitochondrial genes . I . Small subunit rRNA gene sequence and microevolution; Seilhamer JJ et al.; The sequences of the small subunit mitochondrial rRNA genes from two divergent species of Paramecium (primaurelia and tetraurelia) were determined . The gene lies near the center of the linear mitochondrial genome, on the same strand as are all other currently identified genes . The sequences generally resemble their counterparts found in cytoplasmic, procaryotic, and other mitochondrial sources . The rDNA gene boundaries were located by nuclease S1 protection . Small subunit rDNA spans about 1680 nucleotides, including an extraneous 83-base pair sequence very near the 3' end which is unique to Paramecium mitochondria . This "insert" occurs at the apex of the highly variable in length penultimate helix, according to proposed models for small subunit rRNA secondary structure . A discontinuity occurs in isolated rRNA near the start of the insert, resulting in a stable 13 S RNA species and a small segment containing the remaining 3' portion of the gene . The overall rRNA gene sequence was 94% conserved between the two species, and the nucleotide differences consisted of 53% transitions, 37% transversions, and 9% insertions plus deletions . These substitutions were somewhat clustered, and the two most divergent regions coincided with the gene boundaries . The sequence was aligned with Escherichia coli 16 S rRNA for direct comparison of sequence and structure.

J Biol Chem, 1984 Apr 25, 259(8), 5010 - 6
Relative affinities of all Escherichia coli aminoacyl-tRNAs for elongation factor Tu-GTP; Louie A et al.; The relative affinities of all Escherichia coli amino-acyl-tRNAs for E . coli elongation factor (EF) Tu-GTP have been measured by two independent applications of the competition form of the ribonuclease resistance assay . The set of aminoacyl-tRNAs includes at least one tRNA for each of the 20 amino acids as well as purified isoacceptor tRNA species for arginine, glycine, leucine, lysine, and tyrosine . In the first competition study, {3H}Phe-tRNA was used as the competing aminoacyl-tRNA against {14C}aminoacyl-tRNA in the set of all tRNAs; in the second study, {3H}Leu-tRNALeu4 was used as the competing aminoacyl-tRNA . The relative order of aminoacyl-tRNA affinities for EF-Tu-GTP was the same in each study . The results indicate that the affinity of EF-Tu-GTP at 4 degrees C, pH 7.4, is strongest for Gln-tRNA and weakest for Val-tRNA . Both Gly-tRNA and Pro-tRNA bind very strongly to EF-Tu-GTP relative to other aminoacyl-tRNAs . Various models of ternary complex interactions are discussed in light of the new data . Although the properties of the amino acid substituent are primarily responsible for the differences in relative affinities among the noninitiator aminoacyl-tRNAs, the results for the four isoacceptor species of Leu-tRNALeu indicate that the secondary structural features of the tRNA are also influential.

J Biol Chem, 1984 Apr 25, 259(8), 4991 - 7
Zn(II)-113Cd(II) and Zn(II)-Mg(II) hybrids of alkaline phosphatase . 31P and 113Cd NMR; Gettins P et al.; Methods have been developed for the addition of different metal ion species to the three distinct pairs of metal sites (A, B, and C) found in the dimer of apoalkaline phosphatase . This allows the preparation of hybrid alkaline phosphatases in which A and B sites of each monomer contain two different species of metal ion or the A and B sites of one monomer contain the same species of metal ion, while the adjacent monomer contains a second species . The following hybrids have been characterized in detail: (Zn(II)ACd(II)B)2 alkaline phosphatase, (Zn(II)AMg(II)B)2 alkaline phosphatase, (Cd(II)AZn(II)B)2 alkaline phosphatase, and (Zn(II)AZn(II}B)(Cd(II)ACd(II)B) alkaline phosphatase . 31P and, where appropriate, 113Cd NMR have been used to monitor the behavior of the covalent (E-P) and noncovalent (E X P) phosphointermediates and of the A and B metal ions . From the pH dependencies of the E-P in equilibrium E X P in equilibrium E + Pi equilibria, it is clear that A site metal is the dominant influence in dephosphorylation of E-P and may have a coordinated water molecule, which ionizes to ZnOH- at a low pH providing the nucleophile for dephosphorylation . A site metal also serves to coordinate phosphate in the E X P complex . B site metal has a much smaller effect on dephosphorylation rates, although it does dramatically alter the Pi dissociation rate, which is the rate-limiting step for the native enzyme at alkaline pH, and is probably important in neutralizing the charge on the phosphoseryl residue, thus potentiating the nucleophilic attack of the OH- bound at A site . Phosphate dissociation is slowed markedly by replacement of B site zinc by cadmium . There is clear evidence for long range effects of subunit-subunit interactions, since metal ion and phosphate binding at one active center alters the environments of A and B site metal ions and phosphoserine at the other active site.

J Biol Chem, 1984 Apr 25, 259(8), 4964 - 70
Structural study of hinge bending in L-arabinose-binding protein; Mao B et al.; The L-arabinose-binding protein of Escherichia coli is a periplasmic component of the bacterial L-arabinose transport system . The three-dimensional structure of the molecule has been determined by x-ray diffraction and shown to have two globular domains and a connecting hinge . Theoretical study of the flexibility of the hinge using computer simulation showed that the hinge is quite permissive in that only moderate increases in the internal energy are required for opening the cleft where the L-arabinose-binding site is located . In this study, the structural changes that accompany the hinge bending are analyzed . The results show that bending-induced stresses are accommodated by coupled action of covalent and noncovalent forces within the protein molecule . Strains in internal coordinates (bond lengths, bond angles, and torsional angles) are distributed throughout the hinge region after structural relaxation . The pattern of structural changes within a hinge strand upon bending and relaxation depends in large degree on its geometric relationship with the bending axis (e.g . distance and orientation) and the atomic packing of its immediate environment . The distributed structural changes result in a characteristic zigzag pattern for the directional change at each residue in the hinge strands.

J Biol Chem, 1984 Apr 25, 259(8), 4706 - 9
Isoleucyl initiator tRNA does not initiate eucaryotic protein synthesis; Wagner T et al.; Initiator tRNA from yeast (tRNAMeti) was quantitatively misaminoacylated with L-isoleucine using isoleucyl-tRNA synthetase from Escherichia coli . Surprisingly the misaminoacylated Ile-tRNAMeti neither participates in nor inhibits the initiation of globin synthesis in a rabbit reticulocyte lysate, whereas Met-tRNAMeti readily initiates protein synthesis in the same system . The incompetent behavior of Ile-tRNAMeti may be related to the observation that in vitro it does not form a stable complex with eucaryotic initiation factor 2 (eIF-2) and GTP, under conditions which lead to a stable eIF-2 X GTP X Met-tRNAMeti ternary complex . This indicates that eIF-2 can discriminate between the side chains of the aminoacyl adducts of the tRNAMeti during ternary complex formation, the first essential step in initiation of eucaryotic protein synthesis.

Nucleic Acids Res, 1984 Apr 25, 12(8), 3659 - 76
Characterization of the genes coding for the Eco RV restriction and modification system of Escherichia coli; Bougueleret L et al.; A plasmid encoding the recently described Eco RV restriction and modification system has been isolated and characterized . This plasmid, pLB1 , is 6.2 kb long and carries only the Eco RV genes . A subclone of 3 kb has been inserted in pBR322 . The relative positions of the endonuclease and the methylase genes were determined by the construction of a set of overlapping deletions generated by Bal31 resection . The DNA sequence of a 2.2 kb fragment containing the two genes was determined . The two genes are transcribed divergently from a 310 bp region and the assignment of the coding region has been confirmed by direct aminoacid sequence analysis . Possible mechanisms of regulation of the endonuclease gene expression at the translational level are proposed and discussed.

Nucleic Acids Res, 1984 Apr 25, 12(8), 3631 - 42
Complete nucleotide sequence of the fumarase gene fumA, of Escherichia coli; Miles JS et al.; The nucleotide sequence of a 2.41 kb fragment of E . coli DNA containing the fumA gene encoding fumarase was determined using the dideoxy chain termination method . The initiation and termination sites of fumA transcription were located using RNA:DNA hybridisation with single-stranded M13 probes . The length of the fumA transcript was estimated as 1760 +/- 15 nucleotides and the fumA coding region of 1647 base pairs (549 codons) was identified . The deduced molecular weight of the fumarase protein (Mr = 60163) is in good agreement with that of the protein identified by the maxicell procedure . The DNA fragment also contains the 5' end of the manA gene encoding mannose 6-phosphate isomerase, which is transcribed with the opposite polarity to fumA .

Nucleic Acids Res, 1984 Apr 25, 12(8), 3619 - 30
The trfA and trfB promoter regions of broad host range plasmid RK2 share common potential regulatory sequences; Smith CA et al.; The positions of the trfA and trfB promoters of broad host range IncP plasmid RK2 (identical to RP1, RP4, R68 and R18 ) were identified by RNA polymerase protection studies, and the nucleotide sequences of the promoter regions determined . A mutation within the trfA promoter sequence is associated with loss of kilD activity . In addition a probable promoter region for the kilB locus was identified . The three promoter regions share common palindromic sequences which may serve as sites for the coordinate regulation of replication and kil functions.

Nucleic Acids Res, 1984 Apr 25, 12(8), 3445 - 60
Unusual long terminal repeat sequence of a retrovirus transmissible mouse (VL 30) genetic element: identification of functional domains; Norton JD et al.; We have determined the nucleotide sequence and mapped the transcriptional boundaries in the long terminal repeats (LTRs) and adjacent regions of a retrovirus transmissible virus-like 30S ( VL30 ) mouse genetic element . The 572 base pair LTRs contain transcriptional regulatory sequences and are bounded by short imperfect repeats, with a minus strand tRNAgly primer binding site and a purine rich plus strand primer site flanking each of their inner boundaries . The 3' end of each LTR consists of an extensive 80 base pair redundancy of tRNA primer site and inverted repeat sequences while 41 and 47 base pair imperfect tandem repeats are present between the 5' capping site and the putative polyadenylation signal . Comparison with other retrovirus-like LTR sequences suggests possible modes of recombination that could occur between VL30 and other genetic elements.

J Biol Chem, 1984 Apr 25, 259(8), 5232 - 7
Autogenous repression of Escherichia coli threonyl-tRNA synthetase expression in vitro; Lestienne P et al.; Escherichia coli threonyl-tRNA synthetase (EC 6.1.1.3) expression has been examined in an acellular protein-synthesizing system programmed with a plasmid DNA carrying thrS, infC, pheS, and pheT, the gene for threonyl-tRNA synthetase, initiation factor 3, and the two protomers of phenylalanyl-tRNA synthetase (EC 6.1.1.20), respectively . The initial rate of synthesis of L-{35S}methionine-labeled threonyl-tRNA synthetase is markedly reduced by the addition of homogeneous RNase-free threonyl-tRNA synthetase to the assay, not by that of phenylanyl- or tyrosyl-tRNA synthetase (EC 6.1.1.1) . The inhibition is 50% in the presence of 0.25 microM threonyl-tRNA synthetase and reaches 90% with 2 microM enzyme . Synthesis of mRNA in the acellular DNA-dependent protein-synthesizing system has been measured by molecular hybridization to gene-specific lambda DNA probes corresponding to thrS, pheS, and pheT . The addition to the assay of 2 microM threonyl-tRNA synthetase does not affect the extent of mRNA hybridizing to the thrS-specific DNA probe . This result is interpreted as reflecting an effect of the synthetase on its expression at the translational level . Analysis of the DNA sequence of the thrS gene predicts several potential secondary structures capable of forming in the thrS mRNA . One of these potential structures is a cloverleaf . The possible role of such structures in controlling expression of thrS is discussed.

J Mol Biol, 1984 Apr 25, 174(4), 627 - 46
Control of cell division by sex factor F in Escherichia coli . II . Identification of genes for inhibitor protein and trigger protein on the 42.84-43.6 F segment; Miki T et al.; The genetic structure of the 42.84-43.6 F (BamHI-PstI) segment of the F plasmid, which contains all the F DNA sequences necessary for coupling cell division of F+ bacteria with plasmid DNA replication, was analyzed by isolating a series of amber mutants . Two cistrons were found in this region and they were designated letA and letD (an abbreviation for lethal mutation) . The letA and letD cistrons were mapped on the 42.84-43.35 F (BamHI- XmaI ) segment and the 43.07-43.6 F (HincII-PstI) segment, respectively, and are presumed to correspond to the first (43.04-43.26 F) and second (43.26-43.57 F) open reading frames, respectively, which were found in this region by nucleotide sequencing . The letD gene product acts to inhibit cell division of the host bacteria and to induce prophages in lysogenic bacteria, whereas the letA gene product acts to suppress the activity of the letD gene product . Taking into consideration the fact that the 42.84-43.6 F segment carries all the F plasmid genes necessary for coupling cell division with plasmid DNA replication, and that the expression of the genes is likely to be controlled by plasmid DNA replication, we constructed the following hypothesis . Before completion of plasmid DNA replication, LetD protein acts to prevent cell division of the host bacteria . When plasmid DNA replication is completed, synthesis of LetA protein (and also LetD protein) takes place and the LetA protein synthesized acts to suppress the activity of LetD protein and make the cell ready for cell division . Actual cell division will take place when replication of both chromosomal and plasmid DNA is completed and the termination protein of the chromosome and the LetA protein of F plasmid are both synthesized . When cell division takes place LetA protein is consumed, and as a result LetD protein becomes active and prevents cell division until the next round of DNA replication is completed.

J Biol Chem, 1984 Apr 25, 259(8), 5093 - 9
A new DNA-dependent ATPase from Escherichia coli . Purification and characterization of ATPase IV; Meyer RR et al.; A new DNA-dependent ATPase named ATPase IV has been purified to apparent homogeneity from Escherichia coli as a by-product of DNA polymerase III purification . The enzyme has a specific activity of 360 mumol of ATP hydrolyzed per min/mg of protein . The purified enzyme exists as monomer with a molecular weight of 81,000 . It sediments in a glycerol gradient as a single species of 4.5 S . The enzyme has considerable activity at 0 degree C and has a Q10 of 3.8 . In the presence of a DNA effector and magnesium ion, the enzyme will hydrolyze ATP, dATP, GTP, or dGTP to a nucleoside diphosphate plus orthophosphate with a Km of 0.20, 0.50, 0.60, and 1.30 mM, respectively . The guanine nucleotides, however, are only 25-35% as effective as substrates compared with the adenine nucleotides . ATPase IV shows strong substrate inhibition by ATP, but not dATP, above 0.2 mM . The polynucleotide effector requirement can be satisfied by either single-stranded or double-stranded DNA . The enzyme binds the effector very tightly with a Km of 3 X 10(-8) M (nucleotide) for G4 DNA . The enzyme is inhibited by E . coli single-stranded DNA-binding protein, a variety of ATP analogues and N-ethylmaleimide . The relationship of ATPase IV to DNA polymerase III holoenzyme is discussed.

J Biol Chem, 1984 Apr 25, 259(8), 4687 - 90
In vitro biosynthesis and membrane insertion of gamma-glutamyl transpeptidase; Finidori J et al.; gamma-Glutamyl transpeptidase consists of two polypeptide chains anchored to the kidney brush-border membrane only through a short hydrophobic domain near the NH2-terminal end of the heavy subunit . The two subunits were reported to derive from a single polypeptide precursor by tissue labeling experiments . We have investigated the first steps of GGT biosynthesis and processing in a cell-free system . mRNA was prepared from kidney and enriched in specific sequences by a preparative gel electrophoresis . In vitro translation resulted in the synthesis of a single polypeptide (Mr = 63,000) specifically immunoprecipitated by antibodies raised against the mature dimeric enzyme . Incubation with microsomal membranes resulted in the appearance of a glycosylated form of the propeptide (Mr = 78,000) . This latter form was cotranslationally segregated into microsomes and was sensitive to endoglycosidase H . Purified Escherichia coli leader peptidase did not process the primary gamma-glutamyl transpeptidase chain . This ectoprotein therefore appears to be inserted in the phospholipid bilayer without cleavage of a signal peptide, similar to most integral membrane proteins so far studied.

Biochemistry, 1984 Apr 24, 23(9), 2073 - 8
Site-specific modification of Escherichia coli DNA polymerase I large fragment with pyridoxal 5'-phosphate; Hazra AK et al.; Pyridoxal 5'-phosphate (PLP) is an inhibitor of DNA polymerase activity of Escherichia coli DNA polymerase I large fragment . Kinetic studies indicated that overall PLP inhibition was noncompetitive with respect to dNTP, and Hill plot analysis revealed that two molecules of PLP were involved in the inhibition . Reduction of the PLP-treated enzyme with sodium {3H}borohydride resulted in covalent incorporation of 3 mol of PLP/mol of enzyme . This incorporation was at lysine residues exclusively, and the PLP-modified enzyme was not capable of DNA polymerase activity . The presence of dNTP during the modification reaction blocked the incorporation of 1 mol of PLP/mol of enzyme . Similar results were obtained in the presence or absence of template-primer . These data indicate that a PLP target lysine is in or around a dNTP binding site that is essential for polymerase activity and that this binding site is functional in the absence of template-primer . The enzyme modified in the presence of dNTP, containing 2 mol of PLP/mol of enzyme, was capable of DNA polymerase activity but was unable to conduct elongation of product molecules beyond a short oligonucleotide length.

Biochemistry, 1984 Apr 24, 23(9), 1899 - 906
Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme; Srivenugopal KS et al.; A new topoisomerase capable of relaxing negatively supercoiled DNA in Escherichia coli has been identified during chromatography on novobiocin-Sepharose . A simple and reproducible purification procedure is described to obtain this enzyme, called topoisomerase III (topo III), in a homogeneous form . The protein is a single polypeptide with a molecular weight of 74 000 +/- 2000 and is a type I topoisomerase, changing the linking number of DNA circles in steps of one . It is present in deletion strains lacking the topA gene and further differs from the well-studied topoisomerase I (omega protein; Eco topo I) in (1) its requirement for K+ in addition to Mg2+ to exhibit optimal activity and (2) its affinity to novobiocin-Sepharose . Positively supercoiled DNA is not relaxed during exposure to the enzyme . Topo III has no ATPase activity, and ATP does not show any discernible effect on the reduction of superhelical turns . The purified topoisomerase has no supercoiling activity and is unaffected by high concentrations of oxolinic acid and novobiocin in the relaxing reaction . Single-stranded DNA and spermidine strongly inhibit the topoisomerase activity.

Presse Med, 1984 Apr 21, 13(17), 1079 - 81
{Electrophoretic typing of Escherichia coli esterases in septicemia}; Goullet P et al.; Esterases produced by 175 strains of Escherichia coli isolated from 34 patients with septicaemia were subjected to electrophoresis in acrylamide-agarose gel . Forty-four electrophoretic types were identified, and the septicaemias were divided into two groups according to whether or not all the strains isolated in a given patient were of the same electrophoretic type . In the first group (21 patients) the presence of one single electrophoretic type demonstrated that the strains isolated from haemocultures and those isolated from focal specimens were identical and could be held responsible for infection in different foci . In the second group (13 patients) the presence of different electrophoretic types in the same patient either discard an relationship between strain from haemoculture and strain from focal specimen, or demonstrated that different strains could coexist in a focus of infection, or revealed successive and overlapping infections . These results not only provide precise information on the nature of bacteria responsible for infections, but also have interesting pathophysiological and epidemiological implications.

Eur J Biochem, 1984 Apr 16, 140(2), 273 - 80
The size of the pyruvate dehydrogenase complex of Azotobacter vinelandii . Association phenomena; Bosma HJ et al.; Sedimentation analysis and light-scattering studies indicate that the aggregation state of the pyruvate dehydrogenase complex of Azotobacter vinelandii in 50 mM potassium phosphate (pH 7.0) can be described in terms of a monomer-dimer equilibrium with a dissociation constant of 6.8 microM . The apparent molecular mass of the monomeric particle is 750 000-850 000 Da . The equilibrium is shifted to the monomeric species when pressure is applied on the system . Pressure-jump experiments yielded a relaxation time of about 70 ms . In the presence of 3% poly(ethylene glycol) 6000 and 10 mM MgCl2, further association takes place to a system that can be described in terms of dimer-tetramer-octamer equilibria . Upon applying a pressure of 80 MPa to this system these equilibria are shifted to the dimeric state but some monomer formation cannot be excluded . Release of pressure shows that the relaxation time of the dimer-tetramer equilibrium is less than 5 s, that of tetramer-octamer equilibrium is of the order of minutes . The isolated E2 component has a molecular mass of 2 000 000 +/- 100 000 Da; and thus consists of about 30 E2 peptide chains . Electron micrographs are similar to those of the E2 component of the Escherichia coli complex, which were interpreted as cubic structures with an octagonal symmetry . Upon addition of E1 to the pure E2 component, changes in the assembly occur and mixtures of large (E . coli-like, 22-45 S) and small (A . vinelandii-like, 11-18 S) subcomplexes are obtained . The two forms of the subcomplexes are in slow equilibrium (relaxation time 10-30 min) . It is proposed that the E2 tetramer of the intact pyruvate dehydrogenase complex of A . vinelandii is represented by the corner structures of the isolated E2 component.

Eur J Biochem, 1984 Apr 16, 140(2), 249 - 55
Factors necessary for the export process of colicin E1 across cytoplasmic membrane of Escherichia coli; Yamada M et al.; Factors necessary for the export process of colicin E1 across the cytoplasmic membrane of Escherichia coli were investigated . beta-Galactosidase activities from gene fusions between the colicin E1 and lacZ genes were recovered in the inner membrane fraction of E . coli when the region containing the internal signal-like sequence of colicin E1 {M . Yamada et al . (1982) Proc . Natl Acad . Sci . USA 79, 2827-2831} was present, but were found in the soluble fraction when the region was eliminated . The colicin E1 export was reduced upon insertion mutation in a gene that is located downstream from the colicin E1 gene in the same operon and responsible for mitomycin-C-induced killing of the host cell . A frame shift mutation of the colicin E1 plasmid was constructed to direct the protein which had lost the COOH-terminal 13 residues of original colicin E1 and was altered in 6 residues of the new COOH-terminal portion . The aberrant colicin E1 that was inducibly synthesized remained inside the cells . These results indicate that colicin E1 is exported with the aid of a product of the downstream gene and that the COOH-terminal portion is necessary for the export . The binding of colicin E1 to the cytoplasmic membrane through the internal signal-like sequence may be a step in the protein export process.

Eur J Biochem, 1984 Apr 16, 140(2), 343 - 52
Studies on the structure and expression of Escherichia coli pyrC, pyrD, and pyrF using the cloned genes; Jensen KF et al.; The Escherichia coli pyrC, pyrD and pyrF genes were cloned on multicopy plasmids derived from pBR322 and analysed by means of restriction endonucleases . It was found that the pyrC gene is destroyed by cutting with the restriction endonuclease BamHI, that the entire pyrD gene can be isolated on a 1300-base pairs DNA fragment generated by EcoRI cleavage and that cutting with EcoRI removes the promotor and probably also the translational start site from the pyrF gene . More details on the restriction maps are presented . Further, it was found that the presence of a pyr gene in multiple copies on a plasmid does not significantly interfere with the activity of the chromosomal pyr genes . Using the 'minicell' technique, the polypeptides encoded by the three cloned pyr genes were identified . The relative molecular masses for the pyrC-encoded and pyrD-encoded polypeptides are 38 000-40 000 and 36 000-38 000, respectively . Thus in their native form, dihydroorotase and dihydroorotate oxidase appear to be dimeric proteins . The 'minicell' experiments positively identified a protein chain of Mr 23 000-24 000 as being a subunit of OMP decarboxylase encoded by pyrF . Moreover, the coding frame for this polypeptide seems to be expressed as the first gene in the operon with the coding frame for another protein chain of Mr 13 000-14 000 . Since, however, the native OMP decarboxylase during sedimentation and gel filtration behaves as a protein of Mr 45 000 +/- 4000, this latter polypeptide (Mr 13 000-14 000) is hardly a component of the enzyme . Pyr-lac+ operon fusions were constructed by the Mu d1 procedure . By integrating an F'lac episome into the lac part of the fusions and determining the direction of chromosomal transfer from the resultant Hfr strains, the direction of pyrC transcription was found to be counter-clockwise, while pyrD and pyrF were found to be transcribed in a clockwise direction.

Biochem Biophys Res Commun, 1984 Apr 16, 120(1), 74 - 80
Differences in coenzyme specificity of the N5-methyltetrahydrofolate-homocysteine methyltransferases of various species: implications for corrin binding loci; Beck WS et al.; Investigations of the coenzyme specificity of N5-methyltetrahydrofolate-homocysteine methyltransferases of diverse biological origin revealed previously unrecognized differences between Escherichia coli methyltransferase and the corresponding enzymes of other species . Cyanocobalamin (CNCbl) actively supports methyltransferase in extracts of animal tissues and E . coli . Cobinamide is more active than CNCbl with rat liver methyltransferase; however, it is non-competitively inhibitory with E . coli enzyme . E . coli methyltransferase, but not rat liver enzyme, is competitively inhibited by alpha-ribazole 3'-phosphate and 5,6-dimethyl-benzimidazole, two moieties of the nucleotide loop . This suggests that animal enzyme binds its corrinoid coenzyme at a site on the corrin macro-ring, while E . coli enzyme binds to the nucleotide loop as well as the macro-ring.

Biochem Biophys Res Commun, 1984 Apr 16, 120(1), 1 - 8
Enzymatic repair of O-alkylated thymidine residues in DNA: involvement of a O4-methylthymine-DNA methyltransferase and a O2-methylthymine DNA glycosylase; Ahmmed Z et al.; Alkylation of poly(dT) by N-{methyl-3H} (N-nitrosomethylurea) and subsequent annealing with poly(dA) yield a substrate containing O2 and O4-methylthymidine, 3-methylthymidine and phosphotriesters . In an in vitro assay using this substrate, cell extracts from Escherichia coli catalyse i) the transfer of the O4-methyl present in O4 methylthymidine to a protein which becomes alkylated; ii) the release of O2-methylthymine by a glycosylase activity . The two DNA repair activities described above appear to be involved in the adaptive response.

Eur J Biochem, 1984 Apr 16, 140(2), 319 - 24
Outer-membrane protein PhoE from Escherichia coli forms anion-selective pores in lipid-bilayer membranes; Benz R et al.; Porin PhoE of the outer membrane of Escherichia coli was isolated and purified . Reconstitution experiments with lipid bilayer membranes showed that this protein formed pores which had a single channel conductance of 210 pS at 0.1 M KCl . The PhoE pores were obviously not voltage-controlled or regulated . In contrast to pores formed by the OmpF porin from E . coli the PhoE channel was found to be anion-selective at neutral pH . Chloride is about three to ten times more permeable through the pore than alkali ions . On the basis of the observed pH dependence of the permeability ratio of anions and cations, this anionic selectivity is explained by the assumption that the PhoE pore contains an excess of fixed positive charges.

J Mol Biol, 1984 Apr 15, 174(3), 483 - 96
Structural relationship between glutathione reductase and lipoamide dehydrogenase; Rice DW et al.; The elucidation of the primary structure of the Escherichia coli lipoamide dehydrogenase (EC 1.8.1.4) by sequencing the corresponding structural gene (lpd) has enabled a detailed structural comparison between lipoamide dehydrogenase and the related disulphide oxido-reductase, human erythrocyte glutathione reductase (EC 1.6.4.2) . Some 28% of the amino acid residues were found to be identical and a striking degree of homology was apparent throughout the polypeptide chains . It was concluded that the two enzymes possess very similar three-dimensional structures with particularly strong conservation of residues around the FAD and NAD(P) binding sites and at the redox centres of the molecules . Significant amino acid substitutions occur in the substrate binding pocket and these include an extra 18 amino acid residues at the C terminus of lipoamide dehydrogenase . Under physiological conditions, lipoamide dehydrogenase and glutathione reductase act in opposite directions, passing reducing equivalents to NAD+ or from NADPH (respectively), and two key substitutions near the redox centre could be associated with this difference in function . This study represents the first direct structural comparison between two related enzymes that are NADP+-linked (glutathione reductase) and NAD+-linked (lipoamide dehydrogenase) . The differential recognition of these two cofactors could be explained in terms of amino acid substitutions . A divergent evolutionary relationship between the two enzymes including their NAD and NADP binding domains is fully supported by this analysis.

Virology, 1984 Apr 15, 134(1), 238 - 43
Comparison of the primary sequence of spring viremia of carp virus M protein with that of vesicular stomatitis virus; Kiuchi A et al.; The nucleotide sequence of Spring viremia of carp virus mRNA coding for the matrix protein (M) has been determined from a complete DNA copy cloned in the Escherichia coli plasmid pBR322 . The clone is 710 nucleotides long and, excluding the 3' homopolymeric end (representing the mRNA polyadenylic acid sequence), encodes a protein of some 223 amino acids (25,600 Da) . The protein has more basic than acidic amino acids and no significant amino- or carboxy-terminal hydrophobic domains . The predicted amino acid sequence of the M protein of Spring viremia of carp virus can be aligned with that of vesicular stomatitis virus, revealing a significant amount of homology (28%) . The carboxy-terminal regions of the two proteins exhibit much less homology than their amino termini.

J Immunol Methods, 1984 Apr 13, 69(1), 61 - 70
Monoclonal antibodies to human immune interferon and their application for affinity chromatography; Le J et al.; Two IgG1/kappa class monoclonal antibodies specific for human immune interferon (IFN-gamma), designated B1 and B3, were developed . Specific binding of both monoclonal antibodies to natural or Escherichia coli-derived recombinant human IFN-gamma was demonstrated in a solid-phase radioimmunoassay or by immunoprecipitation . Antibody B3 showed potent neutralizing activity against both natural and recombinant IFN-gamma . Antibody B1, which showed neutralizing activity only when very high concentrations were employed, was used for preparing immunosorbents for affinity chromatography of IFN-gamma . When a highly purified preparation of 125I-labeled natural IFN-gamma was loaded onto the affinity column, all of the biological activity was retained on the column . The bulk of 125I-labeled IFN-gamma bound to the affinity column be eluted in biologically active form, suggesting that antibody B1 could be used for the purification of human IFN-gamma . Analysis of IFN-gamma eluted from the column by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE) indicated that both of the known molecular weight subspecies of IFN-gamma (25,000 and 20,000 MW), as well as the presumed dimer of 45,000 MW, were retained by the B1 antibody affinity column.

Clin Chim Acta, 1984 Apr 13, 138(2), 151 - 61
Sensitive enzyme immunoassay for the measurement of platelet factor 4 in blood plasma; Shimizu T et al.; A sandwich enzyme immunoassay method for the measurement of platelet factor 4 (PF4) was developed with the use of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from E . coli . The measurable range was 30 pg to 3 ng of PF4 per tube . Within-run and between-run coefficients of variation were less than 10% . The results obtained with the enzyme immunoassay correlated well with those of a radioimmunoassay (r = 0.952, slope = 0.954, gamma-intercept = 2.43 ng/ml) . Platelets contained large amounts of PF4 (7.21 +/- 1.97 ng/10(6) cells or 2.51 +/- 1.13 ng/mg protein), whereas the PF4 levels in red blood cells and lymphocytes were negligible, confirming the specific localization of PF4 in platelets . The applicability of the immunoassay method was tested to determine the in vitro release of PF4 during preparation and storage of platelet concentrates.

J Immunol Methods, 1984 Apr 13, 69(1), 105 - 13
Phagocytosis and killing of Escherichia coli X43 by individual resident mouse peritoneal macrophages assessed by an autoradiographic technique; Rhodes JM et al.; An autoradiographic technique for the determination of viable bacteria in individual cells is described, based on the incorporation of {3H}thymidine into the DNA of viable Escherichia coli X43, following phagocytosis by resident mouse peritoneal macrophages . The results of the autoradiographic technique were in overall agreement with viable colony counts . Investigation of the killing of E . coli X43 with the autoradiographic technique showed that the percentage viable bacteria tended to be the same irrespective of the number of bacteria ingested per macrophage, although there was a definite correlation between the numbers phagocytosed and the percentage killed in some of the experiments.

Nucleic Acids Res, 1984 Apr 11, 12(7), 3173 - 83
Template-dependent variation in the relative fidelity of DNA polymerase I of Escherichia coli in the presence of Mg2+ versus Mn2+; Hillebrand GG et al.; The fidelity of E . coli DNA polymerase I in the presence of Mg2+ vs Mn2+ was examined at many positions along natural DNA templates, by use of an electrophoretic assay of misincorporation . Although there was an overall greater tendency for misincorporation to occur in Mn2+-activated chain elongation, some specific sites on the template were more prone to misincorporation with Mg2+ and others with Mn2+ . This sequence-dependent effect was seen in spite of the finding that the relative rate of incorporation of the correct nucleotide at different positions on the template was essentially the same with Mg2+ and Mn2+ . In agreement with previous studies, the fidelity of E . coli pol I was higher at activating, than at inhibiting, concentrations of Mg2+ . The results reveal new complexities regarding the role of divalent cation in the control of fidelity in DNA synthesis and attest to the dynamic nature of interactions between DNA polymerase, its substrates and divalent metal activator during the course of polymerization on natural templates.

Nucleic Acids Res, 1984 Apr 11, 12(7), 3373 - 86
Complete nucleotide sequence of the 23S rRNA gene of the Cyanobacterium, Anacystis nidulans; Douglas SE et al.; The nucleotide sequence of the Anacystis nidulans 23S rRNA gene, including the 5'- and 3'-flanking regions has been determined . The gene is 2876 nucleotides long and shows higher primary sequence homology to the 23S rRNAs of plastids (84.5%) than to that of E . coli (79%) . The predicted rRNA transcript also shares many secondary structural features with those of plastids, reinforcing the endosymbiont hypothesis for the origin of these organelles.

Nucleic Acids Res, 1984 Apr 11, 12(7), 3333 - 42
The nucleotide sequence of the cloned nusA gene and its flanking region of Escherichia coli; Ishii S et al.; The nucleotide sequence of the promoter-proximal portion of the nusA operon including the genes for tRNAMetf2, a 15 kilodalton protein and the initial portion of the nusA gene has been determined previously (1) . Here, we report the sequence for the entire nusA gene and its flanking region . The open reading frame, consisting of 1,482 nucleotides, was identified as that of the nusA protein on the basis of agreement of the amino acid sequence deduced from the DNA sequence with the N-terminal sequence of the purified nusA protein . The molecular weight of 54,417 daltons calculated for the 494 amino acid polypeptide is significantly lower than that determined previously by SDS polyacrylamide gel analysis . The nusA gene is immediately followed by another open reading frame encoding a polypeptide of at least 22 amino acids, which was identified as the initial portion of the infB structural gene . In the spacer region of 24 base pairs between the nusA and infB structural genes there is no significant DNA sequence that fits the canonical transcriptional termination signal or promoter sequence . We suggest, therefore, that the genes for tRNAMetf2, a 15 kilodalton protein, the nusA protein and IF2 alpha, aligned in this order, are co-transcribed.

Nucleic Acids Res, 1984 Apr 11, 12(7), 3321 - 32
Initiation signals for complementary strand DNA synthesis in the region of the replication origin of the Escherichia coli chromosome; Stuitje AR et al.; We have used an in vivo plasmid-phi X174 packaging system to detect replication initiation signals in the region of the replication origin (oriC) of the Escherichia coli chromosome . The results obtained are summarized as follows: (i) Neither within nor close to oriC effective signals for initiating complementary strand synthesis could be detected . We conclude that initiation mechanisms for leading and lagging strand synthesis at oriC are not identical to any known priming mechanism of DNA synthesis . (ii) At least five signals that can function as complementary strand origins on ss-plasmid DNA are located in a region about 2000-3300 base pairs away from oriC in the clockwise direction on the chromosome . We suggest that these signals are protein n' like recognition sequences since they are dependent for their activity on dnaB protein and show sequence similarities to other putative n' recognition sequences . Surprisingly, some of the signals are located on the template for leading strand synthesis.

Nucleic Acids Res, 1984 Apr 11, 12(7), 3303 - 19
Complex RNA chain elongation kinetics by wheat germ RNA polymerase II; Job D et al.; Kinetics of RNA chain elongation catalyzed by wheat germ RNA polymerase II have been studied using various synthetic DNA templates in the presence of excess dinucleotide monophosphate primers . With single- or double-stranded homopolymer templates, the double reciprocal plots 1/(velocity) as a function of 1/(nucleotide substrate) exhibit positive, negative or no curvature . With poly(dAT) as template, the mechanism of nucleoside monophosphate incorporation into RNA is not the ping-pong kinetic mechanism which was derived for E . coli RNA polymerase (6) . Noncomplementary nucleoside triphosphates inhibit RNA transcription allosterically . Cordycepin triphosphate behaves as ATP, and not only inhibits AMP incorporation but also that of UMP and GMP on appropriate templates . The reason for this complex kinetic behavior is not yet understood . Possibilities are raised that there are several nucleoside triphosphate binding sites on wheat germ RNA polymerase II, that additional nucleoside triphosphate dependent enzymatic activities are required for reaction to occur or that the Km value for incorporation of a given nucleoside monophosphate into RNA is dependent on the length of the RNA chain and/or the nucleotide sequence surrounding the complementary base on the DNA template.

Nucleic Acids Res, 1984 Apr 11, 12(7), 3295 - 302
Use of complementary DNA oligomers to probe trp leader transcript secondary structures involved in transcription pausing and termination; Fisher R et al.; DNA oligomers were synthesized that are perfectly complementary to different segments of the tryptophan (trp) operon leader transcript . These 15 nucleotide long oligomers were used as probes of the involvement of transcript secondary structures in two processes: transcription pausing at the pause site located near base pair 90 in the leader region, and transcription termination at the attenuator . The 15-mers were complementary to the four segments of the trp leader transcript which have been shown to form the alternative secondary structures that are believed to be responsible for pausing, termination, and antitermination . Oligomers complementary to RNA segments 1 and 3 relieved termination while the 15-mer complementary to RNA segment 1 relieved pausing . 15-mers complementary to segment 2 had no effect on pausing and the oligomer complementary to segment 4 had virtually no effect on termination.

J Biol Chem, 1984 Apr 10, 259(7), 4043 - 8
Glycyl- and alanyl-tRNA synthetases from Bombyx mori . Purification and properties; Dignam SS et al.; Glycyl- and alanyl-tRNA synthetases have been purified from an extract of Bombyx mori posterior silk glands by a procedure that allows for the simultaneous isolation of both enzymes . Glycyl-tRNA synthetase is a dimer of Mr = 160,000 consisting of similar or identical subunits, whereas alanyl-tRNA synthetase is a monomer of Mr = 110,000 to 115,000 . The abundance of these two enzymes in the posterior silk gland is consistent with the observed adaptation of this organ to the production of the silk protein, fibroin . The two enzymes are similar in oligomeric structure to the corresponding enzymes in Saccharomyces cerevisiae, but dissimilar from their counterparts in Escherichia coli.

J Biol Chem, 1984 Apr 10, 259(7), 4521 - 6
The localization of protein L19 on the surface of 50 S subunits of Escherichia coli aided by the use of mutants lacking protein L19; Stoffler G et al.; Three independently isolated mutants of Escherichia coli which apparently lacked protein L19 on their ribosomes, as judged by two-dimensional gels, were analyzed by a range of immunological tests to determine if the protein was indeed lacking . In two of the three, all the tests indicated that protein L19 was absent from both ribosome and supernatant . In the third, a drastically altered form of protein L19 was present on the ribosome . Electron micrographs of ribosomes obtained from the mutants were indistinguishable from those of wild type strains . The location of ribosomal protein L19 on the surface of the large subunit was determined . It was situated at the base of the 50 S particle facing the small subunit, on the side where the rod like appendage originates.

Biochemistry, 1984 Apr 10, 23(8), 1710 - 5
Integrated function of a kinetic proofreading mechanism: dynamic analysis separating the effects of speed and substrate competition on accuracy; Okamoto M et al.; All of the data relating to isoleucyl-tRNA synthetase and its proofreading of valyl-tRNAIle have been integrated into a single model whose dynamic behavior has been determined by numerical solution of the relevant kinetic equations . The results indicate that (1) the system normally operates in vivo with amino acid concentrations slightly above the apparent Km of the system, (2) increases in the displacement of reactants from thermodynamic equilibrium increase the net reaction velocity when the appropriate nominal parameter values are selected, (3) the cost of proofreading decreases with an increase in displacement of reactants from equilibrium, (4) accuracy and reaction velocity tend to be inversely related when substrate competition is unchanged but directly related or unrelated when substrate competition is altered, (5) changes in substrate competition are about twice as effective as changes in reaction velocity in altering the overall accuracy of aminoacylation, and (6) simultaneous changes in substrate competition and reaction velocity have a cumulative but not additive effect upon accuracy . With regard to the temporal development of errors and proofreading costs, we have seen two different patterns . In one, errors or costs gradually change with time following an abrupt alteration; in the other, errors or costs change dramatically in one direction and then more slowly reverse themselves . In all cases, the system responds to change quickly (less than 0.02-0.2 s) but shows no tendency to oscillate.

Biochemistry, 1984 Apr 10, 23(8), 1701 - 9
Integrated function of a kinetic proofreading mechanism: steady-state analysis testing internal consistency of data obtained in vivo and in vitro and predicting parameter values; Okamoto M et al.; Experimental measurements of the kinetic mechanism involving isoleucyl-tRNA synthetase proofreading valyl-tRNAIle in Escherichia coli have been incorporated into the conventional Michaelis-Menten model for this system . The model was subjected to a detailed mathematical analysis in the steady state . The results of this analysis provide an excellent illustration of the value of integrating fragmentary data into a model of the intact system . (1) Such integration provides a rigorous test for consistency of the individual measurements . For the above synthetase system, the published experimental data were found to be internally inconsistent . (2) Such integration predicts which experimental data are most suspect . In this case, one of the three most questionable measurements, the isoleucine pool size in vivo, was found upon reexamination to be in error by 10-15-fold . Correction of this error produced a self-consistent set of parameter values . (3) The integrated analysis provides predictions for various parameter values . In many cases, these predictions provide estimates for parameter values that are difficult to determine directly or that have yet to be measured experimentally . (4) A sensitivity analysis provides an indication of the relative importance of various parameter values and, hence, an indication of where future experimental effort might be focused most profitably.

Biochemistry, 1984 Apr 10, 23(8), 1688 - 95
Escherichia coli ribosomal 5S RNA-protein L25 nucleoprotein complex: effects of RNA binding on the protein structure and the nature of the interaction; Kime MJ et al.; The complexes of three variants of Escherichia coli 5S RNA with ribosomal protein L25 have been studied by high-field proton nuclear magnetic resonance . A spectroscopic method is demonstrated to help distinguish the macromolecular sources of proton resonances in nucleoprotein complexes . The effects of L25 binding on the three RNAs tested were small; the presence of the L25 did not strongly influence the conformation of the RNA . The interaction of L25 with 5S RNA produced modest, but distinctive, alterations in the protein spectrum, in both the aromatic region and the upfield spectrum . As judged by these changes, the mechanism of binding was the same in all three cases . The changes seen in the spectrum of L25 indicate that its conformation is not altered in a major way upon RNA binding . Arginine residues appear to be involved in the binding mechanism . Intercalation of L25 aromatic residues with RNA bases does not appear to play a role in the interaction.

Biochim Biophys Acta, 1984 Apr 10, 798(2), 216 - 25
A common pathway for the activation of several molybdoenzymes in Escherichia coli K12; Giordano G et al.; Three molybdoenzymes, nitrate reductase, formate benzyl-viologen oxidoreductase and trimethylamine-N-oxide reductase which form part of different systems, have been studied in a parental strain of Escherichia coli K12 . When the organism is grown in the presence of 10 mM tungstate, these three enzymes are present in an inactive form which may be activated in vivo by the addition of 1 mM sodium molybdate . The mixing of soluble fractions from chlA and chlB mutants grown under the appropriate conditions leads to the activation of nitrate reductase, formate benzyl-viologen oxidoreductase and trimethylamine-N-oxide reductase . The activation of each enzyme is maximal when the mutants are grown under conditions that lead to the induction of that enzyme in the wild-type strain . The employment of purified proteins, the association factor FA and the Protein PA, which are presumed to be the products of the chlA and chlB genes, has shown that these proteins are responsible for the activation of the three enzymes during the complementation process.

J Biol Chem, 1984 Apr 10, 259(7), 4589 - 95
Adenosine 5'-O-(3-thiotriphosphate) can support the formation of an initiation complex between the DNA polymerase III holoenzyme and primed DNA; Johanson KO et al.; Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) will substitute for ATP in the formation of an initiation complex between the DNA polymerase III holoenzyme of Escherichia coli and primed DNA . The initiation complex formed in the presence of ATP gamma S between the DNA polymerase III holoenzyme and single-stranded DNA-binding protein-encoated primed M13 Gori DNA is stabile and isolable by gel filtration at room temperature . Upon addition of the four required deoxynucleoside triphosphates, this complex is rapidly converted to the duplex replicative form without dissociation of the polymerase . Initiation complexes formed in the presence of either ATP gamma S or ATP are indistinguishable by their resistance to antibody directed against the beta subunit of the holoenzyme and by their ability to elongate without further activation . A 2-fold difference was observed, however, in both the extent of initiation complex formation and in the dissociation of initiation complexes once formed . This difference is discussed in the light of previous proposals regarding a dimeric polymerase capable of replicating both strands at a replication fork concurrently.

J Biol Chem, 1984 Apr 10, 259(7), 4023 - 6
Escherichia coli alpha-ketoglutarate dehydrogenase complex; Steginsky CA et al.; The alpha-ketoglutarate dehydrogenase complex from Escherichia coli catalyzes the hydrolysis of S-succinyl-CoA to succinate and CoASH . The reaction rate is dependent upon the presence of thiamin pyrophosphate and NADH, as well as the functional integrity of the alpha-lipoyl groups associated with the enzyme . The Km value for S-succinyl-CoA is 9.3 X 10(-5) M, and the maximum velocity is 0.02 mumol X min-1 X mg of protein-1 at pH 7 and 25 degrees C . This hydrolysis can be rationalized on the basis that succinyl thiamin pyrophosphate is generated under reductive succinylation conditions . Occasional diversion of succinyl thiamin pyrophosphate to hydrolysis produces succinate.

Biochemistry, 1984 Apr 10, 23(8), 1774 - 9
Reaction mechanism of phosphoenolpyruvate carboxylase . Bicarbonate-dependent dephosphorylation of phosphoenol-alpha-ketobutyrate; Fujita N et al.; Phosphoenolpyruvate carboxylase (EC 4.1.1.31) of Escherichia coli was found to catalyze the cleavage reaction of phosphoenol-alpha-ketobutyrate, a potent competitive inhibitor with the substrate, to yield inorganic phosphate and alpha-ketobutyrate . The rate of phosphate liberation was about 1/20 th of that in the normal reaction with phosphoenolpyruvate . Although HCO3- and Mg2+ were the necessary components in this reaction as in the normal reaction, no CO2 fixation could be detected . When the reaction was carried out in the presence of {18O}HCO3-, multiple incorporations of 18O atoms into the liberated phosphate molecule were observed . The molar proportions of phosphate having one, two, and three 18O atoms were 70, 25, and 5%, respectively . No multiple but only one 18O atom incorporation was observed when phosphoenolpyruvate was used as a substrate . These results suggest that the liberation of phosphate can proceed without CO2 fixation, being not consistent with the concerted mechanism { Maruyama , H., Easterday , R . L., Chang, H . C., & Lane, M . D . (1966) J . Biol . Chem . 241, 2405-2412} but essentially consistent with the current stepwise mechanism {O'Leary, M . H., Rife , J . E., & Slater , J . D . (1981) Biochemistry 20, 7308-7314}.

Biochemistry, 1984 Apr 10, 23(8), 1652 - 6
Detection of ATP-dependent conformational change in the F1 portion and beta subunit of Escherichia coli H+-ATPase using 8-anilinonaphthalene-1-sulfonate; Hirano M et al.; The bindings of ATP to Escherichia coli coupling factor ATPase (F1) and its isolated beta subunit were studied with 8-anilinonaphthalene-1-sulfonate (ANS) . The fluorescence of ANS increased upon addition of F1 or the beta subunit, and this fluorescence was quenched on addition of ATP . Thus, ANS bound to the hydrophobic region of F1 or the beta subunit, and the binding of ATP was detected as a quenching of ANS fluorescence . The quenching of the fluorescence suggested that F1 or the beta subunit underwent a conformational change on binding to ATP . With and without ATP, 5.0 and 3.6 mol of ANS, respectively, bound to 1 mol of F1 . On the other hand, with or without ATP about 1 mol of ANS bound to beta . The fluorescence quenching was specific for ATP and was not observed with GTP or CTP . It was dependent on pH, being higher at acidic pH, but it was not enhanced by MgCl2 . The dissociation constants of F1 and the beta subunit for ATP were estimated to be 10(-4)-10(-5) M . The significance of these binding sites is discussed in relation to the mechanism of the ATPase.

J Biol Chem, 1984 Apr 10, 259(7), 4661 - 6
Molecular cloning of adeno-associated virus variant genomes and generation of infectious virus by recombination in mammalian cells; Senapathy P et al.; Continued passage of the human parvovirus, adeno-associated virus (AAV), at high multiplicity of infection in human cells results in the accumulation of AAV particles containing variant genomes . We have analyzed the structure of individual variant AAV genomes by molecular cloning in the Escherichia coli plasmid, pBR328 . Each of the AAV inserts in six individual recombinant plasmids contained a single internal deletion but in contrast to a previous model, the locations of the deletions were nonrandom . The molecular cloning protocol also generated recombinant plasmids containing the entire AAV2 DNA sequence which yielded infectious AAV particles when transfected into human 293 cells in the presence of helper adenovirus using a DEAE-transfection procedure . Infectious AAV genomes were also generated by recombination when cells were jointly transfected with a mixture of plasmids containing two different mutant AAV genomes . The efficiency of this recombination appear to be influenced by the degree of homology between the mutant AAV genomes.

J Biol Chem, 1984 Apr 10, 259(7), 4576 - 81
Potent catenation of supercoiled and gapped DNA circles by topoisomerase I in the presence of a hydrophilic polymer; Low RL et al.; An exceptionally potent DNA catenation activity, identified in an extract from Escherichia coli, has been purified and partially characterized . Catenation results from the sequential action of the following two polypeptides: beta, 34 kDa and identical to exonuclease III; and alpha, 101 kDa and identical to DNA topoisomerase I (omega protein) . An additional requirement is that a small proportion of the circles be nicked in order to provide the substrate for exonuclease III to generate gaps, estimated to be about 100 nucleotides long . Following exonuclease III digestion, one molecule of topoisomerase I can interlock per minute at 30 degrees C about 20 supercoiled and gapped DNA circles into a massively catenated network . The reaction requires Mg2+ and a hydrophilic polymer (polyvinyl alcohol or polyethylene glycol) at about 7%, but neither ATP nor spermidine . The hydrophilic polymer appears to drive catenation by condensing the DNA; decatenation by topoisomerase I proceeds upon removal of the polymer.

J Biol Chem, 1984 Apr 10, 259(7), 4320 - 6
Nucleotide sequence of the melB gene and characteristics of deduced amino acid sequence of the melibiose carrier in Escherichia coli; Yazyu H et al.; The nucleotide sequence of the melB gene coding for the melibiose carrier in Escherichia coli has been determined . The melibiose carrier is predicted to consist of 469 amino acid residues, resulting in a protein with a molecular weight of 52,029 . The predicted carrier protein is highly hydrophobic (70% nonpolar amino acid residues) . The hydropathic profile suggests that there are 10 long hydrophobic segments in the primary structure of the carrier protein . Most of them seem to traverse the membrane . Although the hydropathic profile of the melibiose carrier is similar to that of the lactose carrier as a whole, homology in the primary structure between the two carriers is very low . Furthermore, no homology in the nucleotide sequence is found in the structural genes for the two carriers . However, the nucleotide sequences of the intergenic regions are very similar between the melibiose operon and the lactose operon . There is a typical intercistronic regulatory sequence in the 3'-flanking region of the melB as well as in that of the lacY, which suggests the presence of another gene downstream of the melB.

J Biol Chem, 1984 Apr 10, 259(7), 4254 - 7
The crystallization of outer membrane proteins from Escherichia coli . Studies on lamB and ompA gene products; Garavito RM et al.; The outer membrane protein LambB from Escherichia coli has been crystallized from detergent-containing solutions . Several different crystal habits can be obtained under the same ionic and precipitant conditions by altering the detergent head group composition of the protein-detergent mixed micelle or by adding polar organic compounds . Two crystal forms have been partially characterized as P1 and C2221, the former diffracting to beyond 4 A resolution and the latter to 6 A . The detergents used were beta-octyl glucoside, octyl tetraoxyethylene, and octyl polyoxyethylene (polydisperse) either alone or as mixtures . In some experiments, the addition of small nonionic amphiphiles having n-butyl alkyl tails significantly influenced crystallization . The experiments suggest that the detergent region of the mixed micelle plays a critical role in crystal formation . Using the methods developed here for LamB and also for matrix porin (Garavito, R . M., Jenkins, J . A., Jansonius, J . N., Karlsson, R., and Rosenbusch, J . P . (1983) J . Mol . Biol . 164, 313-327), an additional protein from the outer membrane, OmpA, has been obtained as a microcrystalline preparation.

Life Sci, 1984 Apr 9, 34(15), 1481 - 6
Plasma levels and biochemical characterisation of circulating met-enkephalin in canine endotoxin shock; Evans SF et al.; Endogenous opioid peptides have been implicated in the pathophysiology of shock (1-5) . In anaesthetised mongrel dogs, administration of E coli endotoxin caused a rise in plasma met-enkephalin-like immunoreactivity (MLI) . Biochemical characterisation of MLI by gel filtration chromatography revealed various molecular forms: 31K, 8K, 3-5K and the native pentapeptide in approximately equal amounts . After enzymatic treatment of column fractions the 31K form predominated (90.7%) . This is the first demonstration of elevated MLI in endotoxin shock.

FEBS Lett, 1984 Apr 9, 169(1), 17 - 20
Hybrid 5 S ribosomal RNA encoded by a multicopy plasmid is incorporated into ribosomes of Escherichia coli; Szeberenyi J et al.; The recombinant plasmid pJR3 delta contains a tandem pair of promoters from rrnA followed by a hybrid 5 S rRNA gene, derived from the two 5 S rRNA genes of the rrnD transcription unit, and a terminator . Escherichia coli cells transformed with this plasmid produce 2-3-times more 5 S rRNA compared to untransformed cells . The growth of cells containing this plasmid is not affected significantly . Although the sequence and the secondary structure of the plasmid-specific 5 S rRNA differ from those of its counterparts (e.g., from 5 S rRNA species encoded by chromosomal genes), it is processed properly and is incorporated into ribosomes.

J Theor Biol, 1984 Apr 7, 107(3), 387 - 403
Enzymic editing mechanisms and the origin of biological information transfer; Lambert GR; Current knowledge of enzymic editing mechanisms in DNA replication, transcription and translation can be used to predict error rates in the absence of editing . Primitive enzymes which possessed synthetic activity but not yet editing mechanisms would have had extremely high error rates resulting in heterogeneous proteins . Based on present knowledge of molecular biology and biochemistry, it is concluded that the evolution of contemporary information transfer systems from primitive systems lacking such editing mechanisms remains an unsolved problem in theoretical biology.

Presse Med, 1984 Apr 7, 13(15), 931 - 4
{Neuro-hormonal regulation of the function of the enterocyte}; Dupont C; The intestinal epithelial cell, or enterocyte, performs a dual function: absorption and secretion . Absorption takes place in the villi and secretion in the crypts (and perhaps also in the villi) . Both functions are controlled by complex extra- and intracellular systems . The extracellular system is mediated by neuromediators released in the subcellular space . Some mediators, like the vasoactive intestinal peptide (VIP) or the prostaglandins, stimulate secretion while others, like enkephalins, stimulate absorption . All these mediators act on intracellular metabolic processes by activating "second messengers" (e.g . amp or intracellular calcium) which in turn regulate the enterocyte transport system . Diarrheogenic processes are frequently due to disturbances in enterocyte control with abnormal stimulation of the secretory processes . Some pancreatic cancers and sympathicoblastomas release VIP into the blood stream in sufficient amounts to stimulate cAMP production by the enterocytes and, consequently, secretion . The cholera toxin and the E-coli thermolabile toxin can also induce stimulation of intracellular cAMP production . Conversely, the most promising antidiarrhoeal drugs are antisecretory agents, like opiates, which stimulate the enterocyte absorption processes.

Biochim Biophys Acta, 1984 Apr 5, 781(3), 279 - 85
Effects of preparative procedures on the sedimentation behaviour of 40-S ribosomal subunits; Manchester KL; The sedimentation characteristics on sucrose gradients of derived 40-S subunits from rat liver depend on the ionic conditions used in their preparation . Following the procedure of Wettenhall, R.E.H., Wool, I.G . and Sherton, C.C . (Biochemistry 12 (1973) 2403-2411) in which 12.5 mM Mg2+ is present throughout the preparation of the subunits, sucrose gradients of the 40-S fraction show two peaks consistent with the presence of a mixed and non-interchanging population of monomers and dimers . By contrast, when ribosomes and subunits are prepared in 2 mM Mg2+ it is shown here that 40-S subunits sediment as a single peak at a rate intermediate between that of monomers and dimers as established by prior fixation with glutaraldehyde or formaldehyde - a phenomenon observed by Spirin, A.S., Belitsina, N.V . and Lishnevskaya, E.B . (FEBS Lett . 24 (1972) 219-224) for the Escherichia coli 30-S--50-S couple but not previously shown for subunits of eukaryotic origin . Under conditions of low {Mg2+} it is suggested that the subunits are of a uniform population capable of dimerization and that equilibrium can continue during sedimentation . In the present experiments it was not possible to find tRNA in 40-S subunits as shown by Wettenhall et al . By fixation before sedimentation the relative proportions of monomers and dimers of 40 S incubated under a variety of conditions was determined . Warming from 0 to 37 degrees C increased the proportion of monomers markedly, but variation of {Mg2+} had little effect . Incubation with albumin or tRNA or spermine also had little effect, but addition of ribosomal salt-wash increased the proportion of monomers . It seems most likely that many of the 40-S subunits prepared in 12.5 mM Mg2+ have initiation factors or other proteins bound to them which prevent dimerization, but that subunits prepared at any stage with buffer containing only 2 mM Mg2+ have lost these proteins and therefore are able to dimerize to a degree depending on concentration . Different relative proportions of monomers and dimers were observed depending on whether glutaraldehyde or formaldehyde was used as fixative . Reasons are suggested for believing that formaldehyde is to be preferred to glutaraldehyde for fixation in experiments of this nature.

J Mol Biol, 1984 Apr 5, 174(2), 265 - 84
Positions of proteins S14, S18 and S20 in the 30 S ribosomal subunit of Escherichia coli; Ramakrishnan V et al.; A map of the 30 S ribosomal subunit is presented giving the positions of 15 of its 21 proteins . The components located in the map are S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S14, S15, S18 and S20.

Biochim Biophys Acta, 1984 Apr 5, 781(3), 225 - 33
Small-angle X-ray study of the 50 S ribosomal subunit of Escherichia coli . A comparison of different models; Meisenberger O et al.; A small angle X-ray scattering curve obtained from Escherichia coli 50 S subunits has been compared with the scattering curves calculated for three-dimensional 50 S models proposed by various authors . The one proposed by Lake, Stoffler and Vasiliev showed to some extent a good agreement with our experimental data . Calculating a large number of possible particle structures we found a model which optimally fits our experimental curves, but it's two-dimensional projections differ a little from the projections of 50 S subunits observed by electron microscopy.

J Mol Biol, 1984 Apr 5, 174(2), 251 - 64
Transcriptional control of IS1 transposition in Escherichia coli; Biel SW et al.; The movement of the bacterial insertion sequence IS1 often generates cointegrate structures in which donor and target replicons are connected by direct repeats of IS1 . The experiments reported here were designed to understand how IS1 transposition is controlled . Our physical characterization of the structures of cointegrates between an F factor ( pOX38 ) and a set of pBR322::Tn9-related plasmids indicate that the relative mobilities of the two IS1 elements of Tn9 are inversely correlated with the strength of promoters upstream in the vector DNA . This implies that transcription across the ends of an IS1 element inhibits its transposition . Transcriptional inhibition may be due to interference with either the synthesis or the action of transposase.

Nature, 1984 Apr 5-11, 308(5959), 509 - 13
The locus of sequence-directed and protein-induced DNA bending; Wu HM et al.; The bending locus of trypanosome kinetoplast DNA, identified by gel electrophoresis, has tracts of a simple repeat sequence (CA5-6 T) symmetrically distributed about it, with a repeat interval of 10 base pairs . The analogous bending induced when catabolite gene activating protein binds to its recognition sequence near the promoter of the Escherichia coli lac operon is centred on a site about 5-7 base pairs away from the centre of the protein binding site.

Microbiologica, 1984 Apr, 7(2), 113 - 20
Different ability of novobiocin and coumermycin A1 to interact with nucleic acids; Masotti L et al.; The possibility of two structurally related antibiotics, Coumermycin A1 and Novobiocin, to interact with nucleic acids was investigated . Only Coumermycin A1 was able to form complexes with DNA showing an apparent affinity constant comparable to that of the interaction with ribosomal RNA . A binding specificity for A + T complementary and repeating sequences was also exhibited by Coumermycin A1 . In view of the different behaviour of the two compounds some considerations are made on their mode of action; although they are acting on the same target enzymes in Escherichia coli, they may affect the functions of eukaryotic cells through a different mechanism not equally specific and probably distinct for each of the two antibiotics.

Biol Reprod, 1984 Apr, 30(3), 679 - 86
Modulation of phospholipase A2 activity associated with human sperm membranes by divalent cations and calcium antagonists; Thakkar JK et al.; Phospholipase A2 activity in sonicates and acid extracts of ejaculated, washed human sperm was measured using {1-14C} oleate-labeled autoclaved E . coli and 1-{1-14C} stearoyl-2-acyl-3-sn- glycerophosphorylethanolamine as substrates . Phospholipase A was optimally active at pH 7.5, was calcium-dependent, and exclusively catalyzed the release of fatty acid from the 2-position of phospholipids . The activity was membrane-associated, and was solubilized by extraction with 0.18 N H2SO4 . Acid extracts of human sperm had the highest specific activity (1709 nmols /h per mg), followed by mouse, rabbit and bull, which were 105, 36 and 1.7 nmols /h per mg, respectively . para-bromophenacyl bromide inhibited human sperm phospholipase A2 activity, but mepacrine was without effect . In the presence of 1.0 mM added CaCl2, phospholipase A2 activity was inhibited by Zn2+ and Mn2+; whereas Cu2+, Cd2+, Mg2+, or Sr2+ had no effect . Zn2+ stimulated activity at low concentrations (10(-6) to 10(-8) M), and inhibited activity in a dose-dependent manner at concentrations of 10(-5) M . The extent of stimulation by low concentrations of Zn2+ was dependent on Ca2+ concentration; at 10(-7) M, Zn2+ activity was stimulated 160% with 0.5 mM CaCl2, and only 120% with 1.0 mM CaCl2 . At low concentrations (10(-5) to 10(-7) M), methoxyverapamil (D600) and trifluoperazine stimulated human sperm phospholipase A2 activity, and trifluoperazine but not D600 produced almost complete inhibition between 10(-5) and 10(-4) M of the drug . The significance of human sperm phospholipase A2 activity and its modulation by Ca2+, Zn2+ and Mn2+ in the sperm acrosome reaction is discussed.

Pediatr Res, 1984 Apr, 18(4), 365 - 9
Hyperimmunoglobulin-E-associated recurrent infection syndrome accompanied by chemotactic inhibition of polymorphonuclear leukocytes and monocytes; Chikazawa S et al.; An 8-yr-old girl with a history of severe recurrent infections including perinephritic, pulmonary, and hepatic abscesses had elevated serum IgE levels . Her serum inhibited chemotaxis of polymorphonuclear leukocytes (PMN) and monocytes . Exchange blood transfusion or plasma exchange at the time of severe infection resulted in normalization of chemotactic activity of PMN shown by the skin window method . Although this effect became negative 1 wk after the treatment, the procedures improved her clinical course . The patient's serum, obtained by exchange blood transfusion, 1) inhibited normal PMN chemotaxis toward cultured supernatant of E . coli, zymosan-activated serum, and formyl methionyl-leucyl-phenylalanine (f . Met-Leu-Phe), a synthetic chemotactic peptide; 2) inhibited monocyte chemotaxis, 3) showed an absence of digestive activity of f . Met-Leu-Phe, 4) was heat stable at 56 degrees C for 30 min and 5) showed an absence of antigenicity of IgE in a partial purified inhibitor with a molecular weight of 30,000-40,000 . The inhibitory effect seemed to be reversible.

Arch Biochem Biophys, 1984 Apr, 230(1), 117 - 28
Specific modification of the condensation domain of fatty acid synthase and the determination of the primary structure of the modified active site peptides; Poulose AJ et al.; Fatty acid synthase from the uropygial gland of goose was inactivated by iodoacetamide with a second-order rate constant of 1.3 M-1 S-1 at pH 6.0 and 25 degrees C . Of the seven component activities of the synthase, only the condensation activity was significantly inhibited by iodoacetamide modification . Since preincubation of the enzyme with acetyl-CoA, but not with malonyl-CoA, protected the enzyme from inactivation by iodoacetamide, it is suggested that iodoacetamide probably modified the primer-binding thiol group at the condensation active site . Determination of the stoichiometry of modification was done using {1-14C}iodoacetamide that was purified by high-performance liquid chromatography . Graphical analysis of the data showed that binding of 1.2 carboxamidomethyl groups per subunit of fatty acid synthase would result in complete inhibition of the enzyme activity, suggesting that there is one condensation domain per subunit of fatty acid synthase . Analysis of the tryptic peptide map of the enzyme that was modified with {1-14C}iodoacetamide in the presence and absence of acetyl-CoA revealed that acetyl-CoA prevented the labeling of a major radioactive peptide and a minor radioactive peptide . These two peptides were purified by high-performance liquid chromatography . Amino acid analysis of these two peptides revealed that the major radioactive peptide contained S-carboxymethylcysteine while the minor radioactive peptide did not . However, the latter peptide contained beta-alanine, suggesting that this peptide was from the acyl carrier protein segment of fatty acid synthase and that the iodoacetamide treatment resulted in modification of the pantetheine thiol, although to a lower extent than the primer-binding thiol . The sequence of the primer-binding active site peptide from the condensation domain was H2N-Gly-Pro-Ser-Leu-Ser-Ile-Asp- Thr-Ala-Cys(carboxamidomethyl)-X-Ser-Ser-Leu-Met-Ala-Leu-Glu-Asn-A la-Tyr-Lys- COOH, the first reported sequence of the condensation active site from a vertebrate fatty acid synthase . The acyl carrier protein segment showed extensive sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the phosphopantetheine attachment, and the sequence was H2N-Asp-Val-Ser-Ser-Leu- Asn-Ala-Asp-Ser-Thr-Leu-Ala-Asp-Leu-Gly-Leu-Asp-Ser(4'-phosphopanteth ein e) -Leu-Met-Gly-Val-Glu-Val-Arg-COOH.

Dev Biol, 1984 Apr, 102(2), 368 - 78
The Caenorhabditis elegans dauer larva: developmental effects of pheromone, food, and temperature; Golden JW et al.; Three environmental cues influence both the entry into and exit from the developmentally arrested dispersal stage called the dauer larva: a dauer-inducing pheromone, food, and temperature . The pheromone, which is a measure of population density, induces dauer larva formation at the second (L2) molt and inhibits recovery in a dose-dependent manner . Food acts competitively to reduce the frequency of dauer larva formation and to enhance recovery . The pheromone causes a specific extension of the second larval stage, coupled with a transient decrease in the growth rate of the L2 . Second-stage larvae grown in the presence of added pheromone are morphologically distinguishable from L2 larvae grown without pheromone . We have named the pre-dauer L2 larva the L2d . Commitment to dauer larva formation can occur at the L2d molt . When L2d larvae are shifted out of pheromone to a lawn of E . coli just before the L2d molt, a few worms complete development into dauer larvae . In contrast, worms are essentially committed to the non-dauer life cycle by the first larval molt if the L1 larvae are not grown in appropriately high levels of pheromone . In the presence of pheromone, the percentage of dauer larva formation is enhanced at higher temperatures within the normal growth range . Temperature down-shifts induce dauer larva recovery . Temperature-shift experiments show that the enhancement of dauer larva formation requires exposure to the higher temperature around the L1 molt . Two sensory mutants defective in thermotaxis are altered in their sensitivity to the dauer-inducing pheromone, but their pheromone response retains temperature dependence . Response of dauer larvae to environmental cues is highly age dependent, with older dauer larvae exhibiting an increased tendency to recover.

J Gen Microbiol, 1984 Apr, 130 ( Pt 4), 887 - 92
The effect of inhibitors of DNA repair on the genetic instability of Streptomyces cattleya; Coyne VE et al.; Various streptomycetes show well defined instabilities that do not appear to be attributable to plasmid loss . The unstable phenotype, in many cases, arises at frequencies too high to be explained by point mutations . The frequency of instability can be enhanced by UV irradiation . Two major repair systems have been found in Escherichia coli: the 'error-free' system which is inhibited by caffeine and the 'error-prone' system which is inhibited by arsenite . Using spores of Streptomyces cattleya NRRL 8057 and the virulent actinophage VC11 we have shown that a caffeine inhibitable, host mediated UV repair system is active in spores during early development . Some evidence was also found for the presence of an arsenite inhibitable UV repair system . The caffeine inhibitable UV repair system was found to be involved in the induction of genetic instability in S . cattleya . The arsenite system may be implicated in the repair of such events . Genetic instability was also induced by single strand breaks in DNA caused by 32P.

Int Surg, 1984 Apr-Jun, 69(2), 107 - 12
Septic shock in colony bred puppies versus random source puppies; Coran AG et al.; Many shock studies utilize unconditioned random source animals . To determine whether unconditioned random source dogs and colony bred dogs differ in response to septic shock, the following experiment was undertaken . Colony bred puppies and unconditioned random source puppies were pretreated with methylprednisolone and were subject to live E . coli induced septic shock . Resuscitation was carried out with Ringer's lactate and 5% albumin . Cardiac output dropped to less than 50% of control values in all animals . The colony bred puppies maintained a significantly higher cardiac output and pulse pressure and required a longer time for their cardiac output to decrease to 50% of control levels than did the unconditioned animals . In addition, the serum acid phosphatase rose significantly (indicating cellular damage) in the unconditioned group and did not change in the colony bred dogs . Marked hyperglycemia occurred in the unconditioned puppies whereas only a mild decrease in serum glucose was seen in the colony bred animals . These data indicate that extreme caution should be exercised in interpreting the physiological response of the unconditioned animal to experimental septic shock.

Zh Mikrobiol Epidemiol Immunobiol, 1984 Apr, (4), 40 - 2
{Nature of the interaction of enteropathogenic Escherichia coli with the surface structures of epithelial cells}; Uvarova VI et al.; The use of scanning electron microscopy has made it possible to reveal the character of changes in the topography of cell surface at different periods after the inoculation of CHO cells with enteropathogenic Escherichia . The process of the adhesion of the strains under study to the surface of epithelial cells in the in vitro system has been found to consist of two phases . The second phase of adhesion is characterized by the appearance of specific structures, ruffles, on the surface of the cells.

Mutat Res, 1984 Apr, 126(2), 121 - 6
Exposure to low concentrations of mutagens alters the mutagenic and lethal effects of 4-nitroquinoline-N-oxide and mitomycin C in Escherichia coli; Alldrick AJ et al.; Mitomycin C and 4-nitroquinoline-N-oxide are two mutagens which can induce the SOS DNA repair system in Escherichia coli . Growth of E . coli B/r strain WP2, or its uvrA- derivative, in very low concentrations of either mutagen led to the increased sensitization to the lethal and/or mutagenic effects of a subsequent challenge with the same mutagen . These phenomena were not observed in either the recA- or lexA- derivatives of the parent strain . Induction of the adaptive response repair pathway by prior cultivation in low concentrations of N-methyl-N'-nitro-N-nitrosoguanidine reduced both the mutagenic and lethal effects of a subsequent challenge with mitomycin C but not 4-nitroquinoline-N-oxide.

J Bacteriol, 1984 Apr, 158(1), 63 - 8
Rapid turnover of mannitol-1-phosphate in Escherichia coli; Rosenberg H et al.; The phosphate moiety of D-mannitol-1-phosphate in Escherichia coli is subject to rapid turnover and is in close equilibrium with Pi and the phosphorus of fructose-1,6-bisphosphate . These three compounds account for the bulk of 32P label found in cells after several minutes of uptake of 32Pi and mannitol-1-phosphate represents some 30% of this label . Mannitol-1-phosphate occurs in E . coli grown on a variety of carbon sources, in the absence of D-mannitol, and is synthesized de novo even in mutants lacking mannitol-1-phosphate dehydrogenase . The mannitol moiety of mannitol-1-phosphate was not affected during the total chase of the P moiety, which exchanged with a half-life of about 30 s . These findings suggest that the rapid equilibration of the phosphorus is a function of an enzyme, possibly a component of the phosphotransferase system, capable of forming a complex that allows the exchange of the phosphate without the equilibration of the mannitol moiety with free mannitol.

Acta Physiol Scand, 1984 Apr, 120(4), 529 - 36
Renal excretion of prostaglandin metabolites, arginine vasopressin, and sodium during endotoxin and endogenous pyrogen induced fever in the goat; Jonasson H et al.; Responses to intravenous injections of an endotoxin (E . coli-lipopolysaccharide, 1 microgram/kg b.wt.) and endogenous pyrogen were studied in euhydrated and hyperhydrated goats . The biphasic febrile response to the endotoxin was associated with a pronounced increase in the renal excretion of measured prostaglandin (PG) metabolites (11-ketotetranor PGF metabolites) . This increase was time-correlated with the elevation of the rectal temperature, and (in hyperhydrated animals) with an inhibition of the water diuresis and an increase in renal excretion of arginine vasopressin (AVP) . Other effects of the endotoxin were an immediate depression of renal Na and K excretion followed by the development of pronounced natriuresis, and a reduction of plasma Fe and Zn concentrations . The appearance of the febrile reactions (peripheral vasoconstriction and shivering) was accompanied by miosis . The maximum elevation of the rectal temperature was significantly greater during euhydration than during hyperhydration . Also endogenous pyrogen elicited miosis concomitant with febrile reactions, and an elevation of the renal excretion of PG metabolites which was closely correlated in time with the monophasic febrile response, and (during hyperhydration) with temporary inhibition of the water diuresis and an increase in the renal AVP excretion . However, the responses were much weaker than the corresponding endotoxin effects . No appreciable changes in renal excretion of Na and K were observed in response to the endogenous pyrogen . It is concluded that the observed effects on renal cation excretion were manifestations of direct endotoxin influences on kidney function.(ABSTRACT TRUNCATED AT 250 WORDS)

J Trop Med Hyg, 1984 Apr, 87(2), 83 - 9
Nutritional status, body size and severity of diarrhoea associated with rotavirus or enterotoxigenic Escherichia coli; Black RE et al.; PIP: This study assessed the effects of undernutrition (measured by weight-for-length) and low body weight on stool output rates in 82 children under 5 years of age with diarrhea associated with rotavirus or enterotoxigenic Escherichia coli . Although weight-for-length status did not affect stool output, children appeared to lose fluid as a greater rate if they had low body weight . Lower-weight children had rates of stool loss 14-61% greater than higher-weight children . Multiple regressionanalysis aimed at determining whether age, weight, or length was the best predictor of stool output found that length was the best predictor . However, weight and age are likely to be indirectly related to diarrheal severity, since malnutrition and young age are reasons for smaller body weight or length . It is hypothesized that the small intestine of an underweight child is greater in proportion to body weight than that of a normal weight child, resulting in larger stool losses per kg/body weight . The finding that children of small body size lose a greater proportion of their body fluid during diarrhea suggests that these children should be regarded as a high risk group during diarrhea . Special attention should be given to water and electrolyte replacement to prevent dehydration and death among these children .

Avian Dis, 1984 Apr-Jun, 28(2), 475 - 81
Immunogenic potency of an oil-emulsified Escherichia coli bacterin; Panigraphy B et al.; Immunogenicity of an oil-emulsified Escherichia coli (O1:K1) bacterin with an aqueous-phase-to-oil-phase ratio of 1:4 was evaluated in chickens . Chickens were vaccinated subcutaneously with 0.5 ml of the bacterin at 4 and 6 weeks of age . At 8 weeks, the vaccinated chickens and unvaccinated controls were challenged via air sacs with 10(4) colony-forming units (CFU) of homologous E . coli . Vaccinated chickens were protected against active respiratory infection in that they (a) gained body weights comparable to those in unvaccinated, unchallenged chickens, (b) suffered no morbidity or mortality, (c) had gross lesions so mild that the scored values were comparable statistically to the 0 lesion scores of the negative controls, and (d) did not yield E . coli when their heart blood, pericardial sacs, livers, and air sacs were cultured . Unvaccinated challenged chickens had severe respiratory distress, suffered 36% mortality, and had average air sac, pericardial sac, and liver lesion scores significantly (P less than or equal to 0.05) different from both the vaccinated and negative control chickens . Also, the challenge strain of E . coli only was isolated from the affected tissues of 5 of 14 chickens . Protection against active respiratory infection was again demonstrated in a second experiment, though the challenge dose was 1.06 X 10(6) CFU of E . coli . The immunity, however, was partially overcome, as the vaccinated chickens gained less body weight and the scored values for lesions in the air sacs, pericardial sacs, and livers were significantly higher than those of the negative controls (P less than or equal to 0.05).

Tohoku J Exp Med, 1984 Apr, 142(4), 453 - 60
Determination of low affinity platelet factor 4 in frozen and thawed human platelets by the newly developed enzyme immunoassay system; Shimizu T et al.; By the use of a newly developed sandwich enzyme immunoassay method for low affinity platelet factor 4 (LA-PF4), the effects of repeated freeze-thawing on the contents of this protein in platelets were determined and compared with those of Triton-X 100 lysed platelets . The assay system consisted of polystyrene balls covered with immobilized antibody fragments F(ab')2 and the same antibody Fab' labeled with beta-D-galactosidase from E . coli . The assay was specific to LA-PF4 with no significant cross-reactivity with platelet factor 4 . Coefficients of variation within-run and between-run for the assay of LA-PF4 were less than 12% . The results obtained with this enzyme immunoassay correlated well with those of a radioimmunoassay of beta-thromboglobulin which is immunologically identical with LA-PF4 (r = 0.961, slope = 1.056, y-intercept = -3.739 ng/ml; n = 22) . The contents of LA-PF4 per 10(9) Triton-X 100 lysed platelets in platelet-rich plasma were 23.65 +/- 3.14 micrograms (mean +/- S.D.) . The contents of LA-PF4 in platelets were increased from 46% to 95% of Triton-X 100 values by repeated freezing and thawing 1 to 7 times . The present data indicate that the freeze-thawing technique should be done carefully to obtain the reliable determination of LA-PF4 in platelets.

J Gen Microbiol, 1984 Apr, 130 ( Pt 4), 779 - 87
Mutants of Escherichia coli with altered hydrogenase activity; Krasna AI; Mutant strains of Escherichia coli which expressed different levels of hydrogenase activity when grown anaerobically under a variety of conditions were obtained by mutagenesis and selective growth and screening procedures . Four classes of mutants were isolated, ranging from those devoid of enzyme activity to those expressing maximal activity under all growth conditions . One class of mutants (A) could not grow on fumarate plus H2 in the presence of active fumarate reductase . Since hydrogenase is essential for growth under these conditions some of these strains may be hydrogenase-negative . Three other classes of mutants were isolated which were all hydrogenase-positive and fully expressed this activity when grown on fumarate plus H2 . They differed in the level of expression of hydrogenase activity when grown anaerobically on glucose, conditions which do not require hydrogenase for growth . Class B mutants expressed less activity, while class C mutants expressed more activity than the parental strain . Class D mutants fully expressed hydrogenase activity and were dependent on the enzyme for growth . The different strains were also assayed for reduction of dyes by hydrogen and for evolution of hydrogen from reduced methyl viologen . Some of the hydrogenase-positive strains showed altered activities in these assays suggesting that mutations may have occurred either in enzymes or proteins required for reaction with dyes or in the hydrogenase enzyme itself.

Gene, 1984 Apr, 28(1), 123 - 5
Correlation of DNA adenine methylase activity with spontaneous mutability in Escherichia coli K-12; Marinus MG et al.; Using a multicopy plasmid in which the tac promoter has been placed in front of the dam gene of Escherichia coli K-12, we show that levels of DNA adenine methylase activity are correlated with the spontaneous mutation frequency.

Antimicrob Agents Chemother, 1984 Apr, 25(4), 443 - 5
Importance of the hydrophobic sulfoxide substituent on nontoxic analogs of sparsomycin; Ash RJ et al.; Nontoxic analogs of sparsomycin were competitive inhibitors of puromycin in the peptidyl transferase assay with Escherichia coli polysomes . The sensitivity of HeLa cells in vitro to the analogs was used as a preliminary index of cellular toxicity . In vitro killing of HeLa cells by this class of compounds correlated well with in vivo 50% lethal doses . The data indicate that modification of the hydrophobic sulfoxide substituent on sparsomycin decreases the toxicity of the molecule for mammalian cells by several hundredfold . Such modifications have less of an effect on the inhibitory activity of the compounds for peptidyl transferase . The differential effects of an analog active against bacterial but not mammalian cells was due to a decreased uptake of the compound by HeLa cells.

Am J Vet Res, 1984 Apr, 45(4), 670 - 7
Effects of Escherichia coli endotoxin on leukocyte and platelet counts, fibrinogen concentrations, and blood clotting in colostrum-fed and colostrum-deficient neonatal calves; Deldar A et al.; Effects of a single IV injection of Escherichia coli endotoxin on hemogram and clotting function were compared in colostrum-fed and colostrum-deficient neonatal calves . Before endotoxin administration, the 2 groups of calves only differed in their prothrombin times . After endotoxin administration, there were significant differences (P less than 0.005) between colostrum-fed and colostrum-deficient calves in total leukocyte, segmented neutrophil, nonsegmented neutrophil, and lymphocyte (P less than 0.05) counts and partial thromboplastin time . Significant time dependent changes were observed in the aforementioned parameters and in platelet count and fibrinogen concentration . Seemingly, colostrum feeding improved the calf's ability to respond to endotoxin challenge exposure probably because of improved granulopoietic activity.

Am J Vet Res, 1984 Apr, 45(4), 652 - 60
Effect of rotavirus and/or Escherichia coli infection on the aggregated lymphoid follicles in the small intestine of neonatal gnotobiotic calves; Torres-Medina A; The effect of rotavirus and/or Escherichia coli infections on the follicle-associated epithelium (FAE or M cells) of the domes of the aggregated lymphoid follicles (ALF, or Peyer's patches) of gnotobiotic calves was evaluated by light, scanning electron, transmission electron, and immunofluorescence microscopies . Calf rotavirus (CRV) infection produced loss of FAE cell microvilli, and virions were observed in cytoplasmic vacuoles of FAE cells, as well as in intercellular spaces between FAE cells and lymphoid cells migrating through the dome epithelium . The CRV particles appeared to have entered the FAE cells by phagocytosis, with no subsequent cytoplasmic replication . Enterotoxigenic E coli (ETEC) induced more severe alterations including marked microvilli loss and ballooning in the FAE cells . There was no adhesion to, or colonization of FAE cells by ETEC, but bacteria were observed free or phagocytized within the dome and the germinal centers of the ALF . There were no ETEC observed in the cytoplasm of FAE cells . The presence of nonenterotoxigenic E coli (NETEC) in the intestine of calves had no effect on the intestinal FAE cells . The addition of NETEC to CRV infections did not enhance or modify in any way the response of FAE cells to the viral infection; however, the combination of CRV + ETEC produced severe necrosis of the FAE cells, and loss of dome epithelium of ALF.

Am J Vet Res, 1984 Apr, 45(4), 643 - 51
Effect of combined rotavirus and Escherichia coli in neonatal gnotobiotic calves; Torres-Medina A; Gnotobiotic calves (24 hours old) were monoinfected with calf rotavirus (CRV) strain NCDV, an enterotoxigenic Escherichia coli (ETEC) strain B44 (K99+), or a nonenterotoxigenic E coli (NETEC) strain 123 (K99-) . Calves also were dually infected with CRV and either ETEC or NETEC . Eighteen calves equally allotted between 6 treatment groups were used in these studies: Noninfected controls--group A; CRV--group B; ETEC--group C; NETEC--group D; CRV + ETEC--group E; and CRV + NETEC--group F . Severe diarrhea and villous atrophy were observed in calves of treatment groups B, C, E, and F . Mortality was present only in treatment groups C and E as result of ETEC infection . There were no significant differences in the clinical responses or enteric lesions between treatments B and F, although a significant increase in the concentrations of NETEC was demonstrated in calves dually infected with CRV + NETEC (group F) as compared with calves monoinfected with NETEC (group D) . Calves inoculated with ETEC (group C) had severe villous atrophy, neutrophilic infiltration of intestinal lumen, and moderate enterocyte necrosis . Calves dually inoculated with CRV + ETEC (group E) had the most extensive and severe lesions, similar to those in group C, plus a pronounced necrotic fibrino-hemorrhagic enteritis . Infection of enterocytes by CRV did not affect in any way the adherence of ETEC to the intestinal mucosa . Dual viral and bacterial infections of the same enterocytes were evident.

Anal Biochem, 1984 Apr, 138(1), 95 - 8
Assay of biological thiols by a combination of high-performance liquid chromatography and postcolumn reaction with 6,6'-dithiodinicotinic acid; Nishiyama J et al.; A simple and rapid method for the determination of nanomole levels of biological thiols is described . The analysis is based on the combination of reverse-phase high-performance liquid chromatography with a postcolumn reaction with 6,6'-dithiodinicotinic acid . Thiols, including cysteine, cysteamine, thiolhistidine, homocysteine, glutathione, penicillamine, ergothioneine, and thiouracil were separated by eluting with 33 mM KH2PO4 at pH 2.2 . Glutathione, cysteine, cysteamine, homocysteine, and penicillamine were quantitatively determined with detection limits of 0.1 nmol, while the quantitative detection of thiolhistidine, ergothioneine, and thiouracil was not successful . The method was applied to the assay of glutathione in human erythrocytes and Escherichia coli.

J Pediatr Surg, 1984 Apr, 19(2), 218 - 20
Epidural abscess complicating Swenson procedure: a case report and a review of the literature; Watters DA et al.; A case is reported in which an anastomotic leak following the Swenson procedure for Hirschsprung's disease was complicated by the development of a pelvic abscess that communicated freely with the epidural space . The child presented with signs and symptoms of an epidural abscess, but his myelogram was normal . The diagnosis was made by urografin enema . The child was treated by defunctioning colostomy and drainage of the pelvic abscess by enlarging the defect at the anastomosis site digitally . The epidural space drained freely to the pelvis and therefore laminectomy was not required . The possible etiology of such a communication is discussed.

Genetika, 1984 Apr, 20(4), 533 - 41
{Induced mutagenesis of plasmid and chromosomal genes inserted into plasmid DNA . I . The mutagenic action of radiation}; Esipova VV et al.; This paper describes the results of treating plasmid DNA in vitro with mutagens, to obtain mutations both in plasmid genes and chromosomal genes comprised within the plasmid, thus avoiding disorganization characteristic of in vivo mutagenesis . The model system is represented by DNA of RSF2124 responsible for colicine E1 synthesis and resistance to ampicillin . Col- mutants were looked for after exposure to UV- and gamma-irradiation . The lethal effect was estimated as inactivation of the ampicillin resistance marker . After reisolation from mutant transformant of the plasmid DNA, the novel character and resistance to ampicillin proved to retain in the course of subsequent transformations and passages of transformed colonies, suggesting the mutational nature of the changes . Exposure of RSF2124 to short-wave UV-irradiation (lambda = 254 nm) produced a pronounced mutagenic effect: the relative quantity of Col- mutants under optimal conditions of mutagenesis increased about 10 times . In the case of W-reactivation (additional UV-irradiation of C600 wild type cells) of lethal lesions, a 95% reliable increase in mutagenic effect was observed . Significant enhancement of mutagenesis (about 4-fold) was detected when only recipient cells were exposed to low doses of UV (the so-called indirect UV mutagenesis) . Thus, with regard to W- and indirect UV mutagenesis, the plasmid DNA behaves like DNA of temperate phages which suggests their evolutionary relationship . Treatment of plasmid DNA with acridine orange prior to UV, only protected from lethal lesions . Gamma-irradiation (60Co) at the dose producing 100-fold inactivation, increased the yield of Col- mutants by one order of magnitude . The presence of RSF2124 plasmid in a cell does not affect its UV sensitivity.

Fundam Appl Toxicol, 1984 Apr, 4(2 Pt 2), S165 - 72
Two enzymes for the detoxication of organophosphorus compounds--sources, similarities, and significance; Hoskin FC et al.; An enzyme in E . coli that hydrolyzes diisopropylphosphorofluoridate (DFP) has now been found to hydrolyze the nerve gas 1,2,2- trimethylpropylmethylphosphonofluoridate (soman) many times faster . With either substrate the E . coli enzyme is stimulated manyfold by 10(-3) M Mn2+ . These criteria are combined and applied to this, and to a superficially similar but distinctly different, enzyme found in squid nerve . The results suggest that while several tissues of the squid contain only this second kind of DFP hydrolyzing enzyme, termed squid type DFPase , many other sources including E . coli contain a mixture of squid type DFPase (the name not strictly indicative of source) and the other DFP hydrolyzing enzyme, now termed Mazur type DFPase . Procedures for the purification of Mazur type DFPase from hog kidney, while increasing the specific activity for DFP hydrolysis may actually have been enriching the purified material in the squid type DFPase . Because E . coli has the highest soman hydrolyzing capacity of any source so far examined, this organism is a promising source for the development of new purification procedures for Mazur type DFPase .

EMBO J, 1984 Apr, 3(4), 909 - 12
A cDNA encoding a small common precursor for human pancreatic polypeptide and pancreatic icosapeptide; Boel E et al.; A cDNA for the hormone, human pancreatic polypeptide (PP), was isolated by oligodeoxynucleotide screening from a cDNA library constructed from normal human pancreatic mRNA . The primary structure of the precursor protein as deduced from the cDNA sequence is 95 amino acids long and is composed of a typical, but rather long signal peptide of 29 residues, followed by the sequence of the 36 amino acid human pancreatic polypeptide, which again is separated from the human pancreatic icosapeptide sequence by a classic cleavage and amidation site, Gly-Lys-Arg . The precursor terminates in a heptapeptide which is cleaved from the icosapeptide at a monobasic processing site . Both the size and the structure of the PP precursor was supported by the results of peptide analysis of biosynthetically labeled pro-PP isolated from canine PP cells in which processing was prevented by the arginine analogue canavanine . It is concluded that the precursor for mammalian PP gives rise to two peptide products, the well preserved, carboxyamidated PP and an icosapeptide which is preserved only in its COOH-terminal end, plus a small highly variable COOH-terminal oligopeptide.

EMBO J, 1984 Apr, 3(4), 757 - 60
pBR322 plasmid DNA modified with 2-acetylaminofluorene derivatives: transforming activity and in vitro strand cleavage by the Escherichia coli uvrABC endonuclease; Fuchs RP et al.; Covalently closed circular plasmid DNA was treated with three reactive derivatives of 2-acetylaminofluorene: N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF), its 7-iodo derivative (N-Aco- AAIF ) and N-hydroxy-N-2-aminofluorene (N-OH-AF), and tested as substrates for the Escherichia coli uvrABC endonuclease and for transformation frequencies on wild-type, uvrA, recA, uvrArecA and polA mutant strains . The uvrABC endonuclease reacted with all three substrates with high efficiency, implicating this enzyme in the repair of DNA containing all three types of adducts . However, only AAF- and AAIF -DNA showed greatly reduced survival on uvrA mutants (five adducts/lethal hit) relative to wild-type (20 adducts/lethal hit) . AF-DNA survived equally well on uvrA mutant and wild-type cells, and at a much higher level of modification (60 adducts/lethal hit) . A mutation in recA had only a minor effect on the survival of either DNA . The polA mutation reduced the survival of the AAF-treated DNA to the same extent as the uvrA mutation (five adducts/lethal hit) . Also AF-DNA showed reduced survival on polA mutant cells versus wild-type . However, many more adducts (20/lethal hit) were tolerated than for AAF-DNA, indicating that AF lesions in the template do not efficiently block replication of DNA.

Biophys J, 1984 Apr, 45(4), 749 - 54
Induction by gamma irradiation of double-strand breaks of Escherichia coli chromosomes and their role in cell lethality; Bresler SE et al.; Viscoelastometric measurements of DNA from gamma-irradiated bacteria were used to identify the induction of double-strand breaks ( DSBs ) in the chromosome of Escherichia coli . It is shown by means of inhibitors of repair endonucleases and different repair mutants that most DSBs in DNA of E . coli, gamma-irradiated in buffer, arise from enzymatic incision of primary gamma-damages; therefore, previous conclusions regarding DSB repair must be reconsidered . Based on these results, much of the reparable damage is single-strand breaks, and this damage can initiate formation of gaps and ultimately, when repair is insufficient, generation of enzymatically caused DSBs . After extensive repair, the first residual DSB in the E . coli chromosome is generated at approximately 160 Gray (Gy), which corresponds to the D37 dose . We propose that DSBs induced directly by gamma-irradiation are not repaired in wild-type strains . In a recently isolated gamma-resistant strain, E . coli Gamr444 , the dose required for observation of DSB after postirradiation incubation is 1,000 Gy, which corresponds to the D37 of the strain . The resistance is proposed to be due to an ability to repair genuine DSBs .

Appl Environ Microbiol, 1984 Apr, 47(4), 868 - 9
Chloramphenicol acetyltransferase should not provide methanogens with resistance to chloramphenicol; Beckler GS et al.; Growth of the four methanogens investigated was inhibited by chloramphenicol-3-acetate; therefore, introduction of chloramphenicol acetyltransferase-encoding genes should not confer chloramphenicol resistance on these methanogens . Reduction of the aryl nitro group of chloramphenicol produced a compound which did not inhibit the growth of these methanogens.

Proc Natl Acad Sci U S A, 1984 Apr, 81(8), 2456 - 60
Differential antibody screening of cloned Plasmodium falciparum sequences expressed in Escherichia coli: procedure for isolation of defined antigens and analysis of human antisera; Stahl HD et al.; We describe a procedure that uses polyspecific human sera for screening Escherichia coli colonies expressing cloned Plasmodium falciparum cDNA sequences in order to detect colonies that react differentially with different sera . This procedure can be used for two distinct purposes . First, it enables the isolation of clones encoding specified antigenic sequences present in the complex mixture, without purification of either antigens or antibodies by conventional procedures . This requires that the antigen can be expressed in E . coli and that antisera are available that differ substantially in their reactivities to the component of interest . To develop the procedure, we used two polyspecific sera that shared many anti-P . falciparum specificities but differed in that only one was reactive to the isolate-specific S antigen of P . falciparum strain FCQ27/PNG (called FC27) . Differential screening with the two sera identified 30 cDNA clones, and colony hybridization confirmed that 25 of these express S-antigen sequences . Second, the procedure identifies defined antibody specificities within polyspecific human sera by virtue of their ability to react with any given cDNA clone . The procedure has been used here to identify antibody specificities that increase dramatically in titer between the acute and convalescent phases of malaria in certain individuals and, hence, to isolate clones encoding the corresponding antigens.

Proc Natl Acad Sci U S A, 1984 Apr, 81(7), 1956 - 60
Strategy for the mass spectrometric verification and correction of the primary structures of proteins deduced from their DNA sequences; Gibson BW et al.; Fast atom bombardment mass spectrometry has been used to confirm and correct regions from the amino acid sequences of three large proteins, glutaminyl- and glycyl-tRNA synthetase from Escherichia coli and methionyl-tRNA synthetase from yeast, whose primary structures had been deduced from the base sequences of their corresponding genes . The strategy is based on a comparison of the molecular weights of the tryptic peptides predicted from all three reading frames of the gene sequences with those determined mass spectrometrically . The experimental molecular weights either match or differ and can be used to assess the correctness of the base sequences, identify errors that lead to frame shifts, premature stop codons, incorrect amino acids, etc., or identify the presence of posttranslational modifications . This method is very fast and requires little material (5-20 nmol).

Mutat Res, 1984 Apr, 139(4), 183 - 7
Mutagenicity studies of human fibroblast interferon (HuIFN-beta); Shimada H et al.; Human fibroblast interferon (HuIFN-beta) was studied for mutagenicity using the Ames method and in vitro cytogenetics . HuIFN-beta had no mutagenic effect on S . typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) and E . coli (WP2 uvrA) at concentrations of 3, 30, 300, 3000, 30 000 or 300 000 IU/plate . In the cytogenetic study, HuIFN-beta had no clastogenic effect on human peripheral blood lymphocytes at concentrations of 3, 30, 300, 3000, or 30 000 IU/ml . These results suggest that HuIFN-beta has no mutagenic potential.

J Clin Microbiol, 1984 Apr, 19(4), 489 - 91
Prevalence of heat-stable II enterotoxigenic Escherichia coli in pigs, water, and people at farms in Thailand as determined by DNA hybridization; Echeverria P et al.; The DNA hybridization assay employing a 460-base-pair fragment of DNA encoding for the methanol-insoluble form of heat-stable toxin (ST-II) was used to determine the prevalence of ST-II enterotoxigenic Escherichia coli (ETEC) in pigs, people, and water at 57 farms in Sri Racha, Thailand . ST-II ETEC was found in 62 (3%) of 2,110 suckling, 181 (32%) of 560 weaned, and 4 (1%) of 457 adult pigs examined . Of 62 suckling pigs with ST-II ETEC infections 21% had diarrhea, but none of 185 infected older pigs had diarrhea . ST-II ETEC was found more frequently in suckling pigs with diarrhea than without diarrhea (13 of 146 versus 49 of 1,964) (P less than 0.001) . ST-II ETEC was detected in water collected from 3 of 57 clay jars containing water used to bathe at three pig farms, in 1 jar used to bathe immediately after working in the barn, and from one farmer who did not have a recent history of diarrhea . Evidence of this organism was not found in 245 other individuals living on the pig farms or in 220 inhabitants and 114 water specimens collected at tapioca farms nearby . In Thailand ST-II ETEC was found in suckling pigs with diarrhea but was infrequently found in humans.

J Bacteriol, 1984 Apr, 158(1), 365 - 8
Cloning of the Escherichia coli release factor 2 gene; Caskey CT et al.; The protein release factor 2 (RF2) participates in Escherichia coli polypeptide chain termination with codon specificity (UAA or UGA) . A colicin E1 recombinant identified in the Carbon and Clarke E . coli bank contains the protein release factor 2 gene . A 1.7-kilobase E . coli fragment has been subcloned into the plasmid pUC9 vector . Bacterial cells, containing the plasmid recombinant, produce elevated levels of protein release factor 2 as detected by an immune precipitation assay and in vitro measurement of UGA-directed peptide chain termination and {3H}UGA codon recognition.

J Bacteriol, 1984 Apr, 158(1), 296 - 9
Independence of buoyant cell density and growth rate in Escherichia coli; Kubitschek HE et al.; The relationship between growth rate and buoyant density was determined for cells from exponential-phase cultures of Escherichia coli B/r NC32 by equilibrium centrifugation in Percoll gradients at growth rates ranging from 0.15 to 2.3 doublings per h . The mean buoyant density did not change significantly with growth rate in any of three sets of experiments in which different gradient conditions were used . In addition, when cultures were allowed to enter the stationary phase of growth, mean cell volumes and buoyant densities usually remained unchanged for extended periods . These and earlier results support the existence of a highly regulated, discrete state of buoyant density during steady-state growth of E . coli and other cells that divide by equatorial fission.

J Bacteriol, 1984 Apr, 158(1), 208 - 15
Phasmid P4: manipulation of plasmid copy number and induction from the integrated state; Lagos R et al.; "Phasmid" P4 is unusual in that it is capable of (i) temperate, (ii) lytic, helper-dependent, and (iii) plasmid modes of propagation . In this report we characterize most of the known P4 genetic functions as to their essential or nonessential roles in the stable maintenance of plasmid P4 vir1 (pP4 vir1 (pP4 vir1) . We also identify growth conditions that can be used to stably maintain pP4 vir1 at any one of several different copy number levels (n = 1 to 3, n = 10 to 15, or n = 30 to 40) . Analyses of a temperature-sensitive alpha derivative of pP4 vir1 show that shifting the temperature from 37 to 42 degrees C allows this mutant to maintain an integrated copy of the plasmid, whereas replication of free copies is repressed because of the nonpermissive condition for their DNA synthesis . Conversely, a shift from 42 to 37 degrees C can be used to reinstate plasmid propagation . The utility of the inducible states of pP4 vir1 is discussed with respect to its attributes as a vector with the potential for cloning inserts of DNA up to 33,000 base pairs in a wide range of bacterial hosts.

J Bacteriol, 1984 Apr, 158(1), 141 - 7
Functional limits of the araIc promoter suggest an additional regulatory site for araBAD expression; Horwitz AH et al.; The araBAD promoter is defined, in part, by two types of cis-acting constitutive mutations, araIc at position -35 and araXc at position -10 . Subcloning experiments demonstrated that the araIc and araIcXc promoters require DNA sequence information out to position -53 to -56 for maximum constitutive expression . This is 8 to 10 base pairs more DNA than is generally thought to be necessary for RNA polymerase interaction . The -53 to -56 region is required for glucose repression, suggesting that an additional factor interacts in this region and is necessary for maximum expression.

J Bacteriol, 1984 Apr, 158(1), 128 - 33
DNA segregation in Escherichia coli cells with 5-bromodeoxyuridine-substituted nucleoids; Canovas JL et al.; The pattern of segregation of DNA in Escherichia coli K-12 was analyzed by labeling replicating DNA with 5-bromodeoxyuridine followed by differential staining of nucleoids . Three types of visible arrangement were found in four-nucleoid groups derived from a native nucleoid after two replication rounds . Type A, segregation of both old strands toward cell poles, appeared with the highest frequency (0.6 to 0.8) . Type B, segregation of one old strand toward the cell pole and the other toward the cell center, was twice as frequent as type C, segregation of both old strands toward the cell center . These results confirm previous data showing that DNA segregation in E . coli is nonrandom while presenting a certain degree of randomness . The proportions of the three indicated types of arrangement suggest a new probabilistic model to explain the observed segregation pattern . It is proposed that DNA strands segregate either nonrandomly, with a probability of between 0 and 1, or randomly . In nonrandom segregation, both old strands are always directed toward cell poles . Experimental data reported here or by other authors fit better with the predictions of this model than with those of other previously proposed proposed deterministic or probabilistic models.

J Bacteriol, 1984 Apr, 158(1), 115 - 20
Metabolism of 4'-phosphopantetheine in Escherichia coli; Jackowski S et al.; Coenzyme A (CoA) and acyl carrier protein (ACP) contain 4'-phosphopantetheine moieties that are metabolically derived from the vitamin pantothenate . The utilization of metabolites in the biosynthetic pathway during growth was investigated by using an Escherichia coli beta-alanine auxotroph to specifically and uniformly label the pathway intermediates . Pantothenate and 4'-phosphopantetheine were the two intermediates detected in the highest concentration, both intracellularly and extracellularly . The specific cellular content of CoA and ACP was not constant during growth of strain SJ16 (panD) on 4 microM beta-{3-3H}alanine, and alterations in the utilization of 4'-phosphopantetheine and pantothenate correlated with the observed fluctuations of the intracellular pool sizes of CoA and ACP . Double-label experiments indicated that extracellular 4'-phosphopantetheine was derived from the degradation of ACP, and the extent that this intermediate was utilized by 4'-phosphopantetheine adenylyltransferase exerted control over the degradative aspect of the pathway . Control over the biosynthetic aspect of the biochemical pathway was exerted at the level of pantothenate utilization by pantothenate kinase . Reduction in the specific cellular content of CoA and ACP by 4'-phosphopantetheine excretion was irreversible since, in contrast to pantothenate, strain SJ16 was unable to assimilate exogenous 4'-phosphopantetheine into CoA or ACP.

Arch Biochem Biophys, 1984 Apr, 230(1), 110 - 6
Acylation of plant acyl carrier proteins by acyl-acyl carrier protein synthetase from Escherichia coli; Kuo TM et al.; The acyl-acyl carrier protein synthetase from Escherichia coli has been examined for its ability to specifically acylate acyl carrier protein (ACP) from higher plants in order to develop an assay for plant ACP, and to prepare labeled acyl-ACP of plant origin . It was found that the E . coli enzyme was able to acylate ACP from spinach, soybean, avocado, corn, and several other plants . The acylation was very specific because, in crude extracts of spinach leaves where ACP represented approximately 0.1% of the total soluble protein, ACP was shown to be the only protein acylated . In contrast to other E . coli enzymes that display 2- to 10-fold lower rates with plant versus bacterial ACP, the kinetic constants (Km and Vmax) for acyl-ACP synthetase were found to be essentially identical for spinach and E . coli ACP when acylated with palmitic acid . Palmitic, myristic, lauric, stearic, and oleic acid could all be esterified to both spinach and E . coli ACP with similar specificity . Procedures are described that allow the assay of ACP in plant extracts at the nanogram level.

Urology, 1984 Apr, 23(4), 343 - 7
Significance of asymptomatic bacteriuria in neurogenic bladder disease; Lewis RI et al.; Whether or not to treat bacteriuria (greater than or equal to 100,000 col/cc) in the asymptomatic patient has long been controversial . Fifty-two patients with uncomplicated neurogenic bladder disease secondary to spinal cord injury and bacteriuria were followed throughout their hospitalization . Antibiotics were reserved only for symptomatic patients . Our results indicate the value of no treatment for chronic bacteriuria as an alternative to chronic suppressive therapy.

Surgery, 1984 Apr, 95(4), 444 - 53
Regional blood flow and pulmonary thromboxane release after sublethal endotoxin infusion in sheep; Schuette AH et al.; Fifteen minutes after intravenous Escherichia coli endotoxin (EN) infusion the sheep lung transiently releases into the systemic circulation up to 12 micrograms of thromboxane A2 (TxA2)/min measured by radioimmunoassay as its metabolite thromboxane B2 (TxB2) . To determine whether lung thromboxane release alters regional blood flow (RBF) we injected microspheres before and 20, 30, and 100 minutes after EN infusion in 13 awake sheep . In seven untreated control sheep (group 1) pulmonary vascular resistance (PVR) increased threefold at 20 and 30 minutes after EN infusion, coinciding with a large transpulmonary blood concentration gradient of TxB2 . There was no measured RBF change to any systemic tissue or organ except the right ventricle at 20 minutes after EN infusion, when blood flow per gram of tissue doubled . In six sheep (group 2) treated with intravenous ibuprofen before EN infusion there was no increase in PVR, plasma TxB2, or blood flow to the right ventricle, and no difference of RBF to any other organ or tissue when compared to group 1 . TxA2 is a potent local pulmonary vasoconstrictor but it rapidly hydrolyzes . Its short half-life and the circulatory delay in systemic arterial delivery prevent TxA2 from acting as a circulating vasoconstrictor.

J Clin Pathol, 1984 Apr, 37(4), 467 - 70
Endotoxaemia as a cause of fever in immunosuppressed patients; Harris RI et al.; Using a recently developed chromogenic substrate assay sensitive to 10 pg/ml Escherichia coli endotoxin in plasma, systemic endotoxaemia was found in 52% of 21 episodes of fever in patients with a haematological malignancy who were infected . Endotoxaemia was also found in 27% of 22 episodes of fever of unknown origin . In 45 afebrile patients neither neutropenia nor cytotoxic chemotherapy was a cause of endotoxaemia . Passage of endotoxin from portal blood into the systemic circulation can contribute to unexplained fever in immunosuppressed patients.

Fed Proc, 1984 Apr, 43(5), 1283 - 8
Role of protein degradation in the regulation of cellular protein content and amino acid pools; Scornik OA; A decrease in the rate of protein degradation contributes to the accumulation of cellular protein during growth or storage of dietary amino acids . Examples of this process in the liver of live mice are reviewed . One aspect of this regulation is the direct effect of amino acids on the rate of protein breakdown . At least in the case of histidine starvation in Chinese hamster ovary cells, the regulatory mechanism recognizes the level of aminoacylation of tRNA.

J Virol, 1984 Apr, 50(1), 213 - 9
Analysis of the coliphage T5 DNA ejection process with free and liposome-associated TonA protein; Tosi F et al.; Outer membrane protein TonA, the receptor for coliphage T5, has been partially purified and incorporated into the phospholipid bilayer of liposomes . Adsorption of the phage to its receptor in either a free or liposome-associated form is fast and sufficient to trigger the ejection of encapsidated DNA . In both in vitro systems the exit of DNA from the phage capsid is a very slow process . Ejected DNA can partially accumulate inside the liposome aqueous compartment, but the transfer from the phage head to the liposome internal space is never complete, perhaps because the liposome volume is too small . The presence of polyamines or divalent cations (magnesium) or both in the incubation medium diminished the extent of DNA ejection, possibly by stabilizing DNA inside the head . DNA movement was slowed as the temperature was decreased from 37 to 18 degrees C . Furthermore, incubation at 4 degrees C totally prevented this DNA movement, even if a large part of the DNA had already exited the capsid.

Can J Microbiol, 1984 Apr, 30(4), 515 - 8
Construction of a shuttle vector and expression in Streptomyces lividans of the sulfonamide-resistance gene derived from Escherichia coli plasmid pSAS1206; Shareck F et al.; We have constructed a hybrid plasmid using Streptomyces lividans plasmid p1J101 and Escherichia coli plasmid pSAS1206 . This plasmid, designated pFSH102, is able to replicate in both hosts and the sulfonamide-resistance gene encoded by pSAS1206 is phenotypically expressed in S . lividans.

J Gen Microbiol, 1984 Apr, 130 ( Pt 4), 861 - 8
Location and direction of transcription of the ptsH and ptsI genes on the Escherichia coli K12 genome; Britton P et al.; Recombinant plasmids were constructed that carried various fragments of the DNA specifying the Escherichia coli genes ptsH and (part of) ptsI, the genes for the common components of the phosphoenolpyruvate: sugar phosphotransferase . Expression of plasmid-specified functions in minicells showed that ptsH and ptsI were transcribed clockwise . Most of the transcription of ptsI was from the ptsH promoter, but some was from a second site within or after ptsH.

Gene, 1984 Apr, 28(1), 37 - 43
Plasmid vectors for positive galactose-resistance selection of cloned DNA in Escherichia coli; Ahmed A; Insertion of a HindIII-EcoRI fragment carrying part of the gal operon from lambda gal+ into pBR322 yields a plasmid (pAA3) which confers strong galactose sensitivity on E . coli strains deleted for the gal operon . Sensitivity to galactose is caused by the expression of kinase and transferase (but not epimerase) genes from a promoter located in the tet gene of pBR322 . Insertion of a DNA fragment carrying Tn9 at the HindIII junction blocks gal expression and produces a galactose-resistant phenotype . Hence, galactose resistance can be used to select DNA fragments cloned at the HindIII site . The system was used efficiently for cloning lambda, yeast, and human DNA . The cloned fragments can be screened directly for the presence of promoters by testing for tetracycline resistance . Alternatively, these plasmids can be used as cosmids for cloning large fragments of DNA at a number of sites . Construction of several related vectors is described.

Gene, 1984 Apr, 28(1), 127 - 32
Plasmid vectors designed for high-efficiency expression controlled by the portable recA promoter-operator of Escherichia coli; Shirakawa M et al.; A novel expression vector using the 236-bp promoter-operator fragment of the recA gene (recApo) of Escherichia coli has been constructed . This DNA fragment contains complete signals for the initiation of RNA synthesis, as well as for regulation by the lexA product, but lacks the coding sequence for the RecA protein . The strength of the recA promoter was examined by assaying beta-galactosidase activity expressed from a cro-lacZ fused gene placed downstream of the promoter . Under noninducing conditions, the promoter was regulated by the LexA protein, and the fused gene was expressed only weakly . Upon induction by nalidixic acid in a recA+ strain, high expression was observed for an extended period . After 5 h under inducing conditions, as much as 11% of the total cellular protein was cro-lacZ product . The expression level was higher than that from promoters of lac, trp, and lambda early genes.

Gene, 1984 Apr, 28(1), 103 - 12
Absence of direct repeats flanking transposons resulting from intramolecular transposition; Thorpe PA et al.; Tn2660 is an ampicillin-resistance-conferring transposon with a high degree of homology for the transposon Tn3 . The nucleotide sequences flanking the termini of Tn2660 have been determined on plasmids inferred to have resulted from both inter- and intramolecular transposition of Tn2660 . In all cases, transposition of Tn2660, as of Tn3, creates 5-bp flanking direct repeats, except following intramolecular transposition resulting from trans ligation . In this case, in R6K replicons, the nucleotide sequence between the two Tn2660 elements is stably inverted from the normal orientation, and 5-bp direct repeats do not flank each transposon, but instead flank opposite ends of the two transposon copies.

Gann, 1984 Apr, 75(4), 325 - 33
Comparison of genome structures among three different strains of avian erythroblastosis virus; Nishida T et al.; The genomic DNAs of two strains of avian erythroblastosis virus (AEV), AEV-R and AEV-H, were molecularly cloned in Escherichia coli using lambda Charon 16A as a vector . Comparison of the restriction maps of the cloned DNAs with that of AEV-ES4 DNA revealed that R strain is identical with ES4 strain, but totally differs from H strain . The erbB gene product of AEV-R and that of AEV-H were shown to be glycoproteins with molecular weights of 68,000 and 72,000, respectively . Nucleotide sequence analysis of the 3' part of the erbB gene showed that the erbB gene in AEV-R is 126 base pairs (bp) shorter than that in AEV-H, which might result in some difference in tumorigenicity between these viruses.

Acta Pathol Microbiol Immunol Scand {B}, 1984 Apr, 92(2), 93 - 9
Cytoskeletal disarrangement in rat intestinal epithelium after in vivo exposure to secretagogues; Hansson HA et al.; Cholera toxin (CT) and E . coli heat-labile enterotoxin (LT) induced hypersecretion in the small intestine, as did cytochalasin B and dibutyryl-cyclic AMP (DB-cAMP) . The cytoskeleton in the apical part of the intestinal epithelial cells was disorganized after challenge with either of the four secretagogues, but not cholera B-subunit toxoid, as revealed by immunofluorescence microscopy using actin and the intermediate filament keratin as markers . Electron microscopic analysis confirmed the re-engagement of the terminal web and the appearance of short microvilli lacking most of their normally observed central core of actin filaments . Pretreatment with chloroquine prevented cytoskeletal disorganization as well as hypersecretion by CT, but not by cytochalasin B . A similar effect was achieved with chloroquine on CT-induced fluid secretion, while chlorpromazine inhibited the fluid response to cytochalasin B as well as to CT . The observed cytoskeletal re-engagement might be one of the reactions behind enterotoxic diarrhoea.

J Antimicrob Chemother, 1984 Apr, 13(4), 383 - 8
Pivmecillinam for bacteriuria in pregnancy; Sanderson P et al.; Pivmecillinam was given to 44 women with bacteriuria in pregnancy . Treatment was successful in 33 (87%) out of the 38 patients assessed . Thirty women subsequently received at random either a low-dose of pivmecillinam for up to three months or acted as a control group . Further bacteriuric episodes during pregnancy were recorded only in three patients in the control group . Thirty-nine out of the 41 women followed to term delivered healthy babies . One infant was stillborn and another child had a cleft palate . Neither was considered to be related to treatment with pivmecillinam.

EMBO J, 1984 Apr, 3(4), 807 - 11
Insertion element IS5 contains a third gene; Rak B et al.; We have previously shown that IS5 contains two genes encoded on opposite DNA strands within the same stretch of DNA . Here we present evidence that a third gene and its promoter are present on IS5 . The newly discovered gene, ins5C , is contained within the longest gene of IS5, ins5A , but encoded by the complementary DNA strand . The three genes comprise a total of 519 codons present on the 1195-bp element . The arrangement of these genes represents a coding structure of unprecedented compactness.

Proc Natl Acad Sci U S A, 1984 Apr, 81(7), 2227 - 31
Isolation and characterization of a stage-specific transforming gene, Tlym-I, from T-cell lymphomas; Lane MA et al.; A cellular transforming gene detected by transfection of mouse T-cell lymphoma DNA has been isolated by molecular cloning . This gene (designated Tlym-I) is homologous to a small conserved family of sequences present in normal mouse and human DNAs but is not related to any of the previously described viral or cellular transforming genes.

Proc Natl Acad Sci U S A, 1984 Apr, 81(7), 2030 - 4
Enzymatic techniques for the isolation of random single-base substitutions in vitro at high frequency; Abarzua P et al.; A general and efficient method has been developed to generate large numbers of single-base substitution mutations simply and rapidly . A unique f1 phage recombinant DNA cloning vector is described, which contains the phi X174 origin of viral strand DNA synthesis and allows one to direct mutagenesis to any specific segment of DNA . Gapped circular DNA is constructed by annealing viral single-stranded circular DNA {ss(c) DNA} with a mixture of linear duplex DNAs that have had their 3'-OH termini processively digested with Escherichia coli exonuclease III under conditions in which the resulting, newly generated 3'-OH termini present in the various hybrid molecules span the region of interest . Base changes are induced by misincorporation of an alpha-thiodeoxynucleoside triphosphate analog onto this primer-template, followed by DNA repair synthesis . The asymmetric segregation of mutants from wild-type sequences is accomplished by double-stranded replicative form DNA----ss(c) DNA synthesis in vitro, initiated from the phi X174 viral strand origin sequence present on the vector DNA . Mutated ss(c) DNA is screened by the dideoxy chain termination method . In one mutagenesis experiment, 21 independent single-base substitutions were isolated in a 72-nucleotide-long target region . DNA sequence analysis showed that all possible base transversions and transitions were represented.

J Surg Oncol, 1984 Apr, 25(4), 263 - 7
Unusual presentation of carcinoma of the vermiform appendix: a report of two cases; Waizbard E et al.; Two unusual presentations of perforated mucoid adenocarcinoma of the appendix are described . One patient presented with a large periappendicular abscess that extended into the gluteal region, the other with a retroperitoneal abscess and abscesses in the right flank and groin . Recognition of the source of the abscess may be difficult but is essential for the prevention and elimination of the sepsis which may be life-threatening . The presence of mucin in the drained pus is highly suggestive for perforated bowel carcinoma . A high index of suspicion and the performance of prompt diagnostic procedures may bring early surgical treatment and better results.

J Bacteriol, 1984 Apr, 158(1), 313 - 6
Direct evidence for specific binding of the replicative origin of the Escherichia coli chromosome to the membrane; Kusano T et al.; The origin of replication of the Escherichia coli chromosomal DNA binds with high affinity to outer membrane preparations . This specific binding requires a 463-base-pair region of origin DNA between positions -45 and +417 of the oriC map . We show that binding does not require the presence of adjacent regions . From further analysis, we conclude that more than one binding site resides within the 325-base-pair fragment between positions +38 (BamHI) and +417 (XhoI) . When this fragment is cut, two pieces bind with high affinity and one binds with lesser affinity . The binding ability of one of the high affinity sites is abolished by cutting it at position +92 with BamHI.

J Bacteriol, 1984 Apr, 158(1), 307 - 12
Complete nucleotide sequence of mini-Rts1 and its copy mutant; Kamio Y et al.; The nucleotide sequence of the whole mini-Rts1 genome consisting of 1,855 base pairs was determined . In addition to the cluster of five 24-base-pair direct repeats previously detected (Y . Kamio and Y . Terawaki, J . Bacteriol . 155:1185-1191, 1983), another cluster of three 21-base-pair direct repeats was found . All eight repeats were located in one direction and contained a consensus sequence TTCCCCPyPyPuPuCACACACC . Between the two clusters, a large open reading frame that could encode a 32,980-dalton polypeptide consisting of 288 amino acids was assigned . The molecular size predicted from the amino acid composition was close to the value of a unique mini-Rts1 product, the RepA protein, newly determined in minicells . A copy mutant of mini-Rts1 obtained in vitro by hydroxylamine treatment contained two-base-pair substitutions, one of which was located in the RepA protein coding region, and the other was close to the region where the oriC homologous sequences exist.

J Bacteriol, 1984 Apr, 158(1), 300 - 6
Overproduction of subunit a of the F0 component of proton-translocating ATPase inhibits growth of Escherichia coli cells; Kanazawa H et al.; A hybrid plasmid, pKY159, carrying the promoter and the proximal region of the gene cluster for proton-translocating ATPase caused growth inhibition of Escherichia coli cells (K . Yamaguchi and M . Yamaguchi, J . Bacteriol . 153:550-554, 1983) . The mechanism of this growth inhibition was studied, especially in terms of the responsible gene(s) . Insertion of IS1, IS5, or gamma delta between the promoter and the gene for a possible component of the ATPase of 14,000 daltons (14K protein) released the inhibitory effect by pKY159 . Deletion of the gene for subunit a also released the effect . However, deletion in the gene for the 14K protein released the effect only with an additional insertion within the gene . These results suggested that overproduction of subunit a is closely related to growth inhibition, whereas the 14K protein is not.

J Bacteriol, 1984 Apr, 158(1), 279 - 85
Relative activities of the transcriptional regulatory sites in the rplKAJLrpoBC gene cluster of Escherichia coli; Ralling G et al.; The pattern of transcription of the rplKAJLrpoBC gene cluster of Escherichia coli appears to be complex . At least four different promoters and a transcriptional attenuator have been identified . To compare the relative effect of each of the putative promoters and the attenuator on transcription of these genes, we fused these regulatory sites to lacZ . These transcriptional fusions were constructed on lambda transducing phages so a single copy of each could be stably integrated into the chromosome . The level of beta-galactosidase in a lysogen of each phage reflects the activity of the transcriptional regulatory site . We find that the promoters preceding rplK (rplKp) and rplJ (rplJp) are indeed the major promoters of this gene cluster . The minor promoter before rplL (rplLp) is much weaker and contributes little to the transcription of the downstream genes . Under these conditions, we find no evidence of a promoter (rpoBp) in the rplL-rpoB intercistronic region . The attenuator (atn) terminates ca . 70% of the transcripts initiated at the promoters preceding it . Although we cannot rule out that some transcripts from rplKp may read through into rplJLrpoBC, we find that rplJp alone is sufficient for high-level expression of these genes.

J Bacteriol, 1984 Apr, 158(1), 246 - 52
Escherichia coli intracellular pH, membrane potential, and cell growth; Zilberstein D et al.; We studied the changes in various cell functions during the shift to alkaline extracellular pH in wild-type Escherichia coli and in strain DZ3, a mutant defective in pH homeostasis . A rapid increase in membrane potential (delta psi) was detected in both the wild type and the mutant immediately upon the shift, when both cell types failed to control intracellular pH . Upon reestablishment of intracellular pH - extracellular pH and growth in the wild type, delta psi decreased to a new steady-state value . The electrochemical proton gradient (delta muH+) was similar in magnitude to that observed before the pH shift . In the mutant DZ3, delta psi remained elevated, and even though delta muH+ was higher than in the wild type, growth was impaired . Cessation of growth in the mutant is not a result of cell death . Hence, the mutant affords an interesting system to explore the intracellular-pH-sensitive steps that arrest growth without affecting viability . In addition to delta muH+, we measured respiration rates, protein synthesis, cell viability, induction of beta-galactosidase, DNA synthesis, and cell elongation upon failure of pH homeostasis . Cell division was the only function arrested after the shift in extracellular pH . The cells formed long chains with no increase in colony-forming capacity.

J Bacteriol, 1984 Apr, 158(1), 216 - 21
Map location of the pcbA mutation and physiology of the mutant; Bryan SK et al.; Many temperature-resistant revertants of a polA1 polB polCts (HS432) strain are PolI+ (by either suppression of the polA1 amber allele or intragenic reversion) but remain polCts (contain a temperature-sensitive DNA polymerase III) . It appears that DNA replication in such temperature-resistant revertants depends on an extragenic mutation, pcbA, already present in the parent strain and not linked to any of the DNA polymerase loci . This allele allows DNA replication dependent on DNA polymerase I and bypasses a temperature-sensitive DNA polymerase III (polC bypass), so that reversion to PolI+ makes the strain temperature resistant . This pathway of DNA replication also supports phage and plasmid DNA replication . At restrictive temperature, these mutants display a normal response to UV irradiation but show increased sensitivity to the alkylating agent methyl methanesulfonate . We have located pcbA linked to dnaA.

J Bacteriol, 1984 Apr, 158(1), 195 - 201
DNA-stimulated ATPase activity on the lon (CapR) protein; Charette MF et al.; The gene product of the pleiotropic lon (also called capR) locus in Escherichia coli, the CapR protein, is an ATP hydrolysis-dependent protease and a nonspecific nucleic acid-binding protein . We demonstrated that it is also a DNA-stimulated adenosine triphosphatase (ATPase) . This new activity is distinct from the protease-associated ATPase activity and occurs in the absence of proteolytic substrate . The reaction requires the presence of a divalent cation and has a pH optimum of 8.0 . The products of the reaction are ADP and inorganic phosphate . No adenylation or phosphorylation of the DNA or proteins was detected . The maximum rate of ATP hydrolysis occurs in the presence of supercoiled (form I) DNA . Relaxed circles (form II), double-stranded DNA, and single-stranded DNA are less effective in promoting ATPase activity, whereas RNA is inactive . The DNA-stimulated ATPase activity is inhibited by a mutationally altered form of the CapR protein called the CapR9 protein . The interaction of the CapR and CapR9 subunits suggests that this enzymatic activity of the CapR protein is oligomeric in the presence of DNA . Our in vitro experiments indicate a possible role for nucleic acids in the regulation of all lon (capR) activity.

J Bacteriol, 1984 Apr, 158(1), 110 - 4
Regulation of cyclic AMP synthesis in Escherichia coli K-12: effects of the rpoD800 sigma mutation, glucose, and chloramphenicol; Grossman AD et al.; An immediate 12-fold inhibition in the rate of beta-galactosidase synthesis occurs in Escherichia coli cells containing the mutant sigma allele rpoD800 after a shift to 42 degrees C . In the present study we characterize the nature of the inhibition . The severe inhibition of beta-galactosidase synthesis was partly relieved by cyclic AMP (cAMP) . We inferred that the inhibition might be mediated by a decreased intracellular concentration of cAMP . Consistent with this inference, the rate of cAMP accumulation in mutant cells after a temperature upshift was depressed relative to that in wild-type cells . Glucose and chloramphenicol, two agents known to inhibit differentially beta-galactosidase mRNA synthesis, caused a similar inhibition in the rate of cAMP accumulation . Thus, three diverse stimuli, glucose, chloramphenicol, and a temperature-sensitive sigma mutation, appear to affect beta-galactosidase synthesis by regulating the synthesis of cAMP.

Exp Parasitol, 1984 Apr, 57(2), 172 - 7
Entamoeba histolytica: virulence enhancement of isoenzyme-stable parasites; Mirelman D et al.; Pathogenic Entamoeba histolytica isolated from patients with clinical amoebiasis can be differentiated from nonpathogenic E . histolytica obtained from asymptomatic carriers on the basis of the electrophoretic pattern of their isoenzymes . Virulence of different strains of axenically grown trophozoites of Entamoeba histolytica, as determined by various laboratory tests, such as damage to tissue culture monolayers, or their ability to cause an hepatic abscess in a hamster, are known to vary considerably . Reassociation of trophozoites of strain HK-9 with certain Escherichia coli strains for short periods of time markedly augmented their virulence, as tested by the above-mentioned methods . The bacterial association, however, did not cause any change in the electrophoretic pattern of amoebic isoenzymes (zymodeme).

Infect Immun, 1984 Apr, 44(1), 16 - 21
Porins of Brucella species; Douglas JT et al.; The outer membrane of Brucella species contains two major proteins, denoted as group 2 and group 3 proteins (Verstreate et al., Infect . Immun . 35:979-989, 1982) . We reconstituted proteoliposomes from the purified proteins and egg phosphatidylcholine and showed that group 2 proteins, but not a group 3 protein, had the porin activity . The influx rates of sugars of various sizes into the proteoliposomes indicated that the porin channels had apparent diameters in a range comparable to that of Escherichia coli OmpF porin and that the channels were predominantly open . Among different Brucella species, there were small but detectable differences in the channel diameter, and it was possible to explain the differential sensitivity of several Brucella species to diagnostic dyes on the basis of these observed differences.

Cell, 1984 Apr, 36(4), 925 - 31
Construction and characterization of autonomously replicating plasmids in the green unicellular alga Chlamydomonas reinhardii; Rochaix JD et al.; Plasmids that replicate autonomously in Chlamydomonas reinhardii were constructed by inserting random DNA fragments from this alga into a plasmid containing the yeast ARG4 locus . Arginine prototrophy was used as a selective marker . The presence of free plasmids in the DNA of the transformants was demonstrated by hybridization with a specific plasmid probe and by recovering these plasmids in E . coli after transformation . Four of them were characterized . Their inserts of 415, 257, 153, and 102 bp all hybridize to chloroplast DNA and were localized on the physical map of the chloroplast genome . One of these plasmids also promotes autonomous replication in yeast . Sequence analysis of the inserts of the plasmids reveals several short direct and inverted repeats and two semiconserved AT-rich elements of 19 and 12 bp that may play a role in promoting autonomous replication in C . reinhardii.

Cell, 1984 Apr, 36(4), 1097 - 103
Multimerization of high copy number plasmids causes instability: CoIE1 encodes a determinant essential for plasmid monomerization and stability; Summers DK et al.; Although the natural multicopy plasmid CoIE1 is maintained stably under most growth conditions, plasmid cloning vectors related to it are relatively unstable, being lost at frequencies of 10(-2)-10(-5) per cell per generation . Evidence suggests that CoIE1 and related plasmids are partitioned randomly at cell division and that plasmid stability is correlated inversely with plasmid multimerization; factors or conditions that reduce multimerization increase stability . Cells containing plasmid multimers segregate plasmid-free cells because the multimers are maintained at lower copy numbers than monomers, as predicted by origin-counting models for copy number control . CoIE1 is stable because it encodes a determinant, cer, that is necessary for recA-, recF-, and recE-independent recombination events that efficiently convert any multimers to monomers . We have localized monomerizing and stability determinants of CoIE1 to within a 0.38 kb region that, when cloned into plasmid vectors, greatly increases their stability.

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