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 identifie