Escherichia coli

Cell, 1983 Jan, 32(1), 141 - 9
Autoregulation of the Escherichia coli crp gene: CRP is a transcriptional repressor for its own gene; Aiba H; The restriction fragments carrying the region preceding the Escherichia coli crp structural gene were transcribed . The 5' end of the crp mRNA was determined by RNAase partial digestion and S1 digestion methods . Thus the crp gene has been shown to possess a 167 bp leader . CRP-cAMP specifically prevents the crp transcription . In other words, the crp gene is regulated autogenously . DNAase foot-printing studies indicated that CRP-cAMP binds to the crp gene at positions +26 to +67 . This region exhibits a striking sequence homology to the CRP-binding sites in other genes . CRP and RNA polymerase bind to the crp regulatory region simultaneously . These results suggest a different mechanism for transcriptional repression of the crp gene by CRP-cAMP from that of a typical operator-repressor model.

Cell, 1983 Jan, 32(1), 131 - 40
The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding; Germino J et al.; We have tagged the replication initiator protein of the plasmid R6K near the C-terminal end by fusion, in the correct reading frame, with the 89 amino acid long N-terminal alpha-donor polypeptide of beta-galactosidase of E . coli . This fusion was carried out with recombinant DNA methods . The protein chimera thus generated retained the activities of both initiation of DNA replication in vivo at the replication origin gamma of R6K and hydrolysis of beta-galactopyranoside when complemented in vivo with the alpha-acceptor polypeptide coded by the lac Z gene containing the M15 deletion . Using the simple and convenient assay for detecting beta-galactosidase, we have partially purified the tagged replication initiator, and have demonstrated that the protein binds to specific DNA sequences of the R6K chromosome . The protein bound to DNA sequences located at two places in the 5' untranslated leader region of the initiator protein cistron.

J Virol, 1983 Jan, 45(1), 478 - 81
Simian virus 40 promoters direct expression of the tetracycline gene in plasmid pACYC184; Jenkins FJ et al.; Insertion of HindIII DNA fragments into the HindIII site of plasmid pACYC184 destroys the promoter of the plasmid tetracycline resistance gene and causes Escherichia coli cells harboring recombinant plasmids to be tetracycline sensitive and chloramphenicol resistant . The HindIII-C DNA fragment of simian virus 40 contains the two virus promoters and the virus origin of replication . We report the isolation of recombinant plasmids that contained the simian virus 40 HindIII-C DNA fragment at the HindIII site but were capable of conferring tetracycline resistance to E . coli cells . The viral promoter sequences contained in the HindIII-C fragment presumably replaced the inactivated tetracycline resistance gene promoter sequences and enabled transcription of the tetracycline resistance gene.

J Bacteriol, 1983 Jan, 153(1), 416 - 22
Escherichia coli mutants defective in the uncH gene; Humbert R et al.; Plasmids carrying cloned segments of the unc operon of Escherichia coli have been used in genetic complementation analyses to identify three independent mutants defective in the uncH gene, which codes for the delta subunit of the ATP synthetase . Mutations in other unc genes have also been mapped by this technique . ATPase activity was present in extracts of the uncH mutants, but the enzyme was not as tightly bound to the membrane as it was in the parental strain . ATP-dependent membrane energization was absent in membranes isolated from the uncH mutants and could not be restored by adding normal F1 ATPase from the wild-type strain . F1 ATPase prepared from uncH mutants could not restore ATP-dependent membrane energization when added to wild-type membranes depleted of F1 . Membranes of the uncH mutants were not rendered proton permeable as a result of washing with low-ionic-strength buffer.

J Bacteriol, 1983 Jan, 153(1), 408 - 15
Characterization of the mgl operon of Escherichia coli by transposon mutagenesis and molecular cloning; Harayama S et al.; We used transposon insertion mutagenesis, molecular cloning, and a novel procedure for in vitro construction of polar and nonpolar insertion mutations to characterize the genetic organization and gene products of the beta-methylgalactoside (Mgl) transport system, which utilizes the galactose-binding protein . The data indicate that the mgl operon contained three genes, which were transcribed in the order mglB, mglA, and mglC . The first gene coded for the 31,000 Mr galactose-binding protein, which was synthesized as a 3,000-dalton-larger precursor form . The mglA product was a 50,000 Mr protein which was tightly associated with the membrane, and the mglC product was a 38,000 Mr protein which was apparently loosely associated with the membrane and was probably located on the internal face of the cytoplasmic membrane . Identification of gene products was facilitated by in vitro insertion of a fragment of Tn5 containing the gene conferring kanamycin resistance into a restriction site in the operon . The fragment proved to have a polar effect on the expression of promoter-distal genes only when inserted in one of the two possible orientations . The three identified gene products were necessary and apparently sufficient for transport activity, but only the binding protein was required for chemotaxis towards galactose . The transport system appeared to contain the minimum number of components for a binding protein-related system: a periplasmic recognition component, a transmembrane protein, and a peripheral membrane protein that may be involved in energy linkage.

J Bacteriol, 1983 Jan, 153(1), 395 - 407
Mapping of chromosomal IS5 elements that mediate type II F-prime plasmid excision in Escherichia coli K-12; Timmons MS et al.; Three IS5 elements were mapped in overlapping chromosomal segments on a series of F-prime plasmids by restriction analysis and hybridization . IS5A was located clockwise of proA near 6 min, IS5B was located clockwise of purE near 12 min, and IS5C was tentatively located near 14 min on the Escherichia coli K-12 map . The physical structures of nine type II F-prime plasmids that contain chromosomal DNA from this region indicated that these plasmids were excised from the chromosome by recombination between pairs of IS5 elements.

J Bacteriol, 1983 Jan, 153(1), 241 - 52
Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins; Nikaido H et al.; Rates of diffusion of uncharged and charged solute molecules through porin channels were determined by using liposomes reconstituted from egg phosphatidylcholine and purified Escherichia coli porins OmpF (protein 1a), OmpC (protein 1b), and PhoE (protein E) . All three porin proteins appeared to produce channels of similar size, although the OmpF channel appeared to be 7 to 9% larger than the OmpC and PhoE channels in an equivalent radius . Hydrophobicity of the solute retarded the penetration through all three channels in a similar manner . The presence of one negative charge on the solute resulted in about a threefold reduction in penetration rates through OmpF and OmpC channels, whereas it produced two- to tenfold acceleration of diffusion through the PhoE channel . The addition of the second negatively charged group to the solutes decreased the diffusion rates through OmpF and OmpC channels further, whereas diffusion through the PhoE channel was not affected much . These results suggest that PhoE specializes in the uptake of negatively charged solutes . At the present level of resolution, no sign of true solute specificity was found in OmpF and OmpC channels; peptides, for example, diffused through both of these channels at rates expected from their molecular size, hydrophobicity, and charge . However, the OmpF porin channel allowed influx of more solute molecules per unit time than did the equivalent weight of the OmpC porin when the flux was driven by a concentration gradient of the same size . This apparent difference in "efficiency" became more pronounced with larger solutes, and it is likely to be the consequence of the difference in the sizes of OmpF and OmpC channels.

J Bacteriol, 1983 Jan, 153(1), 232 - 40
Porin channels in Escherichia coli: studies with beta-lactams in intact cells; Nikaido H et al.; Wild-type Escherichia coli K-12 produces two porins, OmpF (protein 1a) and OmpC (protein 1b) . In mutants deficient in both of these "normal" porins, secondary mutants that produce a "new" porin, protein PhoE (protein E), are selected for . We determined the properties of the channels produced by each of these porins by measuring the rates of diffusion of various cephalosporins through the outer membrane in strains producing only one porin species . We found that all porin channels retarded the diffusion of more hydrophobic cephalosporins and that with monoanionic cephalosporins a 10-fold increase in the octanol-water partition coefficient of the solute produced a 5- to 6-fold decrease in the rate of penetration . Electrical charges of the solutes had different effects on different channels . Thus, with the normal porins (i.e., OmpF and OmpC proteins) additional negative charge drastically reduced the penetration rate through the channels, whereas additional positive charge significantly accelerated the penetration . In contrast, diffusion through the PhoE channel was unaffected by the presence of an additional negative charge . We hypothesize that the relative exclusion of hydrophobic and negatively charged solutes by normal porin channels is of ecological advantage to E . coli, which must exclude hydrophobic and anionic bile salts in its natural habitat . The properties of the PhoE porin are also consistent with the recent finding (M . Argast and W . Boos, J . Bacteriol . 143:142-150, 1980; J . Tommassen and B . Lugtenberg, J . Bacteriol . 143:151-157, 1980) that its biosynthesis is derepressed by phosphate starvation; the channel may thus act as an emergency pore primarily for the uptake of phosphate and phosphorylated compounds.

J Bacteriol, 1983 Jan, 153(1), 191 - 9
Mechanism of CRP-mediated cya suppression in Escherichia coli; Harman JG et al.; Escherichia coli strain NCR30 contains a cya lesion and a second-site cya suppressor mutation that lies in the crp gene . NCR30 shows a pleiotropic phenotypic reversion to the wild-type state in expressing many operons that require the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex for positive control . In vivo beta-galactosidase synthesis in NCR30 was sensitive to glucose-mediated repression, which was relieved not only by cAMP but also by cyclic GMP and cyclic CMP . The CRP isolated from NCR30 differed from the protein isolated from wild-type E . coli in many respects . The mutant protein bound cAMP with four to five times greater affinity than wild-type CRP . Protease digestion studies indicated that native NCR30 CRP exists in the cAMP-CRP complex-like conformation . The protein conferred a degree of cAMP independence on the in vitro synthesis of beta-galactosidase . In addition, the inherent positive control activity of the mutant protein in vitro was enhanced by those nucleotides that stimulate in vivo beta-galactosidase synthesis in NCR30 . The results of this study supported the conclusion that the crp allele of NCR30 codes for a protein having altered effector specificity yet capable of promoting positive control over catabolite-sensitive operons in the absence of an effector molecule.

Methods Enzymol, 1983, 97, 100 - 12
Phage lambda receptor (lamB protein) in Escherichia coli; Schwartz M; The main properties of the lambda receptor are summarized in the table . Because these can be studied by a combination of genetic, biophysical, and biochemical techniques, the lambda receptor now appears to represent one of the best systems for study of structure-function relationships in a membrane protein . In addition, as explained in this volume, it also constitutes a good system for study of the export of proteins to extracytoplasmic locations.

Princess Takamatsu Symp, 1983, 13, 267 - 76
Infidelity of DNA synthesis as a cause of mutagenesis; Loeb LA et al.; The concept underlying these studies is that a major determinant of mutagenesis involves perturbations in the fidelity of DNA replication . i.e., the accuracy by which DNA polymerases copy DNA templates . To investigate this relationship, we have designed in vitro assays to measure the accuracy of DNA replication and used these systems to screen for and to quantitate factors that promote errors in DNA synthesis . Using DNA polymerase from bacteria, the frequency of mistakes with phi X174 DNA as a template approaches 10(-7) and is similar to the spontaneous mutation rates in bacterial cells . In contrast, DNA polymerases from animal cells are more error-prone . The differences in fidelity among mammalian DNA polymerases which lack error-correcting mechanisms suggest that these enzymes enhance accuracy by improving base-selection . Thus, mutants in DNA polymerase-alpha might be altered in base-selection . Chinese hamster V79 cell mutants selected by resistance to aphidicolin, a specific inhibitor of DNA polymerase-alpha, have been reported (Somatic Cell Genet., 7: 235-253, 1981) . DNA polymerase-alpha was purified from mitochondria-free crude extracts of these mutants by sequential column chromatography using DEAE-cellulose and phosphocellulose . DNA polymerase-alpha purified from one of the mutants is 10-fold more resistant to aphidicolin than the same enzyme purified from the parental cells . Moreover, the apparent Km for dCTP is 1.0 +/- 0.4 microM for the mutant polymerase and 10 +/- 4 microM for the parental enzyme . These observed differences are in accord with the known competition between aphidicolin and dCTP, and provide a mechanism for the aphidicolin resistance of the mutant, i.e., the decrease in Km for dCTP . The elevated spontaneous and induced mutation rate exhibited by this mutant could be mediated by the alteration in DNA polymerase-alpha . With DNA replicating enzymes from a variety of sources, enhancement of mutagenesis has been demonstrated by alteration in precursor pools, damage to DNA templates, loss of nucleotide bases on DNA, metal ions that interact with nucleotide bases, and organic compounds that intercalate into DNA . The alterations of deoxynucleoside triphosphate pools also occur after treatment of animal cells with known mutagens . This observation may provide a new mechanism for mutagenesis by these agents independent of alterations in DNA.

Mol Gen Genet, 1983, 192(1-2), 177 - 86
Cloning of the ugp region containing the structural genes for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli; Schweizer H et al.; Using a novel positive selection method for G3P transport activity, lambda phages that carry either all or part of ugp, the genes of the pho regulon-dependent G3P transport system of Escherichia coli were isolated from a library of EcoRI fragments of Escherichia coli established in lambda gt7 . By subcloning EcoRI fragments carried by the different phages into the multicopy plasmids pACYC184 and pUR222, it was shown that two chromosomal fragments of 6.0 and 6.6 kb are required for the expression of ugp, whereas all the structural information is located on the 6.6 kb EcoRI fragment . A restriction map of the cloned DNA was established and the extent of ugp genes determined by Tn5 insertions . Using ugp-lacZ fusions, it could be shown that the ugp region consists of at least two different operons that are transcribed in the same direction (counterclockwise) on the E . coli chromosome.

Mol Gen Genet, 1983, 192(1-2), 10 - 4
Genetic characterization of a gene for prolipoprotein signal peptidase in Escherichia coli; Yamagata H et al.; A mutation (lspA, prolipoprotein signal peptidase) rendering the prolipoprotein signal peptidase temperature-sensitive in Escherichia coli has been analyzed . The mutation was mapped in the dnaJ-rpsT-ileS-dapB region by interrupted mating with various Hfr strains and P1 phage transduction . lambda transducing phage lambda ddapB2 that carries the rpsT-ileS-dapB region was shown to complement the lspA mutation . Plasmid pLC3-13 which had been isolated from Clarke and Carbon's collection as a plasmid carrying the lspA locus was shown to carry the dnaJ and rpsT loci . Complementation analysis with plasmids carrying various DNA fragments derived from pLC3-13 showed that the lspA locus is between the rpsT and ileS loci . The wildtype allele was dominant over the lspA allele.

EMBO J, 1983, 2(11), 1863 - 8
Ham22, a mini-F mutation which is lethal to host cell and promotes recA-dependent induction of lambdoid prophage; Karoui H et al.; A mini-F region 800 bp long, located between the two F origin sites, plays an essential role in the relationship between the F plasmid and its host . This region comprises two sets of overlapping coding sequences: the first set codes for the newly identified H1 and H2 polypeptides; the second set codes for polypeptides G1 and G2 . A mini-F amber mutation (Ham22) causes the virtual disappearance of polypeptides H1 and H2 but only slightly reduces synthesis of polypeptides G1 and G2 . This mutation: (i) renders mini-F hybrids lethal to the host cells (conditional Hos- phenotype for host survival) and (ii) causes the induction of a resident prophage in recA+ strains (conditional Map- phenotype for maintenance of the prophage) . When an additional mutation prevents the synthesis of polypeptides G1 and G2, both the lethal character and the induction of the prophage are abolished . We conclude: (i) that polypeptides G1 and/or G2 are specific mini-F polypeptides involved in the plasmid-mediated killing effect and in the recA-dependent induction of the resident prophage and (ii) that, in normal conditions, polypeptides H1 and/or H2 negatively control (directly or indirectly) the action of polypeptides G1 and/or G2 . In relation to the analysis of indirect induction mediated by u.v.-irradiated lambda mini-F hybrids, we propose that polypeptides G1 and/or G2 are specific mini-F products involved in the activation of the bacterial SOS pathway . The H1/H2 and G1/G2 polypeptides could constitute the controlled mini-F signal enabling the coordination between cell division and F plasmid replication.

Mol Gen Genet, 1983, 191(3), 347 - 52
Molecular characterization of ilvC specialized transducing phages of Escherichia coli K-12; Baylor NW et al.; A series of lambda defective ilvC specialized transducing phage has been isolated which carry regions of isoleucine and valine structural and regulatory genes derived from the ilv cluster at minute 83 on the linkage map of the chromosome of Escherichia coli K-12 . The ilv genes carried by these phages and their order have been determined by transduction of auxotrophs . The ilvC+ lysogen of an ilvC- strain gave rise, after heat induction of the lysogen, to transducing particles which carried the wild-type allele of the cya-marker . Further experiments have shown that the lambda defective ilvC phages were able to cotransduce a rho-15ts mutation as well as a rep-5 mutation . Hence, the order of the clockwise excision of the ilv cluster was found to be ilvC-rho-rep-cya . Enzyme levels in strains carrying the lambda defective ilvC phages indicated the the ilvC gene was not altered by the insertion of lambda into the ilv cluster . The isolation and digestion of lambda defective ilvC DNA by EcoRI and HindIII restriction endonucleases demonstrated that the specialized transducing phages carried part of the genome from the E . coli K-12 chromosome.

Mol Gen Genet, 1983, 190(1), 70 - 9
The products of gene A of the related phages Mu and D108 differ in their specificities; Toussaint A et al.; By recombination between different mutants of mutator phages Mu and D108, we isolated a set of viable hybrids . The structure of the hybrids was analyzed by digestion with different restriction enzymes . Genetic studies show that hybrids which carry the left end of the Mu genome complement a mini-Mu deleted from within the A gene as well as Mu while hybrids with the left end of the D108 genome or D108 do not . Vice versa, hybrids with the left end of the D108 genome or D108, but not hybrids with the left end of the Mu genome or Mu complement a mini-D108 deleted from within the A gene . The nucleotide sequence of the A gene of Mu and its equivalent on D108 are mainly similar except on their left end . These observations demonstrate that the two pA products, although only partially different, have different specificities.

Mol Gen Genet, 1983, 190(1), 101 - 11
DNA repair properties of Escherichia coli tif-1, recAo281 and lexA1 strains deficient in single-strand DNA binding protein; Whittier RF et al.; Mutations affecting single-strand DNA binding protein (SSB) impair induction of mutagenic (SOS) repair . To further investigate the role of SSB in SOS induction and DNA repair, isogenic strains were constructed combining the ssb+, ssb-1 or ssb-113 alleles with one or more mutations known to alter regulation of damage inducible functions . As is true in ssb+ strains tif-1 (recA441) was found to allow thermal induction of prophage lambda + and Weigle reactivation in ssb-1 and ssb-113 strains . Furthermore, tif-1 decreased the UV sensitivity of the ssb-113 strain slightly and permitted UV induction of prophage lambda + at 30 degrees C . Strains carrying the recAo281 allele were also constructed . This mutation causes high constitutive levels of RecA protein synthesis and relieves much of the UV sensitivity conferred by lexA- alleles without restoring SOS (error-prone) repair . In contrast, the recAo281 allele failed to alleviate the UV sensitivity associated with either ssb- mutation . In a lexA1 recAo281 background the ssb-1 mutation increased the extent of postirradiation DNA degradation and concommitantly increased UV sensitivity 20-fold to the level exhibited by a recA1 strain . The ssb-113 mutation also increased UV sensitivity markedly in this background but did so without greatly increasing postirradiation DNA degradation . These results suggest a direct role for SSB in recombinational repair apart from and in addition to its role in facilitating induction of the recA-lexA regulon.

Mol Gen Genet, 1983, 189(2), 226 - 34
Biologically active recombinant formed through DNA pairing by purified recA protein in vitro; Masukata H et al.; We have detected in vitro homologous recombination mediated by purified recA protein of Escherichia coli as a recombinant phage produced by using the DNA packaging system of phage lambda . When double-stranded DNA of phage lambda carrying amber mutations is incubated with double-stranded DNA carrying the wild-type genes in the presence of recA protein, Mg++ and ATP, and the DNA packaged, amber+ recombinant phage is produced at a high frequency . This reaction depends completely upon the function of the wild-type recA protein . After incubation of 32P-labeled linear DNA (Form III) with bromouracil-labeled circular DNA (Form I-Form II mixture) in the presence of recA protein, Mg++ and ATP, about 10% of the 32P-counts band at an intermediate density in CsCl equilibrium gradient . This fraction yields a high percentage of the recombinant phage after DNA packaging and shows the alpha-shaped and sigma-shaped joint molecules of linear and circular DNA under the electron microscope . Furthermore, we demonstrate that a non-homologous region inhibits the recombination reaction when it is between the marker concerned and the closer cos end . Our results indicate that recA protein acts directly in the initial step of recombination to join the homologous double-stranded DNA and that the resulting molecule can be matured into the recombinant DNA.

Mol Gen Genet, 1983, 189(1), 48 - 57
Regulation of the rpsU-dnaG-rpoD macromolecular synthesis operon and the initiation of DNA replication in Escherichia coli K-12; Lupski JR et al.; We have fused the E . coli dnaG 5' regulatory region to the TcR structural gene in the promoter probe plasmids pPV33 and pKK175-6 to demonstrate that a promoter activity is located on a 250 bp SacII-HindIII restriction fragment and that a transcription terminator, previously reported by nucleotide sequence (Smiley et al . 1982) to immediately precede the dnaG gene, acts as such in vivo . We have determined the complete nucleotide sequence of this controlling region and report: 1) tandem promoters on the same SacII-HindIII restriction fragment which promotes tet expression in the gene fusion experiments, 2) an open reading frame between these promoters and the dnaG gene which is the rpsU (ribosomal protein S21) gene, 3) a sequence homologous to the lambda nut site, 4) a possible LexA protein binding site on one of the dnaG promoters . This places the order on the E . coli genetic map at 66 min in the clockwise direction as rpsU-dnaG-rpoD which are all contained in a single macromolecular synthesis operon . We postulate a model for regulation of the initiation of DNA replication based on antitermination of the rpsU-dnaG-rpoD operon.

J Mol Appl Genet, 1983, 2(1), 83 - 99
Structure of the trifunctional trp-1 gene from Neurospora crassa and its aberrant expression in Escherichia coli; Schechtman MG et al.; The trifunctional trp-1 gene from Neurospora crassa was cloned by complementation of a phosphoribosylanthranilate isomerase-deficient mutant of E . coli . A 2.7-kb DNA sequence containing trp-1 was determined . Homology of the deduced trp-1 polypeptide sequence to the corresponding E . coli proteins is striking; the order of functional domains within trp-1 is NH2-glutamine amidotransferase-indoleglycerolphosphate synthase-phosphoribosylanthranilate isomerase-COOH (NH2-trpG-trpC-trpF-COOH) . Whereas trpF complementing activity can be detected in E . coli, trpC activity is absent . It is likely that translation of trp-1 does not proceed from the proper start site in E . coli; the carboxy terminal portion of the trp-1 polypeptide may be the only portion synthesized . Fusion of a bacterial amino terminus and ribosome binding site to the trp-1 coding region results in expression of trpC as well as trpF activity in E . coli . The locations of several startpoints for trp-1 mRNA synthesis were determined by the S1 nuclease mapping technique . DNA immediately 5' to the trp-1 transcription initiation region does not possess a sequence resembling the canonical TATAAA of eukaryotes.

Mutat Res, 1983 Jan, 107(1), 33 - 40
Induction of prophage lambda in Escherichia coli recA- strain by N-methyl-N'-nitro-N-nitrosoguanidine; Yamamoto K et al.; Induction of prophage by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) occurred in a recA- strain lysogenic for lambda phage at a level significantly higher than the spontaneous level although the frequency was much lower than that of induction in a recA+(lambda) strain . The plaque-forming ability of lambda c17 super-infecting the recA-(lambda) strain pretreated with MNNG increased with dose of MNNG as it did for super-infection of the recA+(lambda) strain, indicating that the frequency of maturation of lambda c17 increased owing to a decrease in the immunity of the lambda lysogen with dose of MNNG given to it . Further, the activity of lambda repressor in the recA-(lambda) strain decreased after treatment with MNNG as measured by the decrease of repressor-binding activity to lambda DNA although it decreased at a 3-fold slower rate than that in recA+(lambda) strain . From these results and others previously reported we conclude that inactivation of repressor leading to MNNG-initiated prophage induction takes place through two pathways, one being the recA-dependent normal process and the other a recA-independent process unique to the effect of MNNG.

Mutat Res, 1983 Jan, 119(1), 1 - 6
Repression of UV induction of lambda prophage by caffeine; Muche AA et al.; Caffeine was found to function as a repressor of UV-induced lambda prophage when added to the post-irradiation culture medium with both uvrB+ and uvrB- strains of E . coli K12 . Caffeine functioned as a repressor of lambda induction at high but not low inducing doses of UV . Caffeine alone at concentrations above 10 mg/plate functioned as an inducer of lambda prophage.

J Bacteriol, 1983 Jan, 153(1), 390 - 4
Assessment of a futile cycle involving reconversion of fructose 6-phosphate to fructose 1,6-bisphosphate during gluconeogenic growth of Escherichia coli; Daldal F et al.; In gluconeogenesis, fructose 6-phosphate is formed from fructose 1,6-bisphosphate, and if fructose 1,6-bisphosphate were reformed by the phosphofructokinase reaction there would be a "gluconeogenic futile cycle." We assessed the extent of this cycling in Escherichia coli growing on glycerol 3-phosphate, using a medium containing 32Pi . Fructose 1,6-bisphosphate coming from glycerol 3-phosphate should be unlabeled, but any coming from fructose 6-phosphate should contain label from the gamma-position of ATP . The amount of labeling of the 1-position of fructose 1,6-bisphosphate was only 2 to 10% of that of the gamma-position of ATP in a series of isogenic strains differing in phosphofructokinases (Pfk-1, Pfk-2, or Pfk-2) . In control experiments with glucose 6-phosphate instead of glycerol 3-phosphate, the two positions were equally labeled . Thus, although the presence of Pfk-2 causes gluconeogenic impairment (Daldal et al., Eur . J . Biochem., 126:373-379, 1982), gluconeogenic futile cycling cannot be the reason.

J Bacteriol, 1983 Jan, 153(1), 379 - 83
Distribution and specificity of mutations induced by neocarzinostatin in the lacI gene of Escherichia coli; Foster PL et al.; Although neocarzinostatin (NCS) attacks DNA almost exclusively at adenine and thymine residues in vitro, exposure of Escherichia coli to this antitumor drug resulted in a high frequency of mutations at guanine:cytosine base pairs in the lacI gene . Thus, NCS-induced base substitution mutations do not appear to result from the major DNA lesions that have been biochemically characterized . The overall distribution of nonsense mutations produced by NCS was distinctly nonrandom, consisting in part of a few "hotspots" and a large number of "coldspots." The existence of these coldspots implies that untargeted mutagenesis does not make a significant contribution to the mutations induced by this SOS-dependent mutagen.

J Bacteriol, 1983 Jan, 153(1), 109 - 15
Iron supply to Escherichia coli by synthetic analogs of enterochelin; Heidinger S et al.; Synthetic analogs of enterochelin (enterobactin) were tested for their ability to support the growth of Escherichia coli K-12 under iron-limiting conditions . The cyclic compound MECAM {1,3,5-N.N'; N"-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene} and its N-methyl derivative Me3MECAM promoted growth, whereas the 2,3-dihydroxy-5-sulfonyl derivatives MECAMS and Me3MECAMS were inactive . The same results were obtained with TRIMCAM {1,3,5-tris(2,3-dihydroxybenzoylcarbamido)-benzene} and TRIMCAMS (the 2,3-dihydroxy-5-sulfonyl derivative of TRIMCAM) . However, the sulfonic acid-containing linear compound LICAMS {1,5,10-N,N', N"-tris(5-sulfo-2,3-dihydroxybenzoyl)-triaza-decane} supported growth . In contrast, LIMCAMC, in which the sulfonyl groups at the five position of LICAMS are replaced by carboxyl groups at the four position, was inactive . The uptake of the active analogs required the functions specified by the fepB, fesB, and tonB genes . Surprisingly, growth promotion of mutants lacking the enterochelin receptor protein in the outer membrane was observed . Only MECAM protected cells against colicin B (which kills cells after entering at the enterochelin uptake sites) and transported Fe3+ at about half the enterochelin rate.

Hybridoma, 1983, 2(1), 79 - 86
Monoclonal antibodies directed against human fibroblast interferon: characterization and functional studies; Nyari LJ et al.; Spleen cells from BALB/c mice, immunized with SDS-acrylamide gel purified human fibroblast interferon (HuIFN-beta) were fused with mouse myeloma cells . Culture fluids from the resulting hybrid cells were screened for HuIFN-beta specificity by the following tests: a solid-phase RIA using partially purified HuIFN-beta, a protein-transfer RIA using electrophoretically resolved and immobilized HuIFN-beta, and a bioassay which tests residual HuIFN-beta activity in the supernatant following immunoprecipitation . Six HuIFN-beta-specific hybridomas were identified; two were capable of partially neutralizing HuIFN-beta activity . All six antibodies bind to the beta 1-IFN polypeptide synthesized in E . coli cells containing a cloned beta 1-IFN DNA sequence . All six monoclonal antibodies were found to be IgG3/kappa.

Acta Microbiol Pol, 1983, 32(3), 231 - 6
Cloning of Aspergillus nidulans ribosomal DNA in shotgun experiments using the EK2 vector lambda gtWEST.T5-622; Skalinska BA et al.; EcoRI fragments of Aspergillus nidulans DNA were cloned in a shotgun type experiment using the EK2 vector lambda gtWEST.T5-622 . In situ plaque hybridization was used to screen for hybrid phage particles containing sequences coding for ribosomal RNA . Four clones selected from the whole gene bank were characterized further . Two of them contain sequences homologous to a 3.6 Kb fragment of A . nidulans rDNA.

Acta Derm Venereol, 1983, 63(6), 538 - 40
Fibrin microclot formation in patients with acne; Juhlin L et al.; After the addition of E . coli polysaccharide to blood from patients with deep inflammatory acne, microclots formed in all patients, whereas this was rarely seen in mild acne and never in controls . Furthermore, spontaneous microclot formation without addition of endotoxin was seen in 5 of the 10 patients with the most severe acne.

Acta Microbiol Pol, 1983, 32(2), 197 - 206
A simplified method for coliphage detection in natural waters; Isbister JD et al.; The ARCAT (A Rapid Coliphage Analysis Technique) method for detecting coliphages in water has been modified . Modifications to the original method include media optimization, the use of frozen host cultures, the use of a single agar coliphage assay and optimized plaque resolution with 2, 3, 4-triphenyltetrazolium chloride . Detection of 5 coliphages per 100 ml of water sample is accomplished in 6 hours for rapid estimation of water quality.

Mol Gen Genet, 1983, 192(1-2), 95 - 100
Suppressors of a temperature-sensitive copy-number mutation in plasmid NTP1; Moser DR et al.; A temperature-sensitive high copy-number mutant of plasmid NTP1, first described by Grindley et al . (1978), is lethal to bacterial cells at the non-permissive temperature . This behavior has been used to select mutations in the plasmid replication origin region that suppress the over-replication phenotype . We have identified the site of the original ts lethal mutation and the positions of the reversion mutations . The ts mutation, designated orp, for over-replication, is a single nucleotide change 23 base-pairs upstream from the transcription start site for RNA I, the repressor of plasmid replication . This change simultaneously affects the promoter for RNA I and the precursor of the primer for plasmid replication, RNA II, which is also transcribed from this region . Fusions of the mutant promoter region with the galK gene indicate that transcription is not temperature sensitive . This result suggests that the mutation affects RNA II secondary structure . The reversion mutations are also located within the RNA II coding region more than 200 bp from the site of the original ts mutation . These mutations may also affect RNA II structure.

Mol Gen Genet, 1983, 192(1-2), 5 - 9
Correlation between RNA synthesis and ppGpp content in Escherichia coli during temperature shifts; Mackow ER et al.; Both a correlation and a lack of correlation between guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) level and RNA accumulation have been reported during temperature shifts of E . coli . We have reexamined these phenomena by measuring the total rate of RNA synthesis . After a temperature upshift (23 degrees to 40 degrees C) of E . coli relA+ and relA1 strains, there is an immediate increase in the rate of RNA synthesis which corresponds with the observed in vitro effects of temperature on RNA synthesis (Mangel 1974; Travers 1974) . A subsequent increase in ppGpp level is correlated with a decrease in the rate of RNA synthesis . Conversely, following a temperature downshift (40 degrees to 23 degrees C), both relA+ and relA1 bacteria show an immediate decrease in the rate of RNA synthesis . Subsequently all strains studied decrease ppGpp content and correspondingly increase the rate of RNA synthesis after a downshift . By measuring the rate of RNA synthesis we have separated immediate temperature-induced changes in RNA synthesis, from the apparent effects of ppGpp during temperature shifts . As a result, during temperature upshifts and downshifts of relA+, and relA1 bacteria, an inverse correlation between ppGpp content and the total rate of RNA synthesis does exist . The fact that both relA+ and relA1 strains show similar responses to temperature shifts provides additional evidence for the function of relA-independent basal level ppGpp synthesis in regulating RNA synthesis in E . coli.

Methods Enzymol, 1983, 99, 373 - 8
Characterization of the Abelson murine leukemia virus-encoded tyrosine-specific protein kinase; Wang JY et al.; There is sufficient evidence that the A-MuL V protein is a tyrosine-specific protein kinase . There are methods for detecting this kinase activity and this kinase has been expressed in E . coli . Because the information coding for the tyrosine-specific protein kinase is present in normal mouse cells, such an enzyme must have a normal physiologic function . The elucidation of this physiologic function and the understanding of the role of this enzyme activity in neoplastic transformation is the challenge of the future.

EMBO J, 1983, 2(11), 2019 - 24
A poliovirus type 1 neutralization epitope is located within amino acid residues 93 to 104 of viral capsid polypeptide VP1; Wychowski C et al.; Using nuclease Bal31, deletions were generated within the poliovirus type 1 cDNA sequences, coding for capsid polypeptide VP1, within plasmid pCW119 . The fusion proteins expressed in Escherichia coli by the deleted plasmids reacted with rabbit immune sera directed against poliovirus capsid polypeptide VP1 (alpha VP1 antibodies) . They also reacted with a poliovirus type 1 neutralizing monoclonal antibody C3, but reactivity was lost when the deletion extended up to VP1 amino acids 90-104 . Computer analysis of the protein revealed a high local density of hydrophilic amino acid residues in the region of VP1 amino acids 93-103 . A peptide representing the sequence of this region was chemically synthesized . Once coupled to keyhole limpet hemocyanin, this peptide was specifically immunoprecipitated by C3 antibodies . The peptide also inhibited the neutralization of poliovirus type 1 by C3 antibodies . We thus conclude that the neutralization epitope recognized by C3 is located within the region of amino acids 93-104 of capsid polypeptide VP1.

Int J Biochem, 1983, 15(10), 1231 - 9
In vitro effects of acridine intercalation on RNA polymerase interactions with supercoiled DNA; Greene RS et al.; In vitro transcription of supercoiled DNA by purified E . coli RNA polymerase was inhibited by Acridine Orange in a bimodal manner while N-10 benzyl substituted Acridine Orange is about one-third as inhibitory and effects monophasic inhibition . The inhibition correlates with the supercoil unwinding abilities of these two intercalators with Acridine Orange unwinding supercoiled DNA at 1/3 the concentration required for the substituted acridine orange . Direct visualization of DNA-RNA polymerase complex on agarose gels showed that these intercalators directly interfere with this association and the more effective the drug is in unwinding DNA supercoils the more effective it is in interfering with the DNA-enzyme complex . In addition, specific intercalators differentially affect the stability of DNA-RNA polymerase-RNA ternary complexes.

Mol Gen Genet, 1983, 191(2), 282 - 7
The gyrB gene product functions in both initiation and chain polymerization of Escherichia coli chromosome replication: suppression of the initiation deficiency in gyrB-ts mutants by a class of rpoB mutations; Filutowicz M et al.; A class of rpoB mutations is described which suppresses replication and transcription deficiency in gyrB-ts mutants shifted to a nonpermissive temperature . The compensatory effect of an altered subunit B of RNA polymerase (rpoB) for the gyrB defect, indicates that transcription is a primary target of the B subunit of DNA gyrase . One gyrB mutation (gyrB402-ts) shows deficiency in chromosome elongation at the nonpermissive temperature, both in vivo and in cells permeabilized with toluene . It is therefore concluded that the gyrB polypeptide functions at least dually in replication; first, at the level of transcription initiation and second, at the level of chain polymerization.

Adv Exp Med Biol, 1983, 163, 199 - 214
In vitro methotrexate polyglutamate synthesis by rat liver folylpolyglutamate synthetase and inhibition by bromosulfophthalein; McGuire JJ et al.; We have investigated the properties of the rat liver folylpolyglutamate synthetase using methotrexate (MTX; 4-NH2-10-CH3-PteGlu) as a substrate . Many characteristics of the synthetase (e.g., the apparent Km values for L-glutamate and ATP, and the optimal concentrations of KC1 and 2-mercaptoethanol) are virtually identical whether MTX or tetrahydrofolate is the "folate" substrate . There are, however, several significant differences between the reactions catalyzed with these two substrates . The length of products synthesized from tetrahydrofolate are inversely related to the initial monoglutamate concentration . Low tetrahydrofolate concentrations allow synthesis of longer (n greater than or equal to 3) polyglutamates, up to pentaglutamate length, while high concentrations lead to predominantly diglutamate synthesis . However, 4-NH2-10-CH3-PteGlu2 predominates regardless of the initial MTX concentration, under otherwise identical conditions . Also, tetrahydrofolate can be readily converted to pentaglutamate lengths, the same as predominates in rat liver in vivo . In contrast, MTX forms species containing only up to a total of three glutamates, i.e., 4-NH2-10-CH3-PteGlu3 . Finally, the ultimate product of synthesis from tetrahydrofolate, H4PteGlu5, is a fairly good inhibitor of synthetase activity with either MTX or tetrahydrofolate as the substrate . The ultimate product of MTX synthesis, 4-NH2-10-CH3-PteGlu3, however, is a poor inhibitor of activity with either substrate . We have also investigated the inhibitory effect of bromsulfophthalein (BSP) on the rat liver synthetase . Gewirtz et al., showed that this organic anion inhibited the uptake of MTX and 5-CH3-tetrahydrofolate into hepatocytes, and presented indirect evidence that BSP effected polyglutamate biosynthesis . We have demonstrated that BSP is a potent (Ki = 1 microM) inhibitor of the rat liver synthetase . Inhibition is noncompetitive with rspect to L-glutamate, ATP, and MTX . Pre-incubation and time course experiments demonstrated that inhibition is not stoichiometric and is not caused by slow inactivation of the synthetase . Since BSP is transported into mammalian cells, it is the first inhibitor of polyglutamate biosynthesis which has potential for use in in vivo studies.

Adv Shock Res, 1983, 9, 265 - 74
Serum complement levels in canine endotoxin shock: relation to survival and to corticosteroid therapy; Shatney CH et al.; Recent studies suggest prominent roles of the complement system in endotoxin shock and steroid-complement interactions in its treatment . To assess further the potential importance of the complement system in this condition, we measured serum total complement levels in dogs after an IV bolus of 1.5 mg/kg DIFCO E coli endotoxin . Dogs were assigned to control (C), dextran40 (LMD), or LMD + steroid groups . Corticosteroids, given IV 15 min after endotoxin, were 1) methylprednisolone sodium succinate (MPSS), 30 mg/kg; 2) dexamethasone sodium phosphate (DSP), 6 mg/kg; 3) hydrocortisone sodium succinate (HSS), 150 mg/kg; or 4) hydrocortisone sodium phosphate (HSP), 150 mg/kg . LMD was infused to maintain BP greater than 60% of preshock levels during the 4 h of monitoring . Survival rates at 48 h were C--7/24 (29%); LMD--3/12 (25%), MPSS--11/19 (59%) (P less than 0.1); DSP--9/14 (64%) (P less than 0.05); HSS--3/10 (30%) HSP--5/10 (50%) . Within 15 min of endotoxin administration, serum complement titers fell at least 48% in all groups . Complement levels returned to the preshock range in only the LMD group . Mean complement levels of all survivors and nonsurvivors were nearly identical throughout the acute experiment . The results indicate that survival in canine endotoxin shock, with or without corticosteroid therapy, is not related to normalization of serum total complement titers during the first few hours after endotoxin injection.

Dev Biol Stand, 1983, 53, 183 - 7
Studies on the expression and organization of the K88ac adherence antigen; Dougan G et al.; The genetic determinant for the K88ac adhesion antigen has been cloned on a 6.5 kilobasepair DNA fragment into the multiple copy plasmid pBR322 . The resulting hybrid plasmid named pMK005 was used to study the organization and expression of polypeptides involved in K88ac antigen assembly . Five cistrons named adh A, B, C, D and E were mapped on the cloned DNA and maximal expression of K88ac antigen was found to be dependent on a pBR322 encoded promoter . Four polypeptides of molecular weights 70,000 daltons, 25,000 daltons, 17,000 daltons, and 23,500 daltons (K88ac fimbrial subunit) were identified as the products of the adh A, B, C and D cistrons respectively . The subcellular location of each of these polypeptides was determined by fractioning minicells . pMK005 encodes sufficient information to promote adhesion of a wild-type E . coli strain 09:K13:H19 to the porcine small intestine in vivo.

Mol Gen Genet, 1983, 189(3), 422 - 31
Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome; Hirose S et al.; A new round of chromosomal replication of a temperature-sensitive initiation mutant (dnaC) of Escherichia coli was initiated synchronously by a temperature shift from a nonpermissive to a permissive condition in the presence of arabinosyl cytosine . Increased amounts of nascent DNA fragments with homology for the chromosomal segment containing the replication origin (oriC) were found . The nascent DNA fragments were purified and treated with alkali to hydrolyze putative primer RNA and to expose 5'-hydroxyl DNA ends at the RNA-DNA junctions . The ends were then labeled selectively with T4 polynucleotide kinase and {gamma-32P}ATP at 0 degrees C and the terminally-labeled initiation fragments were purified by hybridization with origin probe DNAs containing one each of the constituent strands of oriC-DNA segment . The 32P-labeled initiation sites were then located at the resolution of single nucleotides in the nucleotide sequence of the oriC segment after cleavage with restriction enzymes . Two initiation sites of DNA synthesis, 37 nucleotides apart, were detected in one of the component strands of the oriC; in other words, in the strand whose 5' to 3' polynucleotide polarity lies counterclockwise on the E . coli genetic map . The results support the involvement of the primer RNA in the initiation of DNA synthesis at the origin of the E . coli genome and suggest that the first initiation event is asymmetric.

Cell, 1983 Jan, 32(1), 119 - 29
The origin of replication of plasmid p15A and comparative studies on the nucleotide sequences around the origin of related plasmids; Selzer G et al.; Replication of Escherichia coli plasmid p15A was examined by use of a cell extract or a mixture of three purified E . coli enzymes: RNA polymerase; RNAase H; and DNA polymerase I . In each system, replication initiates at any of three consecutive nucleotides located at a unique site . Primer transcription starts 508 bp upstream of the replication origin . The region between 294 and 524 bp upstream of the origin determines the incompatibility property . This region specifies an RNA (RNA I) of about 105 nucleotides that is involved in regulation of primer formation . We compare the nucleotide sequences around the origins of related plasmids p15A, ColE1, pBR322, RSF1030 and CloDF13, and discuss the significance of possible RNA secondary structures in primer formation.

J Bacteriol, 1983 Jan, 153(1), 66 - 75
Physiological properties of cold-sensitive suppressor mutations of a temperature-sensitive dnaZ mutant of Escherichia coli; Blinkowa A et al.; Suppressors of a temperature-sensitive dnaZ polymerization mutant of Escherichia coli have been identified by selecting temperature-insensitive revertants . Those suppressed strains which concomitantly became cold sensitive were chosen for further study . Intragenic suppressor mutations, which caused cold-sensitive defects in DNA polymerization, were located in dnaZ by transduction with lambda dnaZ+ phages . Extragenic suppressor mutations were mapped within the initiation gene dnaA . These suppressor-containing strains were defective in initiation at low temperature as determined by measurements of DNA synthesis in vivo and in toluene-treated cells . The occurrence of suppressor mutations of dnaZ(Ts) within the dnaA gene is considered evidence that the dnaA and dnaZ products interact in vivo . A second indication of a dnaA-dnaZ protein-protein interaction was provided by the observation that the introduction of additional copies of the dnaZ+ gene into a strain carrying the dnaA suppressor mutation was lethal {whether the strain was dnaZ+ or dnaZ(Ts)}.

Mol Gen Genet, 1983, 192(3), 378 - 85
Escherichia coli uvrD mutants with thermosensitive DNA-dependent adenosine triphosphatase I (helicase II); Richet E et al.; Three mutants producing thermosensitive DNA-dependent Adenosine triphosphatase (ATPase) I were screened from a collection of temperature-sensitive mutants of Escherichia coli K12 . ATPase I purified to near homogeneity from one of the mutants (JE11000) possesses both thermosensitive DNA-dependent ATPase and DNA helicase activities . We have shown that ATPase I is encoded by the uvrD gene as first suggested by Oeda et al . (1982): (i) the thermosensitive ATPase I mutation present in JE11040 lies in or very close to the uvrD gene, (ii) ATPase I activity is absent in uvrD210, uvrD156, and uvrD252 mutants . Thus the thermosensitive mutations correspond to new uvrD mutations . However, the mutation present in JE11040 confers neither UV sensitivity nor mutator phenotype at high temperature . Evidence is presented that the mutant ATPase I is stabilized in vivo at 42 degrees C.

Mol Gen Genet, 1983, 192(1-2), 104 - 9
Transfer inhibition of RP4 by F factor; Tanimoto K et al.; When RP4 and F factors were brought together into one E . coli cell, the F factor reduced 500-1000-fold the frequency of transfer of RP4 . However, F had almost no effect on the surface exclusion and pilus formation by RP4 . In contrast, RP4 did not affect the transfer of F . Using in vitro recombinant DNA techniques, a gene of F responsible for the above-mentioned transfer inhibition of RP4 was located within the BamHI fragment (40.4-42.8 kb) of the mini-F sequence on F . From the result of product analysis using minicells, the responsible gene in the BamHI fragment was inferred to encode the 33 K protein.

EMBO J, 1983, 2(6), 967 - 71
Nucleotide sequence of the structural gene for dUTPase of Escherichia coli K-12; Lundberg LG et al.; The nucleotide sequence of the dUTPase structural gene, dut, of Escherichia coli has been determined . The DNA sequence predicts a polypeptide chain of 150 amino acid residues (mol . wt . 16 006) corresponding in size and composition to the purified dUTPase subunit . In a tentative promoter region preceding the dut gene, the -35 and -10 regions are separated by a SacI (SstI) site . Cloning of the dut gene utilization this SacI site was previously shown to reduce dut expression dramatically . The nucleotide sequence also contains a 210-codon open reading frame 106 bp downstream of dut and co-directional with dut . Previous protein synthesis experiments using dut plasmids allocated the gene of a polypeptide of mol . wt . 23 500 to this DNA region . The open reading frame thus may correspond to a protein of unknown function co-transcribed with the dut gene.

Folia Microbiol (Praha), 1983, 28(3), 145 - 8
Regulation of inorganic pyrophosphatase in Escherichia coli: relationship between the synthesis of inorganic pyrophosphatase and the thymidine triphosphate pool; Kukko E et al.; The activities of inorganic pyrophosphatase, thymidine kinase and thymidine phosphorylase were measured in Ter-mutants of E . coli K12 which have a higher or a lower dTTP pool than the parent strain . The levels of inorganic pyrophosphatase and thymidine kinase were changed in the same direction and that of thymidine phosphorylase in the opposite direction in these mutants.

Mol Gen Genet, 1983, 190(1), 171 - 5
Nucleotide sequence of the glnA control region of Escherichia coli; Covarrubias AA et al.; The RNA polymerase binding sites present along a DNA segment encompassing the glnA, glnL, and glnG genes have been identified in a hybrid plasmid carrying this chromosomal region of Escherichia coli . The DNA sequence was determined of an 817 base pair segment that contains the region coding for the first 42 amino acids of the NH2-terminal and of the glnA structural gene, as well as its regulatory region . Analysis of this nucleotide sequence revealed three probable RNA polymerase recognition sites, imperfect palindromes, inverted repeats, and direct repeated sequences.

Scand J Infect Dis, 1983, 15(1), 57 - 64
Lack of significance of pili in experimental ascending Escherichia coli pyelonephritis; Guze LB et al.; The role of pili as a bacterial virulence factor has been studied . The model used was acute ascending Escherichia coli pyelonephritis in the mouse . Three strains of E . coli were injected in lightly or more heavily piliated phases into 15 mice each . At sacrifice of 13-15 animals 2 weeks later, no significant difference in severity of pyelonephritis was found as judged by numbers of bacteria in the kidney, nor intensity or frequency of gross abscesses . 27 strains of E . coli were order ranked for severity of pyelonephritis produced and compared with intensity of piliation in vitro under conditions designed to maximize pilus formation . No significant difference was found . 15 strains derived from patients in whom infections were confined to the bladder were compared for degree of piliation with 12 strains infecting the kidney . No significant difference was found . These studies do not support a significant role for the degree of piliation as a virulence factor in pyelonephritis.

Infection, 1983 Jan-Feb, 11(1), 73 - 6
P-fimbriae of pyelonephritogenic Escherichia coli: significance for reflux and renal scarring-a hypothesis; Kallenius G et al.; An experimental pyelonephritis model was developed in monkeys (Macaca fascicularis) using P-fimbriated Escherichia coli as the infecting organism . The relevant receptor molecules for P-fimbriae were also shown to be present in Macaca fascicularis . Atraumatic administration of P-fimbriated E . coli into the ureter induced a ureteritis followed by acute and chronic pyelonephritis . The decisive role of P-fimbriae as an adhesive virulence factor was proven by the receptor blockade of P-fimbriae-mediated bacterial adhesion by a synthetic receptor analogue (alpha-D-Galp-(1-4)-beta-D-Galp-1-OMe), which was administered into the ureter together with the challenge bacteria . On the basis of these and other findings, the role of reflux and pyelonephritis in relation to renal scarring is discussed in this paper . It is proposed that minor transitional vesicoureteral reflux together with the adhesive property of P-fimbriated E . coli and their ability to induce ureteritis might constitute an alternative mechanism to gross reflux by which bacteria ascend to the kidney . These findings and the fact that intestinal colonization with P-fimbriated E . coli coincides with the disease have opened up new prophylactic and therapeutic possibilities.

J Gen Microbiol, 1983 Jan, 129 (Pt 2), 235 - 43
Partial purification and characterization of two non K99 mannose-resistant haemagglutinins of Escherichia coli B41; Chanter N; A K99-variant of Escherichia coli B41 was produced by growing the parent strain in the presence of antiserum to E . coli K12K99 . Two mannose-resistant and eluting (MRE) haemagglutinins with molecular weights greater than 20 x 10(6) were extracted from the cell surface of the variant . One was an anionic antigen, partially purified by ammonium sulphate and isoelectric point precipitation, which adhered to calf intestinal brush borders; it was a protein composed of subunits with mol . wt 34000 . Electron microscopy showed that this material did not have a regular fimbrial appearance, but contained some fine fibrillar structures . A second MRE haemagglutinin which was also partially purified by ammonium sulphate precipitation, had a definite fimbrial structure, being a protein composed of two subunits of mol . wt 49500 and 48000 . This antigen was probably responsible for the fimbrial appearance of the K99-variant, but it was antigenically distinct from the anionic adhesin and did not adhere to calf intestinal brush borders.

J Bacteriol, 1983 Jan, 153(1), 364 - 70
Organization of K88ac-encoded polypeptides in the Escherichia coli cell envelope: use of minicells and outer membrane protein mutants for studying assembly of pili; Dougan G et al.; Escherichia coli K-12 minicells, harboring recombinant plasmids encoding polypeptides involved in the expression of K88ac adhesion pili on the bacterial cell surface, were labeled with {35S}methionine and fractionated by a variety of techniques . A 70,000-dalton polypeptide, the product of the K88ac adhesion cistron adhA, was primarily located in the outer membrane of minicells, although it was less clearly associated with this membrane than the classical outer membrane proteins OmpA and matrix protein . Two polypeptides of molecular weights 26,000 and 17,000 (the products of adhB and adhC, respectively) were located in significant amounts in the periplasmic space . The 29,000-dalton polypeptide was shown to be processed in E . coli minicells . The 23.500-dalton K88ac pilus subunit (the product of adhD) was detected in both inner and outer membrane fractions . E . coli mutants defective in the synthesis of murein lipoprotein or the major outer membrane polypeptide OmpA were found to express normal amounts of K88ac antigen on the cell surface, whereas expression of the K88ac antigen was greatly reduced in perA mutants . The possible functions of the adh cistron products are discussed.

Gene Amplif Anal, 1983, 3, 175 - 200
Expression of the yeast CYC genes and CYC1/GalK fusion genes on yeast plasmids; Zitomer RS et al.; We have presented the results of our studies of the expression of the CYC genes from plasmids . All our data indicate that the levels of expression and the regulation of expression are very similar for the plasmid-borne genes and the chromosomal genes when care is taken to construct the appropriate plasmids . The usefulness of these plasmids has been demonstrated: mutations affecting regulatory sites adjacent to genes of interest have been constructed {such as the Xho I deletion and inversion in the YCpCYC1(2.4) plasmid} and selected {as in the case of the IS1 insertion into the YCpCYC7(2) plasmid}, and these mutations have led us to some tentative conclusions about the location and nature of the regulatory sites of these genes . Furthermore, transformation with plasmids containing modified genes or fusions has permitted isolation of genomic regulatory mutants, as in the selection of lac+ suppressors of the lac- CYC1 1/x inversion carried on the YCpCYC1(2.4) 1/x plasmid . Although we cannot rule out the possibility that use of plasmids might cause us to miss a class of regulatory effects that can be propagated only along a chromosomal structure, we believe that the regulatory effects that we do observe can be more quickly and completely defined by working with plasmids . If any regulatory effects occur only on chromosomes, they can be studied more easily once the basic regulatory phenomena have been analyzed . The regulatory regions of the CYC1, CYC7, and TR2 genes that we have crudely mapped so far all exert their effects 100-300 bp away from the putative transcriptional starting sites . How the information in these regions is transmitted along the DNA is an intriguing question . We are engaged in a mutational analysis of these sites to locate them more precisely, to map second-site mutations that moderate the effects of the original mutations, to obtain genomic mutations that define the genes whose products interact with these sites, and to test combinations of genomic and plasmid mutations to define the sites with which regulatory elements interact . This approach should aid our understanding of the spatial relationships between yeast regulatory sites and transcriptional signals . Ultimately, obtaining mutations in regulatory genes, such as the mutations described here for the anaerobic regulation of TR2, will allow the cloning of these genes by complementation . This will lead to the isolation of the protein encoded and ultimately to an approach to the molecular mechanism of regulation through study of protein-DNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene Amplif Anal, 1983, 3, 103 - 16
Portable Shine-Dalgarno regions; nucleotides between the Shine-Dalgarno sequence and the start codon affect the translation efficiency; de Boer HA et al.; This chapter describes a gene expression system of E . coli that contains a portable Shine-Dalgarno region . Transcription of this system is directed by a hybrid promoter derived from trp and lac-UV5 promoter sequences, which is followed by a region that encodes the portable Shine-Dalgarno region . We used a series of synthetic portable Shine-Dalgarno regions to vary the length (from 4 to 13 bases) of the Shine-Dalgarno by increasing the number of bases on the mRNA that are complementary to the 3' end of 16s rRNA . Increase in the Shine-Dalgarno region to 8 or 13 bases decreased the translation efficiency of the chimeric leukocyte interferon messenger by 40% . In another series of portable Shine-Dalgarno regions, we varied the four bases that follow the Shine-Dalgarno region . The presence of four A residues or four T residues in this region resulted in the highest translation efficiency . The presence of four C residues reduced translation efficiency by 50%, compared with A or T residues . The presence of four G residues after the Shine-Dalgarno region lowered translation efficiency by 75%, compared with A or T residues.

Biochim Biophys Acta, 1982 Dec 31, 699(3), 203 - 10
Ribosomal proteins of Streptomyces aureofaciens producing tetracycline; Mikulik K et al.; Three different two-dimensional polyacrylamide gel electrophoretic systems were employed for identification of individual ribosomal proteins of Streptomyces aureofaciens . Proteins of small subunits were resolved into 21 spots . Larger ribosomal subunits contained 35 proteins . The separated ribosomal proteins from 50 S subunits were transferred on nitrocellulose membranes for immunochemical estimations . Antibodies developed against 50 S proteins of S . aureofaciens and Escherichia coli were used for identification of structural homologies between 50 S proteins of the two species . Results of the experiments indicate that about one half of the 50 S proteins of S . aureofaciens share common immunochemical determinants with corresponding proteins of 50 S subunits of E . coli . Evidence is presented that acidic ribosomal protein SL5 of large ribosomal subunits of S . aureofaciens can be assembled to E . coli P0 cores lacking proteins L7/L12 . Reconstitution of the P0 cores with proteins SL5 or L7/L12 led to restoration of 78% activity in polyphenylalanine synthesis.

Biochim Biophys Acta, 1982 Dec 31, 699(3), 232 - 40
Changes in DNA binding by purified simian RNA polymerase II under transcribing and nontranscribing conditions; Wilson VG et al.; The interaction of RNA polymerase II from African Green Monkey liver tissue with SV40 DNA was examined by a DNAase protection technique . In the absence of nucleoside triphosphates, simian polymerase binds to nicked, linear SV40 DNA and protects 30 bp of binary complex DNA from DNAase I digestion . With the addition of nucleoside triphosphates to initiate transcription, polymerase protects 40 bp of the ternary complex DNA from DNAase I . Thus, a conformational change in either the polymerase, the DNA, or both occurs during the transition from binary to ternary complex, and this altered conformation allows a larger protection of template DNA . Similar results were seen with Escherichia coli RNA polymerase holoenzyme on SV40 DNA.

FEBS Lett, 1982 Dec 27, 150(2), 485 - 8
N-Tosyl-L-phenylalanylchloromethane reacts with cysteine 81 in the molecule of elongation factor Tu from Escherichia coli; Jonak J et al.; Elongation factor EF-Tu from Escherichia coli was labelled with N-{14C}tosyl-L-phenylalanylchloromethane, digested with trypsin and the peptides obtained separated by HPLC . The only radioactive peak recovered corresponded to tryptic peptide containing residues 75-98 . Sequencing of the peptide by automated Edman degradation identified cysteine 81 as the site of N-tosyl-L-phenylalanylchloromethane modification . These results confirm the importance of this residue for the interaction with aminoacyl-tRNAs.

FEBS Lett, 1982 Dec 27, 150(2), 465 - 8
Translocation of colicin E1 through cytoplasmic membrane of Escherichia coli; Yamada M et al.; The product of the malE-lacZ gene fusion was reported to compete with some proteins including outer membrane lipoprotein in the protein translocation across the Escherichia coli membrane . The fusion product also inhibited colicin E1 export . Furthermore, globomycin, which accumulated prolipoprotein in the membrane, inhibited the translocation of colicin E1 in the wild-type cells, but not in lipoprotein-negative mutant cells . Since colicin E1 contains the internal signal-like sequence {Proc . Natl . Acad . Sci . USA (1982) 79, 2827-2831}, these results suggest that colicin E1 is exported by the aid of this sequence at a common site for maltose-binding protein and lipoprotein translocation.

J Biol Chem, 1982 Dec 25, 257(24), 15065 - 71
Renaturation and identification of periplasmic proteins in two-dimensional gels of Escherichia coli; Copeland BR et al.; The locations of the periplasmic proteins of Escherichia coli on standard two-dimensional gel patterns are described . The periplasmic fractions were prepared by osmotic shock of plasmolyzed whole cells and by release during EDTA-lysozyme treatment of whole cells . Within this fraction of proteins, we identify nine binding proteins (leucine-specific, glutamate-aspartate, glutamine, cystine, galactose, maltose, xylose, ribose, and arabinose) in addition to leucine-isoleucine-valine binding protein, which has been previously identified (Bloch, P . L., Phillips, T . A., and Neidhardt, F . C . (1980) J . Bacteriol . 141, 1409-1420) . The identifications are based upon genetic criteria, protein induction, and comigration with purified protein . The levels of these proteins are compared in strains K12, B, and HA12 (a derivative of W) . A technique was developed for renaturation of the ligand binding sites of periplasmic binding proteins in denaturing two-dimensional gels . This technique was used to demonstrate that leucine-specific and cystine binding proteins both have different isoelectric points in different strains . Renaturation was also used to demonstrate that there are two different charged forms for glutamine binding protein.

J Biol Chem, 1982 Dec 25, 257(24), 14826 - 9
A calorimetric investigation of the interaction of the lac repressor with inducer; Donner J et al.; A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG) . The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively . This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values . The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1 . The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG . The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0 . The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant . This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.

J Biol Chem, 1982 Dec 25, 257(24), 15110 - 21
The in vitro transcription-translation of DNA and RNA templates by extracts of Rhodopseudomonas sphaeroides . Optimization and comparison of template specificity with Escherichia coli extracts and in vivo synthesis; Chory J et al.; A DNA-directed coupled transcription-translation system has been developed in cell-free extracts from the facultative phototroph, Rhodopseudomonas sphaeroides . The in vitro protein-synthesizing system was active when prepared from either chemoheterotrophically or photoheterotrophically grown cells . Optimal activity was dependent upon: use of extracts prepared freshly from early exponential phase cells, the method of cell breakage, and the length of preincubation of the extract (S-30), as well as the concentrations of S-30, template, and cations . The R . sphaeroides cell-free system was compared to one prepared from Escherichia coli . DNA templates tested included R . sphaeroides phage RS1 DNA and E . coli phages T4 and T7 DNA, as well as plasmids RSF1010, pBR322, pSL25 (a pBR322 derivative), and a chimeric plasmid of pSL25 and RSF1010 . One RNA template, phage R17, was also employed to test translational fidelity . Transcriptional-translational specificity was observed between R . sphaeroides and E . coli and these observations are discussed in terms of differential gene expression among phylogenetically distinct groups of bacteria.

J Biol Chem, 1982 Dec 25, 257(24), 15167 - 73
Purification and properties of the biotin repressor . A bifunctional protein; Eisenberg MA et al.; Definitive evidence is presented for the bifunctional nature of the biotin repressor protein which possesses both regulatory and enzymatic activities . The repressor protein can activate biotin in the presence of ATP to form biotinyl-5'-adenylate, the co-repressor which remains tightly bound to the repressor protein . This complex can either bind to the operator site and inhibit transcription or transfer the biotinyl moiety to a lysine residue of the apoenzyme of acetyl-CoA carboxylase . The two activities were coincident throughout a purification procedure which resulted in a 3500-fold increase in activity . Gel electrophoresis of the purified preparation, under native or denaturing conditions, showed three proteins with the activity corresponding to the major protein band of apparent Mr = 34,000 . On gel exclusion chromatography, the activity was also associated with a protein of Mr varying fro 37,000-44,000, indicating the protein is monomeric . The occasional appearance of multiple bands with biological activity in the native gels suggests that the repressor protein can also exist in multimeric forms . On chromatofocusing, the repressor activity and the holoenzyme synthetase activity were coincidental, with the peak of activity at pH 7.2, the isoelectric point . Only a single protein band with Mr = 34,000 was observed on SDS gel electrophoresis of all fractions showing activity.

Biochemistry, 1982 Dec 21, 21(26), 6675 - 84
General acid-base catalysis of alpha-glucan phosphorylases: stereospecific glucosyl transfer from D-glucal is a pyridoxal 5'-phosphate and orthophosphate (arsenate) dependent reaction; Klein HW et al.; D-Glucal, containing a highly reactive double bond, can replace glucose 1-phosphate as the glucosyl donor in phosphorylase-catalyzed glucosyl transfer to a suitable oligo- or polysaccharide acceptor: D-glucal + Pi + (glucose)Pi leads to n 2-deoxy-alpha-D-glucosyl(glucose)n in equilibrium 2-deoxy-alpha-D-glucose-1-P + (glucose)n . This reaction is catalyzed by alpha-glucan phosphorylases from rabbit skeletal muscle, potato tuber, and Escherichia coli . D-Glucal is only measurably consumed by alpha-glucan phosphorylases when orthophosphate or arsenate is present . With saturating concentrations of these anions and a glucosyl acceptor, the D-glucal reaction proceeds at rates comparable with the rates of glucosyl transfer from glucose 1-phosphate and of phosphorolysis or arsenolysis of poly- or oligosaccharides . Furthermore, for the reaction to proceed, the enzyme must be in the active conformation containing the cofactor pyridoxal 5'-phosphate in its dianionic form . On the basis of proton nuclear magnetic resonance spectra, it is proposed that protonation at C-2 of D-glucal gives rise to a hypothetical 2-deoxy-beta-D-glucose intermediate, yielding as a final product (2-deoxy-alpha-D-{2(e)-2H}glucose)n alpha (1 leads to 4) saccharides . These 2-deoxy-alpha-D-glucose oligo- or polysaccharides are degraded by alpha-glucan phosphorylases by phosphorolysis or arsenolysis like natural linear and branched alpha-glucans . The absolute requirement of the D-glucal reaction for phosphate (or arsenate) and its dependency on the dianionic form of the pyridoxal 5'-phosphate bound to phosphorylase are rationalized in terms of a proton transfer relay involving juxtaposed phosphates . Phosphate--phosphate interactions were postulated by Withers et al . {Withers, S . G., Madsen, N . B., Sykes, B . D., Takagi, M., Shimomura, S., & Fukui, T . (1981) J . Biol . Chem . 256, 10759-10762}.

Biochemistry, 1982 Dec 21, 21(26), 6656 - 60
A proteolyzed derivative of Escherichia coli phosphofructokinase is no longer sensitive to allosteric effectors and still shows cooperativity in substrate binding; Le Bras G et al.; Limited proteolysis of Escherichia coli phosphofructokinase by subtilisin yields a homogeneous derivative . This proteolyzed protein is composed of four polypeptide chains, with a molecular weight of 32 000 as compared to 37 000 for the original enzyme . Removal on each chain of about 5 kdaltons maintains the enzymatic activity and does not change the apparent affinity for the substrates ATP and fructose 6-phosphate . Limited proteolysis, however, affects the cooperativity of fructose 6-phosphate binding: the Hill coefficient is reduced from almost 4 in the native enzyme to only 2 in its proteolyzed derivative . Also, the proteolyzed protein is no longer sensitive to allosteric effectors, activator, or inhibitor . These changes in regulatory properties upon proteolysis are apparently due to the destruction of the effector binding site . The allosteric effector GDP protects phospho-fructokinase against proteolysis and irreversible thermal inactivation; GDP is, however, inefficient in protecting the proteolyzed protein against thermal denaturation . These results suggest that phosphofructokinase may function as a dimer of dimers, in which homotropic and heterotropic allosteric effects are not mediated by the same sets of quaternary interactions.

Nucleic Acids Res, 1982 Dec 20, 10(24), 8211 - 23
On the cooperative and noncooperative binding of ethidium to DNA; Winkle SA et al.; The equilibrium binding of ethidium bromide to native DNAs and to poly(dG-dC) X poly(dG-dC) has been studied by both phase partition and direct spectrophotometric techniques . The binding isotherms obtained from both experimental techniques show that ethidium binds in a cooperative manner to E . coli DNA . On the other hand, no evidence of cooperative binding was observed in the binding isotherms obtained with calf thymus, C . perfringens, M . lysodeikticus, or poly(dG-dC) X (dG-dC) under the experimental conditions used (0.1 M NaCl).

C R Seances Acad Sci III, 1982 Dec 20, 295(13), 765 - 70
{A protease of the external membrane of Escherichia coli sensitive to environmental conditions . Its relations with the expression of envZ gene}; Cavard D et al.; A mutant strain (called Cpr), devoid of the outer membrane protease that cleaves colicin A, has been isolated . The location on the genetic map of the cpr locus as well as its pleiotropic effects concerning chiefly: the protein composition of the outer membrane, sensitivity to phages and colicins, alteration in protease activity (cpr), are very similar to the location on the genetic map and to phenotypic properties observed in strains tpo, perA or envZ which are altered in the ompB locus . Conditions resulting in inhibition of the colicin A protease activity also result in transcriptional regulation of OmpF, OmpC and LamB proteins synthesis . The possibility for this protease to be an osmosensor of the cell's external environment is discussed.

Nucleic Acids Res, 1982 Dec 20, 10(24), 8031 - 48
The regulatory region of the divergent argECBH operon in Escherichia coli K-12; Piette J et al.; The nucleotide sequence of the control region of the divergent argECBH operon has been established in the wild type and in mutants affecting expression of these genes . The argE and argCBH promoters face each other and overlap with an operator region containing two domains which may act as distinct repressor binding sites . A long leader sequence - not involved in attenuation - precedes argCBH . Overlapping of the argCBH promoter and the region involved in ribosome mobilization for argE translation explains the dual effect of some mutations . Mutations causing semi-constitutive expression of argE improve putative promoter sequences within argC . Implications of these results regarding control mechanisms in amino acid biosynthesis and their evolution are discussed.

Nucleic Acids Res, 1982 Dec 20, 10(24), 7919 - 34
Sequences of four tRNA genes from Caenorhabditis elegans and the expression of C . elegans tRNALeu (anticodon IAG) in Xenopus oocytes; Tranquilla TA et al.; Four tRNA genes have been identified in cloned segments of Caenorhabditis elegans DNA by tRNA hybridisation and expression after injection into Xenopus laevis oocyte nuclei . From DNA sequencing these are (with DNA anticodon sequences) tRNAAsp (GTC), tRNALeu (AAG), tRNALys (CTT) and tRNAPro (TGG) . Their flanking DNA sequences are compared . Two identical tRNALys (CTT) genes from different regions of the genome have quite unrelated 5' flanking sequences . The tRNA synthesised in Xenopus oocytes after injection of the tRNALeu cloned DNA has the modified anticodon IAG . The tRNALeu gene precursor transcript from injected oocytes has short 5' and 3' additional sequences and lacks certain of those modified bases found in the processed tRNA.

Nucleic Acids Res, 1982 Dec 20, 10(24), 7905 - 18
The structure and expression of the preproenkephalin gene; Legon S et al.; Enkephalins are pentapeptides with opioid activity which are found in a wide variety of tissues . Studies of enkephalin-containing peptides from the adrenal gland have established that the mature pentapeptides are derived by proteolytic processing of a precursor protein . We have shown that human adrenal medullary tumours contain mRNA which can be translated in vitro to yield a single major enkephalin precursor . The sequence of cloned cDNA shows that the preproenkephalin mRNA encodes four copies of met-enkephalin, two copies of met-enkephalin extended sequences and one copy of leu-enkephalin; each copy is flanked by paired basic amino acids which are presumably recognised by the processing protease . We have used the cloned human cDNA as a hybridisation probe to detect the corresponding mRNAs in rat adrenal gland and, in smaller amounts, in rat brain . We have been unable to detect in brain any other cross-hybridising mRNAs which might encode other putative precursor proteins.

Nucleic Acids Res, 1982 Dec 20, 10(24), 8311 - 22
A comparison of nucleoside (beta-S)triphosphates and nucleoside (gamma-S)triphosphates as suitable substrates for measuring transcription initiation in preparations of cell nuclei; Washington LD et al.; RNA chains initiated with nucleoside (beta-S)triphosphates and (gamma-S)triphosphates retain the thiol groups and can be separated from thiol-free RNA by chromatography on mercury-Sepharose . Thiol-containing mouse mammary tumor virus (MMTV) RNA synthesized by preparations of nuclei from virus-infected cells was quantitated by nucleic acid filter hybridization . With ATP beta S and GTP beta S, region-specific initiation of MMTV RNA chains was detected in the cell free system . However, with ATP gamma S and GTP gamma S, region-specific initiation was not clearly demonstrable . The nuclear preparations can also transfer thiol groups, presumably in the form of thiophosphate, from ATP gamma S or GTP gamma S onto preexisting RNA molecules; little or no thiol-transfer occurs with the two (beta-S)-analogues . The thiophosphate transfer activity apparently interferes with the measurement of RNA chain initiation with ATP gamma S and GTP gamma S.

Nucleic Acids Res, 1982 Dec 20, 10(24), 8113 - 25
Cloning and expression of the cDNA for human antithrombin III; Bock SC et al.; A partial cDNA clone for human antithrombin III (ATIII) was obtained by screening a cDNA library prepared from size fractionated liver RNA with a pool of eight 16-base long synthetic DNA fragments whose sequence was determined from protein sequence data . A fragment of the partial cDNA clone was used to enrich RNA for ATIII messages, and cDNA clones encoding the entire ATIII structural gene were identified . The complete nucleotide and predicted amino acid sequences of human ATIII and its 32 residue signal peptide are reported, and provide further opportunity to compare the ATIII primary structure with corresponding regions from homologous proteins, alpha 1-antitrypsin and ovalbumin . Plasmids in which the structural genes for mature and pre-ATIII were linked to the E . coli trp promoter-operator support the synthesis of human antithrombin III and pre-antithrombin III in bacteria.

Biochim Biophys Acta, 1982 Dec 17, 719(3), 509 - 17
Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenine nucleoside and nucleotides, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives; Hiratsuka T; The ribose-modified chromophoric and fluorescent analog of ATP 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) adenosine 5'-triphosphate (TNP-ATP) has been synthesized previously (Hiratsuka, T., and Uchida, K . (1973) Biochim . Biophys . Acta 320, 635-647 and Hiratsuka, T . (1976) Biochim . Biophys . Acta 453, 293-297) . In the present study, four TNP-derivatives of ATP, ADP, AMP and adenosine were synthesized and compared for several chemical, spectral and enzymatic properties . Their visible absorption and fluorescent properties were found to be quite similar . Visible absorption and fluorescence spectra of TNP-derivatives were sensitive to solvent polarity . TNP-adenosine and TNP-AMP showed considerable substrate activities with adenosine deaminase and alkaline phosphatase, respectively . TNP-ATP proved to be an excellent substitute for ATP in adenylate kinase and myosin ATPase systems . The results indicate that these analogs are useful as chromophoric and fluorescent probes for hydrophobic regions in adenine nucleoside and nucleotide requiring enzymes.

Science, 1982 Dec 17, 218(4578), 1223 - 5
Fragment spanning the SV40 replication origin is the only DNA sequence required in cis for viral excision; Conrad SE et al.; A 311-base pair fragment containing the SV40 origin of replication was linked to the chicken thymidine kinase gene on a recombinant plasmid . This molecule was transfected into human 143 thymidine kinase-deficient (TK-) cells, and colonies positive for thymidine kinase were selected . When cell lines derived from these colonies were fused to permissive simian cells that produce SV40 T antigen, the recombinant plasmid excised itself from the human cellular genome and replicated with a high copy number per cell . These results show that this segment of the viral genome is the only sequence required in cis to mediate SV40 excision and replication upon fusion to permissive cells . In addition, we have shown that excised plasmids apparently identical to the input DNA can be efficiently rescued in Escherichia coli . SV40 excision and replication may therefore be useful for the recovery of cloned genes from eukaryotic cells.

Eur J Biochem, 1982 Dec 15, 129(2), 409 - 14
Kinetics of phosphatidylglycerol synthesis in isolated membrane vesicles from Escherichia coli containing different amounts of membrane-bound phosphatidic acid; Demant EJ; Cytoplasmic membrane vesicles of Escherichia coli containing various amounts of phosphatidic acid ranging from 0.2% to more than 50% (mol/mol) of the total lipid has been prepared by de novo synthesis of phosphatidic acid in the isolated membranes from acyl-CoA esters and sn-glycerol 3-phosphate . The kinetics of CTP-initiated phosphatidylglycerol synthesis in the phosphatidic-acid-enriched membranes has been studied . Phosphatidic acid pools with high and low reactivity as substrate for phosphatidylglycerol synthesis were present in the membranes . The two pools were found identical with respect to fatty acid composition and content of molecular species . The rate of phosphatidylglycerol synthesis from the highly reactive phosphatidic acid pool was independent of the phosphatidic acid concentration in membranes containing from 0.2-30% (mol/mol) phosphatidic acid . Ca2+-ions were found to inhibit the synthesis of phosphatidylglycerol . On the basis of the findings presented it is suggested that phosphatidic acid probably plays a minor role as a feed-back modulator of sn-glycerol 3-phosphate acylation in E . coli, and that phosphatidylglycerol synthesis can occur at near maximal rate in growing cells.

Eur J Biochem, 1982 Dec 15, 129(2), 257 - 63
Amino-acid sequence of a heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli; Aimoto S et al.; A heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli was extensively purified by reverse-phase high-performance liquid chromatography . The minimum effective dose of the purified toxin to cause fluid accumulation in suckling mice was 2.5 ng . The amino acid sequence of the purified toxin was determined by Edman degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Ser-Ser-Asn-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr . This sequence was identical to that deduced from the nucleotide sequence encoding a human heat-stable enterotoxin, reported by Moseley et al., except for the C-terminal Tyr residue.

FEBS Lett, 1982 Dec 13, 150(1), 181 - 4
Evidence that the acyl-O-esters are intermediates in the catalysis . The mechanism of rabbit mammary fatty acid synthase; McCarthy AD et al.; The sequence acetyl-CoA leads to acetyl-O-enzyme leads to acetyl-S-acyl carrier protein has for the first time been demonstrated directly with a multifunctional (mammalian) fatty acid synthase . This was achieved by blocking of the active-site thiols of rabbit mammary fatty acid synthase with iodoacetamide . The modified enzyme was incubated with {14C}acetyl-CoA to form acetyl-O-enzyme, and acetyl-CoA was removed rapidly by centrifuge desalting . We were then able to demonstrate transfer of the acetyl group from {14C}acetyl-O-enzyme to the pantetheine thiol in a fragment of rabbit mammary fatty acid synthase containing the phosphopantetheine group, and to E . coli acyl carrier protein.

Biochim Biophys Acta, 1982 Dec 13, 713(3), 570 - 80
Characterization of reconstituted partially purified glycerophosphate acyltansferase from Escherichia coli; Kessels JM et al.; A modification of the method of Snider and Kennedy (J . Bacteriol . (1977) 130, 1072-1083) was worked out to solubilize sn-glycero-3-phosphate acyltransferase from whole cells by Triton X-100 . The solubilized preparation was used for a systematic study of the reconstitution of enzymatic activity as observed by addition of phospholipid vesicles . Although enzymatic activity was regained by addition of vesicles and not by addition of multilayered liposomes, subsequent Sepharose 4B chromatography revealed the enzyme to be incorporated in large lipid aggregates of undefined structures . Incorporation of glycerophosphate acyltransferase in single bilayer vesicles composed of phosphatidylcholine and phosphatidylglycerol (4:1) was obtained after removal to Triton X-100 from the enzyme solution, co-dispersion of enzyme and phospholipids with cholate and Sephadex G-50 gel filtration of this mixture to remove cholate . The optimal conditions for this reconstitution procedure with respect to phospholipid/protein and phosphatidylcholine/phosphatidylglycerol ratio were established . The active site of glycero-3-phosphate acyltransferase in the reconstituted system was localized for at least 90% at the outside surface of the vesicle, as revealed by proteolysis experiments under conditions of vesicle intactness as shown by C-NMR experiments . The reconstituted systems produced only lysophosphatidate from sn-{14C}glycero-3-phosphate and palmitoyl-CoA and showed identical apparent Km for sn-glycero-3-phosphate and identical pH- and temperature-dependencies as the enzyme in isolated Escherichia coli membranes.

FEBS Lett, 1982 Dec 13, 150(1), 9 - 18
Mechanism of lactose-proton cotransport in Escherichia coli . Kinetic results in terms of the site exposure model; Lancaster JR Jr; Rigorous kinetic derivations are presented for the Site Exposure mechanism of lactose-proton cotransport in E . coli {J . Theor . Biol . (1978) 75, 35-50} . Proton translocation inwards is solely associated with the external exposure of the galactoside binding site . A symmetric dimer configuration of the transporter is proposed, resulting in two forms corresponding to the cis and the trans orientation of the binding sites . The cis to trans orientation is inherently unfavorable, induced only by transmembrane substrate gradients . Recently reported extensive kinetic data are straightforwardly predicted by this mechanism, including the complicated effects on the apparent affinity and maximal velocity of uptake exhibited by changes in the magnitude of the proton electrochemical gradient.

FEBS Lett, 1982 Dec 13, 150(1), 228 - 32
Effect of ribonuclease H from chick embryo on the covalent-linked poly(A)--poly(dA) complementary to poly(dT) template; Sawai Y et al.; In vitro poly(dA) synthesis on poly(dT) template can be initiated by poly(A) primer . Poly(A) chains are covalently extended by DNA polymerase . The reaction product consists of poly(dA) chain with poly(A) at their 5'-ends, hydrogen bonded to the template poly(dT) . The primer poly(A) is linked to the product poly(dA) via a 3':5'-phosphodiester bond, and can be specifically removed by ribonuclease H from chick embryos, leaving a 5'-phosphate end of poly(dA) . Poly- or oligoriboadenylate longer than the (pA)5 could serve as a priming activity to synthesize poly(A) covalently linked to poly(dA).

Nucleic Acids Res, 1982 Dec 11, 10(23), 7763 - 75
The regulatory region of phage fr replicase cistron . III . Initiation activity of specific fr RNA fragments; Berzin V et al.; RNA fragments from phage fr covering the complete or part of the replicase cistron initiation region have been used as templates in the formation of a ribosomal initiation complex in vitro . The results so obtained together with our earlier findings in a similar approach applied to fragments of the structurally related RNA from phage MS2 have allowed us to pinpoint the boundaries of the replicase cistron initiation region on phage RNA . A structural model of the above initiation region has been provided which shows that besides the minimal initiation region (comprises the Shine-Dalgarno sequence and initiator AUG), the flanking regions are also involved and are responsible for additional interactions with the ribosome . The flanking regions possibly contribute to the stability of specific contact between the ribosome and template realized by the minimal initiation region.

Nucleic Acids Res, 1982 Dec 11, 10(23), 7657 - 76
Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits . Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide; Chiaruttini C et al.; 1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC) was used to cross-link 30S ribosomal proteins to 16S rRNA within the E . coli 3OS ribosomal subunit . Covalently linked complexes containing 30S proteins and 16S rRNA, isolated by sedimentation of dissociated crosslinked 30S subunits through SDS containing sucrose gradients, were digested with RNase T1, and the resulting oligonucleotide-protein complexes were fractionated on SDS containing polyacrylamide gels . Eluted complexes containing 30S proteins S9 and S12 linked to oligonucleotides were obtained in pure form . Oligonucleotide 5'terminal labelling was successful in the case of S12 containing but not of the S9 containing complex and led to identification of the S12 bound oligonucleotide as CAACUCG which is located at positions 1316-1322 in the 16S rRNA sequence . Protein S12 is crosslinked to the terminal G of this heptanucleotide.

Nucleic Acids Res, 1982 Dec 11, 10(23), 7531 - 42
A new RNA polymerase and in vitro transcription of the origin of replication from rat mitochondrial DNA; Yaginuma K et al.; A new RNA polymerase was found in a rat mitochondrial extract . This enzyme showed strong template preference in vitro for the supercoiled recombinant plasmid consisting of pBR322 and the D-loop region of rat mtDNA carrying the origin of heavy-strand replication . The main products synthesized by the D-loop region were two RNAs of different sizes . Both of these products were light-strand products transcribed from the region upstream from the origin of replication . This specific transcription is discussed in relation to initiation of primer RNA synthesis for heavy-strand replication of rat mtDNA.

Nucleic Acids Res, 1982 Dec 11, 10(23), 7609 - 20
Sequence of the chloroplast 16S rRNA gene and its surrounding regions of Chlamydomonas reinhardii; Dron M et al.; The sequence of a 2 kb DNA fragment containing the chloroplast 16S ribosomal RNA gene from Chlamydomonas reinhardii and its flanking regions has been determined . The algal 16S rRNA sequence (1475 nucleotides) and secondary structure are highly related to those found in bacteria and in the chloroplasts of higher plants . In contrast, the flanking regions are very different . In C . reinhardii the 16S rRNA gene is surrounded by AT rich segments of about 180 bases, which are followed by a long stretch of complementary bases separated from each other by 1833 nucleotides . It is likely that these structures play an important role in the folding and processing of the precursor of 16S rRNA . The primary and secondary structures of the binding sites of two ribosomal proteins in the 16SrRNAs of E . coli and C . reinhardii are considerably related.

J Biol Chem, 1982 Dec 10, 257(23), 14203 - 10
Stereochemistry of sodium borohydride reduction of tryptophan synthase of Escherichia coli and its amino acid Schiff's bases; Miles EW et al.; Tryptophan synthase alpha 2 beta 2 complex containing {4'-3H}pyridoxal phosphate was reduced with sodium borohydride in the presence of various substrates and analogs in an attempt to trap reaction intermediates . Reduction in the presence of L-serine gave noncovalently bound radioactive material which was identified as phosphopyridoxylalanine, presumably resulting from reduction of the intermediate Schiff's base formed between pyridoxal phosphate and alpha-aminoacrylate . The tritium in this compound was located in the pro-R position at C-4', indicating that reduction of the Schiff's base double bond had occurred on the Si face at C-4' . On the other hand, analysis of phosphopyridoxyllysine obtained by hydrolysis of the reduced {3H}pyridoxal-P-alpha 2 beta 2 protein showed that the internal Schiff's base had been reduced on the C-4' Re face, suggesting a cofactor reorientation upon substrate binding . Analysis of phosphopyridoxylalanine from a reduction of unlabeled alpha 2 beta 2 complex in the presence of (2S,3R)-{2,3-2H2}serine with tritiated sodium borohydride demonstrated the presence of tritium at C-4' (50%), C-2 (20%), and C-3 (30%) . According to the configuration at C-3, reduction of the phosphopyridoxal-alpha-aminoacrylate Schiff's base has occurred from the same side of the molecule at C-4' and C-3.

J Biol Chem, 1982 Dec 10, 257(23), 13981 - 6
D-loop cycle . A circular reaction sequence which comprises formation and dissociation of D-loops and inactivation and reactivation of superhelical closed circular DNA promoted by recA protein of Escherichia coli; Shibata T et al.; Excess recA protein, a protein essential to general genetic recombination in Escherichia coli, promotes a sequence of formation and dissociation of D-loops from negative superhelical closed circular double-stranded DNA (form I DNA) and homologous single-stranded fragments in the presence of excess ATP, resulting in inactivation of the form I DNA without apparent damage to the DNA . The dissociation of D-loops is accompanied by hydrolysis of ATP to ADP that apparently depends on homologous DNA molecules (homology-dependent ATP hydrolysis) . However, at a lower concentrations of ATP, we observed anomalous kinetics in the formation and dissociation of D-loops; as the concentration of ATP was decreased, there was a progressively smaller dissociation of D-loops and a faster resynthesis in the second phase, without changing the rate of the first formation of D-loops . This anomaly might suggest that, as the increase in the amount of ADP relative to that of ATP, dissociation form I DNA is stimulated before formation of D-loops is inhibited . We found that addition of ADP inhibited competitively both formation and dissociation of D-loops and that the latter process was more sensitive to the inhibition than was the former process . Addition of a sufficient amount of ADP to inhibit both formation and dissociation of D-loops, cessation of homology-dependent hydrolysis of ATP, or incubation at low temperature resulted in reactivation of form I DNA that had been inactivated by the sequence . In the presence of an ATP-regenerating system, we confirmed our previous result that limiting the amount of recA protein also causes anomalous kinetics in the formation and dissociation of D-loops . These observations indicate that the formation and dissociation of D-loops and the inactivation and reactivation of form I DNA make a circular reaction sequence.

J Biol Chem, 1982 Dec 10, 257(23), 13971 - 6
Regulation of Escherichia coli carbamyl phosphate synthetase . Evidence for overlap of the allosteric nucleotide binding sites; Boettcher B et al.; Regulation of Escherichia coli carbamyl phosphate synthetase by UMP and IMP was examined in studies with various analogs of these nucleotides . Whereas UMP inhibits enzyme activity, the arabinose analog of UMP was found to be an activator . dUMP neither activates nor inhibits, but binds to the enzyme in a manner similar to UMP as evaluated by direct binding studies, sedimentation behavior, and ultraviolet difference spectral measurements . dUMP decreases inhibition by UMP and activation by IMP, but has no effect on activation by L-ornithine . The findings are in accord with the view that IMP and UMP bind to the same region of the enzyme; a possible general model for such overlapping binding sites is considered . Additional evidence is presented that inorganic phosphate can modulate regulation of the activity by nucleotides . Phosphate (and arsenate) markedly increase inhibition by UMP, decrease activation by IMP, but do not affect activation by L-ornithine . The extent of activation by IMP and by L-ornithine and that of inhibition by UMP are decreased when Mg2+ concentrations are increased relative to a fixed concentration of ATP . The findings suggest that the allosteric effectors may affect affinity of the enzyme for divalent metal ions as well as, as previously shown, the affinity of the enzyme for Mg-ATP.

J Biol Chem, 1982 Dec 10, 257(23), 13924 - 9
Mutation-induced changes in RNA polymerase-lac ps promoter interactions; Stefano JE et al.; A composite rate assay has been applied to investigate the rate and mechanism of formation of open promoter complexes . Seven DNA templates were studied which were related to the lac ps promoter by single base pair changes in the -10 or -35 region of promoter sequence homology . These small changes induce a nearly 3 order of magnitude variation in the rate of open complex formation . This variation persists over a wide range of concentrations of RNA polymerase . Nevertheless, all promoters direct open complex formation which proceeds through a "closed" or A polymerase:DNA complex which dissociates readily . These data, when taken together with our previous results on the lac "spacer" mutations, demonstrate that mutation of the lac ps promoter leads to changes in the rate of open complex formation predicted by the following rule . Changes which substitute a less conserved element of sequence in the -10 and -35 regions, or of length in the spacer, always decrease the rate in this homologous series of promoters.

Biochemistry, 1982 Dec 7, 21(25), 6614 - 8
Kinetics of cytochrome b oxidation in antimycin-treated submitochondrial particles; Hatefi Y et al.; It has been shown that in bovine heart submitochondrial particles, antimycin and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) inhibit the oxidation of NADH, succinate, and reduced ubiquinone incompletely, the uninhibited rate being about 20-40 nmol of substrate oxidized min-1 (mg of protein)-1 . By contrast, rotenone, cyanide, BAL (2,3-dimercaptopropanol), and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole {Trumpower, B . L., & Haggerty, J . G . (1980) J . Bioenerg . Biomembr . 12, 151-164} caused essentially complete inhibition when added alone or after maximal inhibition by antimycin or HQNO . Having thus ascertained that the electron leak through the antimycin block appeared to follow the normal path through complex III (ubiquinol: cytochrome c oxidoreductase) and cytochrome oxidase, the reduction of the b cytochromes by substrates and their oxidation through the leak in the antimycin block by molecular oxygen were studied . It was shown that at normal electron flux from NADH and succinate, both cytochromes b562 and b566 were reduced in antimycin-treated submitochondrial particles . Their oxidation after substrate exhaustion was biphasic, however . At 565 minus 575 nm, 56% of the total reduced cytochrome b was oxidized through the leak in the antimycin block at a more rapid rate, while the remaining 44% was oxidized about 10 times slower . When electron flux from substrates to complex III was slowed down by the use of inhibitors or substrates at less than or equal to 0.1 Km concentration, then only reduced b562 accumulated in antimycin-treated particles . The oxidation of b562 after substrate exhaustion or inhibition of substrate oxidation by an appropriate inhibitor occurred at a rate comparable to that of the slower reoxidation phase described above . These results indicated, therefore, that cytochromes b566 and b562 are oxidized through the leak in the antimycin block at two different rates, the reoxidation rate of b566 being about 10 times faster than that of b562 . The implications of these findings on the kinetic relationship of these two cytochromes in the respiratory chain have been discussed.

Biochemistry, 1982 Dec 7, 21(25), 6489 - 96
Fluorescence polarization study of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli; Waskiewicz DE et al.; The lipoic acids of the alpha-ketoglutarate dehydrogenase multienzyme complex from Escherichia coli have been modified with two fluorescent probes, N-(1-pyrenyl)-maleimide and 5-{{{(iodoacetyl)amino}ethyl}amino}-naphthylene-1-sulfonic acid . Time-resolved fluorescence polarization of partially labeled complexes (18-77% inhibition of enzyme activity) reveals a complex depolarization process: one component of the anisotropy is characterized by a rotational correlation time much longer than the time scale of the measurements (less than or equal to 400 ns), reflecting the overall rotation of the complex, while a second component of the anisotropy decays with a rotational correlation time of 320 (+/- 50) ns . This decay is essentially independent of viscosity and is consistent with a model in which the depolarization is due to the dissociation from and rotation of lipoic acids between binding sites on the multienzyme complex . The sum of the rate constants characterizing the association and dissociation with the binding sites is approximately 3 x 10(6) s-1 . In addition, approximately 5% of the anisotropy of the N-(1-pyrenyl)maleimide-labeled complex decays with a rotational correlation time of 25 ns; this can be attributed to local motion of the probe . At high extents of N-(1-pyrenyl)maleimide labeling (90-95% inhibition of enzyme activity), the anisotropy decay can be described by a constant term plus a rotational correlation time of about 1 microseconds . The increase in the correlation time probably reflects interactions between pyrene moieties . The N-(1-pyrenyl)maleimide-labeled dihydrolipoyl transsuccinylase core of the multienzyme complex has been isolated, and the anisotropy is constant over the observed time range of 300 ns . This suggests that the native structure is necessary for observation of lipoic acid movement within the complex . Fluorescent-labeled limited trypsin digestion fragments of the alpha-ketoglutarate dehydrogenase complex also have been isolated, and anisotropy measurements reveal substantial mobility of the label within the fragments . The time-resolved anisotropy of FAD in the native complex and in the isolated dihydrolipoyl dehydrogenase indicates some rapid local mobility of the FAD (rotational correlation time of 12 ns) that is viscosity independent, as well as a component of the anisotropy that is constant over the 35-ns time scale of the experiments.

Biochemistry, 1982 Dec 7, 21(25), 6503 - 8
Aerobactin-mediated utilization of transferrin iron; Konopka K et al.; Aerobactin and enterobactin, hydroxamate- and catechol-type siderophores, respectively, were found capable of removing iron (III) from transferrin in buffered solution . Although under these conditions aerobactin displaced the iron much more slowly than did enterobactin, the rate for the former could be accelerated by addition of pyrophosphate as mediator . Transfer of iron (III) from transferrin to aerobactin appeared to proceed via a ternary complex . Cells of Escherichia coli BN 3040 NalR iuc containing transport systems for both enterobactin and aerobactin, the genetic determinants for the latter specified on a ColV-type plasmid, took up iron from {55Fe}transferrin in minimal medium . In this case aerobactin was effective at a much lower concentration, although enterobactin still displayed superior ability to transfer the iron . In serum, however, the rate measured with aerobactin exceeded that found with enterobactin . The results indicate that aerobactin, in spite of its relatively unimpressive affinity for iron (III) as a siderophore, is nonetheless equipped with structural features or properties that enhance its ability to remove the metal ion from transferrin, especially when receptor-bearing cells of E . coli are present to act as a thermodynamic sink for the iron . These attributes of the aerobactin system of iron assimilation may account for its status as a virulence determinant in hospital isolates of E . coli.

Arch Microbiol, 1982 Dec 3, 133(4), 274 - 7
The effect of gyrase inhibitors and cyclic AMP on induction and glucose repression of the 6-hydroxy-nicotine oxidases in Arthrobacter oxidans; Brandsch R et al.; The induction by D,L-nicotine of the enantiozymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase in Arthrobacter oxidans was differently affected by the inhibitors of Escherichia coli gyrase, novobiocin and nalidixic acid . These compounds inhibited 6-hydroxy-L-nicotine oxidase induction slightly, but led to an increase in the level of 6-hydroxy-D-nicotine oxidase activity . Furthermore, the specific repression by glucose of 6-hydroxy-D-nicotine oxidase synthesis was not abolished by the addition of cAMP but by that of novobiocin.

Can J Biochem, 1982 Dec, 60(12), 1132 - 42
Purification and characterization of dihydrofolate reductase from Walker 256 carcinosarcoma; Johnson SJ et al.; Dihydrofolate reductase of Walker 256 carcinosarcoma has been purified to homogeneity by affinity chromatography . The enzyme reduced 28 mumol dihydrofolate (FAH2) . min-1 . mg protein-1 at 22 degrees C and pH 7.3 . Km values with respect to FAH2 and NADPH were 21 and 29 mM, respectively . The IC50 (amount of inhibitor required for 50% loss of enzyme activity) values were 0.2 nM for MTX and aminopterin and 50 and 67 nM, respectively, for N10-formyl FA and triazinate (NSC-139105) . The pH maximum is around pH 7.0 and the isoelectric point is 6.8 . This reductase has an apparent molecular weight of 21 500 . The N-terminal amino acid is valine and the comparison of the N-terminal 20 residues of this reductase shows very high sequence homology with other mammalian reductases . The enzyme contains two cysteine residues and one of these residues is not involved in catalysis . This reductase has four tryptophan residues and modification of one of these residues leads to loss of activity . The intrinsic circular dichroic (CD) spectrum of this reductase is very different from the CD spectra of reductase of Escherichia coli B and L1210/MTX . However, the CD spectra of the enzyme--substrate and enzyme--inhibitor complexes are very similar to that of the L1210/MTX enzyme . This suggests that the ligands may be constrained in similar conformation on the two enzymes . The fluorescence emission maximum at 314 nM when activated at 286 nM is considerably lower than other mammalian enzymes.

J Pediatr, 1982 Dec, 101(6), 901 - 5
Effects of prematurity on the immunologic system in human milk; Goldman AS et al.; A longitudinal study of the effect of prematurity on the development of several components of the immunologic system in human milk was performed . Milk was obtained during the second through the twelfth week after parturition . The mean concentrations of lactoferrin and lysozyme were greater in preterm than in term milk during each interval of lactation . The patterns of change in these components were similar for term and preterm milk . Secretory IgA was the predominant form of IgA in preterm milk . The mean concentrations of IgA were greater in preterm milk throughout the study period . Furthermore, total and secretory IgA levels in preterm milk rose linearly during the sixth through the twelfth week, whereas the concentrations of IgA did not change in term milk during that period . In most preterm mothers, secretory IgA antibodies to Escherichia coli somatic antigens increased as lactation proceeded . These increments in specific antibodies usually did not correlate with changes in total secretory IgA . In addition, leukocyte counts in preterm milk were usually lower at two weeks and higher at 12 weeks than in term milk . Thus the concentrations of certain components of the immunologic system in human milk are altered by premature delivery . A decrease in milk volume may account for some changes, whereas certain alterations may be the result of other consequences of premature delivery or less stimulation by the premature infant.

Proc Natl Acad Sci U S A, 1982 Dec, 79(24), 7644 - 8
Cloning the double-stranded RNA genes of reovirus: sequence of the cloned S2 gene; Cashdollar LW et al.; The genes of the Dearing strain of reovirus serotype 3, which consist of double-stranded RNA, have been cloned into pBR322 by tailing both strands of each gene with poly(A), transcribing them with reverse transcriptase, self-hybridizing the cognate plus and minus cDNA strands, incubating them with Escherichia coli DNA polymerase I to ensure that they are complete, and cloning the double-stranded cDNA molecules by standard procedures . The sequence of the cloned S2 gene has been determined . The sequence at the termini are exactly the same as those at the ends of the native double-stranded RNA gene . The gene is 1,329 nucleotides long and possesses a single long open reading frame that starts at the first initiation codon (residue 19) and extends for 331 codons, sufficient to encode a protein of the same size as the known S2 gene product, protein sigma 2, a major reovirus core component (Mr, 38,000) . A second open reading frame of 85 codons, in a different phase, starts close to where the first ends . The protein translated from this reading frame is unknown.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7258 - 62
In vitro characterization of the fibroin gene promoter by the use of single-base substitution mutants; Hirose S et al.; A highly efficient method for segment-directed mutagenesis has been developed . The method relies on the deamination by sodium nitrite of the bases in the separated strands of a small DNA restriction fragment . The mutagen-treated strands produce transition mutations by the following sequence: (i) hybridization with the complementary strand of the wild-type DNA that had been cloned into a phage fl vector, (ii) repair synthesis in vitro, and (iii) transfection of Escherichia coli . Using this method, we have isolated 14 single-point mutants within a 31-base-pair stretch of the fibroin gene (from the T-A-T-A box at the nucleotide position -30 to the cap site at +1) . In vitro transcription experiments with the HeLa cell or the silk gland cell extract show that single-base transitions at the T-A-T-A box (T to C at -30, A to G at -29, and T to C at -28) and at the -20 region (G to A at -21, T to C at -20, and A to G at -17) result in decreased promoter activities, whereas those at the cap site and the -10 regions have no effect . The initiation site of transcription is the same for five "down" (reduced activity) mutants (T to C at -30, T to C at -28, G to A at -21, T to C at -20, and A to G at -17), the cap site mutant (A to G at +1), and the wild-type genes--position +1 . However, the A-to-G transition at -29 (the second base of the T-A-T-A box) induces an additional transcription start from position +4 . Functions of the T-A-T-A box and the -20 regions are discussed.

Jpn J Pharmacol, 1982 Dec, 32(6), 1135 - 42
Drug interaction of antitumor drugs . III . Antitumor activity of tegafur in lipopolysaccharide-treated mice; Sasaki K et al.; The effect of lipopolysaccharide (obtained from Escherichia coli, LPS) on the antitumor activity, acute toxicity and metabolism of tegafur was investigated in mice in comparison with 5-fluorouracil (5-FU) . It was found that the intravenous administration of LPS (1.25 or 2.5 mg/kg) 24 hr prior to tegafur decreased the antitumor activity of tegafur against the solid form of Sarcoma 180 . On the acute toxicity of tegafur or 5-FU, the lethality of the former was decreased and that of the latter was enhanced by the pretreatment with LPS 24 hr before . In LPS-treated mice, after the administration of tegafur, the level of tegafur in plasma was higher and the elevated level maintained longer than in untreated mice; and a small amount of 5-FU was released . A high level of 5-FU in plasma after the administration of 5-FU was also observed in LPS-treated mice . In the liver and kidneys of LPS-treated mice, the level of 5-FU after the administration of tegafur or 5-FU was higher, and its conversion of 5-FU to fluorouridine (FUR) was lower than that of control mice . On the other hand, LPS inhibited significantly the hepatic drug-metabolizing enzymes 24 hr after . It can, therefore, be presumed that the antitumor activity of tegafur was affected with LPS as a result of inhibition of conversion from tegafur to 5-FU or from 5-FU to FUR mainly according to depression in the hepatic drug-metabolism.

Eur J Biochem, 1982 Dec, 129(1), 127 - 32
Isolation and characterization of Streptomyces aureofaciens protein-synthesis elongation factor Tu in an aggregated state; Weiser J et al.; The ability of EF-Tu to aggregate spontaneously was employed for the purification of homogeneous EF-Tu . GDP from Streptomyces aureofaciens . The formation of filamentous structures in the aggregated EF-Tu was demonstrated in a light microscope . The purified factor, with a specific activity of 19,100 +/- 1,000 units/mg in {3H}GDP exchange, was shown to be active in the translation of poly(U) . Aggregated EF-Tu . GDP exhibited almost eight-times lower GDP-exchange capacity at 2 degrees C than at 30 degrees C . This suggests that GDP-binding sites are not freely accessible at lower temperatures in the aggregated factor, in contrast to Escherichia coli polymerized EF-Tu . Turbidimetric assays revealed that the solubilization of diluted aggregated S . aureofaciens EF-Tu is strongly dependent on temperature and causes an increase in the number of accessible GDP-binding sites.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7152 - 6
Protein D1 preferentially binds A + T-rich DNA in vitro and is a component of Drosophila melanogaster nucleosomes containing A + T-rich satellite DNA; Levinger L et al.; Our previous work {Levinger, L . & Varshavsky, A . (1982) Cell 28, 375-385} has shown that D1, a 50-kilodalton chromosomal protein of Drosophila melanogaster, is specifically associated with isolated nucleosomes that contain a complex A + T-rich satellite DNA with buoyant density of 1.688 g/ml . We show here that D1 is also a component of nucleosomes containing a simple-sequence, pure A + T satellite DNA, buoyant density 1.672 g/ml . Furthermore, using a modification of a protein blotting technique in which proteins are not exposed to dodecyl sulfate denaturation, we have found that D1 preferentially binds to A + T-rich double-stranded DNA in vitro, and it is apparently the only abundant nuclear protein in cultured D . melanogaster cells that possesses this property . Synthetic poly{d(A-T)}.poly{d(A-T)} and poly(dA).poly(dT) duplexes effectively compete in vitro with A + T-rich D . melanogaster satellite DNAs for binding to D1, whereas total Escherichia coli DNA is an extremely poor competitor . These findings strongly suggest that D1 is a specific component of A + T-rich, tandemly repeated, heterochromatic regions, which constitute up to 15-20% of the total D . melanogaster genome . Possible functions of D1 protein include compaction of A + T-rich heterochromatin and participation in microtubule-centromere interactions in mitosis . In addition, D1 may prevent nonspecific binding to A + T-rich satellite DNA of other nuclear proteins that have a preference for AT-DNA, such as RNA polymerase or regulatory proteins, and may also participate in the higher-order chromatin organization outside tandemly repetitive regions by binding to nonrandomly positioned stretches of A + T-rich DNA.

Cancer Res, 1982 Dec, 42(12), 4979 - 84
An unusual transfer RNA (guanine-2-)-methyltransferase from transplantable rat mammary tumors; Brunke KJ et al.; We have previously demonstrated that (guanine-2-)-methyltransferase activity in extracts from 9,10-dimethyl-1,2-benzanthracene-induced rat mammary tumors differs from that of nonneoplastic mammary tissue . In this report, we explore further the nature of these differences by purification and characterization of the two major transfer RNA (tRNA) (guanine-2-)-methyltransferases from transplantable mammary tumors and proliferating mammary glands from pregnant rats . The position 10-specific (guanine-2-)-methyltransferases (2mGI) from proliferating rat mammary gland and mammary tumor were found to have similar properties with respect to molecular weight, substrate specificity, and elution behavior on ion-exchange columns . In addition, no tissue-specific differences were observed when the mammary tumor and mammary gland 2mGI activities were compared with those of purified rat liver enzyme . In contrast, the position 26-specific (guanine-2-)-methyltransferase (2mGII) from mammary tumors was seen to possess properties different from both the nontumorous mammary gland and liver enzyme . The tumor 2mGII activity showed unusual elution behavior on diethylaminoethyl-Sephadex, eluting along with the 2mGI activity . A small difference in molecular weight was detected between tumor and nontumorous 2mGII activities . Examination of the tumor enzyme in comparison with the well-characterized 2mGII from rat liver indicated that the mammary tumor 2mGII methylated a broader range of tRNA substrates . In particular, mature yeast phenylalanine-specific tRNA, which is methylated in vivo at all major eukaryotic methylation sites and should not be a substrate for eukaryotic methylating enzymes in vitro, could be methylated at low levels by the tumor enzyme . Two-dimensional electrophoretic fingerprint maps of T1 RNase-digested phenylalanine-specific tRNA from Escherichia coli methylated in vitro showed the presence of a methylated oligonucleotide which could not be correlated with normal sites of methylation on the tRNA . From these results, it appears that the mammary tumor 2mGII can methylate at some unusual site(s) on the tRNA molecule.

J Gen Microbiol, 1982 Dec, 128 (pt 12), 3067 - 70
Effect of acriflavine on the plasma membrane of Escherichia coli K12; Nakamura H et al.; Plasma membranes of acriflavine-sensitive mutant (acrA) and acriflavine-resistant (acrA+, wild-type and true revertant) Escherichia coli K12 strains treated with acriflavine were observed under the electron microscope by means of the freeze-fracture technique . The plasma membrane of the acrA mutant exhibited a complex lamellar structure at the end of the cell when treated with 20 micrograms acriflavine ml-1 . However, the membrane of the acrA+ cells also gave the lamellar complex when treated with a very high concentration of acriflavine (100 micrograms ml-1) . The size of the intramembranous particles was not affected by the acriflavine treatments.

J Gen Microbiol, 1982 Dec, 128 (pt 12), 2877 - 92
Cell cycle dynamics inferred from the static properties of cells in balanced growth; Koch AL et al.; The duration of a morphological phase of the cell cycle is reflected in the steady state distribution of the sizes of cells in that phase . Relationships presented here provide a method for estimating the timing and variability of any cell cycle phase . It is shown that the mean size of cells initiating and finishing any phase can be estimated from (1) the frequency of cells exhibiting the distinguishing morphological or autoradiographic features of the phase; (2) the mean size of cells in the phase; and (3) their coefficient of variation . The calculations are based on a submodel of the Koch-Schaechter Growth Controlled Model which assumes that (i) the distribution of division sizes is Gaussian; (ii) there is no correlation in division sizes between successive generations; and (iii) every cell division gives rise to two daughter cells of equal size . The calculations should be useful for a wider range of models, however, because the extrapolation factors are not sensitive to the chosen model . Criteria are proposed to allow the user to check the method's applicability for any experimental case . The method also provides a more efficient test of the dependence of growth on cell size than does the Collins-Richmond method . This is because the method uses the mean and coefficient of variation of the size of the total population, in conjunction with those of the cells in a final phase of the cell cycle, to test potential growth laws . For Escherichia coli populations studied by electron microscopy, an exponential growth model provided much better agreement than did a linear growth model . The computer simulations were used to generate rules for three types of cell phases: those that end at cell division, those that start at cell division, and those totally contained within a single cell cycle . For the last type, additional criteria are proposed to establish if the phase is well enough contained for the formulae and graphs to be used . The most useful rule emerging from these computer studies is that the fraction of the cell cycle time occupied by a phase is the product of the frequency of the phase and the ratio of the mean size of cells in that phase to the mean size of all cells in the population . A further advantage of the techniques presented here is that they use the 'extant' distributions that were actually measured, and not hypothesized distributions nor the special distributions needed for Collins-Richmond method that can only be calculated from the observed distributions of dividing or newborn cells on the basis of an assumed growth law.

J Gen Microbiol, 1982 Dec, 128 (pt 12), 2865 - 76
Variation of generation times in Escherichia coli populations: its cause and implications; Bremer H; The cell cycle of Escherichia coli contains a period of indeterminate length that reflects a stochastic reaction, beginning at some time after a round of chromosome replication, and ending before the cell divides . Although the chemical nature of this reaction is not known, the time of its onset and the single statistical 'half-life parameter' required for its quantitative description have been measured previously . Here it is shown that this parameter implies the distribution of generation times and the age distribution, as well as the distributions of replication initiation and termination ages; these distributions are derived from this half-life parameter for exponentially growing populations of E . coli . It is also shown that the stochastic reaction affects the results and interpretation of any experiments involving synchronous growth of bacteria.

Bull Assoc Anat (Nancy), 1982 Dec, 66(195), 519 - 29
{Infectious diarrhea: weakening of mucosal protection caused by a pathogenic Escherichia coli in ileal loops in the rabbit}; Rateau JG et al.; The histological, enzymatic and water-electrolyte modifications induced by a toxinogenic strain of E . coli (O128B12) were studied . The observed water loss was due to the action of the bacterial toxins on the ionic pumps of the epithelial cells . After 4 hours of contact with the bacterial strain, one-third of the goblet cells of the intestinal epithelium were totally degranulated . This indicated an increase in the destruction of the mucus and therefore the weakening of mucosa protection through action of bacterial enzymes and toxins.

Aust J Exp Biol Med Sci, 1982 Dec, 60(6), 675 - 86
Superhelical turns of closed circular DNA in solution and in a gel: evidence for a conformational difference; Dougherty G; We have compared the number of superhelical turns, tau, in circular covalently closed plasmid pBR beta G DNA obtained by four different methods, each based on one particular distinguishing principle . Three of the methods allow an unequivocal determination of tau under gel electrophoresis conditions, whilst the fourth enables us to determine its value in solution . We were able to detect a significant difference between the two environments, corresponding to an unwinding of the DNA duplex angle by 0.3 degrees when a sample is transferred from solution to gel . The possible existence of such an effect has been generally overlooked by previous investigators . Our result suggests that the previously reported value for the number of base-pairs per helical turn should be adjusted downwards by about 0.10, so that it applies to conditions in solution.

Gene, 1982 Dec, 20(3), 471 - 5
Hfr-mediated conjugative transfer of pBR322 vector carrying the chromosomal DNA of Escherichia coli; Yamada M et al.; Hybrid plasmids consisting of a non-mobilized plasmid, pBR322, and a segment of chromosomal DNA of Escherichia coli could be transferred from an Hfr donor to recipient cells by a bacterial mating . When the chromosomal DNA in the plasmid corresponded to the early transfer region of the Hfr, the frequency of the transfer was high . The recA function of both donor and recipient cells was required in the transfer . The physical association of the hybrid plasmid with the transferring Hfr chromosome through the homologous sequences may mediate the transfer of the non-mobilized plasmid . This phenomenon is useful for the determination of the chromosomal location of an unidentified fragment cloned in a non-mobilized plasmid.

Gene, 1982 Dec, 20(2), 169 - 76
Selective deletion of large segments of Balbiani ring DNA during molecular cloning; Case ST; Transcription units in Balbiani ring 1 (BR1) and Balbiani ring 2 (BR2) of Chironomus tenans salivary glands are comprised of about 40 kb of repetitive DNA sequences organized in a satellite-like array . Because of this sequence organization, it was possible to prepare 30 to 40-kb target DNA fragments for cloning by performing limit restriction endonuclease digestion of high-Mr genomic DNA . These fragments were ligated to cohesive termini of the linearized cosmid, pHC79, packaged in vitro, and used to transduce Escherichia coli . Alternatively, target fragments were randomly sheared to a mean length of 8-10 kb, annealed to plasmid pBR322 using homopolymeric tails, and used for bacterial transformation . Recombinant cosmids and plasmids generally contained inserts which were proportional to the length of target fragments used in ligation reactions . However, the subset of recombinants that hybridized to 32P-labeled 75S RNA (highly enriched in BR1 + BR2 sequences) had disproportionately smaller inserts . With the exception of one metastable clone with a 2.1-kb insert, all others had inserts of 0.8 or 0.4 kb . Similar results were obtained in host cells that were recA- or recBC- . The most likely conclusion is that repetitive BR sequences are highly unstable during replication in E . coli and are selectively deleted.

Can J Physiol Pharmacol, 1982 Dec, 60(12), 1680 - 5
Effects of epinephrine, clonidine, L-phenylephrine, and morphine on intestinal secretion mediated by Escherichia coli heat-stable enterotoxin in pig jejunum; Ahrens FA et al.; Perfusion of pig jejunum with Escherichia coli heat-stable enterotoxin (strain 1261) reversed net absorption of water and electrolytes to net secretion . Addition of the alpha-adrenergic agonists clonidine (5 X 10(-7) M) or L-phenylephrine (5 X 10(-6) M), or the opiate agonist morphine (3.6 X 10(-6) M) to the perfusate reduced the secretory response to enterotoxin and stimulated absorption in normal jejunum . Epinephrine (5 X 10(-5) M) did not stimulate absorption in controls but reduced chloride loss in the presence of enterotoxin . Mucosal sodium--potassium adenosine triphosphatase was unchanged but disaccharidase activity was decreased in the presence of enterotoxin . The results suggest that alpha-adrenergic agonists and opiate agonists may exert an antidiarrheal action by increasing net transport across intestinal epithelium.

Can J Biochem, 1982 Dec, 60(12), 1143 - 7
Large-scale production of citrate synthase from a cloned gene; Duckworth HW et al.; Starting with a colicin E1 resistance recombinant plasmid which contains gltA, the gene for citrate synthase in Escherichia coli, we have constructed an ampicillin-resistance plasmid containing the gltA region as a 2.9-kilobase-pair insert in the tetracycline-resistance region of pBR322 . Escherichia coli HB101 harbouring this plasmid, when grown on rich medium containing ampicillin, contains citrate synthase as about 8% of its soluble protein . The enzyme has been purified from this rich source and is identical to the chromosomal enzyme prepared previously in every property tested, except for specific activity, which is 64 U . mg-1 as compared with 45-50 U . mg-1 previously obtained . The N-terminal sequences of both enzymes are reported, and they are identical up to residue 16 at least . The overall yield of pure enzyme, starting with the cells grown in 15 L of medium, is 600-800 mg.

Can J Biochem, 1982 Dec, 60(12), 1095 - 100
Analysis of the expression of cloned genes using an Escherichia coli cell-free system; See YP et al.; An Escherichia coli coupled transcription-translation cell-free system, which is efficient in the synthesis of proteins directed by exogenously added DNA, is described . These cell-free extracts direct protein synthesis against a low background of endogenous protein synthesis providing a means for analyzing the expression of isolated genes . This is especially important when using restriction enzyme-linearized DNAs which are less efficient templates than circular DNAs . This cell-free system has been used to study the expression of the proteins coded by plasmids pBR322 and pBL101.

Biochem J, 1982 Dec 1, 207(3), 529 - 33
The determination by radiochemical assay of argininosuccinase produced in an Escherichia coli system in vitro; Zidwick MJ et al.; A highly sensitive radiochemical assay used to measure the synthesis and regulation of the product of the argH gene, argininosuccinase, in an Escherichia coli system in vitro is described . With L-{guanidino-14C}argininosuccinic acid as a substrate, and in the presence of excess arginase and urease, 14CO2 is collected in a simply designed micro-vessel . With this method less than 1 nmol of product can be measured in the presence of various concentrations of L-arginine.

Am J Vet Res, 1982 Dec, 43(12), 2138 - 42
Enhancement of the chemotactic response of bovine polymorphonuclear leukocytes by levamisole; Jayappa HG et al.; The effects of levamisole on random migration, chemotaxis, phagocytosis, and intracellular killing by bovine polymorphonuclear leukocytes (PMN) were investigated . Chemotactic assays were performed, using Micropore filters in modified Boyden chambers . Phagocytosis and intracellular killing of Escherichia coli were analyzed by standard viable bacterial counts . Results indicated that levamisole enhanced the chemotactic response of PMN at concentrations ranging from 10(-4) to 5 x 10(-3)M . Phagocytes collected from cows at 90 minutes after IM injection of levamisole also showed enhanced random migration and chemotaxis . Freshly prepared serum was shown to enhance the levamisole-induced stimulation of chemotaxis . Levamisole had no effect on phagocytosis or intracellular killing of E coli by bovine PMN.

J Clin Microbiol, 1982 Dec, 16(6), 1086 - 90
Detection of enterotoxigenic Escherichia coli in water by filter hybridization with three enterotoxin gene probes; Echeverria P et al.; The DNA hybridization assay for genes encoding for Escherichia coli enterotoxins was used to examine water specimens in Thailand . In a reconstruction experiment, the DNA hybridization assay was 10(4) times more sensitive than testing random E . coli in the Y-1 adrenal and suckling mouse assays in identifying enterotoxigenic E . coli (ETEC) in water . Drinking and bathing water collected from 2 of 10 different homes of individuals with ETEC-associated diarrhea and 6% (5 of 78) and 11% (11 of 78) of drinking and bathing water samples collected from homes of individuals with diarrhea without ETEC infections, as well as 6% (5 of 77) and 8% (6 of 77) of drinking and bathing water collected from homes in which no inhabitants had diarrhea, were homologous with the DNA probes . Ten E . coli from each of the 31 water specimens which contained bacteria which were homologous with the DNA probes were tested in the Y-1 adrenal and suckling mouse assay . In only 2 of these 31 specimens could ETEC be identified with the standard assays . The DNA hybridization assay is a much more sensitive means of detecting organisms carrying genes coding for enterotoxin production than testing 10 individual colonies in the Y-1 adrenal and suckling mouse assays . This novel application of recombinant DNA technology provides a sensitive method of detecting organisms carrying genes coding for enterotoxin, and this method will be useful in defining the epidemiology of ETEC.

Eur J Biochem, 1982 Dec, 129(1), 51 - 6
Small-angle X-ray scattering studies of tryptophan synthase from Escherichia coli and its alpha and beta 2 subunits; Wilhelm P et al.; The alpha and beta 2 subunits of tryptophan synthase were investigated by small-angle X-ray scattering . The molecular parameters are: radius of gyration, alpha: 1.95 nm, beta 2: 3.01 nm; maximum particle diameter, alpha: 5.8 nm, beta 2: 10.5 nm; and hydrated volume, alpha: 60 nm3, beta 2 160 nm3 . The shape of the alpha subunit can best be described by a circular cylinder, slightly tapered at one end . An elongated elliptical cylinder with its cross section larger in the middle than at the ends was found to be a model equivalent in scattering to the beta 2 subunits . The alpha 2 beta 2 enzyme complex was found to have a radius of gyration of 4.01 nm, a maximum length of 13.5 nm, and a hydrated volume of 270 nm3 . No satisfactory fit of the scattering data was obtainable by mere apposition of the models of the alpha and beta 2 subunits . Two cylinders overlapping laterally fit the experimental data considerably better, suggesting changes in the conformation of the subunits on forming the alpha 2 beta 2 complex.

Eur J Biochem, 1982 Dec, 129(1), 233 - 9
Signal peptide digestion in Escherichia coli . Effect of protease inhibitors on hydrolysis of the cleaved signal peptide of the major outer-membrane lipoprotein; Hussain M et al.; Upon incubation of the envelope fraction of Escherichia coli a precursor of the major outer membrane lipoprotein that accumulates in the cytoplasmic membrane of the globomycin-treated cell is processed to the mature form {Hussain, M., Ichihara, S., and Mizushima, S . (1980) J . Biol . Chem . 255, 3707-3712; (1982) J . Biol . Chem . 257, 5177-5182} . When this precursor-containing envelope fraction was incubated in the presence of protease inhibitors such as antipain, leupeptin, chymostatin and elastatinal, a new peptide appeared on a polyacrylamide gel at the position where the signal peptide was expected to appear . This was proved to be the signal peptide of the lipoprotein from the following facts: (a) its appearance is in proportion to the appearance of the lipoprotein and disappearance of the precursor; (b) when the cleavage of the signal peptide from the precursor was inhibited by globomycin, the peptide did not appear on the gel; and (c) the results of labeling of the peptide with {3H}leucine, {35S}methionine and {3H}arginine were consistent with the amino acid composition of the signal peptide . The signal peptide thus accumulated in the envelope fraction was hydrolyzed by an enzyme named 'signal peptide peptidase' when the envelope fraction was washed to remove the inhibitors . The hydrolysis was inhibited by re-addition of these inhibitors . The signal peptide peptidase hydrolyzed the signal peptide only after its cleavage from the lipoprotein precursor.

Antimicrob Agents Chemother, 1982 Dec, 22(6), 942 - 8
Role of porin proteins OmpF and OmpC in the permeation of beta-lactams; Jaffe A et al.; Mutants of Escherichia coli K-12 lacking major outer membrane proteins were obtained by selecting for resistance to the beta-lactam cefoxitin . Three classes of resistant strains were found: mutants in ompB, a regulatory locus for proteins OmpF and OmpC; mutants in ompF; and one mutant in tpo . The OmpF and OmpC proteins facilitate penetration of beta-lactams through the outer membrane.

Tsitologiia, 1982 Dec, 24(12), 1417 - 23
{Postreplication DNA repair in Escherichia coli cells . I . Defect of inducible repair in the thermosensitive mutant AX727 dnaZts at a nonpermissive temperature}; Zhestianikov VD et al.; After UV-irradiation at the non-permissive temperature 43 degrees C the survival of the thermosensitive mutant Escherichia coli AX727 dnaZts (the dnaZ gene controls the synthesis of gamma-subunit of DNA polymerase III holoenzyme) appeared somewhat lower and postreplication perair of its DNA less effective by 5-15% than at the permissive temperature 30 degrees . The survival of chloramphenicol treated cells decreased both at 43 and 30 degrees C . Under the influence of chloramphenicol, the postreplication repair of DNA in AX727 dnaZts cells at 30 degrees lower to the level of this process seen at 43 degrees C . The same process in thermoresistant revertant AX729 dnaZtr at 30 degrees and 43 degrees C decreased to the level of the process in AX727 cells at 43 degrees C . The data indicate that dnaZ gene product is necessary for the inducible branch of postreplication DNA repair . By this process (presumably together with prereplication excision repair), the cell eliminates 20% of UV-inducible lethal damages . Besides, in AX727 dnaZts cells there functions protein synthesis dependent repair not associated with the postreplication repair of DNA; this latter process eliminates about 60% lethal damages of the cell.

Proc Natl Acad Sci U S A, 1982 Dec, 79(24), 7689 - 93
Identification of a sequence responsible for periodic synthesis of yeast histone 2A mRNA; Osley MA et al.; Sequences required for the regulated expression of a yeast histone 2A (H2A) gene have been investigated by using fusions between this gene and the Escherichia coli lacZ gene . Fusions containing the entire spacer region in which divergent transcription of the H2A and H2B genes is initiated result in low-level constitutive synthesis of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in yeast . Regulated expression (which is characterized by periodic synthesis during the S phase of the cell cycle) is restored when a 1.3-kilobase HindIII fragment containing a small region of the 3' end of the H2B gene is present in either orientation . The regulatory activity in this region appears to be coincident with a sequence that supports autonomous replication in yeast.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7385 - 9
The yeast his3 promoter contains at least two distinct elements; Struhl K; Phenotypic analysis of 65 mutations indicates that the yeast his3 promoter is composed of at least two separate regions of DNA . Each is necessary, but neither is sufficient for wild-type levels of his3 expression . Deletion mutations that destroy either promoter element express his3 poorly or not at all . The upstream element is located between 112 and 155 base pairs before the site of transcriptional initiation (nucleotides -112 to -155) . A comparison of derivatives strongly suggests that the downstream element maps somewhere between nucleotides -32 and -52 and includes a sequence between nucleotides -45 and -52 . This location coincides with sequences conserved before most eukaryotic genes(the TATA box region) . By using derivatives in which his3 sequences are replaced by a small fragment of coliphage M13 DNA, three properties of the his3 promoter were established . First, his3 TATA box deletions fail to express his3 because they lack specific sequences and not because they disrupt spacing relationships between other sequences . Second, the TATA box region can be replaced functionally by the one orientation of the M13 DNA fragment that contains a TATA-like sequence . Third, the distance between the two elements (normally 90 base pairs) can be varied between 40 and 160 base pairs without markedly affecting promoter function . These results strongly suggest that yeast RNA polymerase II, unlike its Escherichia coli counterpart, does not bind simultaneously to both promoter elements, and they add further support to the view that the upstream element is not part of a transcriptionally competent binding site . This ability of the his3 upstream promotor element to act at a long and variable distance is similar to properties of viral enhancer sequences and is reminiscent of position effects in yeast.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7248 - 52
Identification of the origins of T4 DNA replication; King GJ et al.; Two physical origins of T4 DNA replication were determined by hybridization of viral DNA prepared 2.5 min after infection to a display of total T4 DNA . This is the earliest time after T4 infection of Escherichia coli at 37 degrees C that labeled and hybridizable DNA can be detected . The two origins, separated by about 25 kilobases, were identified and localized in the early region of the T4 map . One of them is located in a 5.6-kilobase EcoRI fragment containing genes 62-46 . The other is located between genes rI and e in a 1.9-kilobase EcoRI fragment . Both of these T4 fragments have been cloned and their interactions with the host cell are discussed.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7238 - 42
Initiator proteins for the assembly of the 50S subunit from Escherichia coli ribosomes; Nowotny V et al.; An rRNA-binding protein that binds to the rRNA independently of other proteins during the course of ribosomal assembly is termed "assembly initiator protein." In spite of the large number of rRNA-binding proteins (more than 17 out of 32 proteins have been identified in the case of the large ribosomal subunit), only a very small number of proteins should actually initiate ribosomal assembly for theoretical reasons . Here we demonstrate that only two of the L proteins derived from the large subunit (50S) function as assembly initiator proteins . Two different techniques are used to identify these initiator proteins: reconstitution experiments with purified proteins and pulse-chase experiments during in vitro assembly . Both methods independently identify L24 and L3 as initiator proteins for the 50S assembly . The existence of two initiator proteins (not just one) resolves an apparent contradiction--namely, that on the one hand, rRNA is synthesized in excess under unfavorable growth conditions, whereas on the other hand, rRNA-binding proteins should be available for translational control.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7214 - 7
Local mutagenesis within deletion loops of DNA heteroduplexes; Peden KW et al.; An efficient method has been developed to generate base substitution mutations within deletion loops of DNA heteroduplexes . This method utilizes a heteroduplex formed between a deletion mutant cloned in a plasmid vector and its wild-type counterpart from which two restriction sites had been removed from the vector . The heteroduplex is exposed to sodium bisulfite to deaminate cytosine residues in the single-stranded loop, and the mutagenized plasmid DNA is used to transform a strain of bacteria lacking the enzyme uracil N-glycosylase . Pooled progeny DNA is digested with the two restriction enzymes, whose sites had been mutated in the wild-type plasmid, to eliminate the original deletion mutant DNA . Point mutants with C . G-to-T . A transitions are obtained at high frequency after a second transformation . To test the feasibility of the approach, the tetracycline resistance gene of pBR322 was chosen as the target sequence . It was found that the proportion of tetracycline-sensitive transformants increased as both the size of the heteroduplex loop and the time of incubation with the mutagen increased and this varied from 20% up to 70% . Nucleotide sequence analysis of several tetracycline-sensitive mutants confirmed that C-to-T transitions had been produced in the segment of DNA corresponding to the deletion loop.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7171 - 4
Interactions of metal-nucleotide complexes with aspartate carbamoyltransferase in the crystalline state; Honzatko RB et al.; We report the results of crystallographic difference maps at 3.0-A resolution of complexes of metal-nucleoside triphosphates with aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli . The complexes Gd3+-ATP, Al3+-ATP, and Gd3+-CTP bind to the allosteric effector domain of the enzyme in nearly the same orientation as the metal-free nucleotides . The result is consistent with kinetic observations of nearly identical allosteric efficacy of ATP and CTP and their complexes with cations . The effector Gd3+-GTP, however, binds in a distinctly different conformation and location than does 8-bromoguanosine 5'-triphosphate, reported in a separate investigation {Honzatko, R . B . & Lipscomb, W.N . (1982) J . Mol . Biol . 160, 265-286} . The difference in the binding modes of Gd3+-GTP and the bromo derivative suggests a possible mechanism for the relief of allosteric inhibition of GTP due to metal cations . We observe no binding of metal-nucleoside triphosphates in the region of the phosphate crevice of aspartate carbamoyltransferase, consistent with the reduced ability of metal nucleotides to compete with carbamoyl phosphate for the active site . However, a single Gd3+ ion binds in the region of the active site as evidenced by strong density . The binding of cations near the active site probably causes the inhibition of catalysis observed in kinetics experiments reported earlier {Honzatko, R.B., Lauritzen, A.M . & Lipscomb, W.N . (1981) Proc . Natl . Acad . Sci . USA 78, 898-902}.

J Pharmacol Methods, 1982 Dec, 8(4), 241 - 53
Intrarenal distribution of ampicillin in the healthy and the infected rat kidney: a comparison of methods; Thonus IP et al.; The ampicillin concentration in the cortex of the normal and the infected rat kidney was determined by means of three different methods: tissue homogenate analysis, renal lymph analysis, and autoradiography . Both tissue homogenate analysis and autoradiography revealed that the cortical ampicillin concentration was about five times as high as the serum concentration . Renal lymph analysis disclosed that the ampicillin concentration in this fluid amounted to about 93% of the serum concentration . The discrepancy between these results can be explained on the basis of the binding of ampicillin to cellular components in the cortex . The results of the study of ampicillin distribution in infected rat kidneys with the aid of tissue homogenate analysis and autoradiography indicate that the ampicillin concentration was lower in the affected than in the intact tissue . Renal lymph analysis showed that the ampicillin concentration in the lymph was not affected by the presence of the infection . Quantitative autoradiography demonstrated that the ampicillin concentration in the cortical foci of infection was of the same order of magnitude as the serum concentration . For this kind of study autoradiography together with lymph analysis are recommended.

J Urol, 1982 Dec, 128(6), 1394 - 400
Immunology of pyelonephritis in the primate model . V . Effect of superoxide dismutase; Roberts JA et al.; Ascending acute pyelonephritis was produced in monkeys by infusion of bacteria through a ureteral catheter to the point of intrarenal reflux . This led to a significant inflammatory response with death of renal tubular cells in the area of the tubular granulocytes and bacteria . We gave superoxide dismutase, and found that the inflammatory response was decreased and fewer tubular cells were killed . Ultrastructural change was also decreased in tubular cells adjoining phagocytosing neutrophils . This suggests that renal damage following a bacterial infection may be due to the production and release of superoxide into the tubular lumen during phagocytosis . We believe that it is the initial event which may lead to the eventual loss of renal tissue and function called chronic pyelonephritis.

J Biochem Biophys Methods, 1982 Dec, 7(1), 35 - 46
Interference of electron donors in the measurement of the proton gradient and membrane potential by flow dialysis; Setty OH et al.; The three most commonly used electron donors for flow dialysis measurements of membrane potential lead to the development of an apparent but artifactual membrane potential with the interior negative in the presence or absence of membrane vesicles . The same three electron donors used in flow dialysis determinations of delta pH in the presence or absence of membrane vesicles lead to the development of an apparent but artifactual delta pH with the interior acidic . These artifacts have been evaluated using two probes for membrane potential, namely, TPP+ and rubidium in the presence of valinomycin and for two probes of delta pH, namely, acetate and DMO . Measurements were made over a range of ionic strengths.

J Cell Biol, 1982 Dec, 95(3), 689 - 96
Molecular components of the signal sequence that function in the initiation of protein export; Emr SD et al.; We are studying the mechanism by which the LamB protein is exported to the outer membrane of Escherichia coli . Using two selection procedures based on gene fusions, we have identified a number of mutations that cause alterations in the LamB signal sequence . Characterization of the mutant strains revealed that although many such mutations block LamB export to greater than 95%, others have essentially no effect . These results allow an analysis of the functions performed by the various molecular components of the signal sequence . Our results suggest that a critical subset of four amino acids is contained within the central hydrophobic core of the LamB signal sequence . If this core can assume an alpha-helical conformation, these four amino acids comprise a recognition site that interacts with a component of the cellular export machinery . Since mechanisms of protein localization appear to have been conserved during evolution, the principles established by these results should be applicable to similar studies in eukaryotic cells.

Infect Immun, 1982 Dec, 38(3), 1073 - 7
Relationship between phagocytosis and immunoglobulin A release from human colostral macrophages; Weaver EA et al.; Macrophages and neutrophils that contain mainly secretory immunoglobulin A (IgA) comprise the majority of cells in human colostrum . These cell populations were separated and analyzed for their ability to release total IgA and secretory IgA when stimulated to phagocytose . Colostral macrophages phagocytosed opsonized bacteria and nonopsonized latex particles; at the same time, IgA was released . Neutrophils poorly phagocytosed opsonized bacteria but actively phagocytosed latex particles . In contrast to the macrophages, the neutrophils did not release IgA, even after active phagocytosis of latex . Consequently, colostral macrophages are the main source of IgA released from colostral leukocytes when these cells are exposed to organisms or particles that are phagocytosed . A function for colostral neutrophils which sequester IgA is proposed.

Biull Eksp Biol Med, 1982 Dec, 94(12), 107 - 10
{Ultrastructural changes in the hepatic mast and plasma cells of pregnant rats with endotoxic shock}; Nikulin OV et al.; Ultrastructural alterations in mast and plasma cells of pregnant rat liver were studied in the initial period of endotoxin shock . A significant increase in functional activity of mast and plasma cells and the presence of intercellular contacts and contacts with cells of perisinusoidal space were found . The data obtained suggest that endotoxin stimulates connective tissue cells, leading to the release of mediators and biologically active substances responsible for hepatic injury during endotoxemia.

Mutat Res, 1982 Dec, 102(4), 401 - 12
Photobiological studies with dictamnine, a furoquinoline alkaloid; Ashwood-Smith MJ et al.; Dictamnine, a naturally occurring furoquinoline, produces bacterial frameshift mutations in the dark . It does not form DNA interstrand crosslinks in bacterial cells in the presence of near-ultraviolet light (300-380 nm) . It is more active than angelicin but slightly less active than 8-MOP as a phototoxic agent with E . coli . It is however a more active mutagen than 8-MOP at equivalent concentration . Dictamnine is slightly more potent than the same concentration of angelicin in producing photosensitized lethality in Chinese hamster cells . It does, however, produce almost twice as many sister-chromatid exchanges per lethal event than angelicin . The concept of 'unit dose' relating the observable photoinduced damage by the photosensitizer and the total irradiation appears to apply reasonably well to the actions of dictamnine in killing bacterial and mammalian cells, in the formation of sister-chromatid exchanges, but not to the induction of bacterial mutations.

Mutat Res, 1982 Dec, 102(4), 309 - 12
Lack of genotoxic activity of 2-(2',4'-diaminophenoxy)ethanol in Escherichia coli strains WP2, WP2uvrA and WP2uvrA/recA; Hastwell RM et al.; A new hair-dyeing ingredient, 2-(2',4'-diaminophenoxy)ethanol, was tested for its genotoxic potential with Escherichia coli WP2, WP2uvrA and WP2uvrA/recA . The tests were conducted in the presence and absence of a post-mitochondrial supernatant fraction from the livers of male rats induced with Aroclor 1254 . Tests for DNA damage and repair and tests for mutagenic activity failed to demonstrate any genotoxic potential associated with the compound.

J Virol, 1982 Dec, 44(3), 1068 - 71
Genetic studies of coliphage 186 . II . Genes associated with phage replication and host cell lysis; Hocking SM et al.; DNA synthesis in coliphage 186-infected cells was investigated . Phage 186 appeared to inhibit host DNA synthesis early in infection . The subsequent synthesis of phage 186 DNA was dependent on the product of 186 gene A . The product of gene B controlled both the production of late 186 proteins and the cessation of 186 DNA synthesis, and the products of genes O and P had no influence on 186 DNA synthesis . The product of gene P controlled host cell lysis, and the product of gene O may have some regulatory function.

J Virol, 1982 Dec, 44(3), 1056 - 67
Genetic studies of coliphage 186 . I . Genes associated with phage morphogenesis; Hocking SM et al.; In coliphage 186, 22 essential genes were defined by complementation studies with amber mutants . Eighteen genes were associated with phage morphogenesis: 11 with phage tail formation, and 7 with phage head formation . The remaining four genes are discussed in the accompanying paper (S . M . Hocking and J . B . Egan, J . Virol . 44:1068-1071, 1982).

J Infect Dis, 1982 Dec, 146(6), 746 - 50
Possible alteration of normal mechanisms of endotoxin toxicity in vivo by actinomycin D; Brown DE et al.; The effect of actinomycin D on the sensitivity of endotoxin-responsive (C3HeB/FeJ) and endotoxin-unresponsive (C3H/HeJ) mice to challenge with purified lipopolysaccharide (LPS) of Escherichia coli strain O55:B5 was examined using an experimental protocol of adoptive transfer of lymphoid cells into lethally irradiated recipients . Earlier results--that in the absence of actinomycin D, the ability of LPS to cause a lethal response in the immunologically chimeric mice reflected the phenotypic response of the donor lymphoid cells--were confirmed . Simultaneous administration of actinomycin D to endotoxin-responsive C3HeB/FeJ mice increased by several orders of magnitude the sensitivity of these mice to the lethal effects of LPS . Determinations of 50% lethal doses in the presence of actinomycin D indicated that immunologic chimeras were sensitive to lethal effects of LPS if either the donor or the recipient phenotype was LPS-responsive . Thus, the mechanism(s) of host response to LPS in the presence of actinomycin D may not be identical to those elicited in untreated mice.

J Bacteriol, 1982 Dec, 152(3), 1298 - 300
Mixed disulfides of acyl carrier protein and coenzyme A with specific soluble proteins in Escherichia coli; Rock CO; Three soluble proteins in Escherichia coli specifically from mixed disulfides with either acyl carrier protein or coenzyme A . Coenzyme A was attached to one of these proteins, and the amount bound depended on the cellular coenzyme A concentration . The other two proteins were mixed disulfides between acyl carrier protein and each of the two 3-ketoacyl-acyl carrier protein synthases.

J Bacteriol, 1982 Dec, 152(3), 1276 - 9
Cloning and characterization of Escherichia coli K-12 regulator gene tyrR; Cornish EC et al.; The Escherichia coli K-12 regulator gene tyrR was cloned into the multicopy plasmid pBR322 from a lambda(Tn10)tyrR+ specialized transducing phage . Further subcloning localized the gene within a 2.1-kilobase region . Analysis of plasmid-coded proteins showed that the tyrR gene codes for a 63,000-dalton polypeptide.

J Bacteriol, 1982 Dec, 152(3), 1241 - 7
Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli; Berger H et al.; We have cloned the chromosomal hemolysin determinants from Escherichia coli strains belonging to the four O-serotypes O4, O6, O18, and O75 . The hemolysin-producing clones were isolated from gene banks of these strains which were constructed by inserting partial Sau3A fragments of chromosomal DNA into the cosmid pJC74 . The hemolytic cosmid clones were relatively stable . The inserts were further subcloned either as SalI fragments in pACYC184 or as BamHI-SalI fragments in a recombinant plasmid (pANN202) containing cistron C (hlyC) of the plasmid-encoded hemolysin determinant . Detailed restriction maps of each of these determinants were constructed, and it was found that, despite sharing overall homology, the determinants exhibited minor specific differences in their structure . These appeared to be restricted to cistron A (hlyA), which is the structural gene for hemolysin . In the gene banks of two of these hemolytic strains, we could also identify clones which carried the genetic determinants for the mannose-resistant hemagglutination antigens Vb and VIc . Both of these fimbrial antigens were expressed in the E . coli K-12 clones to an extent similar to that observed in the wild-type strains . These recombinant cosmids were rather unstable, and, in the absence of selection, segregated at a high frequency.

J Bacteriol, 1982 Dec, 152(3), 1163 - 8
Temperature-sensitive processing of outer membrane lipoprotein in an Escherichia coli mutant; Yamagata H et al.; A mutant of Escherichia coli that accumulated prolipoprotein, a secretory precursor of the outer membrane lipoprotein, was isolated . The prolipoprotein accumulated in this mutant was modified by glyceride, but the in vitro cleavage of the signal peptide of the accumulated prolipoprotein was found to be temperature sensitive . The mutation appears to be located outside the gene for the lipoprotein, thus suggesting that the gene for the signal peptidase for the prolipoprotein was mutated.

J Bacteriol, 1982 Dec, 152(3), 1126 - 31
Identification of membrane anchor polypeptides of Escherichia coli fumarate reductase; Lemire BD et al.; Fumarate reductase of Escherichia coli has been shown to be a membrane-bound enzyme composed of a 69,000-dalton catalytic-flavin-containing subunit and a 27,000-dalton nonheme-iron-containing subunit . Using gene cloning and amplification techniques, we have observed two additional polypeptides encoded by the frd operon, with apparent molecular weights of 15,000 and 14,000, which are expressed when E . coli is grown anaerobically on glycerol plus fumarate . Expression of these two small polypeptides is necessary for the two large subunits to associate with the membrane . The four subunits remain associated in Triton X-100 extracts of the membrane, and a holoenzyme form of fumarate reductase containing one copy of each of the four polypeptides has been isolated . Unlike the well-characterized two-subunit form, the holoenzyme is not dependent on anions for activity and is not labile at alkaline pH . In these respects, it more closely resembles the membrane-bound activity.

J Bacteriol, 1982 Dec, 152(3), 1078 - 90
Multiple mechanisms for expression of incompatibility by broad-host-range plasmid RK2; Meyer R et al.; By cloning fragments of plasmid DNA, we have shown that RK2 expresses incompatibility by more than one mechanism . One previously identified (R . J . Meyer, Mol . Gen, Genet . 177:155--161, 1979; Thomas et al., Mol . Gen . Genet . 181:1--7, 1981) determinant for incompatibility is linked to the origin of plasmid DNA replication . When cloned into a plasmid vector, this determinant prevents the stable inheritance of a coresident RK2 . However, susceptibility to this mechanism of incompatibility requires an active RK2 replicon and is abolished if another replicator is provided . We have also cloned a second incompatibility determinant, encoded within the 54.1- to 56.4-kilobase region of RK2 DNA, which we call IncP-1(II) . An RK2 derivative remains sensitive to IncP-1(II), even when it is not replicating by means of the RK2 replicon . The 54.1- to 56.4-kilobase DNA does not confer susceptibility to the IncP-1(II) mechanism, nor does it encode a detectable system for efficient plasmid partitioning . The incompatibility may be related to the expression of genes mapping in the 54.1- to 56.4-kilobase region, which are required for plasmid maintenance and suppression of plasmid-encoded killing functions.

J Bacteriol, 1982 Dec, 152(3), 1033 - 41
Membrane assembly: movement of phosphatidylserine between the cytoplasmic and outer membranes of Escherichia coli; Langley KE et al.; Phosphatidylserine, normally a trace phospholipid in Escherichia coli, accumulates at high levels in temperature-sensitive phosphatidylserine decarboxylase mutants at nonpermissive temperatures . The intracellular localization of this phospholipid has now been determined . All of the accumulated phosphatidylserine is membrane bound and is distributed about equally between the inner and outer membrane fractions of E . coli as determined by isopycnic sucrose gradient fractionation . Phosphatidylserine is therefore effectively translocated from the inner to the outer membrane . Furthermore, this movement is bidirectional . Outer membrane phosphatidylserine can return to the inner membrane, as shown by the complete conversion of accumulated radioactive phosphatidylserine to phosphatidylethanolamine by inner membrane phosphatidylserine decarboxylase during chase periods . Pulse-chase experiments indicated the newly made phosphatidylserine appears first in the inner membrane and then equilibrates between the inner and outer membranes with a half-time of 12 to 13 min.

J Clin Endocrinol Metab, 1982 Dec, 55(6), 1209 - 11
Natural and biosynthetic insulin stimulates the growth of human erythroid progenitors in vitro; Bersch N et al.; High concentrations of insulin are known to augment the growth of various cell types in vitro . We examined the effect of a purified porcine insulin and biosynthetic human insulin produced in E . coli on the growth of human erythroid progenitors in vitro . Both insulins stimulated peripheral blood erythroid colony formation within the physiological range . An approximately 2-fold augmentation in colony formation was seen at insulin concentrations of 8 ng/ml, and as little as 0.1 ng/ml (0.17 nM) caused detectable stimulation of colony formation . The effect of subnanomolar concentrations of insulin or erythropoiesis in vitro suggests that insulin could modulate erythropoiesis in vitro . Human responsiveness to insulin's growth-promoting activity can be directly assayed in vitro using peripheral blood.

Gene, 1982 Dec, 20(3), 481 - 4
Restriction map of the hepatitis B virus DNA cloned in Escherichia coli; Bichko VV et al.; A plasmid carrying the complete genome of hepatitis B virus (HBV) inserted into the BamHI site of the pBR322 plasmid vector has been constructed . The physical map of HBV DNA established for 13 restriction endonucleases allows to conclude that the cloned DNA is similar, but not identical to the HBV DNA of ayw subtype.

Gene, 1982 Dec, 20(2), 267 - 79
Identification of sequences necessary for packaging DNA into lambda phage heads; Miwa T et al.; Several species of DNA molecules are packaged into lambda phage heads if they carry the region around the cohesive end site of lambda phage (cos lambda) . The minimal functional sequence around cos lambda needed for packaging was examined by cloning in pBR322 . The results showed that the minimal region contained 85 bp around cos lambda; 45 bp of the left arm of lambda phage and 40 bp of the right arm . A 75-bp region located to the right of the minimal region seems to enhance packaging . A 223-bp fragment containing these regions can be used as a portable element for plasmid DNA packaging into lambda phage heads . Plasmid ppBest 322, a derivative of pBR322 carrying this portable packager and both amp and tet genes, was constructed . This plasmid is useful for cloning of large DNA fragments.

Gene, 1982 Dec, 20(2), 255 - 65
Preparation of product-specific antisera by gene fusion: antibodies specific for the product of the yeast cell-division-cycle gene CDC28; Reed SI; Antisera with specificity for the product of a yeast cell-division-cycle (CDC) gene were prepared by immunizing rabbits to a novel hybrid polypeptide . A segment of the yeast gene CDC28 was fused to the Escherichia coli lacZ gene, which encodes beta-galactosidase, by insertion of yeast sequences into the plasmid pBGF1 . pBGF1 contains the lac promoter-operator and most of the lacZ gene . An EcoRI site, 16 codons upstream from the carboxyterminus of the beta-galactosidase coding region, served as a convenient splicing site for the heterologous sequences . To insure that an open reading frame be maintained between the two gene segments for some portion of the fusions, the CDC28-encoding segments were first subjected to limited digestion with nuclease BAL31 to produce random junction points . A hybrid polypeptide encoded by such a continuous open reading frame was purified from E . coli by preparative SDS-polyacrylamide gel electrophoresis and used to immunize rabbits . The resulting antisera were shown to have specificity for CDC28 gene product synthesized by cell-free translation of yeast mRNA.

Gene, 1982 Dec, 20(2), 219 - 29
Expression of the Eco RI restriction-modification system and the construction of positive-selection cloning vectors; O'Connor CD et al.; The genes encoding the Eco RI restriction-modification (R/M) system have been separately cloned onto compatible plasmids . We have shown that the Eco RI restriction gene is expressed in the total absence of methylase enzyme and confirmed that a temperature-sensitive mutant is defective in Eco RI modification activity at higher temperatures . Insertion of transcriptional terminators into the restriction gene had no detectable effect on Eco RI modification activity . This strongly suggests that a separate promoter exists for the methylase gene . Analysis of the published sequence shows that the methylase gene promoter may overlap with the COOH-terminal region of the endonuclease structural gene . The temperature-sensitive Eco RI system has been exploited in the construction of two plasmid cloning vectors, pLV57 and pLV59, which can be used to select positively for transformants bearing recombinant plasmids; cloning of a DNA fragment into pLV57 or pLV59 at the unique HindIII, Bg/II, or PstI sites inactivates the Eco RI restriction gene and permits the hybrid plasmid to survive at 37 degrees C . The temperature-sensitive modification activity of these vectors should also facilitate the introduction of Eco RI linkers into DNA cloned in this way.

Cell, 1982 Dec, 31(2 Pt 1), 319 - 25
Nucleotide sequence of yeast LEU2 shows 5'-noncoding region has sequences cognate to leucine; Andreadis A et al.; The LEU2 structural gene and its regulatory sequences were isolated on a 2200 bp Xho I-Sal I fragment . Sequencing of the 5'-noncoding region showed that at -151 there is an open reading frame of 23 codons of which six are for leucine . The leucine codon usage in this reading frame follows exactly that of other yeast genes . At the carboxy-terminal end and immediately after the peptide reading frame, a 14 bp hairpin (followed by a T-rich segment) can form in the putative mRNA; this arrangement closely resembles an RNA polymerase terminator . These and other features suggest a model for regulation . Preceding this is a gene (which starts at -463) for tRNALeu3, the major tRNALeu isoacceptor . RNA polymerase III transcription start and termination signals flank 5' and 3' ends, respectively, of the structural gene . The features noted above are in the same DNA strand that codes for the LEU2 gene product.

Proc Natl Acad Sci U S A, 1982 Dec, 79(24), 7729 - 33
Isolation of Drosophila proteins that bind selectively to left-handed Z-DNA; Nordheim A et al.; An affinity column for isolating Z-DNA binding proteins was made by attaching brominated poly(dG-dC) to Sephadex . Proteins from Drosophila nuclei were prepared and those that could bind to Escherichia coli B-DNA were removed from the solution . The remaining proteins were passed over the Z-DNA affinity column and then eluted with NaCl . Using both direct and competitive filter binding assays, we found that the eluted proteins bind to brominated poly(dG-dC) (Z-DNA) and poly(dG-m5dC) but not to poly(dG-dC) (B-DNA), native or denatured E . coli or calf thymus DNA, or brominated oligonucleotides . The proteins also bind to negatively supercoiled plasmids carrying Z-DNA sequences but not to relaxed or linearized plasmids in which the Z-DNA conformation is no longer present . Gel analysis reveals a mixture of several large proteins up to approximately 150,000 daltons.

Proc Natl Acad Sci U S A, 1982 Dec, 79(24), 7679 - 83
The cAMP-binding domains of the regulatory subunit of cAMP-dependent protein kinase and the catabolite gene activator protein are homologous; Weber IT et al.; Comparison of the recently determined amino acid sequences of the regulatory subunit of cAMP-dependent protein kinase (RII) from bovine cardiac muscle and the Escherichia coli catabolite gene activator protein (CAP) shows significant homology . This homology extends over most of the amino-terminal domain in CAP and is particularly good for the region of the beta-roll structure . The RII sequence contains two adjacent and internally homologous regions, both of which have high resemblance to the cAMP-binding domain in CAP . This suggests that the protein kinase regulatory subunit contains two cAMP-binding domains in the carboxyl-terminal region, each having a beta-roll structure similar to that in CAP . The cAMP molecule is expected to bind to the RII within a pocket formed by residues from the beta-roll, as is the case with CAP . One cAMP molecule would interact with residues from about 163 to 220, and the other cAMP would interact with amino acids in the stretch 285-350 of the RII protein kinase sequence . As the carboxyl-terminal domain of CAP shows homologies to the DNA-binding domains of other regulatory proteins, the protein appears to be of modular construction: a DNA-binding domain joined to a cAMP-binding domain.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7450 - 4
Control of transposon Tn5 transposition in Escherichia coli; Sasakawa C et al.; Tn5 is a composite transposable element in which the insertion sequences IS50R and IS50L bracket a central region encoding kanamycin resistance (kanr) . IS50R encodes a functional transposase, whereas IS50L contains the promoter of the kanr gene . To determine the relative activities of IS50R and IS50L in transposition we examined the structures of chimeric DNA molecules generated by insertion of segments of pBR322::Tn5 dimeric plasmids into red- lambda phage in recA- Escherichia coli . Restriction endonuclease analyses showed that the inserted sequences contained direct terminal repeats of pairs of IS50R or of IS50L elements and that the frequencies of usage of IS50R vs . IS50L depended on the position and orientation of Tn5 in the plasmid vector: IS50R was used preferentially when Tn5 was in transcriptionally quiescent regions of the vector (in either orientation) or when IS50L was immediately downstream from a strong promoter in the vector . In contrast, IS50L was used preferentially when IS50R was downstream from a strong promoter . We conclude IS50R tends to be used preferentially but that when transcription impinges on the end of an IS50 element the participation of that element in transposition is inhibited.

J Gen Virol, 1982 Dec, 63(2), 277 - 95
Thymidine kinase deletion mutants of herpes simplex virus type 1; Sanders PG et al.; Deletions in the cloned thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1), strain 17 syn+, were produced by two methods . Removal of a 506 base pair fragment from between the unique SstI and Bg/II restriction endonuclease sites of pTK1 (HSV-1 BamHI p cloned in pAT153) and subsequent transformation of Escherichia coli resulted in the isolation of 50 deleted plasmids . Sequential digestion of pTK1 with Bg/II and nuclease BAL 31 followed by ligation and recleavage with Bg/II resulted in the isolation of 31 deleted plasmids . Three clones, pTK2, pTK3 and pTK4, obtained following Bg/II and SstI treatment of pTK1 were recombined with wild-type (wt) HSV-1 (17) syn+ DNA in baby hamster kidney (BHK) cells to produce TK- deletion mutants HSV-1 (17) TK 1301, HSV-1 (17) TK 1302 and HSV-1 (17) TK 1303 respectively . 5-Bromo-2'-deoxyuridine, 5-bromo-2'-deoxycytidine and 9-(2-hydroxyethoxymethyl)guanine were used to reduce the background of TK+ virus in heterogeneous recombinant stocks analysed for the presence of TK- recombinants . All recombinant clones isolated produced a small syncytial plaque morphology in BHK cells . The mutants HSV-1 (17) TK 1301 and HSV-1 (17) TK 1302 were TK-, failed to produce polypeptides of molecular weights 43000 and 19000 found in wt-infected cells and demonstrated one-step growth curves different from wt virus and the TK- mutant HSV-1 (17) dPyk-7 . Superinfection studies with HSV-1 (17) TK 1301, HSV-1 (17) TK 1302, HSV-1 (MDK) and HSV-1 (17) dPyk-7 indicated that all TK- mutants except dPyK-7 produce a trans-acting gene product which can switch on the transforming HSV-1 TK gene.

Infect Immun, 1982 Dec, 38(3), 848 - 54
Polymorphonuclear leukocyte activation by a synthetic muramyl dipeptide analog; Osada Y et al.; N alpha-(N-Acetylmuramyl-L-alanyl-D-isoglutaminyl)-N epsilon -stearoyl-L-lysine, a synthetic derivative of muramyl dipeptide, stimulated the chemotactic mobility, phagocytic activity, and superoxide anion (O2-) productivity of peritoneal polymorphonuclear cells in mice . The chemotactic mobility of both cells preincubated with the adjuvant in vitro and those derived from the mice previously treated with the adjuvant was significantly enhanced . The phagocytic activity of cells preincubated in vitro with the adjuvant was also enhanced transiently, and that of the cells derived from the mice treated subcutaneously with the adjuvant 24 h before intraperitoneal inoculation with Escherichia coli was significantly greater than that of cells from the mice given phosphate-buffered saline instead of the adjuvant . The release of O2- from the cells derived from the adjuvant-treated mice was also greater than that from the cells of untreated control mice . However, the exposure of the cells derived from untreated mice to the adjuvant in vitro did not stimulate O2- generation by the cells.

Genetika, 1982 Dec, 18(12), 1938 - 44
{Isolation of mutants with an increased frequency of transposon Tn1 translocations in Escherichia coli K-12 cells}; Ismailov ZF et al.; Two het mutations (high efficiency of transposition) were isolated and characterized preliminarily . The mutations lead to increasing the frequency of ampicillin transposon Tn1 translocation into different replicons in Escherichia coli cells . Both mutations increase the frequency of transposition by the same degree . One of het mutations has been localized within Tn1 transposition element . This mutation seems to be similar to mutations isolated earlier (Gill et al., 1978) in the transposon Tn3 repressor gene which occurred at a high frequency and led to an increase in transposition . The other het mutation is situated in the bacterial chromosome.

J Virol, 1982 Dec, 44(3), 993 - 1002
Lytic action of cloned phi X174 gene E; Young KD et al.; The phi X174 lysis gene E was placed under control of the lac promoter by cloning into the multicopy plasmid pBH20 . Other phi X174 gene sequences were removed by nuclease digestion . Expression of gene E was shown to be necessary and sufficient to produce lysis phenomena exhibited by infection with intact phage . Lysis, its inhibition by MgSO4 and spermine, its progression through a spheroplasting stage, and its dependence on an early chloramphenicol-sensitive step were reproduced in clones induced for expression of the E gene product . Escherichia coli clones carrying the E gene not under lac control, and clones under lac control but only minimally induced for gene E expression, exhibited morphological aberrations consistent with the view that the mechanism by which gene E mediates cell lysis is related to host cell division processes.

J Virol, 1982 Dec, 44(3), 823 - 33
Identification and nucleotide sequences of two similar tandem direct repeats in Epstein-Barr virus DNA; Dambaugh TR et al.; Epstein-Barr virus DNA is known to have partially homologous segments, designated DL and DR, near the left and right ends of the long unique region (Raab-Traub et al., Cell 22:257-267, 1980) . DL and DR are each partially composed of tandem direct repeat sequences . DL contains 11 to 14 repeats of a 124-base-pair sequence designated IR2 . DR contains approximately 30 direct repeats of a 103-base-pair sequence designated IR4 . The DL and DR sequences have colinear partial homology for approximately 2.4 and 1.5 kilobase pairs to the right of IR2 and IR4, respectively . IR2 and IR4 are similar sequences and evolved in part from a common ancestor . Both sequences are 84% guanine and cytosine and have limited homology to Epstein-Barr virus IR1 and to the herpes simplex virus type 1 inverted terminal repeat "a" sequence . IR2 encodes part of an abundant 2.5-kilobase persistent early EBV RNA expressed in productively infected cells, but does not encode part of the 3-kilobase Epstein-Barr virus RNA which is transcribed from the adjacent IR1-U2 region of the Epstein-Barr virus genome in latently infected cells.

J Exp Med, 1982 Dec 1, 156(6), 1677 - 90
Characterization of a membrane pore-forming protein from Entamoeba histolytica; Young JD et al.; We describe the partial purification and characterization of a pore-forming material (PEM) from Entamoeba histolytica . The formation of ion channels by PFM was examined in three systems . (a) PFM depolarizes J774 macrophages and mouse spleen lymphocytes as measured by {3H}TPP+ uptake . (b) PFM induces rapid monovalent cation flux across the membrane of phosphatidylcholine-cholesterol vesicles . (c) PFM confers a voltage-dependent conductance to artificial planar bilayers, which is resolved as a summation of opening of individually conducting steps of 67 pS in 0.1 M KCl . Monomers of PFM are functional; however, a preferential aggregation occurs in the planar bilayer . Activity is pronase, trypsin, and heat sensitive and is stable between pH 5-8 . PFM is not secreted by unstimulated amoebae but after exposure to the calcium ionophore A23187, concanavalin A, and E . coli lipopolysaccharide, 5-10% of the total cell content of PFM is released into the medium within 5-10 min . High-performance gel filtration results in an approximately 1,000-fold purification of PFM and gives an Mr of 30,000 . This protein may play a role in the cytotoxicity mediated by E . histolytica.

J Immunol, 1982 Dec, 129(6), 2570 - 2
Cyclic GMP as the second messenger in helper cell requirement for gamma-interferon production; Johnson HM et al.; Cyclic GMP and activators (acetylcholine, E . coli heat-stable toxin) of guanylate cyclase were capable of completely replacing the helper cell or interleukin 2 requirement for gamma-interferon (IFN gamma) production by Lyt-1-,2+ cells from C57BL/6 mouse spleen cells . The cyclic GMP help was independent of DNA synthesis or proliferation in the IFN gamma-producing cells, because cyclic GMP reversed mitomycin C blockage of IFN gamma production but did not reverse the inhibition of DNA synthesis . Thus, the findings presented here are unrelated to the question of the second messenger role of cyclic GMP in the activation of lymphocytes for DNA synthesis and cellular proliferation . The cyclic GMP help for IFN gamma production was antagonized by cyclic AMP and inducers (isoproterenol) of adenylate cyclase.

J Bacteriol, 1982 Dec, 152(3), 1022 - 32
Insertions of transposon Tn5 into ribosomal protein PNA polymerase operons; Hui I et al.; The genetic organization and interrelationships between the two ribosomal protein transcription units (the L11 and L10 operons) from near 89 min on the Escherichia coli chromosome were studied by using insertional mutations generated by the kanamycin-resistant transposable element Tn5 . The polar effects of Tn5 insertions on the expression of the L11, L1, L10, and L12 ribosomal protein genes and the beta RNA polymerase subunit gene were examined (i) by the level of beta-galactosidase activity generated from L10-lacZ and beta-lacZ gene fusions, (ii) by direct sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins specified by plasmid ribosomal protein genes in UV-irradiated maxicells, and (iii) by urea-polyacrylamide gel electrophoresis of plasmid- and chromosome-specified L12 protein . The results confirmed the organization of these genes into two transcription units as follows: PL11, rplK (L11), rplA (L1), PL10, rplJ (L10), rplL (L12), rpoB (beta) . . .; they also localized the position of the PL10 promoter within an 80-nucleotide region near the end of the L1 gene . The results also support the idea that the translational regulatory proteins for the L11 and L10 operons are L1 and L10, respectively, and that the expression of the L12 gene is closely linked to L10 gene expression.

J Bioenerg Biomembr, 1982 Dec, 14(5-6), 499 - 512
Properties of a novel ATPase enzyme in chromaffin granules; Cidon S et al.; Membranes were isolated from mitochondria and chromaffin granules of bovine adrenal medullae . The cross-contamination between the two membranes was examined by comparing the radioactive bands on autoradiograms of gels after phosphorylation of the membranes with {gamma-32P}-ATP and decoration with {125I}concanavalin A and {125I}protein A with antibody that was raised against chromaffin-granule membranes . It was found that the membranes cross-contaminated each other by less than 10% . The technique of immunodecoration with antibodies against beta subunits of proton-ATPases from yeast mitochondria, spinach chloroplasts, and E . coli membranes was used for quantitative estimation of proton-ATPase complexes in chromaffin granules and mitochondrial membranes . It was found that chromaffin-granule membranes contain less than 10% of the amount of proton-ATPase complex in mitochondrial membranes . The specific ATPase activity of chromaffin-granule membranes was on the order of 30 to 50% of the mitochondrial membranes . The ATPase activity of the chromaffin-granule membranes was more sensitive to 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene and 4-chloro-7-nitrobenzofurazan . It was much less sensitive than the mitochondrial membranes to antibody against beta subunit of proton-ATPase from E . coli membranes . After solubilization of chromaffin-granule membranes by octyglucoside and cholate and subsequent centrifugation on sucrose gradient, two different ATPase enzymes were separated . The heavier enzyme was identical to the mitochondrial-ATPase complex, while the lighter enzyme was identified as a novel ATPase, which might be responsible for the special properties of the ATPase activity of chromaffin-granule membranes.

Cell, 1982 Dec, 31(3 Pt 2), 509 - 20
Intron within the large rRNA gene of N . crassa mitochondria: a long open reading frame and a consensus sequence possibly important in splicing; Burke JM et al.; We describe the sequence of the 2295 nucleotide long intron and 245 nucleotides of the flanking exon sequences within the large (24S) rRNA gene of Neurospora crassa mitochondria . The intron contains a long open reading frame, which could correspond to ribosomal protein S5 . Comparison with the corresponding intron of the large rRNA gene of yeast mitochondria reveals a single highly homologous 57 nucleotide long sequence, including the sequence (formula; see text), which is present in virtually all the sequenced introns of yeast, Aspergillus nidulans and Zea mays mitochondrial genes, and which may be important for their processing . Sequences closely related to this consensus sequence are also present within all four of the introns of nuclear rRNA genes which have been sequenced . The intron is located within a highly conserved region of the large rRNA sequence and at exactly the same site as in the corresponding introns in yeast mitochondria and also in Physarum polycephalum nuclei.

Proc Natl Acad Sci U S A, 1982 Dec, 79(24), 7639 - 43
Replication of lambda dv plasmid in vitro promoted by purified lambda O and P proteins; Tsurimoto T et al.; An in vitro system for replication of lambda dv plasmid DNA has been constructed . This system consists of an ammonium sulfate fraction from Escherichia coli extract, exogenously added purified lambda O and P proteins, and lambda dv DNA in closed circular form . More than 85% of the added template DNA replicated semiconservatively . In the same system, another plasmid, pBR322, also replicated, but less efficiently than lambda dv . Furthermore, its replication was independent of O and P proteins . Inhibitors of DNA gyrase entirely blocked the replication activity, whereas rifampicin, an inhibitor of RNA polymerase, showed a significant effect only when added prior to initiation of the DNA replication . DNA replication was initiated from a region near to or within the four direct repeats in lambda origin (lambda ori) and proceeded bidirectionally, as examined by DNA chain elongation termination with dideoxy CTP . A cloned DNA carrying a 350-base-pair region including the initiation site also initiated replication, dependent on O and P proteins, and its initiation occurred at the same position as with native lambda dv DNA . An A + T-rich structure neighboring the repeats was found to be essential for lambda DNA replication . Regions corresponding to ice and oop were not required for O,P-dependent initiation.

Gene, 1982 Dec, 20(2), 139 - 44
Sensitive detection of RNA using strand-specific M13 probes; Brown DM et al.; We have extended the method of Hu and Messing (Gene 17 (1982) 271-277) to prepare highly radioactive M13 probes suitable for use in RNA-DNA hybridization experiments . Single strands of M13 DNA carrying cloned sequences are rendered partially double-stranded by primed synthesis using a synthetic oligonucleotide primer complementary to a region 5' to the cloning site . The newly synthesized radioactive complementary strand is then covalently cross-linked to the M13 phage DNA by UV irradiation in the presence of 4,5,8-trimethylpsoralen (trioxsalen) . Since the cross-linked probe is stable to heat denaturation, and the region of cloned sequence is kept single-stranded, these complexes may be used as strand-specific hybridization probes to detect RNA sequences under conditions which would denature DNA-DNA duplexes.

Eur J Biochem, 1982 Dec, 129(1), 211 - 9
Distance measurement by energy transfer: the 3' end of 16-S RNA and proteins S4 and S17 of the ribosome of Escherichia coli; Epe B et al.; Escherichia coli ribosomal proteins S4 and S17 were specifically labelled at their thiol groups with the acetylaminoethyl-dansyl and/or bimane fluorophores . Each formed a complex with 16-S RNA and, when the other 30-S ribosomal proteins were added, a complete 30-S subunit with at least partial activity . If the 3' end of the RNA was also labelled (with fluorescein) then the distance between the two fluorophores could be measured by Forster-type energy transfer . The result for S4 was 6.0 nm (60 A) in the ribonucleoprotein complex and 5.6 nm (56 A) in the 30-S subunit, and for S17 6.3 nm (63 A) in the complex and 6.2 nm (62 A) in the subunit . There is no evidence for a major change in the relative disposition of the 3' and 5' ends of the 16-S RNA during formation of the 30-S subunit . Sources of error are discussed, including the question of multiple labelling . In order to measure more accurately the extent of energy transfer a procedure based upon enzymic digestion was developed and is detailed in this paper.

Cell, 1982 Dec, 31(3 Pt 2), 575 - 83
The importance of RNA secondary structure in CoIE1 primer formation; Tomizawa JI et al.; Formation of the RNA primer for CoIE1 DNA replication is inhibited by random substitution of less than one tenth of G residues by I residues during in vitro transcription . Substitution in any one of several regions of the transcript is inhibitory, even in the region more than 400 nucleotides upstream of the origin of DNA replication . The inhibition results from interference with hybrid formation between nascent RNA II (primer transcript) and the template DNA near the replication origin . Association of RNA I with RNA II, which has been known to inhibit primer formation, enhances pausing of transcription of RNA II at a site far downstream of the region where RNA I hybridizes to the transcript . A large deletion in the region which specifies both RNA I and RNA II suppresses primer formation and also enhances pausing of transcription at the same site . These results show that the secondary structure of RNA II during transcription is important for primer formation and that alteration in the structure of the nascent transcript can change transcriptional events far downstream.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7082 - 6
Specific cleavage of the p15A primer precursor by ribonuclease H at the origin of DNA replication; Selzer G et al.; We report studies on the mechanism of initiation of DNA replication by p15A, a small plasmid whose origin of replication is known to function much as does that of ColE1 . Previous work has shown that an RNA primer for DNA synthesis is generated by the action of RNase H (EC 3.1.26.4) on a precursor transcript . The precursor initiates well upstream of the origin of replication and somehow forms a hybrid with its template during transcription . Here we show that when RNase H cleaves the hybrid at 0 degrees C, an additional cleavage product besides the primer can be identified . Using two-dimensional RNA sequencing techniques, we have established the sequence of this product to within a few nucleotides of each end . The position of the 5' end indicates that the nuclease introduces a nick or very small gap in the precursor at the origin . This suggests that some sequence or structure directs the enzyme to the origin . The position of its 3' end indicates that the precursor terminates at or near a series of six dAs in the template strand about 190 nucleotides from the origin of replication . The data indicate that hybrid formation may be necessary for termination of the precursor at this downstream site.

Mutat Res, 1982 Dec, 106(2), 209 - 16
Sensitivity of Escherichia coli acrA mutants to psoralen plus near-ultraviolet radiation; Hansen MT; The sensitivity to psoralen plus near-ultraviolet radiation (PUVA) was compared in a pair of E . coli strains differing at the acrA locus . Survival was determined for both bacteria and phage lambda . AcrA mutant cells were 40 times more sensitive than wild type to the lethal effect of PUVA . Free lambda phage exposed to PUVA survived as well when plated on acrA mutants as on wild type . In contrast, prophage lambda CI857 ind carried in lysogenic acrA strains was hypersensitive to PUVA . The enhanced sensitivity of bacterial and lambda DNA, when inside acrA cells, was paralleled by an increased photobinding of radiolabelled psoralens in the mutant . Binding was increased specifically to DNA rather than to nucleic acids in general . The difference in psoralen-binding ability determined by the acrA gene persisted after permeabilizing treatment of the cells . The results suggest that the acrA mutation causes an alteration specifically in the environment of the cellular DNA so as to allow increased intercalation and photobinding of psoralens.

J Virol, 1982 Dec, 44(3), 782 - 93
Molecular cloning of DNA complementary to mRNA of the baculovirus Autographa californica nuclear polyhedrosis virus: location and gene products of RNA transcripts found late in infection; Adang MJ et al.; DNAs complementary to late Autographa californica nuclear polyhedrosis virus (AcNPV) mRNA were synthesized by reverse transcription and cloned in Escherichia coli by using pBR322 as a vector . Eleven different cDNAs were distinguished in our screening of 45 AcNPV-homologous clones . Location of the regions of cDNA homology with respect to the AcNPV physical map showed that the 11 cDNAs were dispersed throughout the genome . The most abundant cDNA insertion, representing approximately one-third of the late viral mRNAs, was homologous to the AcNPV HindIII-P,Q and EcoRI-P fragments . The direction of transcription in this region was from left to right on a linearized AcNPV physical map . Hybridization selection followed by in vitro translation showed that this region encoded a 7,200-dalton (7.2K) protein which comigrated with a minor protein found in the extracellular nonoccluded form of the virus (NOV) . Similarly, the gene for polyhedrin, the major structural protein of the occluded virus form, was located, at least in part, in the HindIII-V/EcoRI-I region of the AcNPV map . The polyhedrin transcript represented approximately one-quarter of the viral polyadenylic acid-containing RNAs at 27 h postinfection . Another relatively abundant cDNA was homologous to the HindIII-A/EcoRI-C/SstI-G region, and RNA selected by this cDNA directed the synthesis of two proteins (31K and 30K) . The protein products of five other cDNA-selected RNAs were identified . The HindIII-D/EcoRI-O, HindIII-C/EcoRI-D, HindIII-B1/EcoRI-E, and HindIII-B2/EcoRI-H regions of the AcNPV L-1 genome were homologous to RNAs which directed the synthesis of a 57K protein, a 25K protein, a 61K protein, and a 37K protein (plus a minor 26K protein), respectively . Late mRNA selected by a cDNA homologous to the HindIII-P/EcoRI-B region of the AcNPV map directed the synthesis of 31K and 30K proteins which comigrated with the 31K and 30K proteins translated from RNA selected by the HindIII-A/EcoRI-C/SstI-G cDNA . Three other cDNAs have not been correlated yet with specific protein products.

J Bacteriol, 1982 Dec, 152(3), 1211 - 9
Identification of two genes immediately downstream from the polA gene of Escherichia coli; Joyce CM et al.; We have identified two genes within a 1-kilobase region immediately following the polA gene of Escherichia coli . The first, whose transcription is initiated about 150 base pairs beyond the end of the polA coding sequence, is the gene corresponding to the previously sequenced "spot 42 RNA" (B . G . Sahagan and J . E . Dahlberg, J . Mol . Biol . 131:573--592, 1979) . The second, located further downstream and transcribed towards polA, is the structural gene for a 22-kilodalton polypeptide, which we have detected by using plasmid-directed protein synthesis in maxicells . Sequence analysis of this region of the E . coli genome suggests that it contains little, if any, redundant DNA.

J Gen Microbiol, 1982 Dec, 128 (pt 12), 2991 - 6
Serological classification of conjugative plasmids by indirect haemagglutination; Feilberg Jorgensen NH et al.; The indirect haemagglutination reaction was evaluated in the classification of conjugative plasmids . A simple and sensitive method was worked out using pili and in some cases whole bacteria as antigens . Antibodies were prepared against pili coded for by plasmids from incompatibility groups IncFI, IncFII, IncI alpha and IncN . The antisera were tested against pili from 35 strains harbouring plasmids . The test differentiated clearly between plasmids from unrelated incompatibility groups, whereas cross-reaction occurred with closely related groups such as FI and FII . Minor antigenic variation could be seen within the IncFII group.

Appl Environ Microbiol, 1982 Dec, 44(6), 1444 - 8
Cloning of a gene responsible for the biosynthesis of glutathione in Escherichia coli B; Murata K et al.; A gene (gshI) responsible for gamma-glutamylcysteine synthetase (GSH-I) activity was cloned to construct an Escherichia coli B strain having high glutathione synthesizing activity . For this purpose, two E . coli B mutants (strains C912 and RC912) were used . C912 was deficient in GSH-I activity . RC912, a revertant of C912, had a GSH-I activity that was desensitized to feedback inhibition of reduced glutathione . To clone gshI, chromosomal DNAs of RC912 and plasmid vector pBR322 were digested with various restriction endonucleases and then ligated with T4 DNA ligase . The whole ligation mixture was used to transform C912, and the transformants were selected as tetramethylthiuramdisulfide-resistant colonies . Of about 20 resistant colonies, 2 or 3 became red when treated with nitroprusside and showed appreciably high GSH-I activities . The chimeric plasmid DNA, designated pBR322-gshI, was isolated from the strain having the highest GSH-I activity and transformed into RC912 . The structure and molecular size of pBR322-gshI in RC912 were determined . The molecular size of this plasmid was 6.2 megadaltons, and the plasmid contained a 3.4-megadalton segment derived from RC912 chromosomal DNA, which included gshI gene . The GSH-I activity of RC912 cells containing pBR322-gshI was fourfold higher than that of RC912 cells without pBR322-gshI.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7253 - 7
Deletion mutants defining the Escherichia coli replication factor Y effector site sequences in pBR322 DNA; Soeller WC et al.; The Escherichia coli DNA replication factor Y, along with other genetically undefined replication proteins, is involved in a dnaB-, dnaC-, and dnaG-dependent pathway of primer formation on phi X174 single-stranded circular DNA . In addition, replication factor Y has a site-specific, single-stranded DNA-dependent ATPase activity . We have previously demonstrated the presence of two factor Y effector sites on pBR322 DNA . When inserted into the filamentous phage f1R229, these sites can function as rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent origins of DNA replication . We report here the construction of deletion mutants of the two pBR322 factor Y effector sites . These deleted sites no longer function as effectors for factor Y ATPase activity nor as templates for rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent DNA synthesis . We conclude that the DNA sequences required for factor Y ATPase activity and origin function are likely to be identical.

J Infect Dis, 1982 Dec, 146(6), 751 - 7
Interaction between human polymorphonuclear leukocytes and two different strains of type 1 fimbriae-bearing Escherichia coli; Ohman L et al.; Two type 1 fimbriae-bearing strains of Escherichia coli, ABU2 (ON:K14) and PN7 (O1:K1), with different underlying physicochemical surface properties were compared for their capacity to interact with human polymorphonuclear leukocytes . Both strains attached strongly to neutrophils in a mannose-sensitive manner . One of the strains, ABU2, with a surface exposing weak negative charge and liability to hydrophobic interaction, was efficiently ingested (65%) and caused a release of reactive oxidative metabolites (chemiluminescence) and lysosomal enzymes . The other strains, PN7, exposing a hydrophilic, negatively charged K antigen and hydrophilic uncharged smooth lipopolysaccharide, resisted phagocytosis (only 25% were ingested) but nevertheless caused a release of reactive oxidative metabolites and lysosomal enzymes to a greater extent than did phagocytosed strain ABU2 . These results show that attachment mediated by type 1 fimbriae does not ultimately lead to ingestion . Whether the attached bacteria are being ingested or not depends on their underlying physicochemical surface properties . Furthermore, if the bacteria remain extracellularly attached, they may potentiate the inflammatory process.

Biochim Biophys Acta, 1982 Nov 30, 699(2), 149 - 54
The reaction of the mutagen 1,1'-hexamethylene-bis-{(5-p-chlorophenyl)-biguanide} with guanosine and cysteine; Ackermann-Schmidt B et al.; The mutagen 1,1'-hexamethylene-bis{(5-p-chlorophenyl)-biguanide} reacts at 37 degrees C with guanosine and guanine to yield xanthosine or xanthine and oxidizes cysteine to cystine . After treatment of a guanosine-labelled DNA sample from Escherichia coli with the mutagen xanthine could be detected as a reaction product . At a slow rate the mutagen is hydrolysed spontaneously yielding urea, 1.6-hexanediol and 4-chloroaniline . The reaction mechanisms both of the hydrolysis and of the reaction with cysteine and guanosine are discussed.

FEBS Lett, 1982 Nov 29, 149(2), 328 - 33
Structural domains of ribosomal protein S8 and their relationship to ribosomal RNA binding; Paterakis K et al.; Escherichia coli ribosomal protein S8 has been subjected to mild proteolytic digestion in order to search for structural domains within the protein {1} . A characteristic fragment produced in high yield after chymotrypsin treatment has been located with the protein sequence . Circular dichroism has shown this domain to be rich in alpha helix . However, the fragment loses its ability to bind to 16S rRNA as does a similar fragment produced by trypsin cleavage . The intact protein is required for rRNA binding and is highly protected against proteolytic digestion when bound to the RNA.

Mol Cell Biochem, 1982 Nov 26, 49(2), 87 - 96
beta-Galactosidase alpha-complementation . A model of protein-protein interaction; Zabin I; Studies on beta-galactosidase alpha-complementation are reviewed . The isolation and structure of two beta-galactosidase fragments that form an enzymically active complex are described . One of these is a cyanogen bromide peptide from whole beta-galactosidase; the other is a dimeric protein from a lacZ deletion mutant of Escherichia coli . The mechanism most likely involves an initial binding of two cyanogen bromide peptides to the dimer, followed by formation of a tetramer, and finally a slow conformational change of the complex to a native-like enzyme . The overall reaction is essentially irreversible . A region of the polypeptide chain involved in dimer-dimer contact must be supplied by the cyanogen bromide peptide . alpha-Complemented enzyme contains overlapping sequences . Proteolytic experiments were carried out to determine the origin of the functionally important segment . The effect on alpha-complementation of amino acid substitutions at four positions in the polypeptide chain was investigated . The implications of these results for beta-galactosidase structure and for proteins in general are discussed.

J Immunol Methods, 1982 Nov 26, 55(1), 63 - 72
Immunoradiometric assay of lipid A: a test for detecting and quantitating endotoxins of various origins; Nolan JP et al.; The ability to measure circulating endotoxin in various disease states has been hampered by the lack of a specific and quantitative assay . The test most commonly used has been the Limulus gelation assay, which measures an enzymatic effect of endotoxin rather than the substance itself . Based on a solid-phase immunoradiometric assay previously developed to detect the specific lipopolysaccharide from Escherichia coli 026, a similar assay has been developed for the lipid A moiety of endotoxins . The assay uses rabbit antibodies to lipid A which do not react with ketodeoxyoctonate, myristic or beta-hydroxymyristic acids, and detects lipid A obtained from endotoxins of various origins after acid hydrolysis of lipopolysaccharide . Experiments in rats given exogenous endotoxin suggest that this assay can be useful for quantitation of bacterial endotoxins in serum and for studying the pathophysiology of experimental endotoxemia.

Nucleic Acids Res, 1982 Nov 25, 10(22), 7211 - 29
Precise localisation of three intra-RNA cross-links in 23S RNA and one in 5S RNA, induced by treatment of Escherichia coli 50S ribosomal subunits with bis-(2-chloroethyl)-methylamine; Stiege W et al.; Treatment of E . coli 50S ribosomal subunits with low doses of bis-(2-chloroethyl)-methylamine ("nitrogen mustard") leads to formation of a number of intra-RNA and RNA-protein cross-links . After partial digestion of the cross-linked subunits with cobra venom nuclease, followed by destruction of the protein moiety with proteinase K, complexes containing the intra-RNA cross-links were isolated by two-dimensional gel electrophoresis . The individual complexes were subjected to oligonucleotide analysis, either directly or after a second partial digestion procedure using ribonuclease T1, and the cross-link sites determined . In 23S RNA, the cross-links found were between bases 763 and 1567, 1210 and 1236, 1482 and 1501; in 5S RNA, base 69 was cross-linked to base 107 . The significance of these cross-links in relation to the three-dimensional organization of the ribosomal RNA is discussed.

J Biol Chem, 1982 Nov 25, 257(22), 13776 - 80
Repair of alkylated DNA in Escherichia coli . Physical properties of O6-methylguanine-DNA methyltransferase; Demple B et al.; An inducible methyltransferase of Escherichia coli acts on O6-methylguanine in DNA by conveying the methyl group to one of its own cysteine residues . The protein has now been purified to apparent homogeneity from a constitutively expressing strain . The homogeneous methyltransferase exhibits no DNA glycosylase or endonuclease activity on alkylated DNA . Further, the methyltransferase activity is strikingly resistant to heat inactivation under reducing conditions . The protein has Mr = 18,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sedimentation coefficient and Stokes radius of the native enzyme yield Mr = 18,400 . The amino acid composition of the purified protein shows 4 to 5 cysteine residues/transferase molecule . The methylated, inactive form of the transferase has an unaltered molecular weight.

J Biol Chem, 1982 Nov 25, 257(22), 13205 - 7
Demonstration of specific high affinity binding sites in plasmid DNA by photoaffinity labeling with an ethidium analog; Coffman GL et al.; We have used photoaffinity labeling of pBR322 DNA with 8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride to demonstrate high affinity ethidium-binding sites . Plasmid equilibrated with as little as 1 drug/DNA molecule was photoactivated, freed of uncomplexed drug by ethanol precipitation, and subjected to restriction analysis . There was highly specific, rather than random, blockage of HhaI sites (d(GCGC)) at low drug concentrations . Furthermore, the same 7 new digestion fragments were generated at drug to nucleotide ratios ranging from 1:100 to 1:8000 . All the new DNA fragments had chain lengths greater than the largest HhaI fragment (393 base pairs) . At higher ligand concentrations closely approximating those needed for equilibrium binding studies, detection of the high affinity sites was greatly masked . Drug binding to HhaI restriction fragments which had been prepared prior to the action of drug did not induce new bands . Furthermore, the larger DNA fragments from drug-labeled plasmid were resistant to HhaI digestion over a wide range of enzyme concentrations . These findings suggest that ligand binding can be highly selective even between sites which have the same tetranucleotide sequence . Therefore, selective drug binding must be dictated not only by local base sequence preference, but also by other long range parameters.

Nucleic Acids Res, 1982 Nov 25, 10(22), 7409 - 24
Evidence that rifampicin can stimulate readthrough of transcriptional terminators in Escherichia coli, including the attenuator of the rpoBC operon; Newman AJ et al.; The genes encoding the beta and beta' subunits of RNA polymerase in E.coli, rpoB and rpoC, lie downstream of at least two ribosomal protein genes, rplJ (encoding L10) and rplL (L7/12), in a common operon . All four genes are served by promoter PL10, and an attenuator (partial terminator) of transcription, t1, lies between rplJL and rpoBC . Treatment of E.coli with rifampicin, under conditions producing partial inhibition of general RNA synthesis, can stimulate transcription of rpoBC . We have investigated the locus of this effect by fusing PL10 and t1 separately to galK, in suitable plasmids . Our studies of these fusions, and similar fusions involving transcriptional terminators derived from coliphage T7, indicate that low concentrations of rifampicin cause increased readthrough of several different transcriptional terminators in E.coli in vivo, including rpo t1 . We discuss whether or not this unspecific mechanism is solely responsible for the observed stimulatory effects of the drug on rpoBC transcription.

Nucleic Acids Res, 1982 Nov 25, 10(22), 7373 - 85
The nucleotide sequence of the dnaA gene and the first part of the dnaN gene of Escherichia coli K-12; Hansen EB et al.; The nucleotide sequence of the dnaA gene and the first 10% of the dnaN gene was determined . From the nucleotide sequence the amino acid sequence of the dnaA gene product was derived . It is a basic protein of 467 amino acid residues with a molecular weight of 52.5 kD . The expression of the dnaA gene is in the counterclockwise direction like the one of the dnaN gene, for which potential startsites were found.

Nucleic Acids Res, 1982 Nov 25, 10(22), 7295 - 311
Mapping of RNA polymerase binding sites in R12 derived plasmids carrying the replication-incompatibility region and the insertion element IS1; Chan PT et al.; Interactions between Escherichia coli RNA polymerase holoenzyme and three small plasmid DNAs (pSM1, pSM2, and pSM15) derived from the drug resistant factor R12 have been studied . These plasmids carry the copy number and incompatibility determinants, the origin of DNA replication and the rep gene(s) necessary for plasmid replication . They also contain the insertion element IS1 and the putative finO cistron . Thirteen DNA segments within the largest of the three plasmids (pSM2) were able to form either a binary and/or ternary complex with RNA polymerase . A unique strong binding site was mapped within the left end of IS1 . Five binding sites were found within the rep-cop-inc region . Four of these are weak binding sites whereas the fifth does not form a stable binary complex and was detected by ternary complex formation . A strong binding site was located in the putative finO region whereas the remaining six binding sites are located in regions with unidentified genetic functions.

Nucleic Acids Res, 1982 Nov 25, 10(22), 7273 - 82
Selective binding of actinomycin D and distamycin A to DNA; Wilkins RJ; The exact sites at which a number of drugs inhibit the nick translation of DNA by E.coli DNA polymerase-I have been pinpointed . In order to do this, a method has been developed for sequencing double-stranded plasmid DNA from the site of a specifically induced nick . The initial experiments have concentrated on analysis of drug inhibition of nick translation in a 200 nucleotide region near the Eco Rl origin of pBR313 . Many drugs were found to inhibit nick translation in a highly sequence specific manner . For actinomycin D, significant inhibition occurred at just four sites in the nucleotide sequence under test and only one sequence (pGpCpGpCpGpGp) gave really strong inhibition . Distamycin A gave a different pattern of inhibition with particularly strong stops in just two of the many A-T rich regions in the DNA . Experiments with caffeine suggest that factors in addition to primary sequence are important in determining where major inhibition occurs.

J Biol Chem, 1982 Nov 25, 257(22), 13823 - 7
The nucleotide sequence of the replication origin beta of the plasmid R6K; Shon M et al.; We h ave identified by molecular cloning a region of 283 base pairs of the HindIII 2 fragment of R6K which corresponds to the region of the replication origin beta . This 283 base-pair DNA fragment, when present contiguously with the structural gene for the replication initiation protein of R6K, encoded in the HindIII 9-15 and part of HindIII 2 restriction fragments, will support the replication of a plasmid chimera containing the pBR322 replicon in a pol Ats host at the nonpermissive temperature . The nucleotide sequence of the region of replication origin beta has been determined . The nucleotide sequence has some homology with the ori gamma region of R6K; it has a 15-base-pair homology with the replication origin of Escherichia coli.

J Biol Chem, 1982 Nov 25, 257(22), 13770 - 5
The Escherichia coli dnaC gene product . III . Properties of the dnaB-dnaC protein complex; Kobori JA et al.; The Escherichia coli dnaB and dnaC proteins form a tight complex in the presence of ATP (Wickner, S., and Hurwitz, J., (1975) Proc . Natl . Acad . Sci . U . S . A . 72, 921-925) . The complexed dnaC protein is resistant to inhibition by the sulfhydryl reagent, N-ethylmaleimide . This protection is not observed when ATP is substituted by AMP, ADP, adenyl 5'-yl imidodiphosphate, or adenosine-5'-O-(3-thiotriphosphate); dATP provides partial protection . A sedimentation coefficient of 15.2 S determined by glycerol gradient sedimentation and a Stokes radius of 64 A determined by gel filtration suggests a molecular weight in the range of 400,000 . The complex isolated by DEAE-cellulose chromatography contains six dnaC protein monomers of 29,000 daltons contains six dnaC protein monomers of 29,000 daltons contains six dnaC protein monomers of 29,000 daltons per dnaB protein hexamer (300,000 daltons) consistent with a calculated weight of 474,000 . The isolated dnaB-dnaC protein complex functions in vitro in the replication of phage phi X174 single-stranded DNA to the duplex replicative form . Tritium-labeled dnaC protein, absent from an isolated prepriming com-dnaC protein, absent from an isolated prepriming complex intermediate, was nevertheless bound to the phiX replicative form DNA synthesized in vitro . These results suggest that stable inclusion od dnaC protein in the priming complex bound to DNA requires a completely assembled primosome.

J Biol Chem, 1982 Nov 25, 257(22), 13763 - 9
The Escherichia coli dnaC gene product . II . Purification, physical properties, and role in replication; Kobori JA et al.; The Escherichia coli dnaC protein, purified to homogeneity from overproducing plasmid strains, is a polypeptide of 31,000 daltons (determined on a denaturing gel) . The native molecular weight as calculated from the sedimentation coefficient of 2.75 S and Stokes radius of 24.5 A is 29,000 . dnaC protein is N-ethylmaleimide sensitive (Wickner, S., Berkower, L., Wright, M., and Hurwitz J . (1973) Proc . Natl . Acad . Sci . U . S . A . 70, 2369-2373), and has 3 sulfhydryl groups as determined with {14C}p-chloromercuribenzoate . The activity was assayed by complementation of a mutant dnaC extract or by reconstitution of a purified protein system which converts phi X174 single-stranded DNA to the duplex replicative form . In this conversion the dnaC protein is required during the initial prepriming stage of phi X174 DNA replication . Antiserum against dnaC protein specifically inhibits this stage but not the subsequent priming and elongation steps carried out by primase and the PolIII holoenzyme . Requirement for dnaC protein was also manifested in the in vitro replication of a plasmid DNA containing the E . coli origin of replication (oriC) by complementation of a mutant extract and specific inhibition by dnaC antiserum.

J Biol Chem, 1982 Nov 25, 257(22), 13685 - 91
Osmoregulation of gene expression . I . DNA sequence of the ompR gene of the ompB operon of Escherichia coli and characterization of its gene product; Wurtzel ET et al.; The ompB region on the Escherichia coli chromosome codes for two genes, ompR and envZ, which are required for the osmolarity sensitive biosynthetic regulation of the outer membrane matrix proteins (porins), OmpF and ompC . A part of the ompB region containing the ompR gene has been cloned (Wurtzel, E . T., Movva, N . R., Ross, F . L., and Inouye, M . (1981) J . Mol . Appl . Genet . 1, 61-69) . We have determined the DNA sequence, including the promoter and structural regions encompassed in a 1.3-kilobase pair Ava I-Eco RI subfragment . This fragment codes for the entire ompR gene as well as the 5' end of the envZ gene . The ompR gene codes for a protein of 32,489 daltons, consisting of 284 amino acid residues . This was confirmed by identifying the gene product by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determining a partial amino acid sequence of the NH2-terminal region of the gene product . A sequence of 57 amino acid residues located in the COOH-terminal region of the protein is extremely basic . It contains 10 arginine plus lysine residues in contrast to 1 glutamic acid residue in this region . In vitro transcription of the DNA from this region indicates that ampR and envZ are co-transcribed as a polycistronic mRNA from a promoter located 5' to the ompR gene . Translation of the am pR gene terminates at two tandem TAS codons and translation of the envZ gene initiates 29 nucleotides downstream . Cloning of the promoter region of ompB at a site 5' to the structural portion of the beta-galactosidase gene indicates that transcription of ompB is under positive control by cAMP.

J Biol Chem, 1982 Nov 25, 257(22), 13291 - 6
Interaction of interferon with cellular receptors . Internalization and degradation of cell-bound interferon; Branca AA et al.; Human interferon alpha A, produced in Escherichia coli by recombinant DNA technology, was labeled with 125I to study its binding to receptors on human lymphoblastoid Daudi cells . This binding showed a marked temperature dependency, with maximum binding obtained at 30-37 degrees C . About 60% of the cell-bound radioactivity was released upon subsequent addition of unlabeled interferon, indicating that only part of the cell-bound interferon could be displaced by competitor . Moreover, about 30-50% of cell-bound interferon was not released by treating the cells with 0.2 N acetic acid, a procedure which removes polypeptide hormones on the cell surface, indicating that part of the interferon bound at 37 degrees C was internalized . This interferon was slowly degraded to acid-soluble products, which were released into the culture medium . Treatment of DAudi cells with the lysosomotropic amines chloroquine and methylamine inhibited the degradation of interferon . Methylamine, however, also inhibited the internalization of interferon . Daudi cells treated with interferon in the presence of chloroquine showed an increase in the interferon-induced enzyme 2',5'-oligo(A) polymerase comparable to that of cells treated with interferon alone . This enzyme increased to a similar extent in cells treated with interferon and cytochalasin, a drug which inhibited internalization of interferon by 50% . These results suggest that degradation and possibly internalization of interferon are not required for at least some of its biological activities.

J Biol Chem, 1982 Nov 25, 257(22), 13181 - 4
Expression of an Abelson murine leukemia virus-encoded protein in Escherichia coli causes extensive phosphorylation of tyrosine residues; Wang JY et al.; A segment of the Abelson murine leukemia virus (A-MuLV) genome was inserted into an Escherichia coli plasmid designed to allow the expression of the protein encoded by the viral gene . Bacteria expressing the A-MuLV-encoded protein were isolated; they had new phosphorylated proteins in which the phosphate was linked to tyrosine residues . These proteins included many that must be E . coli protein . One phosphotyrosine-containing protein of 62,000 molecular weight had reactivity with antiserum specific for authentic A-MuLV protein . The A-MuLV protein thus appears to be a tyrosine-specific protein kinase which is active in E . coli.

Nucleic Acids Res, 1982 Nov 25, 10(22), 7181 - 96
Immobilization of denatured DNA to macroporous supports: II . Steric and kinetic parameters of heterogeneous hybridization reactions; Bunemann H; The accessibility of immobilized DNA has been shown to depend more crucially on the method of immobilization than on the type of support used for fixation . When sonicated denatured DNA is coupled via diazotization or via cyanogen bromide reaction to solid Sephadex G-25 and Cellex 410 or to macroporous Sephacryl S-500 and Sepharose C1-6B its accessibility varies from 100 to 24 percent . Generally the loss of accessibility is linked to a depression of the melting temperature of DNA helices formed on the support . This correlation shows a characteristic course for a particular coupling method . DNA coupled under denaturing conditions may become totally inaccessible when only 3 percent of its bases are involved in the covalent linkage . Kinetic experiments with sonicated E.coli DNA have shown that the rate constants for renaturation or hybridization reactions are very similar for DNA immobilized by different methods to solid or macroporous supports . Generally the second order rate constant for a heterogeneous reaction (between mobile and immobilized DNA) is about one order of magnitude smaller than that of the analogous homogeneous reaction (in solution).

Nucleic Acids Res, 1982 Nov 25, 10(22), 7163 - 80
Immobilization of denatured DNA to macroporous supports: I . Efficiency of different coupling procedures; Bunemann H et al.; Methods commonly used for covalent immobilization of single stranded DNA have been applied to several solid supports (Sephadex G-25 and Cellex 410) as well as to a number of macroporous materials (Sepharose C1-6B, C1-2B; Sephacryl S-500 and S-1000) . Coupling efficiencies and stability of covalently bound DNA are compared for both classes of materials . The yields of the immobilization reaction for sonicated DNA are only 10-40% for G-25 and Cellex 410 in contrast to 60-80% for C1-6B and S-500 . Under optimal conditions, up to 0.5 mg of DNA can be coupled initially per g of wet macroporous material . The immobilized DNAs are lost from the supports in a biphasic manner, with about 10-20% loss per day during the first 2-3 days at 45 degrees C, followed by only about 1% loss per day at the same temperature thereafter . The influence of the coupling procedure on the generation of mismatch effects has been studied in 2.4 M tetraethylammonium chloride solution for the hybrid formation between immobilized and mobile DNA . The degree of mismatch ranged from 0-3% and depended on the method of immobilization . The unspecific absorption of DNA on macroporous materials is sufficiently low to allow efficient hybrid selection . No size limitations have been observed when plastid mRNAs are selected by cloned fragments of plastid DNA immobilized to macroporous Sephacryl S-500.

J Biol Chem, 1982 Nov 25, 257(22), 13169 - 72
Mouse interferons enhance the accumulation of a human HLA RNA and protein in transfected mouse and hamster cells; Yoshie O et al.; A recombinant clone (pJY150R1.1) encoding the human major histocompatibility antigen (HLA-B7) was introduced into mouse cells and hamster cells by cotransformation with selectable genes . The exposure to mouse interferon of the cells transformed to HLA-B7+ resulted in a severalfold increase in the level of HLA antigen and RNA . The HLA-B7 clone used for the transfection includes a 670-base pair DNA sequence upstream from the coding segment . It remains to be established if the 670-base pair segment is necessary and/or sufficient to make the transcription of the HLA gene responsive to interferon.

Biochemistry, 1982 Nov 23, 21(24), 6066 - 72
Direct observation of complexes formed between recA protein and a fluorescent single-stranded deoxyribonucleic acid derivative; Silver MS et al.; The reaction of chloroacetaldehyde with single-stranded DNA (ssDNA) yields epsilon DNA, a highly fluorescent substance . The binding of recA protein to epsilon DNA nearly doubles its fluorescence yield . The enhanced fluorescence signals the formation of a recA-epsilon DNA complex . This complex exhibits an ATPase activity as great as that of the corresponding recA-ssDNA complex . Addition of a saturating concentration of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) to a solution of the recA-epsilon DNA complex yields a further rise in fluorescence . Saturation with ATP produces the same rise . The nucleotide triphosphates have converted the recA-epsilon DNA complex into the respective ATP gamma S-recA-epsilon DNA and ATP-recA-epsilon DNA complexes . The fluorescence changes that accompany the formation of the three complexes have enabled us to (1) establish by titration that recA protein binds to 6.0 +/- 0.3 nucleotides of epsilon DNA, (2) show that the binding of ATP to the recA-epsilon DNA complex is highly cooperative under various conditions, with a Hill coefficient of 2.4-4.9 and Kapp = 25 +/- 2 micro M, (3) show that the binding of ATP gamma S is also highly cooperative, with a Hill coefficient of 3.3-4.2 and Kapp congruent to 0.5 micro M, and (4) perform initial measurements on the rate at which recA protein transfers between polynucleotides . The experiments provide the first direct observation of an ATP-recA-ssDNA-like complex, and they illuminate some of the properties of such complexes.

FEBS Lett, 1982 Nov 22, 149(1), 51 - 4
Membrane potential (delta psi) depolarizing agents inhibit maturation; Pages JM et al.; Precursor forms of exported proteins were first accumulated in the envelope of phenethyl alcohol (PEA)-treated cells . After removal of PEA, a complete processing could be obtained in a few minutes . In this work, we demonstrate that colicins A and E1, that act on the electrical gradient in the cytoplasmic membrane, prevent the processing of precursor forms previously accumulated . Concentrations of colicins accounting for approximately 1 killing unit (50--3000 molecules/cell) were found to be sufficient for inhibition of processing . Therefore our results strongly suggest that in intact cells the electrical gradient across the cytoplasmic membrane is required for maturation of exported proteins.

C R Seances Acad Sci III, 1982 Nov 22, 295(10), 587 - 98
{Cloning and expression of the genetic determinants encoding for the serological variants of the adhesive factor K 88 carried by enterotoxinogenic Escherichia coli strains of porcine origin}; Cerf A et al.; The existence of homologous sequences on plasmids carried by 3 enterotoxinogenic strains of Escherichia coli of porcine origin, and encoding for adhesive factors of different antigenic specificities: K 88 ab, K 88 ac, K 88 ad; has been demonstrated by DNA-DNA hybridization . The use as probe of a DNA fragment carrying a piece of structural genes of the adhesive factor K 88 ab taken out of a recombinant plasmid expressing this factor, has allowed us to localize the genetic determinants which encode for the adhesive factors K 88 ac and K 88 ad . Cloned in the plasmid pBR 322, these determinants have allowed us to obtain the expression, by the host bacteria of the corresponding adhesive factors.

Life Sci, 1982 Nov 15-22, 31(20-21), 2209 - 12
M 154,129, a putative delta antagonist, reverses endotoxic shock without altering morphine analgesia; Holaday JW et al.; Studies were conducted with the putative delta opiate receptor antagonist M 154,129 to evaluate its potential for reversing circulatory shock without altering nociceptive processes . M 154,129 (30 mg/kg iv) did not alter tail flick or hot plate latencies by itself, nor did it alter the antinociceptive effects of morphine (4 mg/kg iv) . Following endotoxic shock hypotension in conscious rats, M 154,129 (30 or 60 mg/kg iv) produced a rapid return of arterial pressure to pretreatment levels . These data indicate that circulatory shock may be mediated at delta opioid receptors . Moreover, delta antagonists may have therapeutic benefit in reversing shock without intensification of pain.

Eur J Biochem, 1982 Nov 15, 128(2-3), 515 - 20
Secondary structure of the lac repressor headpiece . Possibilities and limitations of a joint infrared and circular dichroism study; Schnarr M et al.; The secondary structure of the short tryptic headpiece of the lac repressor has been investigated by the analysis of its infrared and circular dichroic spectra . For the latter we used the method of Provencher and Glockner {Biochemistry (1981) 20, 33-37}, which seems to be at present the most successful for the determination of the beta content of proteins . Nevertheless our results indicate that in the case of the lac repressor headpiece this method overestimates the amount of beta structure . We find that the headpiece contains an important helical content of about 50%, depending slightly on the ionic strength . A decomposition of the infrared spectrum in a sum of Gaussian curves reveals clearly the absence of a vibrational band around 1630 cm-1, excluding thus the presence of a multi-stranded beta-pleated sheet . The only beta structure compatible with the infrared results seems to be a two-stranded antiparallel beta sheet, as judged from our results on the beta-sheet model-compound gramicidin S . The unusually strong intensity of the amide I' band is in favour of the existence of such a structure . The quantitative analysis of both infrared and circular dichroism spectra indicates the presence of a certain (but different) amount of beta structure . Comparing these results with several secondary structure predictions, part of the helical residues should be located between Leu-45 and (at least) Arg-35, and an eventual two-stranded beta sheet should be situated in the N-terminal part of the headpiece.

Eur J Biochem, 1982 Nov 15, 128(2-3), 445 - 9
An improved purification method and a physical characterization of phage T7 DNA polymerase; Randahl H et al.; The immunoadsorbent used to purify T7 DNA polymerase contains antibodies directed towards thioredoxin . Elution of the enzyme is made by a pulse of buffer at pH 12.0 . This decreases the binding capacity of the column . Binding experiments with {3H}thioredoxin showed that the effect was caused by reduction of the antibodies by thiols in alkaline buffers . T7 DNA polymerase aggregated and irreversibly lost activity in buffers of low ionic strength . Experiments with gel chromatography and glycerol density gradient centrifugation showed that 0.2 M sodium chloride was required to keep the enzyme in its monomeric form . The sedimentation coefficient and the Stokes' radius are 5.3 S and 4.6 nm respectively, evaluated by gel chromatography and glycerol density gradient centrifugation techniques . The frictional ratio of 1.49 indicates that the T7 DNA polymerase is an asymmetrical protein.

Eur J Biochem, 1982 Nov 15, 128(2-3), 427 - 33
Crosslinking of N-acetyl-phenylalanyl {s4U}tRNAPhe to protein S10 in the ribosomal P site; Riehl N et al.; In order to identify ribosomal components involved in the peptidyl-tRNA binding site on the ribosome, tRNAPhe molecules were prepared in which cytidine residues had been chemically converted into 4-thiouridine (S4U) . This nucleoside is photoactive at 335 nm and able to form covalent bonds with nearby nucleophilic groups . The thiolated AcPhe-tRNAPhe was bound to the ribosomal P site in the presence of poly(U) as verified by puromycin reactivity . Direct irradiation of the AcPhe-{s4U}tRNAPhe poly(U) 70-S ribosome complex induced crosslinking of the tRNA molecule exclusively to 30-S subunits . Analysis of the covalent complex revealed that AcPhe-{s4U}tRNAPhe was specifically crosslinked to protein S10.

Eur J Biochem, 1982 Nov 15, 128(2-3), 371 - 5
Characterization of the region on protein L7/L12 involved in binding to ribosomal particles; Schop EN et al.; Tryptic digestion of reductively methylated protein L7/L12 yields a large tryptic fragment, which comprises amino acids 1-59 . At the most, two molecules of this fragment can bind to a 50-S ribosomal particle, deprived of protein L7/L12 . Besides, binding of each single 1-59 fragment competes with binding of one dimeric L7/L12 molecule . Molecular weight studies on the fragment reveal a monomeric structure . Digestion of the 1-59 fragment with carboxypeptidase Y leads to the formation of a 1-55 fragment . The binding characteristics of the latter fragment are similar to those of the 1-59 fragment . The results suggest that a monomeric stretch of L7/L12, comprising the first 55 amino acids, is sufficient for attaching L7/L12 to the ribosome.

Eur J Biochem, 1982 Nov 15, 128(2-3), 297 - 307
Characterization of the secondary structure features of Escherichia coli, Caldariella acidophila and mammalian ribosomal RNA species by chemical modification of sterically exposed bases; Cammarano P et al.; The helix content of rRNA species (Escherichia coli, Caldariella acidophila, rat liver) and the G . C content of their bihelical domains have been investigated by chemical modification of uracil and cytosine residues with probes specific for sterically exposed bases . By using radioactively labelled rRNA, G . C base pairs and the sum of A . U plus G . U base pairs have been quantified assuming that they are numerically identical with the unreactive cytosine and uracil rings, respectively . Exposed uracil bases were probed by their conversion to alkali-labile, nonultraviolet-absorbing sulphonated adducts, with 1.33 M bisulfite pH 7, at 20 degrees C; the adducts can be separated from unreacted uracil, and quantified, by cation-exchange chromatography of RNase T2 plus pancreatic RNase digests of bisulfite-modified rRNA . Exposed cytosines were probed by their conversion to methoxyaminated, alkali-stable, derivatives with 1 M methoxyamine, pH 5.5, at 37 degrees C, and quantified by monitoring the CMP/AMP radioactivity ratio after alkaline hydrolysis of modified rRNA . Exposed uracil rings can also be estimated spectrophotometrically by the alkali-catalyzed reversal of the non-ultraviolet-absorbing sulphonated adducts after separation of the latter from unreacted uracil . The cytosine deamination reaction, catalyzed by bisulfite at pH 6, has also been investigated and found to exhibit little specificity for sterically exposed bases of rRNA, the (G + C)-richer rRNA species of C . acidophila being considerably less susceptible to non-specific deamination than the (G + C)-poorer rRNA of E . coli . A high degree of congruence is shown to exist between results obtained by chemical modification and melting hyperchromicity experiments.

Eur J Biochem, 1982 Nov 15, 128(2-3), 315 - 29
Isoleucyl-tRNA synthetase from Escherichia coli MRE 600 . Different pathways of the aminoacylation reaction depending on presence of pyrophosphatase, order of substrate addition in the pyrophosphate exchange, and substrate specificity with regard to ATP analogs; Freist W et al.; The substrate specificity of isoleucyl-tRNA synthetase from Escherichia coli MRE 600 with regard to ATP analogs has been compared with the results obtained with isoleucyl-tRNA synthetase from yeast . The enzyme from E . coli is less specific, the two enzymes exhibit different topographies of their active centres . The order of substrate addition to isoleucyl-tRNA synthetase from E . coli MRE 600 has been investigated by bisubstrate kinetics, product inhibition and inhibition by substrate analogs . The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase . As found for isoleucyl-tRNA synthetase from yeast, the results of the pyrophosphate exchange studies indicate the possibility of formation of E . Ile-AMP . ATP complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, release of pyrophosphate and subsequent addition of a second molecule of ATP . For the aminoacylation in the absence of pyrophosphatase, a rapid-equilibrium random ter addition of the substrates is found whereas the enzyme from yeast exhibits a steady-state ordered ter-ter mechanism; in the presence of pyrophosphatase the mechanism is bi-uni uni-bi ping-pong similarly as observed for the yeast enzyme . A comparison of inhibition patterns obtained with N(6)-benzyladenosine 5'-triphosphate under different assay conditions (spermine or magnesium ions, addition of pyrophosphatase) indicates that even more than two pathways of the aminoacylation may exist . The catalytic cycles of the two mechanisms derived from the observed orders of substrate addition and product release include the same enzyme substrate complex (E . tRNA . Ile-AMP) for the aminoacyl transfer reaction . The kcat values, however, are considerably different: kcat of the sequential pathway is about 40% lower than kcat of the ping-pong mechanism.

J Am Vet Med Assoc, 1982 Nov 15, 181(10), 992 - 9
Recombinant DNA technology for the preparation of subunit vaccines; Bachrach HL; Recombinant DNA technology appears to be on the verge of producing safe and effective protein vaccines for animal and human diseases . The procedure is applicable to most viruses because their isolated surface proteins generally possess immunogenic activity . Strategies used for the preparation and cloning of the appropriate genes depend on the characteristics of the viral genomes: whether DNA or RNA; their size, strandedness, and segmentation; and whether messenger RNA are monocistronic or polycistronic . Cloned surface proteins of foot-and-mouth disease and hepatitis B viruses are being tested for possible use as practical vaccines . Two doses of the cloned foot-and-mouth disease viral protein have elicited large amounts of neutralizing antibody and have protected cattle and swine against challenge exposure with the virus . Surface proteins have also been cloned for the viruses of fowl plague, influenza, vesicular stomatitis, rabies, and herpes simplex . Cloning is in progress for surface proteins of viruses causing canine parvovirus gastroenteritis, human papillomas, infectious bovine rhinotracheitis, Rift Valley fever, and paramyxovirus diseases . In addition, advances in recombinant DNA and other facilitating technologies have rekindled interest in the chemical synthesis of polypeptide vaccines for viral diseases . The bioengineering of bacterial vaccines is also under way . Proteinaceous pili of enterotoxigenic Escherichia coli are being produced in E coli K-12 strains for use as vaccines against neonatal diarrheal diseases of livestock.

Biochim Biophys Acta, 1982 Nov 12, 713(2), 285 - 91
Localization of glycerophosphate acyltransferase in Escherichia coli; Kessels JM et al.; sn-Glycero-3-phosphate acyltransferase (EC 2.3.1.15) the first enzyme involved in phospholipid biosynthesis, is known to be associated with the cytoplasmic membrane of Escherichia coli . The localization of this enzyme in the transverse plane of the membrane was investigated by proteolysis of intact and lysed spheroplasts and by inhibition of glycerol 3-phosphate transport into intact cells in the presence of azide . Glycerophosphate acyltransferase was found to be resistant to proteolysis by trypsin in intact spheroplasts, whereas its enzymatic activity could be destroyed completely by trypsin in lysed spheroplasts . These results are in line with a localization of the acyltransferase at the inner aspect of the cytoplasmic membrane . Sodium azide was shown to have no inhibitory effect on glycerophosphate acyltransferase activity . Lack of incorporation of glycero phosphate into the phospholipids of glycerol phosphate transport-negative cells and inhibition of this incorporation in wild-type and glycerol 3-phosphate transport-constitutive cells by azide support a cytoplasmic-oriented localization of the glycerophosphate acyltransferase.

Science, 1982 Nov 12, 218(4573), 646 - 52
Translational efficiency of transfer RNA's: uses of an extended anticodon; Yarus M; Transfer RNA's are probably very strongly selected for translational efficiency . In this article, the argument is presented that the coding performance of the triplet anticodon is enhanced by selection of a matching anticodon loop and stem sequence . the anticodon plus these nearby sequence features (the extended anticodon) therefore contains more coding information than the anticodon alone and can perform more efficiently and accurately at the ribosome . This idea successfully accounts for the relative efficiencies of many transfer RNA's.

Nucleic Acids Res, 1982 Nov 11, 10(21), 6787 - 96
Synthesis of 8-(2-4 dinitrophenyl 2-6 aminohexyl) amino-adenosine 5' triphosphate: biological properties and potential uses; Vincent C et al.; We have synthetised 8-(2-4 dinitrophenyl 2-6 aminohexyl) amino-adenosine 5' triphosphate (in short : rATP-DNP), a derivative of ATP which carries a dinitrophenyl group . We show that rATP-DNP is a substrate for calf thymus deoxynucleotidyl terminal transferase (EC 2.7.7.31) and E . coli DNA polymerase I (Kornberg polymerase EC 2.7.7.7.) . It can therefore be incorporated into DNA molecules by elongation from 3' ends or by nick translation . The incorporated dinitrophenyl group can be recognized by specific antibodies which can then be detected by anti-antibodies coupled to an enzyme . DNP groups could also be introduced into DNA after enzymatic incorporation of 8-aminohexyl adenosine 5' triphosphate and reaction with 1-fluoro-2-4-dinitrobenzene . Thus, DNA molecules carrying DNP groups can ultimately be revealed by enzymatic coloured reactions . Potential uses of this enzymatic labelling as a substitute to the radioactive detection of nucleic acids, are discussed.

Nucleic Acids Res, 1982 Nov 11, 10(21), 6957 - 68
Primary structure of the ompF gene that codes for a major outer membrane protein of Escherichia coli K-12; Inokuchi K et al.; The nucleotide sequence of the ompF gene coding for a major outer membrane protein of Escherichia coli K-12 has been determined and the amino acid sequence of the OmpF protein was deduced from it . The OmpF protein contains 340 amino acid residues, and is produced from a precursor having 22 extra amino acid residues, the signal peptide, at the amino terminus . The expected secondary structure of the OmpF protein had a high beta-sheet content with a low alpha-helix content . The promoter region and the transcription termination region of the ompF gene had a significantly high AT content, while the AT content of the coding region was about the same as the average AT content of the E . coli chromosome . Following the termination codon, a typical rho-independent transcription termination signal was observed . The codon usage in the ompF gene was highly nonrandom; the codons preferably utilized are those recognized by the most abundant species of isoaccepting tRNAs or those, among synonymous codons recognized by the same tRNA, that can interact more properly with the anticodon.

Nucleic Acids Res, 1982 Nov 11, 10(21), 6865 - 78
Structure and transcription of the spinach chloroplast rDNA leader region; Briat JF et al.; A cloned fragment of spinach chloroplast DNA carrying 140 bp of the 16S rRNA gene and 691 bp upstream this gene has been analysed by DNA sequencing, by in vitro transcription, by S1 mapping with chloroplast RNAs and purified 16S rRNA from 30S ribosomal subunits . A tRNAVal gene has been located between the position 394 and 465 . Crude chloroplast RNA polymerase has been purified by heparin sepharose chromatography of a 80 000 g supernatant from pure lysed spinach plastids and used to transcribe the cloned Bg1 II-Pvu II DNA fragment . Four in vitro transcripts of about 830, 550, 350 and 260 bases were obtained whatever RNA polymerase used: the chloroplast or the E . coli enzyme . The transcripts of 550 and 260 bases are initiated by ATP . S1 mapping with in vivo chloroplasts RNAs on 5' labelled separated strands from Bg1 II-Pvu II fragments indicates 2 protected DNA fragments respectively of 140 and 260 bases on the strand which codes for rRNAs and possibly one protected DNA fragment of 550 bases on the other strand . The start site of the 260 bases transcript might correspond to the initiation site of transcription of the rRNA genes . The possibility that the 550 bases transcription of the non coding strand for rRNA genes corresponds to the beginning of a mRNA is discussed.

Nucleic Acids Res, 1982 Nov 11, 10(21), 6833 - 47
The binding of gyrase to DNA: analysis by retention by nitrocellulose filters; Higgins NP et al.; Three distinct Escherichia coli DNA gyrase complexes with DNA can be identified using a nitrocellulose filter-binding assay . One complex consists of an ensemble of two subunit A and two subunit B protomers bound noncovalently to specific sequences of DNA . High levels of each subunit alone are inactive but a single gyrase molecule binds DNA to a filter . At 23 degrees, the complex has a dissociation constant of approximately 10(-10) M and a half-time of decay of about 60 h . It is sufficiently stable that it can be purified by gel filtration and retain full supercoiling activity . Gyrase binds preferentially to relaxed DNA over supercoiled DNA by a factor of about 10 . On addition of oxolinic acid, a second complex is formed that is distinguished by its stability in high ionic strength solutions and by efficient conversion to a third form upon addition of protein denaturants . The first and second complexes require Mg++ for optimal formation . The third form has been shown previously to contain denatured A protomers covalently linked to DNA that is broken at the site of attachment.

Nucleic Acids Res, 1982 Nov 11, 10(21), 6597 - 618
Gene amplification in methotrexate-resistant mouse cells . IV . Different DNA sequences are amplified in different resistant lines; Caizzi R et al.; DNA was purified from double minutes isolated from MTX-resistant EL4/8 mouse lymphoma cells, digested to completion with Bam H1 restriction endonuclease and cloned in lambda-1059 . The properties of the library suggest that the DNA from which it was made was not detectably contaminated with non-dm chromosome material, and that the library is essentially complete for sequences contained in Bam H1 restriction fragments between 9 and 19 kb . The inserts of some selected lambda-recombinants were subcloned in pBR328 or pAT153 to separate sequences of differing repetition frequency . Clones representative of different classes of sequences were used as probes to Southern transfers of Bam H1 digested total nuclear DNAs of various MTX-resistant cell lines . The results clearly show that the amplified unit of each cell line has a unique structure, and that different amplified units differ widely in their sequence composition.

Nucleic Acids Res, 1982 Nov 11, 10(21), 6639 - 57
The construction of a synthetic Escherichia coli trp promoter and its use in the expression of a synthetic interferon gene; Windass JD et al.; An 82 base pair DNA fragment has been synthesised which contains the E . coli trp promoter and operator sequences and also encodes the first Shine Dalgarno sequence of the trp operon . This DNA fragment is flanked by EcoRI and ClaI/TaqI cohesive ends and is thus easy to clone, transfer between vector systems and couple to genes to drive their expression . It has been cloned into plasmid pAT153, producing a convenient trp promoter vector . We have also joined the fragment to a synthetic IFN-alpha 1 gene, using synthetic oligonucleotides to generate a completely natural, highly efficient bacterial translation initiation signal on the promoter proximal side of the IFN gene . Plasmids carrying this construction enable E . coli cells to express IFN-alpha 1 almost constitutively and with significantly higher efficiency than from a lacUV5 promoter based system.

J Biol Chem, 1982 Nov 10, 257(21), 12970 - 8
Effects of neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter . III . High resolution thermal denaturation and circular dichroism studies; Goodman TC et al.; High resolution thermal denaturation and circular dichroism studies were performed on a series of six recombinant DNA restriction fragments . The fragments varied in size from 132 to 193 bp and contained Escherichia coli wild type and UV5 lactose promoters both with and without homopolymer insertions of poly(dA).poly(dT) and poly(dG).poly(dC) . A differential thermal destabilization of the wild type promoter region, as compared to the UV5 promoter, was observed when dA70.dT70 was inserted into the -60 region or both DNAs . This effect may depend, in part, on the differences in the base composition between adjoining cooperative units in the fragments . The relatively larger effect of the AT sequence on the wild type promoter region may be correlated with the increased levels of in vitro transcription activity described in the preceding paper (Klein, R . D., and Wells, R . D . (1982) J . Biol . Chem . 257, 12962-12969) . Stretches of homopolymeric GC base pairs stabilized the wild type and UV5 promoter regions by over 2.5 degrees C . CD studies could not detect conformational differences between DNAs containing the wild type or UV5 promoter . The presence of homopolymers had a marked effect on the CD spectra of the fragments.

J Biol Chem, 1982 Nov 10, 257(21), 12929 - 34
Q beta replicase containing altered forms of ribosomal protein S1; Cole PE et al.; Transcription of Q beta RNA replicase requires that the host-encoded Escherichia coli ribosomal protein S1 be present as a subunit of the replicase . To determine whether the activities of S1 in protein synthesis are operational in Q beta RNA transcription, we formed altered replicase enzymes by reconstituting replicase lacking S1 (R(-S1)) with modified S1 species whose properties in nucleic acid binding and protein synthesis had been previously established . S1 derivatized with N-ethylmaleimide reconstitutes a modified replicase that is 81% as active as replicase reconstituted with unmodified S1 . S1 derivatized with N-ethylmaleimide and unmodified S1 bind with comparable affinity to R(-S1) (1 X 10(8) M-1) . These results indicate that the helix-unwinding properties of S1, which are known to be inactivated by N-ethylmaleimide modification, are not required for Q beta RNA transcription and that the sulfhydryl-derivatized region of S1 is not utilized in replicase subunit contacts . In contrast with its established ability to replace E . coli S1 on the ribosome, Caulobacter crescentus S1 does not reconstitute Q beta RNA transcription activity when added to R(-S1) . Our results suggest this inactivity may be due to poor binding to R(-S1).

J Biol Chem, 1982 Nov 10, 257(21), 12887 - 92
Studies of the flavin adenine dinucleotide binding region in Escherichia coli pyruvate oxidase; Mather M et al.; Experiments have been performed to probe the flavin adenine dinucleotide (FAD) binding region in Escherichia coli pyruvate oxidase . This enzyme functions as a membrane-associated flavoprotein coupled to the aerobic E . coli respiratory chain . The FAD moiety is noncovalently bound to pyruvate oxidase and can be removed reversibly to form apopyruvate oxidase . The addition of free FAD to apoenzyme results in the stoichiometric re-formation of the active flavoprotein . Using this technique, synthetic analogs of FAD were substituted in the flavin binding site and used as structural probes . Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-hydroxy-FAD are stabilized in the enzyme-binding site . This is consistent with the fact that the native flavoprotein forms a red (anion) radical upon photoreduction . These data suggest that the isoalloxazine ring may be poised for reduction via position N-5 by a carbanionic intermediate . The alpha-carbanion of hydroxyethylthiamin pyrophosphate, formed following the decarboxylation of pyruvate, is a likely candidate . The highly resolved visible spectrum of the native flavoprotein suggests that the flavin is buried in a hydrophobic environment . Reactivity studies using 8-chloro-FAD-pyruvate oxidase and 2-thio-FAD-pyruvate oxidase suggest that the C-8 position and C-2 position of the isoalloxazine ring may not be accessible to the solvent . Spectral perturbations observed with 6-hydroxy-FAD-pyruvate oxidase indicate, however, that the isoalloxazine C-6 position may be located near the binding site for the cofactor thiamin pyrophosphate . Restrictions to the accessibility of the active site of the enzyme are suggested by the fact that sulfite does not form an adduct with the flavin in the native enzyme.

J Biol Chem, 1982 Nov 10, 257(21), 12878 - 86
Reconstitution of native Escherichia coli pyruvate oxidase from apoenzyme monomers and FAD; Recny MA et al.; Pyruvate oxidase, a tetrameric enzyme consisting of 4 identical subunits, dissociates into apoenzyme monomers and free FAD when treated with acid ammonium sulfate in the presence of high concentrations of potassium bromide . Reconstitution of the native enzymatically active protein can be accomplished by incubating equimolar concentrations of apomonomers and FAD at pH 6.5 . The kinetics of the reconstitution reaction have been measured by 1) enzyme activity assays, 2) spectrophotometric assays to measure FAD binding, and 3) high performance liquid chromatography analysis measuring the distribution of monomeric, dimeric, and tetrameric species during reconstitution . The kinetic analysis indicates that the second order reaction of apomonomers with FAD to form an initial monomer-FAD complex is fast . The rate-limiting step for enzymatic reactivation appears to be the folding of the polypeptide chain in the monomer-FAD complex to reconstitute the three-dimensional FAD binding site prior to subunit reassociation . The subsequent formation of native tetramers appears to proceed via an essentially irreversible dimer assembly pathway.

J Biol Chem, 1982 Nov 10, 257(21), 12475 - 7
Role of phosphatidylethanolamine in the biosynthesis of pyrophosphoethanolamine residues in the lipopolysaccharide of Escherichia coli; Hasin M et al.; The biosynthesis of pyrophosphoethanolamine residues linked to the core oligosaccharide region of the lipopolysaccharide of Escherichia coli K2 strain BB 26-36 has been investigated by means of isotope tracer experiments in living cells . Phosphoethanolamine was isolated from the pyrophosphoethanolamine residues after hydrolysis in 1 N HCl at 100 degrees C . The kinetics of labeling of the phosphoethanolamine from {3H}serine or sn-glycero-3-32P during pulse-chase experiments revealed that the biosynthetic precursor of the phosphoethanolamine must be a large, relatively stable pool, and not a small, rapidly metabolized pool such as that of free serine, or seryl-tRNA . Labeling of the pyrophosphoethanolamine residues of the lipopolysaccharide from the two isotopes was closely parallel, and the isotope ratio 3H/32P was closely similar to that in phosphatidylethanolamine at the same time intervals . These experiments offer strong evidence that phosphatidylethanolamine functions in the biosynthesis of pyrophosphoethanolamine residues in lipopolysaccharide in a reaction in which the phosphoethanolamine head-group of the phospholipid is transferred as a unit to a lipopolysaccharide acceptor.

J Biol Chem, 1982 Nov 10, 257(21), 12962 - 9
Effects of Neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter . II . In vitro transcription analyses; Klein RD et al.; Transcription studies were conducted on the primary promoter (which initiates at +1) of lac wild type (wt) and UV5 that had insertions of poly(dA).poly(dT) or poly(dG).poly(dC) in their -60 region . This series of recombinant DNAs was designed to study systematically the effect of neighboring DNA sequences on the lac promoter . Investigations using linear templates showed that wild type expression was enhanced by the poly(dA).poly(dT) insertion, whereas UV5 activity was not affected by this polymer . Neither the wt nor the UV5 promoter was influenced by the presence of poly(dG).poly(dC) . Quantitation of abortive transcripts from supercoiled templates showed that the influence of the polymers on promoter activity was small . Comparative studies of the primary promoter with a secondary promoter (which initiates near -22), activated by 25% glycerol, were performed.

J Biol Chem, 1982 Nov 10, 257(21), 12954 - 61
Effects of neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter . I . Cloning and characterization studies; Klein RD et al.; To assess the role of neighboring DNA sequences in gene regulation, poly(dA).poly(dT) and poly(dG).poly(dC) were cloned adjacent to promoters of the lactose control region . Recombinant plasmids were constructed which were suitable for large scale purification of restriction fragments containing these promoters, 95-base pair (bp) AluI fragments containing the lack operator and promoter for the lac wild type and for the catabolite gene activating the protein-independent mutant, lac UV5, were cloned into pBR322 . Homopolymers of varying lengths were inserted into the -60 region of these promoters using recombinant DNA techniques . Six of the recombinant plasmids were chosen for detailed analysis: wild type (wt); wt-AT, containing 70 bp of poly(dA).poly(dT); wt-GC, containing 23 bp of poly(dG).poly(dC); UV5; UV5-AT, containing 70 bp of poly(dA).poly(dT) and finally UV5-GC, containing 43 bp of poly(dG).poly(dC) . These plasmids were characterized by restriction mapping and DNA sequencing . The effects of the DNA homopolymers on the interaction of the Escherichia coli RNA polymerase with the promoters were studied using nitrocellulose filter binding . The results show that poly(dA).poly(dT) increases the level of RNA polymerase binding, whereas poly(dG).poly(dC) has no detectable effect.

J Biol Chem, 1982 Nov 10, 257(21), 12503 - 8
Gene for Escherichia coli glycyl-tRNA synthetase has tandem subunit coding regions in the same reading frame; Keng T et al.; Glycyl-tRNA synthetase is one of two Escherichia coli aminoacyl tRNA synthetases which has two different subunits . A 5.1-kilobase pair HindIII chromosomal DNA fragment was isolated, cloned into pBR322 (to give plasmid pTK201), and shown to direct synthesis in maxicells of both subunits (Mr = 35,000 (alpha) and Mr = 65,000 (beta) of glycyl-tRNA synthetase . Locations of alpha- and beta-subunit coding regions were established by introduction of Tn5 insertions into various positions within the 5.1-kilobase pair HindIII segment of pTK201 and by determining the effect of each Tn5 insertion on synthesis of alpha- and beta-subunits and on enzymatic activity . From the Tn5 insertion analysis, regions encoding the NH2 terminus of the alpha-subunit and of the beta-subunit were approximately defined and these regions were sequenced . To locate rigorously the respective NH2-terminal encoding sections in the DNA sequence, NH2-terminal amino acid sequences of alpha- and beta-subunits were established by standard Edman degradations and these sequences were aligned with the DNA sequence . This analysis established the following: 1) coding regions for the subunits are in tandem; 2) a single promoter is used for transcription of both coding sections and the order of transcription is from alpha to beta; 3) in the 500 nucleotides 5' to the start of the alpha-subunit coding section, there is no sequence arrangement like that found for regulatory regions of bacterial amino acid biosynthetic operons; 4) nine nucleotides serve as the spacer between the TAA stop of the alpha- and the ATG start of the beta-subunit coding regions, thus making both coding regions in the same reading frame; and 5) the TAA stop of the alpha-subunit and the next for nucleotides associated with the intersubunit region are complementary to the 3'-end of 16 S rRNA; this arrangement suggests ribosome re-initiation in the spacer region gives balanced synthesis of both subunits.

Biochemistry, 1982 Nov 9, 21(23), 5805 - 10
Direct measurement of lactose/proton symport in Escherichia coli membrane vesicles: further evidence for the involvement of histidine residue(s); Patel L et al.; Addition of lactose to Escherichia coli ML 308-225 membrane vesicles under nonenergized conditions induces transient alkalinization of the medium, and the initial rate of proton influx is stimulated by valinomycin and abolished by nigericin or carbonyl cyanide m-chlorophenylhydrazone . A functional lac y gene product is absolutely required as the effect is not observed in ML 308-225 vesicles treated with N-ethylmaleimide nor with vesicles from uninduced Escherichia coli ML 30 . Furthermore, the magnitude of the phenomenon is enhanced about 3-fold in vesicles from Escherichia coli T206, which contain amplified levels of the lac carrier protein . Kinetic parameters for lactose-induced proton influx are the same as those determined for lactose-facilitated diffusion, and quantitative comparison of the initial rates of the two fluxes indicates that the stoichiometry between protons and lactose is 1:1 . Treatment of ML 308-225 vesicles with diethyl pyrocarbonate causes inactivation of lactose-induced proton influx . Remarkably, however, treatment with the histidine reagent enhances the rate of lactose-facilitated diffusion in a manner suggesting that the altered lac carrier catalyzes lactose influx without the symport of protons . The results are consistent with the hypothesis that acylation of a histidyl residue(s) in the lac carrier protein dissociates lactose influx from proton influx and indicate that this residue(s) play(s) an important role in the pathway of proton translocation.

Biochemistry, 1982 Nov 9, 21(23), 5849 - 56
Function of transcription termination factor rho in a model transcription system using synthetic deoxyribonucleic acid as template; Shigesada K et al.; The function of a transcription termination factor, rho, has been studied by using several synthetic DNAs with simple repetitive base sequences as templates for transcription . rho actually exhibits various effects on transcription depending on the base sequence of the template: (1) rho terminates poly(A) synthesis with poly(dA) x poly(dT), poly(dT), or oligo(dT), leading to release of RNA from RNA polymerase . rho also inhibits the synthesis of other homoribopolymers such as poly(U) directed by poly(dA) x poly(dT) and poly(C) and poly(I) directed by poly(dG) x poly(dC), presumably by a similar mechanism . (2) rho inhibits the synthesis of another homoribopolymer, poly(G), directed by poly(dG) x poly(dC) at the step of initiation rather than propagation of transcription . (3) rho stimulates rather than inhibits the synthesis of poly(A-C) and poly(G-U) directed by poly{d(A-C)} x poly{d(G-T)}, presumably by enhancing the dissociation of transcription complexes . (4) rho has no influence on the synthesis of poly(A-U) and poly(G-C) directed by poly{d(A-T)} and poly{d(G-C)}, respectively . In the first case, but not otherwise, the effect of rho is coupled with its RNA-dependent nucleosidetriphosphate phosphohydrolase activity, as is rho-mediated transcription termination on natural templates . The implication of these results is discussed in reference to the current view that rho acts on transcription complexes that have ceased elongation and causes release of RNA in an energy-requiring reaction.

Biochemistry, 1982 Nov 9, 21(23), 5839 - 49
High-resolution phosphorus nuclear magnetic resonance spectroscopy of transfer ribonucleic acids: multiple conformations in the anticodon loop; Gorenstein DG et al.; The temperature dependence of the 31P NMR spectra of yeast phenylalanine tRNA, E . coli tyrosine, glutamate (2), and formylmethionine tRNA is presented . The major difference between the 31P NMR spectra of the different acceptor tRNAs is in the main cluster region between -0.5 and -1.3 ppm . This confirms an earlier assignment of the main cluster region to the undistorted phosphate diesters in the hairpin loops and helical stems . In addition the 31P NMR spectra for all tRNAs reveal approximately 16 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester . In the presence of 10 mM Mg2+ most scattered and main cluster signals do not shift between 22 and 66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles . At greater than 70 degrees C, all of the signals merge into a single random-coil conformation signal . A number of the scattered peaks are shifted (0.2-1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ and spermine as a result of a conformational transition in the anticodon loop . The 31P NMR spectrum of the dimer formed between yeast tRNAPhe and E . coli tRNA 2Glu is reported . This dimer simulates codon-anticodon interaction since the anticodon triplets of the two tRNAs are complementary . Evidence is presented that the anticodon-anticodon interaction alters the anticodon conformation and partially disrupts the tertiary structure of the tRNA.

FEBS Lett, 1982 Nov 8, 148(2), 251 - 4
The presence of alkali-labile sites in DNA daughter chains of UV-irradiated Escherichia coli; Slezarikova V et al.; DNA newly synthesized in UV irradiated Escherichia coli B/r Hcr+ was 2 min pulse-labeled at various periods, then denatured and analysed by sucrose gradient centrifugation either in neutral or in alkaline conditions . Data indicate that in DNA of damaged cells alkali-labile sites are produced . In cells saturated with inducible proteins production of alkali-labile sites disappears in approximately 1 h . In the absence of inducible proteins production of alkali-labile sites continues.

FEBS Lett, 1982 Nov 8, 148(2), 247 - 50
Carbodiimide-induced protein--RNA crosslinking in mammalian ribosomal subunits; Buisson M et al.; RNA--protein interactions in the 60 S subunits of rat liver ribosomes were studied using 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC) under conditions which neither changed the sedimentation coefficient of subunits nor the intactness of their rRNA . EDC induced RNA--protein and protein--protein crosslinkings . Proteins crosslinked to 28 S RNA were identified by two-dimensional gel electrophoreses as L17, L19, L23a and L37a, shown to react with 28 S RNA when using a low dose of UV radiation . Attempts have also been made to use EDC for the studies of RNA--protein interactions in 40 S ribosomal subunits.

Ann Neurol, 1982 Nov, 12(5), 445 - 8
Selective reduction of blood flow to white matter during hypotension in newborn dogs: a possible mechanism of periventricular leukomalacia; Young RS et al.; The cerebrovascular response of newborn animals to hypotension has not been defined on a regional basis . Using an autoradiographic technique employing 14C-iodoantipyrine as indicator, we studied the cerebral physiological responses of newborn dogs to hypotension induced by exsanguination or by administration of Escherichia coli endotoxin . Regional cerebral blood flow (rCBF) to all gray matter structures was preserved, even at mean arterial pressures as low as 20 mm Hg . In contrast, rCBF to periventricular and occipital white matter decreased significantly during severe hypotension . The selective hypoperfusion of cerebral white matter during severe hypotension may provide a mechanistic explanation for the pathogenesis of periventricular leukomalacia.

Gene, 1982 Nov, 20(1), 91 - 102
The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase; Shinozaki K et al.; The gene for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase/Oase) from tobacco has been cloned in pBR322 and sequenced . The coding region contains 1431 bp (477 codons) . The deduced amino acid sequence of tobacco LS protein shows 90% homology with those of maize and spinach LS . The positions in the gene corresponding to the 5' and the 3' ends of tobacco LS mRNA have been located on the DNA sequence by the S1 nuclease mapping procedure . The LS gene promoter sequence has homology with Escherichia coli promoter sequences; its terminator sequence is capable of forming a stem-and-loop structure . A sequence GGAGG, which is complementary to a sequence near the 3' end of tobacco chloroplast 16S rRNA and a putative ribosome binding site, occurs 6-10 bp upstream from the initiation codon.

Infect Immun, 1982 Nov, 38(2), 444 - 8
Molecular heterogeneity of heat-labile enterotoxins from human and porcine enterotoxigenic Escherichia coli; Tsuji T et al.; The heat-labile enterotoxins produced by human enterotoxigenic Escherichia coli (LTh) and porcine enterotoxigenic E . coli (LTp) were purified to homogeneity, and their molecular properties were compared with those of purified cholera enterotoxin (CT) . On polyacrylamide gel disk electrophoresis without sodium dodecyl sulfate, LTh, LTp, and CT differed in mobility, suggesting differences in their ionic charges . The pI values of LTh, LTp, and CT were 7.50, 8.10, and 6.80, respectively . On sodium dodecyl sulfate-polyacrylamide gel slab electrophoresis, the B subunit and A1 and A2 fragments of LTh, LTp, and CT differed in mobility, suggesting that they differed in molecular size . Their molecular sizes seemed to decrease in the following order: B subunit, LTh greater than LTp congruent to CT; A1 fragment, LTp greater than LTh congruent to CT; A2 fragment, LTh congruent to CT greater than LTp . Amino acid compositions of LTh, LTp, and CT were also compared.

Fed Proc, 1982 Nov, 41(13), 2966 - 73
113Cd NMR of Cd(II)-substituted Zn(II) metalloenzymes; Gettins P et al.; Cadmium can replace zinc and magnesium in alkaline phosphatase from Escherichia coli, which permits the characterization of the catalytically important metal-binding sites by 113Cd NMR . At pH 6.5, in the absence of phosphate, two equivalents of cadmium are bound in identical sites (A), one in each monomer . Either raising the pH or phosphorylation of Cd2AP (AP is apoalkaline phosphatase) results in migration of Cd(II) from the site A in one monomer to the opposite monomer to occupy a second site (B) adjacent to the A site in the first monomer, a site stabilized by phosphorylation or high pH . At pH 6.5 in the presence of phosphate, the 113Cd NMR spectrum of Cd6AP consists of three narrow resonances from three pairs of fully occupied sites, A, B, and C . The resonances at 153 and 70 ppm represent two metal sites (A and B) 3.9 A apart at each active center and adjacent to the serine phosphorylated during turnover . At this pH the enzyme exists almost exclusively as the covalent phosphoseryl form E-P with a 31P resonance at approximately 9 ppm . As the pH is raised a 31P signal from the noncovalent E.P complex appears at approximately 13 ppm . This is reflected in the 113Cd spectrum by a split of both the A- and B-site resonances into pairs, a set at 137 and 65 ppm for E.P, and 153 and 70 ppm for the E-P species . 113Cd-O-31P coupling of 30 Hz on the 31P resonance of E.P shows the noncovalently bound phosphate to be coordinated to one but not both metal ions at each active site . The resonance of E-P is not coupled and thus the phosphoseryl residue appears to shift out of the coordination sphere of the active site metal ion.

Biochimie, 1982 Nov-Dec, 64(11-12), 1035 - 40
Stabilization of the ternary complex EF-Tu.GTP.valyl-tRNAval by ammonium salts; Antonsson B et al.; In a search for crystallizing conditions for the ternary complex EF-Tu.GTP.valyl-tRNAval, the influence of various salts on its stability has been examined by measuring the rate of deacylation of the aminoacyl-tRNA in the complex . The most striking result is the general higher stability in solutions of ammonium salts and, in particular, the enhancement of this effect by sulphate and citrate . Thus sodium sulphate and citrate lead to destabilization of the complex, as expected from conventional considerations of adding salt, whereas the corresponding ammonium salts stabilize the complex as shown, for example, by an increase in the half-life of the valyl-tRNAval in the complex from about 20 hours to at least 300 hours in the presence of 1.2 M ammonium sulphate . These results suggest that ammonium sulphate and ammonium citrate might be very suitable precipitants for crystallization studies of the ternary complex.

Proc Natl Acad Sci U S A, 1982 Nov, 79(21), 6661 - 5
Cloning and sequence determination of the gene for the human immunoglobulin epsilon chain expressed in a myeloma cell line; Kenten JH et al.; Messenger RNA has been isolated from cells of the human myeloma line 266BL which synthesizes IgE of the myeloma ND . A fraction enriched in mRNA for the epsilon heavy chain was copied into DNA and the DNA was cloned in Escherichia coli . A chemically synthesized oligonucleotide probe, based on the experimentally determined sequence of the specific message, was used to screen colonies . The largest epsilon chain cDNA cloned, 2.0 kilobases, was characterized by restriction endonuclease mapping and DNA sequence analysis . It appears to encode the complete amino acid sequence of the epsilon chain, including a signal peptide at the NH2 terminus as well as untranslated sequences at the 5' and 3' ends of the mRNA . The missing part of the previously published amino acid sequence of the ND epsilon chain was determined from the DNA sequence.

Environ Health Perspect, 1982 Nov, 45, 119 - 25
Mutagenic/carcinogenic potential of DEHP and MEHP; Tomita I et al.; The mutagenic/carcinogenic activities of DEHP and MEHP were studied in bacteria and mammalian cells . MEHP but not DEHP exerted a dose-dependent DNA damaging effect to B . subtilis in Rec-assay . DEHP and MEHP showed mutagenic activities to S . typhimurium TA-100, with and without S-9 mix, respectively . MEHP produced not only the mutation in E . coli WP2B/r but also sister chromatid exchanges (SCE) in Chinese hamster V79 cells . It also induced 8AG/6TG-resistant gene mutations and chromosomal aberrations in the V79 cells . Transplacental administration of DEHP or MEHP to the Syrian golden hamster embryos was carried out by administering DEHP or MEHP to gravid animals on day 11 of gestation, followed by the cultivation of embryonic cells for 15-20 days . Both DEHP and MEHP induced 8AG/6TG-resistant mutation, chromosomal aberrations and morphological transformation in the embryonic cells of the Syrian golden hamster.

J Bacteriol, 1982 Nov, 152(2), 822 - 8
Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Brucella cell envelope; Moriyon I et al.; Cell envelopes prepared from smooth and rough strains of Brucella were characterized on the basis of lipopolysaccharide and protein content . The action of three kinds of detergents on Brucella cell envelopes and Escherichia coli control cell envelopes was examined on the basis of the proteins and lipopolysaccharides that were extracted . As compared with those of E . coli, Brucella cell envelopes were resistant to nonionic detergents . Zwittergents 312 and 316 were most effective in extracting E . coli cell envelopes, and Zwittergent 316 was most effective in extracting Brucella cell envelopes . Sarkosyl extracted proteins but extracted only trace amounts of lipopolysaccharides from cell envelopes of both bacteria . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the Sarkosyl-resistant proteins revealed a composition similar to that of the proteins exposed on the surfaces of viable cells, as determined by the lactoperoxidase-125I radioiodination method . EDTA, with either Tris-HCl or Tris-HCl-Triton X-100, did not have detectable effects on Brucella cell envelopes . Ultracentrifugation of purified lipopolysaccharides in detergents and EDTA demonstrate that, in contrast to that of E . coli, Brucella lipopolysaccharide was not stabilized by divalent cations . Sarkosyl was ineffective in dispersing lipopolysaccharides, whereas the action of Zwittergents was related to the length of their alkyl chains.

J Bacteriol, 1982 Nov, 152(2), 702 - 5
L-Serine deaminase activity is induced by exposure of Escherichia coli K-12 to DNA-damaging agents; Newman EB et al.; The synthesis of L-serine deaminase in Escherichia coli K-12 was induced after exposure of cells to a variety of DNA-damaging agents, including UV irradiation, nalidixic acid, and mitomycin C . Synthesis was also induced during growth at high temperature . A mutant constitutive for SOS functions showed an elevated level of L-serine deaminase activity . The response to DNA-damaging agents thus may be mediated via the SOS system.

Diabetes Care, 1982 Nov-Dec, 5 Suppl 2, 67 - 70
Pharmacodynamics of human insulin (recombinant DNA)--regular, NPH, and mixtures--obtained by the Gerritzen method in healthy volunteers; Weinges K et al.; The study was concerned with the comparison of the action profile of regular and NPH preparations of human insulin (recombinant DNA) and of PPI (pork purified insulin) . In addition, the action profiles of some mixtures (10:90, 15:85, 20:80, 25:75, 30:70) of regular and NPH human insulin were evaluated . The comparisons were based on the Gerritzen test . There was no statistically significant difference in the time-course of the blood glucose levels after administration of NPH human insulin and NPH PPI . A tendency was noted, however, that NPH human insulin has a faster onset of action and that the serum glucose minimum for NPH human insulin lasts longer . The serum glucose curves after the application of regular and NPH human insulin initially lie closer together than after the respective PPI preparations . In the later phase, regular human insulin interferes less with the NPH curves . This means that combinations of regular and NPH human insulin may have a clinically useful action profile . No skin reaction or other adverse reaction was detected after application of human insulin and no antibodies against human insulin and Escherichia coli protein were found.

Diabetes Care, 1982 Nov-Dec, 5 Suppl 2, 135 - 9
The U.S . "new patient" and "transfer" studies; Galloway JA et al.; The large-scale clinical trials of human insulin (recombinant DNA) in the United States consisted of a "New Patient" study and a "Transfer" study . The "New Patient" study involved 101 patients (38% type I) who have never received insulin and who were treated with human insulin and followed for 6 mo using NPH insulin alone or in combination with Neutral Regular Insulin (NRI) . Shortly after treatment, serum glucose and total glycohemoglobin concentration fell . No patients developed insulin lipoatrophy or insulin allergy . Two patients developed insulin hypertrophy; in one, it was transient . Intradermal tests to varying dilutions of human insulin did not change over 6 mo . In addition, there was no evidence of development of antibodies to Escherichia coli polypeptide . Two-hundred-and-forty-three patients, 91% of whom had type I diabetes, were transferred in a controlled double-blind study from mixed beef-pork or purified pork insulin (PPI) either to human insulin or back to their previous insulin treatment and followed for 3 mo . While insulin dosage did not change, there was a slight increase in fasting serum glucose and a statistically significant increase in fasting ketonuria . There was no change in the frequency of the complications of insulin treatment . These limited data are consistent with the conclusion that NPH human insulin is slightly shorter acting than its animal insulin counterparts . Overall, human insulin is a safe, effective insulin.

Diabetes Care, 1982 Nov-Dec, 5 Suppl 2, 129 - 34
A double-blind crossover trial comparing human insulin (recombinant DNA) with animal insulins in the treatment of previously insulin-treated diabetic patients; Clark AJ et al.; Ninety-four diabetic patients established on treatment with pork (N = 47) or beef insulin (N = 47) took part in a double-blind crossover trial in which 6-wk treatment periods of their animal insulin were compared with similar periods on human insulin (recombinant DNA) . Six patients withdrew during the trial--in three cases for hypoglycemia while taking human insulin . In patients initially treated with beef insulin there was no significant change in the mean blood glucose, the 'M' index, the total daily insulin dose, or the frequency of hypoglycemic attacks after the change to human insulin . Home blood glucose sample values were greater before the morning and evening insulin injection on human insulin (morning: 12.8 mmol/L {beef} versus 14.2 mmol/L {human insulin} {P less than 0.05}; evening: 10.0 mmol/L versus 11.6 mmol/L {P = 0.05}) . In pork insulin-treated patients greater values while on human insulin were found for mean glucose (9.0 mmol/L {pork} versus 9.7 mmol/L {human insulin}, P = 0.05), 'M' index (65.0 {pork} versus 79.6 {human insulin}, P less than 0.025), and total daily insulin dose (50.9 U/day {pork} versus 52.5 U/day {human insulin}, P less than 0.001) . The early morning glucose sample was also greater on human insulin (9.6 mmol/L {pork} versus 12.1 mmol/L {human insulin}, P less than 0.001) . No significant differences in either insulin antibody levels or E . coli protein antibody levels were found between either of the animal-insulin treatment periods and human insulin treatment periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell, 1982 Nov, 31(1), 137 - 46
Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes; Hudziak RM et al.; We describe the generation of mammalian cell lines carrying amber suppressor genes . Nonsense mutants in the herpes simplex virus thymidine kinase (HSV tk) gene, the Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco-gpt) gene and the aminoglycoside 3' phosphotransferase gene of the Tn5 transposon (NPT-II) were isolated and characterized . Each gene was engineered with the appropriate control signals to allow expression in both E . coli and mammalian cells . Expression in E . coli made possible the use of well developed bacterial and phage genetic manipulations to isolate and characterize the nonsense mutants . Once characterized, the nonsense mutants were transferred into mammalian cells by microinjection and used, in turn, to select for amber suppressor genes . Xenopus laevis amber suppressor genes, prepared by site-specific mutagenesis of a normal X . laevis tRNA gene, were microinjected into the above cell lines and selected for the expression of one or more of the amber mutant gene products . The resulting cell lines, containing functional amber suppressor genes, are stable and exhibit normal growth rates.

Biosci Rep, 1982 Nov, 2(11), 929 - 39
Viroid RNA is accepted as a template for in vitro transcription by DNA-dependent DNA polymerase I and RNA polymerase from Escherichia coli; Rohde W et al.; The RNA genome of potato spindle tuber viroid (PSTV) is transcribed in vitro into complementary DNA and RNA by DNA-dependent DNA polymerase I and RNA polymerase, respectively, from Escherichia coli . In vitro synthesis of complementary RNA produces distinct transcripts larger than unit length thus reflecting the in vivo mechanism of viroid replication . The influence of varying experimental conditions on the transcription process is studied; actinomycin D is found to drastically reduce complementary RNA synthesis from the PSTV RNA template by RNA polymerase.

Biofizika, 1982 Nov-Dec, 27(6), 995 - 9
{Effect of antioxidants on photoaddition of 8-methoxypsoralen to thymine, double-helix DNA and photosensitized inactivation of Escherichia coli}; Potapenko AIa et al.; Effect of antioxidants--alpha-tocopherol and 2,6-di-tert-butyl-hydroxytoluene (BHT) on 2-methoxypsoralen (8-MOP) photoaddition was investigated . Spectrophotometric experiments have shown that under anaerobic conditions alpha-tocopherol inhibits photodimerization of 8-MOP and photoaddition of 8-MOP to thymine (in 80% ethanol solution) . 50% inhibition is obtained at alpha-tocopherol concentrations of about 10(-5) M . The antioxidants (in concentrations from 10(-8) to 10(-4) M) displayed no effect on photoaddition of 8-MOP to double-stranded DNA in solution . The reactions of 8-MOP photoaddition to double-stranded DNA were registered by formation of fluorescent monoadducts and interstrand crosslinks . The antioxidants also did not inhibit 8-MOP-photosensitized inactivation of E . coli . Perhaps, 8-MOP addition to DNA cannot be inhibited by antioxidants due to steric hindrance . The role of 8-MOP photoaddition to DNA in the induction of PUVA-erythema is discussed.

Biofizika, 1982 Nov-Dec, 27(6), 1000 - 4
{Distribution of large ligands on DNA macromolecules of different GC-content}; Novoseler MA; The complexes of calf thymus DNA, E-coli DNA, phage T2 and T7 DNA with protamine and poli-lysine (mol . wt . 52 000 and 20 000) were studied by thermal denaturation method . The average lengths of free and ligand-covered regions of DNA were determined from the melting curves by means of the formulae derived in the theoretical works by Akhrem A . A . and oths . {5} . The calculations show that poly-l-lysine (PL20000) and protamine are distributed along macromolecules of all the studied DNAs by clusters containing in the average four ligand molecules when NH2/P less than 0.25 . The number of ligands in the clusters is doubled when NH2/P greater than or equal to 0.5 and increased three-sixteen times depending on the types of DNA when NH2/P greater than or equal to 0.75 . The molecules of PL52000 are distributed along the DNA macromolecules isolated from each other, every one covering 120 base pairs . The clusters average lengths and their distribution along the DNA macromolecules do not depend directly on GC-content of DNA, but are defined by the concrete pair of the ligand-lattice . Possible cause of cooperative binding of large ligands with DNA is discussed.

Mol Biol (Mosk), 1982 Nov-Dec, 16(6), 1234 - 44
{A thermodynamic approach to the study of the structural organization of 5S RNA from Escherichia coli ribosomes . 1 . Analysis of scanned microcalorimetry data}; Matveev SV et al.; Melting of the 5S RNA from E . coli ribosomes has been studied by differential scanning microcalorimetry . It has been shown that: (1) heat capacity temperature functions of the "native" and A-forms of the 5S RNA coincide in all the conditions studied; (2) heat capacity temperature functions of the B-form of the 5S RNA differ in a low-temperature region from the heat capacity functions of the A-form, the complete melting enthalpy of the A-form being higher by 125 +/- 30 kJ X M-1; (3) the teritary structure of the 5S RNA does not unite secondary structure elements into a single cooperative unit even in the presence of 10 mM MgCl2; (4) the results of the analysis of the "equilibrium" part of heat capacity temperature functions and the A- and B-forms of the 5S RNA can be explained by melting of four cooperative blocks which interact with each other.

Mol Biol (Mosk), 1982 Nov-Dec, 16(6), 1165 - 72
{The separation of subunits of phenylalanyl-tRNA-synthetase from Escherichia coli MRE-600 by means of affinity chromatography in dissociation conditions}; Zykova NA et al.; The method of affinity chromatography on sepharose with immobilized tRNA in the presence of urea was developed for separating the subunits of phenylalanyl-tRNA synthetase from E . coli MRE-600 (subunit structure alpha 2 beta 2) . Specific binding of large beta-subunits of the enzyme on immobilized tRNA testifies the localization of the tRNA-binding center on the beta-subunit of phenylalanyl-tRNA synthetase . Separately alpha- and beta-subunits of the enzyme exhibit no catalytic activity . Incubation of the mixture of alpha- and beta-subunits in conditions leading to reassociation of the oligomeric structure results in restoration of catalytic activity of the enzyme . In the presence of urea resin with immobilised analogs of ATP binds alpha- and beta-subunits of the enzyme . This testifies the presence of nucleotide-binding sites on both subunits . The possibility of using the affinity chromatography method to separate non-identical subunits of different enzymes is discussed.

Mikrobiologiia, 1982 Nov-Dec, 51(6), 983 - 5
{DNA content in Escherichia coli mutants resistant to rifampicin and nalidixic acid}; Korotiaev AI et al.; DNA content was studied in Escherichia coli strains resistant to rifampicin and nalidixic acid . When the strain resistant to the acid was grown in the M-9 medium with glucose, it was found to contain 14.6 X 10(-15) to 17.8 X 10(-15) g of DNA per cell, the rifampicin-resistant strain contained 10.2 X 10(-15) to 34.8 X 10(-15) g of DNA per cell, and the parent sensitive strain contained 7.5 X 10(-15) to 9,2 X 10(-15) g of DNA per cell under the identical conditions . The morphology and certain physiological characteristics of the mutants were also modified.

Mikrobiologiia, 1982 Nov-Dec, 51(6), 937 - 40
{Action of acrylamide on Escherichia coli cells}; Lusta KA et al.; The action of acrylamide on Escherichia coli B was studied: short-term action at high concentrations, long-term action at low doses under the normal conditions of growth and in the process of cell immobilization in polyacrylamide gel . Such a treatment was found to cause considerable structural changes in the cells . Division of the cells was inhibited and they reached giant sizes when grown in media containing 1-2% of acrylamide . The phenomenon might be used for differentiation of living and dead E . coli cells.

J Gen Microbiol, 1982 Nov, 128 (Pt 11), 2791 - 5
Plasmid R394 is a cointegrate; Hauman JH et al.; Plasmids R394a and R394b which cointegrate to form R394 are described . They have molecular masses of 102 +/- 4 and 11.0 +/- 0.4 MDal, belong to the T and N incompatibility groups and confer resistance to kanamycin and ampicillin, respectively . R394a is self transmissible and mobilizes R394b, which is non-self transmissible . These findings clarify anomalies in the behaviour of R394 and support suggestions based on the properties of variant phages derived from R394.

J Biochem (Tokyo), 1982 Nov, 92(5), 1671 - 4
Studies on the metabolism of unsaturated fatty acids . X . Purification and some properties of 2,4-dienoyl-CoA reductase from Escherichia coli; Mizugaki M et al.; 2,4-Dienoyl-CoA reductase has been separated from Escherichia coli grown in the presence of linoleic acid and purified to homogeneity . The enzyme has a molecular weight close to 50,000 as determined by gel filtration on Sephacryl S-200 Super-fine . The reductase was rather stable in a buffer containing citric acid and kept its full activity on heating at 55 degrees C for 10 min in the pH range of 5.5 to 6.5, but was completely inactivated on heating at 58 degrees C for 10 min . Phosphocellulose column chromatography revealed that the reductase was not involved in the multi-enzyme complex (molecular weight of 260,000) of fatty acid oxidation.

J Biochem (Tokyo), 1982 Nov, 92(5), 1649 - 54
Studies on the metabolism of unsaturated fatty acids . IX . Stereochemical studies of the reaction catalyzed by trans-2-enoyl-coenzyme A reductase of Escherichia coli; Mizugaki M et al.; The steric course of the reaction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase from Escherichia coli was investigated . trans-2-{6,7,8-2H7}Octenoyl-CoA was synthesized as a substrate and incubated with partially purified trans-2-enoyl-CoA reductase in the presence of 4R- or 4S-{4-2H1}NADPH . The deuterium-labeled octenoyl-CoA was also incubated with the reductase in the presence of NADPH in 2H2O . Aliquots of octanoic acids formed were analyzed, after esterification, by gas chromatography-mass spectrometry (GC-MS) to demonstrate that the pro-4R hydrogen of NADPH was incorporated into the C-3 position of octenoyl-CoA and that hydrogen from water was introduced into the C-2 position of octenoyl-CoA . The remaining portions of octanoic acids isolated from the incubation mixtures were converted to their CoA esters by the action of acyl-CoA synthetase, and they were dehydrogenated by treatment with acyl-CoA oxidase, which had previously been shown to catalyze the anti-elimination of the pro-2R and pro-3R hydrogen atoms of acyl-CoA . The deuterium contents of the products were also analyzed by GC-MS, and the results indicated that the reduction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase occurred by an anti-addition of hydrogen via a 2-Re, 3-Re attack on the trans-double bond of the substrate.

Antimicrob Agents Chemother, 1982 Nov, 22(5), 912 - 4
Nonenzymatic chloramphenicol resistance determinants specified by plasmids R26 and R55-1 in Escherichia coli K-12 do not confer high-level resistance to fluorinated analogs; Dorman CJ et al.; Plasmids R26 (Inc P) and R55-1 (Inc C) specify inducible nonenzymatic resistance to chloramphenicol . Escherichia coli K-12 strains harboring these plasmids encoded low-level resistance to thiamphenicol analogs Sch 25298 and Sch 25393 but failed to specify resistance to the fluorinated chloramphenicol analog Sch 24893 . The analogs were efficient inducers of high-level chloramphenicol resistance.

Antimicrob Agents Chemother, 1982 Nov, 22(5), 791 - 9
Transport of the lipophilic analog minocycline differs from that of tetracycline in susceptible and resistant Escherichia coli strains; McMurry LM et al.; Plasmids which specify resistance to tetracycline offer much less resistance to its more lipophilic analog, minocycline . Resistance to minocycline varies for different plasmids . In the case of plasmid R222 (bearing the class B tetracycline resistance determinant on Tn10), minocycline resistance is comparatively high (10 microgram/ml, or 6% of the tetracycline resistance level) . For plasmid pIP7 (bearing the class A determinant), minocycline resistance is only 1% of the tetracycline resistance level . To understand the basis for these differences, we compared the transport of the two tetracyclines by susceptible cells and by resistant cells . Uptake of minocycline by susceptible cells was 10 to 20 times more rapid than uptake of tetracycline and occurred largely via an energy-dependent route . This host-mediated energy-dependent uptake of both analogs was still present in tetracycline-resistant cells . In resistant cells, the same plasmid-mediated active efflux system previously described for tetracycline also exported minocycline . The 15-fold greater susceptibility of tetracycline-resistant R222-bearing cells to minocycline as compared with tetracycline could be explained at least in part by the more rapid influx of minocycline, which more easily overcame the efflux system . The particularly low minocycline resistance offered by pIP7 was due to a weak efflux for minocycline, 10-fold less effective than that mediated by R222 . The rate-limiting step for uptake of both analogs appeared to be the outer membrane . That the lipophilic minocycline should cross this membrane more rapidly than tetracycline stands in contrast with other studies which show the outer membrane to be a barrier for entry of lipophilic substances.

Am J Vet Res, 1982 Nov, 43(11), 2010 - 2
Socialization as a factor in resistance to infection, feed efficiency, and response to antigen in chickens; Gross WB et al.; Chickens were habituated to human beings (socialized) by being talked to, offered food, and handled gently . After 7 weeks of socialization, the birds were challenge exposed with Escherichia coli . When compared with ignored groups, the socialized birds showed more than a 60% reduction in the prevalence of death and pericarditis . Furthermore, small flocks of socialized birds were more uniform in their response to E coli than were similar nonsocialized flocks . Socialized chickens also had improved feed efficiency and increased antibody response to canine RBC . Socialization was also applied easily to larger flocks of chickens.

Proc Natl Acad Sci U S A, 1982 Nov, 79(22), 6894 - 8
Preparation, characterization, and properties of monoclonal antibodies against the lac carrier protein from Escherichia coli; Carrasco N et al.; Monoclonal antibodies directed against the lac carrier protein purified from the membrane of Escherichia coli were prepared by somatic cell fusion of mouse myeloma cells with splenocytes from an immunized mouse . Several clones produce antibodies that react with the purified protein as demonstrated by solid-phase radioimmunoassay and by immunoblotting experiments; culture supernatants from the clones inhibit active transport of lactose in isolated membrane vesicles . Five stable clones were selected for expansion, formal cloning, and production of ascites fluid, and the antibodies secreted in vivo by each clone also were found to inhibit lactose transport . Antibody from hybridoma 4B1, an IgG2a immunoglobulin, inhibits active transport of lactose in proteoliposomes reconstituted with purified lac carrier and in right-side-out membrane vesicles . In contrast, the antibody has no effect on the generation of the proton electrochemical gradient by membrane vesicles nor does it alter the ability of vesicles containing the lac carrier to bind p-nitrophenyl-alpha-D-galactopyranoside . In order to achieve 50% inhibition of transport activity, a 2- to 3-fold molar excess of antibody to lac carrier is required, regardless of the amount of lac carrier in the membrane . Thus, the concentration of antibody required for a given degree of inhibition is proportional to the amount of lac carrier in the membrane . Finally, antibody-induced inhibition occurs within seconds, an observation suggesting that the epitope is accessible on the surface of the membrane.

J Inorg Biochem, 1982 Nov, 17(3), 185 - 203
Preliminary studies on the inhibitory effect of novel Pd(II) complexes of vitamin B6 on cell divisions; Moussa NM et al.; Two novel complexes of Pd(II) involving vitamin B6 compounds have been synthesized . They are compatible with the compositions Pd(P.H.)2 C2(P = pyridoxol) and Pd(PL.H)2 C2(PL = pyridoxal) . The complexes inhibited the growth as well as the biosynthesis of RNA, DNA, and protein of E . coli B-766 . Photoacoustic spectral (PAS) measurements showed that the complexes bound to DNA of the bacteria and were present only in the kidney of treated mice . The complexes inhibited the incorporation of 3H-thymidine as well as 14C-leucine in the DNA and protein, respectively, of liver cell cultures (BL8L) . The inhibition of cell division of Walker-S-cells and human lymphocytes by the complexes was highly significant.

Eur J Biochem, 1982 Nov, 128(1), 41 - 6
Removal of the tightly bound zinc from Escherichia coli trypsin-modified methionyl-tRNA synthetase; Mayaux JF et al.; The study of the behaviour of Escherichia coli methionyl-tRNA synthetase with chelating agents has shown that only 1,10-phenanthroline has an inhibitory effect on the tRNAMet aminoacylation activity . Under identical buffer conditions the isotopic {32P}PPi-ATP exchange activity is insensitive . Dialysis of the enzyme against 1,10-phenanthroline causes a slow loss of zinc from the enzyme which is paralleled by an irreversible loss of both the aminoacylation and isotopic exchange activities . The loss of zinc becomes faster upon the addition of small amounts of guanidine hydrochloride to the dialysis buffer containing phenanthroline, presumably by partially unfolding the protein . Studies of the reversible denaturation of the enzyme by 5 M guanidine hydrochloride shows that the inclusion of EDTA produces an enzyme species that has lost both zinc and activity . The inactive apoenzyme prepared in guanidine and EDTA can regain activity by dilution in a zinc-containing buffer.

Am Rev Respir Dis, 1982 Nov, 126(5), 932 - 5
Ischemic necrosis of both lower extremities as a result of the microembolism syndrome complicating the adult respiratory distress syndrome caused by Escherichia coli pneumonia and septicemia; Rinaldo JE et al.; Publication Types:

Case Reports

What Is Protein?, What Is Listeria Monocytogenes?, What Is MIC?, What Is Amino Acid?, What Is Fermentation?, e, Microbes, e, Bacteria, a, Microorganisms, e, Bacteriology, e, Microbe, i, Clostridia, n, S. cerevisiae, r, Ciprofloxacin, e, Salmonella typhimurium, r, Microorganism, a, Cell cultures, i, Salmonella, i, Antibiotic treatment, o, Escherichia coli, c, Candida albicans, c, Yeasts, c, S. cerevisiae, i, Denitrificans, c, Pseudomonas, r, Haemophilus, n, Agrobacterium, i, Microflora, r, Bacteria, i, Bacteria, r, Pichia, i, Yeasts

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005