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