Eur J Biochem, 1982 Apr, 123(3), 483 - 8 Expression of the gene for Escherichia coli initiation factor IE-3 in vivo and in vitro; Lestienne P et al.; Expression of protein synthesis initiation factor IF-3 in vivo was studied by measuring its level in exponentially growing cells as a function of gene dosage . A strain haploid for infC, the gene for IF-3, was modified to carry one or two additional infC genes giving diploid and triploid strains . Polyploid strains were achieved by the presence of multicopy plasmids expressing the infC gene . When IF-3 levels were measured by quantitative immunoblotting they were found to be proportional to the gene dosage; the presence of a multicopy plasmid thus causes considerable overproduction of IF-3, enabling large quantities to be purified . When lysates were prepared from freshly grown cells, only IF-3 alpha (the long form) was detected; however when IF-3 was purified from a strain containing a multicopy plasmid which overproduced it, the major product found was IF-3 beta (the short form, lacking six amino acids from the N terminus) . The synthesis of the two IF-3 forms was also studied by using a cell-free coupled transcription-translation system dependent on exogenous DNA: the IF-3 gene was found to be very efficiently expressed . IF-3 alpha increased more rapidly than IF-3 beta but following the cessation of protein synthesis IF-3 alpha decreased while IF-3 beta still increased . The results suggest that IF-3 alpha is slowly degraded to the beta form . Addition of non-radioactive IF-3 alpha, up to fivefold molar excess over ribosomes, to the synthesizing system in vitro did not inhibit IF-3 synthesis . Synthesis of IF-3 in vitro appears to be sensitive to guanosine 3'-diphosphate 5'-diphosphate. Eur J Biochem, 1982 Apr, 123(3), 477 - 82 Regulation of formation of threonyl-tRNA synthetase, phenylalanyl-tRNA synthetase and protein synthesis initiation factor 3 from Escherichia coli in vivo and in vitro; Elhardt D et al.; The expression of the structural genes for the protein synthesis initiation factor 3 (IF-3), threonyl-tRNA synthetase and phenylalanyl-tRNA synthetase carried by the transducing phage lambda p2 was studied in a DNA-dependent transcription-translation system in vitro and the results were compared to the regulatory pattern in vivo . In vitro, the DNA of the phage lambda p2 gives rise to the formation of the two forms of IF-3 (IF-31 and IF-3S) which are known to be present in vivo . The kinetics of synthesis indicate an interconversion of IF-31 into IF-3S . Addition of excess purified IF-31 does not significantly repress IF-3 synthesis but does stimulate the rate of conversion of IF-31 into IF-3S . This apparent lack of autoregulation in vitro is in accordance with gene-dosage-dependent synthesis in vivo . The fact that strains with more than one copy of the IF-3 structural gene contain a higher relative amount of IF-3S than do haploid ones suggests that the proteolytic conversion of IF-31 into IF-3S may occur predominantly in the free (non-ribosome-bound) state . In vivo, the amount of IF-3 varies with the growth rate much like elongation factor Tu or aminoacyl-tRNA synthetases . As with the aminoacyl-tRNA synthetases, IF-3 synthesis is not significantly subject to a stringent control system . This coordinated regulatory response in vivo, however, is not paralleled by the susceptibility of synthesis in vitro to guanosine 3'-diphosphate 5'-diphosphate (ppGpp), since IF-3 formation is inhibited by ppGpp whereas that of threonyl-tRNA synthetase and phenylalanyl-tRNA synthetase is stimulated. Biophys Chem, 1982 Apr, 15(1), 1 - 8 Reconstitution of the isolated beta2-subunit of tryptophan synthase from Escherichia coli after dissociation induced by high hydrostatic pressure . Equilibrium and kinetic studies; Seifert T et al.; The isolated beta2-subunit of Escherichia coli tryptophan synthase can be reversibly dissociated into enzymically inactive monomers under high hydrostatic pressure . Deactivation at 1.5 kbar which shows a half-time of 11 min (rate constant k=10 (-3) s (-1) is paralleled by dissociation with a small lag phase of about 5 min . Pressure release leads to 95 +/- 5% recovery of specific activity and complete restoration of the hydrodynamic and spectral properties which specify the native dimer . Over the concentration range 1-100 micrograms/ml (0.02-2.3 micrograms M) the kinetics of reactivation can be fitted by one apparent first-order rate constant (k=6.5 +/- 0.6 X 10 (-4) s (-1), half-time = 17.5 min) . The reconstitution of catalytic activity is paralleled by alterations in tryptophan fluorescence at 327 nm, thus presenting direct evidence for conformational changes in the direct vicinity of the active center (k1 = 1.9 X 10 (-3) s(-1), k2 = 6.5 +/- 0.6 X 10(-4) s (-1) ) . On the other hand, a definite mechanism of reactivation requires the association of the refolding monomers to be included . The kinetics of dimerization have been followed via hybridization between native and chemically modified beta-chains, yielding an apparent first-order rate constant of 6.3 +/- 0.6 X 10 (-4) s (-1) . As a consequence, we propose a sequential uni-uni-bimolecular mechanism, which is characterized by a minimum of two conformational changes in substantially structured monomers followed by a fast dimerization reaction to yield the active beta2-subunit. Am J Vet Res, 1982 Apr, 43(4), 724 - 8 Plasma zinc and iron concentrations as measurements for evaluating the influence of endotoxin-neutralizing agents in Escherichia coli endotoxin-induced mastitis; Verheijden JH et al.; The influence of bacterial lipopolysaccharides on plasma Zn and Fe concentrations and the endotoxin-neutralizing capacity of polymyxin B was studied in 3 dairy cows . Also, the feasibility of using plasma Zn and Fe concentrations as measurements for evaluating the influence of endotoxin-neutralizing agents in Escherichia coli endotoxin-induced mastitis was studied . A positive relationship was ascertained between the dose of endotoxin given intramammarily (IMM) and the effect on mean values of plasma Zn and Fe concentrations . Seemingly, IMM administration of polymyxin B-sulfate before the IMM administration of E coli endotoxin partially blocked the effects of endotoxin, as shown by decreases in plasma Zn and Fe. J Med Chem, 1982 Apr, 25(4), 386 - 92 Use of adenine nucleotide derivatives to assess the potential of exo-active-site-directed reagents as species- or isozyme-specific enzyme inactivators . 5 . Interactions of adenosine 5'-triphosphate derivatives with rat pyruvate kinases, Escherichia coli thymidine kinase, and yeast and rat hexokinases; Hampton A et al.; Adenosine 5'-triphosphate (ATP) derivatives of the types N6-R-ATP {R = (CH2)nNHCOCH2I, (CH2)nNHCO-(CH2)mNHCOCH2I, or (CH2)nCON(Me)(CH2)mN(Me)CO(CH2)nNHCOCH2I}, N6-Me-N6-R-ATP {R = (CH2)nN-(Me)CO(CH2)mNHCOCH2I}, and 8-R-ATP {R = NM(CH2)nNHCOCH2I} with 5--19 spacer atoms between N6 or C-8 and iodine have been evaluated as potential exo-ATP-site-directed reagents for phosphokinases . Substrate and inhibitor properties indicated that the compounds possessed affinity for the ATP sites of the muscle (M), kidney (K), and liver (L) isozymes of rat pyruvate kinase (PK), of E . coli thymidine kinase (TK), and of yeast hexokinase (HK) and rat KH I, II, and III isozymes . Tests for time-dependent loss of enzyme activity (inactivation) were performed under conditions in which a large proportion of each phosphokinase was present as an enzyme-inhibitor complex . No ATP-site-directed inactivations resulted when the M, L, or K isozymes of PK were exposed for 8 h, 22 degrees C, to 5 mM levels of 18 ATP derivatives or 6 analogous ADP derivatives or when yeast HK or rat KH I, II, or III was exposed for 6 h, 22 degrees C, to 5 mM levels of 28 ATP derivatives . Escherichia coli TK was inactivated by 6 of 25 ATP derivatives tested at 10 mM, 6 h, 0 degrees C; inactivation was slowed by MgATP in the case of N6-CH3-N6-R-ATP {R = (CH2)4N(CH3)CO(CH2)5NHCOCH2I} . Only 1% of 298 enzyme-inhibitor combinations exhibited ATP-site-directed inactivation, signifying that few suitably positioned and sufficiently reactive nucleophilic groups were present near the enzymic ATP sites . Studies have now shown that exo-active-site-directed reagents can act as isozyme- or species-selective enzyme inhibitors . The present survey indicates that in many cases such reagents may be difficult of access when data are not available regarding structural or physicochemical features of the target enzyme adjacent to its catalytic site. J Clin Microbiol, 1982 Apr, 15(4), 554 - 7 Use of a hemadsorption technique to evaluate the stability of the hemagglutination reaction of Escherichia coli cultures possessing human colonization factor antigens; Sedlock DM et al.; Two strains of Escherichia coli, producing different colonization factor antigens (CFA), were monitored for the population density of CFA-producing bacteria after repeated subculture . The production of CFA was estimated by flooding agar plates containing isolated colonies with suspensions of human or bovine erythrocytes . The erythrocytes were suspended in a low-ionic-strength buffer and were fixed to CFA-positive colonies with a 1.0% tannic acid solution . Strain H-10407, possessing CFA/I fimbriae, showed a rapid loss of the hemagglutinin when subcultured, whereas strain CL-9699, producing CFA/II, was very stable . By using the hemadsorption assay, we could rapidly and easily distinguish CFA- positive colonies from the CFA-negative variants . A survey of additional E . coli strains demonstrated the utility and specificity of the hemadsorption technique used. J Bacteriol, 1982 Apr, 150(1), 433 - 5 The product of the lexC gene of Escherichia coli is single-stranded DNA-binding protein; Meyer RR et al.; Extracts from lexC113 cells could not support phage G4 DNA-dependent replication unless supplemented with single-stranded DNA-binding protein . Purified lexC113 binding protein supported synthesis in a reconstituted replication assay, using purified proteins at 30 but not at 42 degrees C, indicating that the product of the lexC113 gene is an altered single-stranded DNA-binding protein. J Bacteriol, 1982 Apr, 150(1), 425 - 8 Gene cpxA is a new addition to the linkage map of Escherichia coli K-12; Silverman PM; The cpxA gene of E . coli K-12 lies between genes glpK and tpi, closely linked to the latter at 87.8 min on the linkage map . Since no other gene has been mapped in this interval, cpxA is a new addition to the linkage map. J Bacteriol, 1982 Apr, 150(1), 385 - 8 Resident enhanced repair: novel repair process action on plasmid DNA transformed into Escherichia coli K-12; Strike P et al.; The survival of UV-irradiated DNA of plasmid NTP16 was monitored after its transformation into recipient cells containing an essentially homologous undamaged plasmid, pLV9 . The presence of pLV9 resulted in a substantial increase in the fraction of damaged NTP16 molecules which survived in the recipient cells . This enhanced survival requires the host uvrA+ and uvrB+ gene products, but not the host recA+ gene product . The requirement for both homologous DNA and the uvrA+ and uvrB+ gene products suggests that a novel repair process may act on plasmid DNA . Possible mechanisms for this process are considered. J Bacteriol, 1982 Apr, 150(1), 312 - 8 The catabolite-sensitive promoter for the chloramphenicol acetyl transferase gene is preceded by two binding sites for the catabolite gene activator protein; Le Grice SF et al.; DNase I protection experiments have indicated that the cyclic AMP-catabolite gene activator protein complex binds to two regions preceding the chloramphenicol acetyl transferase (cat) gene in Escherichia coli . One of these lies adjacent to the RNA polymerase binding site, whereas the second lies approximately 130 base pairs upstream from the starting point of transcription . Additional DNase protection experiments and in vitro transcription experiments with modified templates indicate that the catabolite gene activator protein site proximal to the cat promoter functions independently of the distal site, indicating that in vitro the second of these sites is not required for transcriptional activation of the cat gene. Invest Ophthalmol Vis Sci, 1982 Apr, 22(4), 494 - 501 Mononuclear cells in the corneal response to endotoxin; Howes EL et al.; A severe keratitis can be produced after the direct injection of bacterial endotoxin, or lipopolysaccharide (LPS), in rabbits . Corneal inflammation can progress to scarring and vascularization within a 2 to 3 week period . Pretreatment with systemic adrenal corticosteroids (triamcinolone) prevents this response . Limbal cellular and vascular events were studied during the first 20 hr after injection of LPS in treated and nontreated rabbits . Perivascular limbal inflammatory cells were counted and limbal vascular permeability was assessed by extravasation of 131I-albumin and 125I-fibrinogen in the cornea . Corticosteroids decreased but did not prevent the early protein extravasation and profoundly altered the inflammatory cell population around blood vessels at the limbus . Mononuclear cells, particularly mononuclear phagocytes, were sharply reduced . It is proposed that these cell types play an important role in the perpetuation and amplification of the inflammatory response in this reaction. Immunopharmacology, 1982 Apr, 4(2), 95 - 104 Exogenous additions of prostaglandins variably alter the blastogenic response of B and T lymphocytes from different mice lymphoid organs; Rojo JM et al.; Prostaglandins A1 and E2 enhance the 3H-thymidine (TdR) uptake in spleen cell cultures stimulated by two B-lymphocyte mitogens (E.coli lipopolysaccharide (LPS) and dextran sulfate (DXS) at concentrations ranging between 10(-8) and 10(-6) M . The same prostaglandin (PG) concentrations inhibited concanavalin A (Con A) activation of mouse spleen cells depleted or not from a glass-adherent prostaglandin-producing population of suppressor cells . After fractionation of spleen cells by nylon wool adherence, PGs diminished 3H-TdR uptake by nylon nonadherent (T-enriched) cells cultured in the presence of Con A and enhanced the activation of nylon adherent (B-enriched) cells by LPS . The proliferative response of thymic lymphocytes to Con A was drastically inhibited (70-95% by PGE2 10(-8)-10(-6) M . In cultures of purified bone marrow lymphocytes, PGE2 induced a dose-dependent inhibition of either LPS- or DXS-induced activation reaching a 30-40% inhibition at a PGE2 concentration of 10(-6) M . In the spleen, treatment of cells with anti-T sera and complement resulted in abrogation of PG-induced enhancement . Nevertheless, no inhibition of B-cell mitogenesis was observed in the presence of 10(-6) PGE2 . From these results, it can be concluded that a different sensitivity of the proliferative response of lymphoid cells to exogenous PGs exists, depending on the subset (T or B) affected and/or the organ used as a source of these cells. J Pediatr, 1982 Apr, 100(4), 563 - 7 Immunologic factors in human milk during the first year of lactation; Goldman AS et al.; The effects of the duration of lactation upon lactoferrin, lysozyme, total IgA, SIgA, SIgA antibodies to Escherichia coli somatic antigens and leukocytes in human milk were investigated . Longitudinal and cross-sectional studies were performed with milk collected from women 20 to 35 years of age during te first year of lactation . Collection and storage conditions and immunologic analyses were controlled to minimize confounding variables . The concentrations of lactoferrin, total IgA, and leukocytes and the uptake of 3H-thymidine by phytohemagglutinin-stimulated lymphocytes fell during the first several weeks of lactation; afterward, the levels of lactoferrin and IgA stabilized . Approximately 90% of total IgA in human milk during the year was SIgA . Secretory IgA antibody titers to E . coli increased in some individuals studied longitudinally suggesting that the enteromammary gland pathway of SIgA antibody production was active after several weeks of lactation . Moreover, the concentrations of lysozyme, after falling to a nadir of 20 to 30 micrograms/ml at 2 to 4 weeks, rose to 200 to 300 micrograms/ml by six months and remained elevated . The immunologic system in human milk undergoes remarkable changes which may represent adaptations for the recipient infant. Gan To Kagaku Ryoho, 1982 Apr, 9(4), 694 - 701 {Phase II study of CIS-diamminedichloroplatinum (II) by collaborative study}; Kato T; Since the discovery of platinum compounds by Rosenberg, et al . in 1965 on the mitosis inhibitory action against Escherichia coli, the anticancer action of cis-diamminedichloroplatinum (II) (cisplatin) one of the platinum compounds, has called attention, and recently clinical trials of this compounds have been actively performed in the treatment of testicular and ovarian malignancies not only in the U.S.A . but also in Japan . We carried out a joint controlled trial of cisplatin (NK-801, Nippon Kayaku Co.) in the treatment of ovarian malignancies . The eligibility of the patients and evaluation of response were assessed according to the general response criteria proposed by Drs . Koyama and Saito . Fifty-seven patients were evaluable for the tumor response . Complete response were 11%, partial response 47%, and overall response rates 58% in ovarian malignancies, respectively . Toxicities were noted in the gastro-intestinal tract . Myelosupression and renal dysfunction were other prominent adverse effects. Gann, 1982 Apr, 73(2), 255 - 63 Antimutagenic effect of isocyanates and related compounds in Escherichia coli; Kawazoe Y et al.; Isocyanates and isothiocyanates have been suggested to inactivate enzymes involved in the metabolic activation of chemical carcinogens and the repair of DNA damage . These compounds decrease the mutability of a tester strain of Escherichia coli B under UV irradiation . This paper deals with the antimutagenicity of acylating agents, including isocyanates and isothiocyanates, and some anti-oxidants which are suspected to be anticarcinogenic . The results can be summarized as follows . (1) The antimutagenic effect observed in the present study operates on UV-induced mutagenesis but not on X-ray-induced mutagenesis . (2) This effect operates only on the wild-type strain, H/r30R, but not on Hs30R deficient in the excision repair system . (3) This effect may function through giving the irradiated cells a greater chance to carry out excision repair by prolonging the lag-period before entry into the S-phase . (4) The carbamoylating ability of isocyanates and isothiocyanates may be responsible for the antimutagenicity, but other type of reactivities may also be involved . These antimutagens also participate in inactivating enzymes relevant to the metabolic activation of mutagens, resulting in a decrease in the frequency of chemically induced mutagenesis. Zentralbl Bakteriol Mikrobiol Hyg {B}, 1982 Apr, 176(1), 63 - 71 Standard-suspension-test: influence of bovine albumin, saline and water of standard hardness on M.E.-values; van Klingeren B; Notwithstanding considerable international agreement upon the principle of the quantitative suspension test differences still exist between the main variants of this test, in particular on the diluent to be used in the test mixture . For this reason the influence of the main variables - i.e . bovine albumin (0.03%), NaCl (physiol . saline) and divalent ions (water of standard hardness) - on the activity of the different classes of disinfectants have been investigated . Using so-called borderline concentrations only a small protein effect of 0.03% bovine albumin was found with a phenolic product, quaternary ammonium compounds and aldehydes . A substantial protein effect was observed only with chlorine compounds . The chlorine consumption in the present Dutch Standard Suspension Test is approx . 20 ppm . In tests without a protein load as well as in the presence of 0.03% bovine albumin M.E.-values using saline as the diluent were usually higher than in tests using water of standard hardness (WSH) or distilled water (DW) . M.E.-values of quaternary ammonium compounds in WSH were significantly lower than those in DW, presumably due to the antagonizing effect of divalent cations (Mg++ and Ca++) . The different views regarding the aim of quantitative suspension tests are discussed . In The Netherlands the opinion prevails that these tests should have at least some relationship with practical conditions . The use of water of standard hardness as a diluent and the incorporation of a slight protein challenge are in agreement with this standpoint. Immunobiology, 1982 Apr, 161(3-4), 345 - 51 Membrane stimulation and intracellular killing by monocytes; Leijh PC et al.; The present paper reviews our recent studies concerning the intracellular killing of bacteria by human monocytes and compares the results with those found for circulating granulocytes . The findings show that optimal intracellular killing of catalase-positive and catalase-negative bacteria is only reached during interaction of the Fc part of IgG and complement component C3b with their specific receptors in the membrane of the phagocytes . Similar stimulation was found during interaction of plant lectins and their specific sugar moieties in the cell membrane . The studies permit the general conclusion that optimal intracellular killing of micro-organisms requires membrane stimulation, which occurs after triggering of certain receptors. Proc Natl Acad Sci U S A, 1982 Apr, 79(8), 2523 - 7 A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos; Conaway RC et al.; Preparations of DNA polymerase alpha from early embryos of Drosophila melanogaster catalyze the ATP-dependent synthesis of DNA with single-stranded M13 DNA or poly(dT) templates . In the case of M13 DNA, GTP, but not UTP or CTP, can replace ATP . The reaction is completely dependent on added template and is not inhibited by alpha-amanitin . Alkaline hydrolysis of the product synthesized in the presence of {alpha-32P}dATP and poly(dT) generates 32P-labeled 3'(2') adenylate, showing that a covalent ribo-deoxynucleotide linkage is formed . Furthermore, incorporation of ribonucleotides occurs at the 5' end of the newly synthesized polynucleotide chain . These findings are consistent with the hypothesis that a ribo-oligonucleotide primer is synthesized by primase action and subsequently elongated by DNA polymerase . Under the appropriate conditions, DNA polymerase I from Escherichia coli can elongate primers formed by primase in the presence of ATP and poly(dT) . Primase activity copurifies with DNA polymerase alpha and may be part of the multisubunit polymerase molecule. J Bacteriol, 1982 Apr, 150(1), 132 - 40 Transgalactosylation activity of ebg beta-galactosidase synthesizes allolactose from lactose; Hall BG; ebg enzyme, the second beta-galactosidase of Escherichia coli, does not normally convert lactose into an inducer of the lac operon . We previously reported the existence of a mutant ebg enzyme that does make such an inducer in vivo (Rolseth et al., J . Bacteriol . 142:1036-1039, 1980) . Here I report that the mutant enzyme makes inducer from lactose in vitro and that the inducer is allolactose . Allolactose is made from lactose by direct transgalactosylation at a rate that is 8 to 10% of the rate of lactose hydrolysis . Galactose is also transferred to glucose free in solution, but the resulting indirect transgalactosylation products are not allolactose or lactose . The ability to efficiently synthesize allolactose is a general property of class IV mutant ebg enzymes, whereas other classes of ebg mutant enzymes are unable to synthesize allolactose efficiently . The evolutionary implications of this new function are discussed. Eur J Clin Microbiol, 1982 Apr, 1(2), 107 - 11 Serological subtypes of Escherichia coli colonization factor antigen II; Svennerholm AM et al.; Many of the enterotoxin-producing Escherichia coli strains causing diarrhea in humans possess one of two types of pili adhesins, the colonization factor antigens CFA/I and CFA/II . We identified two subtypes of CFA/II in two CFA/II bearing Escherichia coli strains (serotypes O6:H16 and O78:H12) by means of immunodiffusion analyses of heated extracts and hemagglutination inhibition tests . In addition to a shared antigenic structure on CFA/II from both strains, a specific antigenic determinant could be demonstrated on CFA/II from the O6:H16 strain . A protective effect against intestinal loop challenge with homologous bacteria in rabbits was noted for antibodies to both the shared and the subtype-specific structures of CFA/II. J Gen Microbiol, 1982 Apr, 128 (Pt 4), 689 - 92 Tetracycline resistance determinants from groups A to D vary in their ability to confer decreased accumulation of tetracycline derivatives by Escherichia coli; Chopra I et al.; The ability of four genetically distinct plasmid-located tetracycline resistance determinants (TetA, B, C and D) to confer decreased accumulation of tetracycline and some of its analogues by Escherichia coli K12 was examined . Accumulation of oxytetracycline, tetracycline, demethylchlorotetracycline, 6-demethyl-6-deoxy-5-hydroxy-6-methylene-tetracycline, chlorotetracycline, doxycycline and 6-demethyl-6-deoxytetracycline was examined by fluorescence spectroscopy . The determinants varied in their ability to promote decreased accumulation of tetracyclines, defined as an R+/R- fluorescence ratio of less than 0.85 . Plasmid pIP7 (TetA) caused reduced accumulation of only oxytetracycline, tetracycline and chlorotetracycline, but plasmid pDU301 (TetB) promoted reduced accumulation of all the compounds tested except 6-demethyl-6-deoxytetracycline . The TetC determinant of pBR322 caused decreased uptake of five derivatives, but not doxycycline or 6-demethyl-6-deoxytetracycline . Plasmid RA1 (TetD) encoded reduced accumulation of oxytetracycline, tetracycline, 6-demethyl-6-deoxy-5-hydroxy-6-methylenetetracycline and chlorotetracycline . In general, the resistance determinants were more efficient in promoting decreased accumulation of hydrophilic tetracyclines . These accumulation studies provide a satisfactory method for the phenotypic identification of Tet resistance determinants. J Gen Microbiol, 1982 Apr, 128 (Pt 4), 705 - 12 Cyclic AMP and cyclic GMP control of synthesis of constitutive enzymes in Escherichia coli; Calcott PH; Escherichia coli was grown in chemostat culture under glycerol-limited and ammonium-limited conditions at growth rates between 0.1 and 0.5 h-1 . At steady state, the concentrations of cyclic AMP and cyclic GMP and the activities of four constitutive enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, NADH oxidase and cyclic phosphodiesterase) were determined in the organism . Addition of exogenous cyclic AMP, cyclic GMP or phencyclidine perturbed the steady state and caused inhibition or stimulation of synthesis of phosphodiesterase and isocitrate dehydrogenase . A novel hypothesis is proposed to account for the ability of bacteria to regulate the synthesis of constitutive enzymes with cyclic nucleotides and possibly other small molecules. Gene, 1982 Apr, 18(1), 77 - 85 Effect of lexA and ssb genes, present on a uvrA recombinant plasmid, on the UV survival of Escherichia coli K-12; Brandsma JA et al.; The recombinant plasmid pJA01 contains, besides the uvrA gene, the genes lexA, ubiA and ssb . This plasmid does not fully complement a uvrA mutation in a Rec+ background . Plasmids which contain the uvrA and ssg genes, but not the lexA gene, show a higher but still only partial complementation . Full complementation achieved when the ssb gene us inactivated by insertion of Tn5 . Furthermore, it appears that the presence of the ssb gene on a multicopy plasmid sensitizes wild-type cells to UV light . The effect of Ssb (single-strand DNA binding protein) overproduction on UV survival is discussed. Gene, 1982 Apr, 18(1), 21 - 8 The site-specific deletion in plasmid pBR322; Garaev MM et al.; The formation of a deletion derivative of plasmid pBR322, designated pBR322 delta 1, was observed during cloning of various eukaryotic DNAs, when the BamHI site of the plasmid vector was used for construction of the recombinant molecules . The restriction analysis of six independently isolated pBR322 delta 1 plasmids allowed establishment of their complete identity . Similar deletion derivatives were also formed as a result of transformation of Escherichia coli cells by the linear form of vector pBR322 produced by BamHI cleavage, but not by SalI or HindIII . The endpoints of the deletion in one of the pBR322 delta 1 plasmids occurred at positions 375 and 16666 bp from the EcoRI site, as determined by sequence analysis . Formation of pBR322 delta 1 is most probably due to site-specific recombination between the sequence in the 1666-1670 bp region and the BamHI end of the linear pBR322 molecule . THe deletion was not controlled by the recA system of the host bacteria. Proc Natl Acad Sci U S A, 1982 Apr, 79(7), 2240 - 4 Cloning of a yeast gene coding for arginine-specific carbamoyl-phosphate synthetase; Lusty CJ et al.; Several recombinant plasmids containing cpaII, the gene that encodes the large subunit of yeast arginine-specific carbamoyl-phosphate synthetase {carbamoyl-phosphate synthetase (glutamine-hydrolyzing), carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.3.5}, have been isolated . The plasmids were selected by transformation of a yeast strain with a mutation in the structural gene of the large subunit of carbamoyl-phosphate synthetase . By using a recombinant pool with inserts of yeast nuclear DNA of 5-20 kilobase pairs, we obtained 13 transformants . Of five transformants studied, three have been found to have stable plasmid inserts . These plasmids could be amplified in Escherichia coli and transferred back into the yeast carbamoyl-phosphate synthetase-deficient strains with concomitant complementation of the nuclear mutation . Plasmids pJL2/T1 and pJL2/T5 contain identical nuclear DNA inserts of 5.9 kilobase pairs . Although the insert of plasmid pJL2/T3 is also 5.9 kilobase pairs long, the sequence overlap with pJL2/T1 and pJL2/T5 is only 4.5 kilobase pairs long . The T3 insert has an orientation in the vector opposite to that of the T1 and T5 inserts . The recombinant plasmids with the yeast cpaII gene fail to cross-hybridize with a cloned fragment of E . coli DNA containing the carA and carB genes for the bacterial carbamoyl-phosphate synthetase. Proc Natl Acad Sci U S A, 1982 Apr, 79(7), 2152 - 6 Construction of plasmids for expression of Rous sarcoma virus transforming protein, p60src, in Escherichia coli; Gilmer TM et al.; We have constructed plasmids that direct the synthesis of the Rous sarcoma virus transforming gene (src) product (p60src) in Escherichia coli . A 203-base-pair lac promoter-operator DNA encoding the first eight amino acids of beta-galactosidase was ligated to the 5' end of the src gene from the Prague A strain of Rous sarcoma virus (PrA-RSV) which had been cloned in pBR325 . Antiserum, from a tumor-bearing rabbit, directed against pp60src was used to screen bacteria containing the recombinant plasmid for a protein of approximately 60,000 daltons, and several colonies producing a protein immunologically related to pp60src were detected . Partial proteolytic cleavage analysis revealed that the src-related protein produced in bacteria is structurally similar to pp60src immunoprecipitated from PrA-RSV-infected chicken cells . Partially purified src protein from E . coli can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase . Tryptic phosphopeptide analysis demonstrated that the catalytic subunit phosphorylated a serine-containing tryptic peptide in the bacterial src protein that comigrated with the phosphoserine-containing tryptic peptide of pp60src immunoprecipitated from 32P-labeled PrA-RSV-infected chicken cells. Proc Natl Acad Sci U S A, 1982 Apr, 79(8), 2632 - 5 Inverted repeats of Tn5 are transposable elements; Berg DE et al.; Experiments presented here show that each of the 1.5-kilobase inverted repeats of the kanamycin-resistance transposon Tn5 is transposable; we designate them IS50-L (left) and IS50-R (right) . By DNA sequence analyses, IS50 is 1533 base pairs (bp) long and generates 9-bp direct repeats of target sequences . The ends of IS50 comprise a hyphenated 8-of-9-bp inverted repeat and are not used with equal efficiency; the outside ends are more active than the inside ends, suggesting that a strong transposase recognition site at the outside ends, suggesting that a strong transposase recognition site at the outside end extends beyond the 8 bp common to both ends. Proc Natl Acad Sci U S A, 1982 Apr, 79(8), 2427 - 31 Sequence of galR gene indicates a common evolutionary origin of lac and gal repressor in Escherichia coli; von Wilcken-Bergmann B et al.; The nucleotide sequence of the galR gene of Escherichia coli, which codes for galactose repressor, has been determined . The subunits of gal repressor are predicted to consist of 343 residues, including the NH2-terminal methionine . Twenty-six of the predicted NH2-terminal 55 residues of gal repressor are identical to the NH2-terminal residues of lac repressor . Additional homologies appear between residues 165 and 200, between residues 235 and 255, and around residue 325. J Virol, 1982 Apr, 42(1), 346 - 51 Restriction endonuclease and nucleotide sequence analyses of molecularly cloned unintegrated avian tumor virus DNA: structure of large terminal repeats in circle junctions; Katz RA et al.; Avian tumor virus supercoiled DNA was isolated from infected quail tumor cells and molecularly cloned in pBR322 . Four different recombinant clones denoted pATV-6, pATV-7, pATV-8, and pATV-9 were characterized in detail by restriction endonuclease mapping and by DNA sequencing . The results of these studies indicate that (i) the two large terminal repeats (LTRs) present in PATV-6, are different sizes, (ii) pATV-8 and pATV-9 contain only one LTR, (iii) pATV-7 contains an inversion of 0.6 kilobase in the env gene and a deletion of the U3 region and the src gene, and (iv) the src gene is deleted in pATV-6 and pATV-9 . Circle formation from linear molecules was also examined in several of the clones by DNA sequencing through the circle joint . pATV-6 is an example of one class of circular molecules and contains a partially repeated LTR similar to that reported by Ju and Skalka (Cell 22:379-386, 1980) . A second class of circles was exemplified by pATV-8 and pATV-9, which contain a single copy of the LTR with no base changes or deletions . This is in contrast to a class of circles containing a complete double LTR structure described by Swanstrom et al . (Proc . Natl . Acad . Sci . U.S.A . 78:124-128, 1981) and suggests that circles containing a single intact LTR may be formed by a homologous recombinational event in which an entire LTR or complementary regions from both LTRs are removed from the linear DNA molecule during circularization. Biokhimiia, 1982 Apr, 47(4), 619 - 25 {Isolation, purification and properties of restriction endonuclease EcoRII}; Kosykh VG et al.; The restriction endonuclease Eco RII was isolated and purified to homogeneity . The isolation procedure involved the use of the E . coli strain B834/pSK323, containing the recombinant plasmide pSK323 which provides for the oversynthesis of Eco RII enzymes . Data from gel filtration and Na-DS electrophoresis suggest that the restriction endonuclease Eco RII is a protein made up of two subunits, each with molecular weight of 44 000. Pediatr Res, 1982 Apr, 16(4 Pt 1), 329 - 30 Rotavirus viral RNA electrophoresis in hospitalized infants with diarrhea in Santiago, Chile; Avendano LF et al.; Viral RNA electrophoresis technique was used to detect rotavirus in 226 children under 2 years of age with acute diarrhea, admitted to the Roberto del Rio Hospital in Santiago, Chile, during the period of June 1979 through May 1980 . A group of 50 children included in the aforementioned sample, admitted in winter, was compared with a control group of 25 infants without digestive pathology . In these groups, rotavirus was detected in 20 out of 50 children with diarrhea (40%) but not in the controls (0%) . A positive diagnosis of rotavirus was found in 66 out of the total of 226 patients (29.2%); its monthly distribution ranged between a maximum of 83.3% (June) and a minimum of 11.1% (October). J Med Chem, 1982 Apr, 25(4), 382 - 6 Use of adenine nucleotide derivatives to assess the potential of exo-active-site-directed reagents as species- or isozyme-specific enzyme inactivators . 4 . Interactions of adenosine 5'-triphosphate derivatives with adenylate kinases from Escherichia coli and rat tissues; Hampton A et al.; Adenosine 5'-triphosphate (ATP) derivatives of the types N6-R-ATP {R = (CH2)nHNCOCH2I, (CH2)nNHCO-(CH2)mHNCOCH2I, or (CH2)nCON(Me)(CH2)mN(Me)CO(CH2)nNHCOCH2I}, N6-Me-N6-R-ATP {R = (CH2)nN-(Me)CO(CH2)mNHCOCH2I}, and 8-R-ATP {R = NH(CH2)nNHCOCH2I} with 5--19 spacer atoms between N6 or C-8 and iodine have been evaluated as substrates, reversible inhibitors, and inactivators of adenylate kinase (AK) . With Escherichia coli AK, the derivatives were noncompetitive inhibitors, Ki = 4.7--7.3 mM, with little affinity for the ATP site, and N6-(CH2)nNHCOCH2{-ATP (n = 5 or 6) effected progressive inhibitions that were not ATP site directed . With rat muscle AK (M-AK), some compounds had slight affinity for the ATP site as evidenced by weak substrate activity with as much as 8 spacer atoms, but all compounds tested were weak noncompetitive inhibitors; Ki = 6--12 mM vs . ATP . The ATP derivatives, notably N6-(CH2)8NHCOCH2I-ATP, mediated a progressive inhibition of M-AK, which was abolished by substitution of hydrogen for the iodine and thus presumably involves alkylation of the enzyme . The inhibition appeared not to be ATP site directed because kinetic analysis indicated a random bimolecular enzyme-inhibitor reaction and because N6-(CH2)8NHCOCH2I-AMP and its adenosine counterpart, which have relatively low affinity for the ATP site, were more effective than N6-(CH2)8NHCOCH2I-ATP . The ATP derivatives were substrates (KM = 0.4--1.6 mM) and/or competitive inhibitors (Ki = 0.3--6.2 mM) vs . ATP of rat isozymes AK II or III . Exposure of AK II or III for 6 h, 22 degrees C, at pH 7.6 to 10 mM levels of the 1:1 Mg complexes of 25 of the ATP derivatives led in no case to progressive enzyme inhibition, suggesting the absence near the ATP sites of nucleophilic groups suitably positioned for alkylation. J Bacteriol, 1982 Apr, 150(1), 89 - 99 Promoter-distal region of the tra operon of F-like sex factor R100 in Escherichia coli K-12; Hansen BS et al.; The distal region of the tra (transfer) operon of F-like plasmid R100 was investigated, using small plasmids derived from R100, primarily the plasmid pSM6 . The transposon Tn5 (which confers kanamycin resistance) was inserted at different positions into pSM6, and the transposition derivatives were tested for ability to complement defined tra mutants of the F sex factor . Thus, the tra genes traH, G, T, and D were localized on the plasmid R100 . A restriction map of pSM6 was constructed, and the locations of the insertions were mapped, using restriction endonuclease digestion of the plasmid DNA and exploiting the fact that several restriction sites are localized in the inverted repeat regions of the transposon . The gene products of the genes traG, S, T, and D were identified by radioactive labeling of proteins synthesized in minicells carrying the various insertion plasmids followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The presence of another transfer gene, traI, was inferred from these data . Another protein, the r2-A protein, was also identified, and its gene was mapped . On the basis of the data, a best-fit physical map of this region of the tra operon of R100 was constructed . The results confirmed that the general order and size of the distal transfer genes is as in the F sex factor, but showed that differences exist with respect to all of the gene products . The significance of these differences are discussed in the light of the genetic and physical homology (Manning et al., J . Bacteriol . 150:76-88) of the transfer regions. J Bacteriol, 1982 Apr, 150(1), 76 - 88 Analysis of the promoter-distal region of the tra operon of the F sex factor of Escherichia coli K-12 encoded by EcoRI restriction fragments f17, f19, and f2; Manning PA et al.; The promoter-distal region of the tra operon of the F sex factor Escherichia coli K-12 was analyzed, using the chimeric plasmid pRS31, which contains the F EcoRI restriction fragments f17, f19, and f2 cloned into the EcoRI site of pSC101 . A series of deletion plasmids of pRS31, extending increasing distances from a site in f17 through f19 and ending in f2, were isolated . These plasmids were examined by heteroduplex analysis with the parent DNA, and a restriction map of this region of DNA was constructed . A series of Tn5 insertion derivatives of pRS31 were also isolated and mapped, using both heteroduplex analysis and restriction mapping . Both the insertion and deletion mutants were tested in minicells for the synthesis of radioactively labeled proteins . This allowed the identification of the individual gene products and mapping of the genes . The result is a saturated physical map of this region of DNA from fragment f17 through to the IS3 insertion sequence near the promoter-distal end of f2. J Bacteriol, 1982 Apr, 150(1), 389 - 94 DNA homology of the promoter-distal regions of the tra operons of sex factors F and R100 in Escherichia coli K-12; Manning PA et al.; The promoter-distal regions of the tra operons of F and R100-1 were analyzed by heteroduplex analysis, and the regions of nonhomology were identified . A common EcoRI restriction site was shown to be present, and this has allowed the physical maps to be aligned. J Bacteriol, 1982 Apr, 150(1), 36 - 45 Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane; Kranz RG et al.; Isoelectric focusing (IEF), agarose electrophoresis, and crossed immunoelectrophoresis (CIE) were used to resolve the heme-containing proteins of the Escherichia coli cytoplasmic membrane after solubilization by Triton X-100 . Two bands in IEF stained for heme with pI values of 4.7 and 5.3 . One of the bands, with an isoelectric point of pH 5.3, was present only when the cells were grown to late log or stationary phase and possessed N,N,N,'N'-tetramethyl-p-phenylene-diamine (TMPD) oxidase activity . The pI 4.7 band was present in cells harvested in both mid-log and stationary phases . Agarose electrophoresis, using larger samples, revealed the same two components apparent by IEF, and, in addition, a third component . The heme-containing fractions were extracted after agarose electrophoresis and subjected to further study . The component which was present in cells grown to stationary phase contained hemes b, a1, and d . The other two fractions contained only b heme . One of these corresponded to the component with pI 4.7 in IEF and had catalase activity . Antisera were raised against Triton X-100-solubilized cytoplasmic membranes and against the focused TMPD oxidase complex . With these anti-sera, CIE in the presence of Triton X-100 revealed four precipitin complexes containing heme . Three of these corresponded to the components identified by IEF and agarose electrophoresis . We demonstrate that the combined use of IEF and CIE is valuable for analysis of membrane proteins . In particular, this work represents a substantial initial step toward a structural elucidation of the E . coli aerobic respiratory chain. J Bacteriol, 1982 Apr, 150(1), 286 - 92 dnaC-dependent reconstitution of replication forks in Escherichia coli lysates; Nusslein-Crystalla V et al.; Lysates of Escherichia coli exhibit a DNA-synthesizing activity that depends on the presence of replication forks and of replication proteins . Replicative activity was reconstituted in vitro by mixing lysates prepared from temperature-sensitive dnaB mutants with wild-type dnaB protein . Lysates of double mutants deficient in both dnaB and dnaC genes could only be complemented by the addition of both dnaB and dnaC proteins, whereas lysates deficient in dnaC protein did not require the addition of any exogenous factor . This shows that the replication machinery, once it is running along the chromosome, is independent of dnaC protein, dnaC activity, however, is required for the replacement of defective dnaB protein at running replication forks. Proc Natl Acad Sci U S A, 1982 Apr, 79(7), 2236 - 9 Mutant lambda phage repressor with a specific defect in its positive control function; Guarente L et al.; The lambda phage repressor is both a positive and a negative regulator of gene transcription . We describe a mutant lambda phage repressor that has specifically lost its activator function . The mutant binds to the lambda phage operator sites and represses the lambda phage promoters PR and PL . However, it fails to stimulate transcription from the promoter PRM . The mutation lies in that portion of repressor--namely, the amino-terminal domain--that has been shown {Sauer, R . T., Pabo, C . O., Meyer, B . J., Ptashne, M . & Backman, K . C . (1979) Nature (London) 279, 396-400} to mediate stimulation of PRM . We suggest that the mutation has altered that region of repressor which, in the wild-type, contacts RNA polymerase to activate transcription from PRM. J Biochem (Tokyo), 1982 Apr, 91(4), 1155 - 62 ColE1 vectors for direct selection of cells carrying a hybrid plasmid; Ozaki LS et al.; pKY2289, a ColE1::Tn3 derivative, useful for direct selection of cells carrying a hybrid plasmid, was deleted of its mobility functions and parts of the transposon including the left inverted repeat . This deletion mutant, named pKY2700 expresses low levels of colicin E1 synthesis even in recA cells . A nitrosoguanidine mutant of pKY2289 which shows a high level of constitutive colicin E1 synthesis was also deleted of the same sequences as pKY2700 . The second plasmid, named pKY2800, has the same molecular weight (3.8 megadaltons) and almost the same structure as pKY2700, but produces colicin E1 at much higher levels and has a copy number 10 times higher . pKY2800 requires no colicin E1 induction for the direct selection of hybrid clones, while pKY2700 requires mitonmycin C at a concentration of 10 ng per ml . These two colE1 derivatives are useful as safe and convenient vectors for cloning DNA fragments at the cleavage sites of EcoRI, XmaI, and SstII of plasmid. Cell, 1982 Apr, 28(4), 747 - 56 Visualization of recA protein and its association with DNA: a priming effect of single-strand-binding protein; Flory J et al.; A stoichiometric interaction of RecA protein with single-stranded DNA promotes homologous pairing of the single strand with duplex DNA and subsequent polar formation of a heteroduplex joint . Escherichia coli single-strand-binding (SSB) protein augments these reactions . Electron microscopic observations suggest structural bases for these interactions . Without triphosphates or DNA, RecA protein forms short linear filaments . With added circular single-stranded DNA, it forms extended circular filaments as well as collapsed and aggregated complexes of protein and DNA . The extended circular filaments are stiff and regular in appearance, contrasting with the convoluted structure formed by SSB protein and single-stranded DNA . Together, these two proteins form mixed filaments, which mostly resemble the extended structures containing RecA protein; moreover, SSB protein accelerates formation of extended filaments more than 50-fold, increasing the yield of these structures at the expense of heterogeneous aggregates . Other observations further define the interactions of RecA protein with partially single-stranded DNA, and the effects of ATP gamma S on the tendency of RecA protein to form polymeric structures even in the absence of DNA. Acta Pathol Microbiol Immunol Scand {B}, 1982 Apr, 90(2), 113 - 8 Effect of a homologous beta-interferon preparation on degradation of Escherichia coli and on lysosomal enzyme activities in mouse peritoneal macrophages; Rollag H et al.; The degradation of 32P-labelled E . coli and the activity of three lysosomal enzymes, acid phosphatase, cathepsin D and beta-glucuronidase, in mouse peritoneal macrophages (MPM) were tested after cultivation of the cells for 24 or 48 hours with 10(1) - 10(4.7) U per ml of a homologous beta-interferon preparation (IFN-beta) . Low to moderate concentrations of IFN-beta did not influence bacterial degradation in MPM . However, a reduction in bacterial degradation by 20 per cent or more was seen when the MPM were pre-treated with 10(4.7) U per ml for 24 hours or 10(3) U per ml of IFN-beta for 48 hours . Cultivation of the MPM with 10(2) U per ml of IFN-beta suppressed the activities of the lysosomal enzymes, provided that the cells were treated for 48 hours . The beta--glucuronidase activity was significantly reduced also after 24 hours . Increased release of beta-glucuronidase from MPM to the medium during cultivation with 10(2) U per ml of IFN-beta was also observed . Specific anti-IFN-beta globulin abolished the suppression by IFN-beta on the lysosomal enzyme activities . A human IFN-alpha preparation did not influence bacterial degradation or lysosomal enzyme activities in MPM. Infect Immun, 1982 Apr, 36(1), 215 - 20 Comparison of heat-labile enterotoxins from porcine and human strains of Escherichia coli; Geary SJ et al.; Heat-labile enterotoxins (LTs) from porcine EWD299) and human (H74-114) enterotoxigenic strains of Escherichia coli were isolated by a single-step galactose elution procedure . Although both strains had similar amounts of LT in their whole-cell lysates, H74-114 yielded a smaller quantity of purified LT than did EWD299 . Immunodiffusion studies with specific antisera revealed that although the two LTs shared major antigenic determinants each also had unique antigens . Both also had shared and unique specificities in comparison with the cholera enterotoxin (choleragen) . Differences also exist in the apparent molecular weights of their B-subunit oligomers (coligenoid) as well as in the B-subunit monomers . The monomer molecular weights are 11,500 for EWD299 porcine LT and 12,700 for H74-114 human LT . The results suggest that either this isolated human LT has a tetrameric coligenoid or it moves differently in sodium dodecyl sulfate gels for other reasons . The A-subunits of both LTs were similar in size (28,000 daltons), and both LTs were activated by mild proteolytic processing . Amino acid analysis showed a threefold increase in the level of tryptophan and two- and fourfold decreases in the levels of glutamic acid and methionine, respectively, in H74-114 LT compared with EWD299 LT . These structural and antigenic differences may prove to be significant in immunoprophylaxis of the cholera-coli family of enterotoxins . Further studies to define the extent of evolutionary drift of these toxins are needed. J Bacteriol, 1982 Apr, 150(1), 168 - 79 relA-dependent RNA polymerase activity in Escherichia coli; Ryals J et al.; Parameters relating to RNA synthesis were measured after a temperature shift from 30 to 42 degrees C, in a relA+ and relA- isogenic pair of Escherichia coli strains containing a temperature-sensitive valyl tRNA synthetase . The following results were obtained: (i) the rRNA chain growth rate increased 2-fold in both strains; (ii) newly synthesized rRNA became unstable in both strains; (iii) the stable RNA gene activity (rRNA and tRNA, measured as stable RNA synthesis rate relative to the total instantaneous rate of RNA synthesis) decreased 1.7-fold in the relA+ strain and increased 1.9-fold in the relA mutant; and (iv) the RNA polymerase activity (measured by the percentage of total RNA polymerase enzyme active in transcription an any instant) decreased from 20 to 3.6% in the relA+ strain and remained unchanged (or increased at most to 22%) in the relA mutant . It is suggested that both rRNA gene activity and the RNA polymerase activity depend on the intracellular concentration of guanosine tetraphosphate, whereas the altered chain elongation rate and stability of rRNA are temperature or amino acid starvation effects, respectively, without involvement of relA function. Cancer Res, 1982 Apr, 42(4), 1312 - 6 Stimulation of natural killer cell activity and inhibition of proliferation of various leukemic cells by purified human leukocyte interferon subtypes; Lee SH et al.; One of several human leukocyte interferon subtypes A (LeIF-A), obtained in purified form from a gene cloned in Escherichia coli, stimulated human peripheral blood natural killer cell activity, whereas another human leukocyte interferon subtype D (LeIF-D) had no effect with the use of K562 as target cells . With Daudi as target cells, both LeIF-A and LeIF-D stimulated natural killer cell activity . A hybrid human leukocyte interferon, NH2-terminal 61 amino acids and COOH-terminal 104 residues of LeIF-A and LeIF-D, respectively (LeIF-AD) showed greater stimulation than did LeIF-A, but the stimulation did not exceed that of natural buffy coat interferon . A mixture of equal antiviral units of LeIF-A and LeIF-D was no more effective than was LeIF-A alone . The cloned interferon subtypes showed differential effects on the proliferation of three human leukemic cell lines: Daudi (B-cell lymphoblastoid leukemia); BALL 1 (B-cell acute lymphoblastic leukemia); CCRF-HSB-2 (T-cell acute lymphoblastoid leukemia) . Growth of Daudi cells was generally most sensitive to all the interferons tested, LeIF-A, -D, -AD, and a buffy coat preparation; no viable cells remained after 120-hr exposure to 1000-unit/ml doses of the interferons . BALL 1 was relatively resistant to the interferon subtypes tested including LeIF-AD, but this cell lines was very sensitive to a preparation of natural buffy coat interferon . CCRF-HSB-2 showed some sensitivity to all the interferons with greatest sensitivity to LeIF-A (10% of the viable cells were detected after 1000 units/ml exposure for 120 hr) . In contrast to the leukemic cell lines tested, human amnion cells (WISH) and the human erythroid leukemia, K562, were resistant to the antiproliferative activity of the interferons. J Bacteriol, 1982 Apr, 150(1), 70 - 5 Autoregulation of the tyrR gene; Camakaris H et al.; Strains of Escherichia coli K-12 in which the transcription of lacZ is initiated from the tyrR promoter have been constructed by use of the Mu d (Apr lac) phage of Casadaban and Cohen (Proc . Natl . Acad . Sci . U.S.A . 76:4530-4533, 1979) . These strains have been used to examine the regulation of expression from the tyrR promoter, with the synthesis of beta-galactosidase used as an index of expression . The specific activity of beta-galactosidase fell to 51% upon introduction of lambda (Tn10) tyrR+; to 39% upon introduction of F123, an F-prime carrying tyrR+; to 29% upon introduction of pMU309, a derivative of the plasmid RP4 carrying tyrR+; and to 13.6% upon introduction of pMU352, a derivative of the multicopy plasmid pBR322 carrying tyrR+ . These results indicate that the tyrR gene product interacts with its own promoter-operator region, decreasing synthesis of beta galactosidase in the tyrR::Mu d (Apr lac) strains . The increasing extent of repression of beta-galactosidase synthesis with increasing tyrR+ gene dosage was accompanied by increasing repression of the synthesis of tyrosine- and phenylalanine-repressible 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetases . The interaction of the repressor with tyrRo appears unusual in the sense that aporepressor alone is probably one of the repressing species . The levels of beta-galactosidase synthesized in the tyrR::Mu d (Apr lac) strains indicate that tyrR has a relatively efficient promoter, the maximum levels representing on the order of a relatively efficient promoter, the maximum levels representing on the order of 1,000 monomers of beta-galactosidase per cell in the tyrR strain and about 500 monomers in the tyrR+ haploid strain. J Bacteriol, 1982 Apr, 150(1), 202 - 13 Complex glnA-glnL-glnG operon of Escherichia coli; Pahel G et al.; The glnG gene product is both a positive regulator and a negative regulator of the expression of glnA, the structural gene for glutamine synthetase, as well as a positive regulator of the expression of a number of genes whose products are involved in the uptake and degradation of nitrogen-containing compounds . The regulation of beta-galactosidase in various strains containing a Mu d1 (lac bla) insertion within glnG leads to the following conclusions regarding the expression of this gene: (i) like the synthesis of glutamine synthetase, the synthesis of the glnG product is regulated in response to the nitrogen source; (ii) high-level expression of glnG under nitrogen-limiting growth conditions depends on transcription initiated at the glnA promoter; and (iii) there is a second, glnA-distal promoter for glnG, whose activity is negatively controlled by the glnG product . Thus, the glnG product regulates the synthesis of the glnG product at two distinct promoters (positively and negatively at the glnA promoter and negatively at the glnA-distal promoter) . In addition, a high level of glnG product, corresponding to the level produced by initiation of transcription at the glnA promoter under nitrogen-limiting conditions, is necessary for activation of histidase synthesis . The lower level of glnG product originating from transcription initiated at the glnA-distal promoter is not sufficient to activate histidase synthesis, but is sufficient to activate fully and to repress glnA expression. J Bacteriol, 1982 Apr, 150(1), 156 - 60 Another gene affecting sexual expression of Escherichia coli; Lerner TJ et al.; We have examined the relationship between two chromosomal mutations of Escherichia coli K-12, fexA (0 min) and fexB (85 min), in regulating expression of the F sex factor . Together, fexA and fexB exert a pleiotropic effect on the expression of the F tra genes . F pilus synthesis, conjugal donor activity, and surface exclusion activity are all inhibited in the fexA fexB mutant . Either fex mutation alone is cryptic. Biochemistry, 1982 Mar 30, 21(7), 1471 - 8 Formamide-induced dissociation and inactivation of Escherichia coli alkaline phosphatase . Metal-dependent reassociation and restoration of activity from isolated subunits; Falk MC et al.; Alkaline phosphatase from Escherichia coli has been reversibly dissociated by treatment with low concentrations of formamide . The monomer retains the capacity to bind metals and to regenerate catalytically active dimer that is identical with the native dimeric enzyme . The rate and extent of dissociation of dimer to monomer depend upon pH, ionic strength, temperature, formamide concentration, and enzyme-bound metal . Under appropriate experimental conditions, reassociation can be greatly slowed, allowing the properties of the monomer to be examined in solution . The formamide-induced apo monomer has a conformation distinct from that of the dimer and zinc- or cobalt-containing monomers . The monomer tightly binds 1 mol of zinc or cobalt in a metal-binding site altered from those of the dimer but is catalytically inactive . pH, ionic strength, and formamide concentration all influence reassociation . Hydrophobic forces are implicated as important in subunit interactions . The effect of metal content on the dissociation--reassociation process underscores the essential role that metals play in maintaining enzyme tertiary structure and reveals a new role in stabilizing the quaternary structure. Biochim Biophys Acta, 1982 Mar 29, 696(3), 231 - 8 A transcriptionally active plasmid-protein complex isolated from Escherichia coli; Wu FY et al.; A stable transcriptionally active plasmid-protein complex has been isolated in high yield from Escherichia coli containing the thermally-inducible plasmid pKN 402A . The complexes which have a protein/DNA weight ratio of approx . 1 contain more than 11 polypeptide species . The weight percents of the three known proteins in the complex H1, RNA polymerase and HU, are 23, 23 and 5%, respectively . In vitro RNA synthesis by this complex proceeds for several hours and is inhibited by rifampicin and actinomycin to 33 and 98%, respectively, suggesting that most of the observed nucleotide incorporation is due to elongation of preinitiated RNA chains . Exogenous E . coli RNA polymerase but not exogenous DNA stimulates the in vitro transcription indicating that RNA polymerase is limiting and binds tightly to the plasmid . Stimulation of the in vitro transcription by the addition of exogenous E . coli core polymerase suggests that sigma subunit may be released in the RNA synthesis . This transcriptionally active complex should prove to be useful to study the mechanism of transcription and regulation in vivo. Nucleic Acids Res, 1982 Mar 25, 10(6), 1877 - 94 The preparative synthesis of oligodeoxyribonucleotides using RNA ligase; Hinton DM et al.; The synthesis of nmol quantities of defined sequences of oligodeoxyribonucleotides using T4 RNA ligase has been demonstrated . Reacting using from 18 to 200 nmol of substrates in which a single 2'-deoxyribonucleoside 3',5'-bisphosphate was added to an oligodeoxyribonucleotide resulted in yields from 13 to 95% . When two oligodeoxyribonucleotides were similarly joined using RNA ligase, the yields ranged from 10 to 50% . Although the reactions contained high concentrations of enzyme and were incubated from 5 to 21 days, there was little degradation of either substrates or products . We have also characterized an unusual product which arises when 3'-phosphate terminated oligodeoxyribonucleotides are incubated with RNA ligase and high concentrations of ATP . This product has an adenylyl group linked to the 3'-phosphate by an anhydride bond . The mechanistic and synthetic implications of forming this product are discussed. J Biol Chem, 1982 Mar 25, 257(6), 3110 - 2 13C nuclear magnetic resonance study of the pyruvate dehydrogenase-catalyzed acetylation of dihydrolipoamide; O'Connor TP et al.; The dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex catalyzes a reversible reaction between acetyl-CoA and dihydrolipoamide that results in the formation of S-acetyldihydrolipoamide . We have used 13C nuclear magnetic resonance to investigate this reaction using exogenous forms of dihydrolipoamide in place of the protein-bound substrate . With substrate levels of dihydrolipoamide and enzymatically generated {1-13C}acetyl-CoA, both 6-S-{1-13C}acetyl- and 8-S-{1-13C}acetyldihydrolipoamide were formed in the transacetylation reaction and both species participated in the reverse reaction to yield {1-13C}acetyl-CoA and free dihydrolipoamide . The 8-S-acetyl derivative was the principal product . It is suggested that acetylation of both the 6- and 8-thiols of dihydrolipoamide results as a consequence of intramolecular migration following acetylation at a single site . After longer periods of reaction, some 6,8-S,S-{1-13C}diacetyldihydrolipoamide also accumulated . We have also found that {1-13C}acetyl-CoA reacts slowly with dihydrolipoamide in a nonenzymatic reaction to yield the two monoacetylated and some diacetylated derivative . In the reverse reaction catalyzed by the dihydrolipoyl transacetylase, it was clear that monoacetyl derivatives were depleted much more rapidly than the diacetyl derivatives, although we could not quantitate the change in the low concentration of the diacetyl derivative. Nucleic Acids Res, 1982 Mar 25, 10(6), 2065 - 84 New rapid methods for DNA sequencing based in exonuclease III digestion followed by repair synthesis; Guo LH et al.; We describe improve enzymatic methods for sequencing method for sequencing DNA . They are based on partial digestion of duplex DNA with exonuclease III to produce DNA molecules with 3' ends shortened to varying lengths, followed by repair synthesis to extend and label the 3' ends . After asymmetrical cleavage of the DNA with a restriction enzyme, the labeled products are separated by gel electrophoresis and the sequence read from the autoradiogram . The entire procedures, beginning with unrestricted DNA and followed through gel electrophoresis, takes only one day for sequencing both strands of the DNA molecule . These methods are especially suitable for sequencing DNA cloned in plasmid vectors, and they greatly extend the usefulness of the dideoxynucleotide chain termination method of Sanger et al . (Proc . Natl . Acad . Sci . USA 74, 5463, 1977) . Using these methods we have determined the sequence of a 410 base pair fragment which includes the yeast SUP3 tyrosine tRNA gene. Nucleic Acids Res, 1982 Mar 25, 10(6), 1963 - 79 Cloning and determination of the transcription termination site of ribosomal RNA gene of the mouse; Kominami R et al.; A Eco RI 6.6 kb DNA fragment containing the 3'-end of 28S ribosomal RNA gene of the mouse was detected by Southern blot hybridization, and cloned in a lambda-phage vector . The site of transcription termination and the processed 3'-end of 28S RNA were determined on the cloned fragment and the surrounding nucleotide sequence determined . The 3'-terminal nucleotides of mouse 28S RNA are similar to those of yeast, Drosophila and Xenopus although the homology was lost drastically beyond the 3'-end of 28S RNA . 45S precursor RNA terminated at 30 nucleotides downstream from the 3'-end of 28S RNA gene . A structure of a dyad symmetry with a loop was found immediately prior to the termination site of 45S RNA . The rDNA termination site thus shares some common features with termination sites recognized by other RNA polymerases. Nucleic Acids Res, 1982 Mar 25, 10(6), 1913 - 28 The nucleotide sequence of the bacteriocin promoters of plasmids Clo DF13 and Co1 E1: role of lexA repressor and cAMP in the regulation of promoter activity; van den Elzen PJ et al.; Treatment of cells, harbouring the bacteriocinogenic plasmic Clo DF13 with mitomycin-C, which induces the cellular SOS response, results in a significantly increased transcription of the operon encoding the bacteriocin cloacin DF13, the immunity protein and the lysis protein H . The nucleotide sequences of the promoter regions and N-terminal parts of the bacteriocin genes of Clo DF13, Col E1 and the pMB1 derivative pBR324 have been determined . A comparison of these sequences with those of corresponding regions of the lexA, recA and uvrB genes revealed that the promoter regions of the bacteriocin genes studied contain binding sites for the lexA protein, which is the repressor of the E . coli DNA-repair system . Using both, a thermosensitive lexA host strain and a host with pACYC184 into which the lexA gene had been cloned, we were able to demonstrate, that in vivo the lexA protein is involved in the regulation of bacteriocin synthesis . From the data presented, we conclude that bacteriocin synthesis is controlled at least by the lexA repressor . It has been reported that also catabolite repression might play an essential role in the control of bacteriocin synthesis . Computer analysis of the DNA sequence data indicated that the promoter regions of both, the cloacin DF13 and colicin E1 genes contain potential binding sites for the cyclic AMP-cyclic AMP Receptor Protein complex. Nucleic Acids Res, 1982 Mar 25, 10(6), 1857 - 65 The DNA sequence of the gene rpsA of Escherichia coli coding for ribosomal protein S1; Schnier J et al.; The DNA sequence of the gene rpsA as well as of its neighboring regions has been determined using the dideoxyribonucleotide method . It was found that there is an "open-reading-frame" of 350 bp which precedes the gene rpsA . Furthermore, an extensive internal repeats of nucleotide sequence have been found in this gene. J Biol Chem, 1982 Mar 25, 257(6), 3258 - 64 Euglena gracilis chloroplast transfer RNA transcription units . I . Physical map of the transfer RNA gene loci; Orozco EM Jr et al.; The locations of transfer RNA genes with respect to the restriction endonuclease cleavage map of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA have been determined . Purified chloroplast tRNAs were treated with snake venom phosphodiesterase to remove the 3'-CCA terminus, and radioactively labeled by the action of Escherichia coli tRNA nucleotidyltransferase in the presence of {alpha-32P}CTP . Chloroplast DNA was treated individually and with combinations of the enzymes Bal I, Bam HI, Eco RI, Pst I, Pvu II, Sal I, and Xho I . The location of tRNA genes with respect to the cleavage sites for these enzymes was determined by hybridization of the 32P-labeled tRNAs to membrane filter blots of the chloroplast DNA restriction nuclease fragments following gel electrophoresis . The 145-kilobase pair genome was resolved into nine areas of strong tRNA hybridization, separated by areas of weak or no tRNA hybridization . The loci of tRNA genes are within the Eco RI fragments Eco A, B, G, H, I, J', P, Q, and V. J Biol Chem, 1982 Mar 25, 257(6), 2848 - 55 On the recognition and cleavage mechanism of Escherichia coli endodeoxyribonuclease V, a possible DNA repair enzyme; Demple B et al.; Escherichia coli endodeoxyribonuclease V acts at many sites of damage in duplex DNA, including apurinic/apyrimidinic sites, lesions induced by ultraviolet light which are not pyrimidine dimers, adducts of 7-bromomethylbenz{a}anthracene, and, as demonstrated earlier (Gates, F . T., and Linn, S . (1977a) J . Biol . Chem . 252 . 1647-1653), it degrades uracil-containing duplex DNA most efficiently . The cleavage rate increases with increasing substitution of uracil for thymine in T5 DNA, with a replacement of one-eight of thymine generating the apparent maximum cleavage rate . However, the apparent reaction limit with DNA containing 3.8% of thymine replaced by uracil corresponds to cleavage at only 6% of the dUMP residues . Evidently, the enzyme recognizes some peculiarities of abnormal DNA structure, but not simply distortions, since some lesions, including pyrimidine dimers, are not substrates . Endonuclease V generates double strand breaks in a constant ratio to single strand nicks, regardless of the substrate . It degrades DNA processively, completing the digestion of one substrate molecule before proceeding to the next . The enzyme also appears to act cooperatively . Cleavage at methylbenz{a}anthracene adducts is usually or always 5' to the lesion . Endonuclease V seems well suited to act as a DNA repair enzyme, surveying the genome for structural distortions generated by lesions for which specific repair systems might not exist. Nucleic Acids Res, 1982 Mar 25, 10(6), 1947 - 62 Length requirements for tRNA-specific enzymes and cleavage specificity at the 3' end of turnip yellow mosaic virus RNA; Joshi S et al.; This paper describes the minimum length of the turnip yellow mosaic virus (TYMV) RNA necessary to fulfill the tRNA-like properties of the viral RNA: 50 to 75 nucleotides and 86 nucleotides from the 3' end of TYMV RNA are sufficient for adenylation and valylation respectively by the Escherichia coli system . The size of the tRNA-like fragments obtained in vitro in the presence of an E . coli, a reticulocyte or a chinese cabbage leaf extract has also been determined . Among the major fragments liberated from the 3' end of TYMV RNA by the three systems are fragments of 117 and 112 nucleotides . In addition, the E . coli extract liberates fragments of 139 and 61 nucleotides, and the reticulocyte lysate fragments of 109, 94, 84, 73 and 46 nucleotides . The cleavage of the viral RNA by several systems in vitro to yield RNA fragments encompassing the tRNA-like sequence suggests that such fragments might also be liberated in vivo. Biochemistry, 1982 Mar 16, 21(6), 1279 - 84 Structure of the Escherichia coli K2 capsular antigen . Stereochemical configuration of the glycerophosphate and distribution of galactopyranosyl and galactofuranosyl residues; Fischer W et al.; The Escherichia coli K2 capsular antigen is known to be composed of alpha-D-galactopyranosyl(1--2)glycerophosphate and alpha-D-galactofuranosyl(1--2)glycerophosphate units which are connected by phosphodiester bonds to C-4 of the galactopyranosyl and C-5 or C-6 of the galactofuranosyl moieties . In the present study the glycerophosphates were released by two different procedures and shown to have the sn-glycero-3-phosphate stereochemical configuration . In the first, the chain was fragmented by Smith degradation to glycerophosphothreitol from which the glycerophosphate was released by alkali hydrolysis . The structure-dependent low recovery of alpha-glycerophosphate (less than 10%) initiated the development of another degradative sequence which consisted of periodate oxidation, beta elimination, hydrazinolysis, and alkaline treatment . This way, approximately 90% of the glycerophosphate was released as sn-glycero-3-phosphate . beta elimination revealed in addition that most of the galactofuranosyl residues carry the phosphodiester bond at position 5 . Separation by gel permeation chromatography and analysis of the fragments obtained by beta elimination showed that pyranosidic and furanosidic galactosyl residues alternate in the same chain and suggested the sequences Galf(p)GroP-(GalpGroP)n-Galf- and -GalfGroP-(GalpGroP)n-Galf-, where n is 6, 4, and 3, respectively. Biochemistry, 1982 Mar 16, 21(6), 1162 - 9 Two DNA glycosylases in Escherichia coli which release primarily 3-methyladenine; Thomas L et al.; Two enzymes have been partially purified from Escherichia coli and designated 3-methyladenine DNA glycosylases I and II . The glycosylase I is that described by Riazuddin & Lindahl {Riazuddin, S., & Lindahl, T . (1978) Biochemistry 17, 2110-2118} . The apparent molecular weight of glycosylase I is 20 000, and that of II is 27 000 . Glycosylase I releases 3-methyladenine (3-MeA) while II releases 3-MeA, 3-methylguanine (3-MeG), 7-methylguanine (7-MeG), and 7-methyladenine (7-MeA) . The rate of release of 3-MeA by glycosylase II is 30 times that of 7-MeG . Glycosylase I is missing in mutants tag 1 and tag 2 {Karran, P., Lindahl, T., Ofsteng, I., Evenson, G . B., & Seeberg, E . (1980) J . Mol . Biol . 140, 101-127} . In crude extracts, the 3-MeA activity of II is approximately 10% of the total 3-MeA activity . A 50% inactivation at 48 degrees C required 5 min for I and 65 min for II . The apparent Km for 3-MeA residues for glycosylase I was 1.4 x 10(-8) M . The enzyme was inhibited noncompetitively by 3-MeA with an average apparent Ki of 1.6 mM . The apparent Km for 3-MeA, for glycosylase II, was 9.2 x 10(-9)M, and it was not inhibited by 3-MeA . The 3-MeA and 7-MeG activities of the glycosylase II preparation could not be separated by isoelectric focusing, by chromatography on DEAE, Sephadex G-100, phosphocellulose, DNA-cellulose, or carboxymethylcellulose, or by heating at 50 degrees C . The apparent Km for 7-MeG was 1.1 x 10(-8)M . Glycosylase II released N1-(carboxyethyl)adenine and N7-(carboxymethyl)guanine from DNA treated with beta-{3H}propiolactone but did not release the aflatoxin B-1 adduct at N-7 of guanine. Biochemistry, 1982 Mar 16, 21(6), 1357 - 63 Specificity of the photoreaction of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen with ribonucleic acid . Identification of reactive sites in Escherichia coli phenylalanine-accepting transfer ribonucleic acid; Bachellerie JP et al.; In order to test the potential of psoralen photoaddition for the probing of RNA conformation at sequence resolution, we have analyzed the specificity of the reaction of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) with Escherichia coli tRNAPhe . The sites of HMT covalent addition have been identified by a combination of analytical techniques involving chemical cleavage of the tRNAPhe molecule at the m7G site and gel electrophoresis of RNase T1 digests together with paper electrophoretic characterization of HMT-modified nucleotides and oligonucleotides . In addition, we have taken advantage of the alteration of the cleavage rate of pancreatic RNase adjacent to a photoadduct . HMT photoaddition shows a very high preference for uracil residues . However, important differences in HMT photoreactivity are observed for various U sites of the tRNAPhe molecule . Reactivity of specific bases has been correlated with partial melting of the molecule . Data available so far indicate a strong preference of the photoreactive probe for a "loose" helical conformation as compared with a tight helix, whereas a random coil appears poorly reactive. Nucleic Acids Res, 1982 Mar 11, 10(5), 1771 - 80 A pseudogene cluster in the leader region of the Euglena chloroplast 16S-23S rRNA genes; Miyata T et al.; The nucleotide sequence of a region (leader region) preceding the 5'-end of 16S-23S rRNA gene region of Euglena gracilis chloroplast DNA was compared with the homologous sequences that code for the 16S-23S rRNA operons of Euglena and E . coli . The leader region shows close homology in sequence to the 16S-23S rRNA gene region of Euglena (Orozco et al . (1980) J . Biol.Chem . 255, 10997-11003) as well as to the rrnD operon of E . coli, suggesting that it was derived from the 16S-23S rRNA gene region by gene duplication . It was shown that the leader region had accumulated nucleotide substitutions at an extremely rapid rate in its entirety, similar to the rate of tRNAIle pseudogene identified in the leader region . In addition, the leader region shows an unique base content which is quite distinct from those of 16S-23S rRNA gene regions of Euglena and E . coli, but again is similar to that of the tRNAIle pseudogene . The above two results strongly suggest that the leader region contains a pseudogene cluster which was derived from a gene cluster coding for the functional 16S-23S rRNA operon possibly by imperfect duplication during evolution of Euglena chloroplast DNA. Nucleic Acids Res, 1982 Mar 11, 10(5), 1425 - 37 Sealing of gaps in duplex DNA by T4 DNA ligase; Nilsson SV et al.; Single-strand gaps in DNA molecules were found to be a substrate for T4 DNA ligase . Sealing of the gaps was optimal at the same conditions as ligation of blunt-ended DNA molecules . Spermidine at a concentration of 2 mM stimulated the ligation of gaps, as well as the joining of DNA molecules with cohesive and blunt ends . In addition, spermidine reduced the optimal ATP concentration . The ligation of single-stranded gaps was a slow process, reaching a plateau after several hours at 25 degrees C . Approximately 10% of circular duplex plasmid pBR322 DNA molecules with a gap of 1-5 nucleotides could be converted to a covalently closed form . When such molecules were used for transformation of E . coli cells deletion mutants were obtained at a high frequency . The size and position of the gaps and the deletions were equivalent, confirming that T4 DNA ligase was sealing the gaps. Nucleic Acids Res, 1982 Mar 11, 10(5), 1733 - 40 Replication of lambda dv DNA in vitro; Anderl A et al.; Exogenous lambda dv DNA was replicated in extracts prepared from E . coli cells carrying plasmids with inducible lambda O and /or P genes . Extracts from cells carrying only one of the two lambda replication functions complement each other in this reaction . The reaction further requires ribonucleotide triphosphates, an ATP regenerating system, DNA gyrase and RNA polymerase functions . Density labelling of the superhelical reaction products results in hybrid density indicating that one complete round of replication has taken place in vitro. Nucleic Acids Res, 1982 Mar 11, 10(5), 1741 - 54 Expression in Escherichia coli of chemically synthesized gene for a novel opiate peptide alpha-neo-endorphin; Tanaka S et al.; Chemically synthesized alpha-neo-endorphin gene was fused to the Escherichia coli beta-galactosidase gene on the plasmid pKO13 . The resulting recombinant DNA was used to transform E . coli cells . Radioimmunoassay for alpha-neo-endorphin in CNBr-treated bacterial cells showed that alpha-neo-endorphin was synthesized at approximately 5 x 10(5) molecules per single E . coli cell . One of the transformants, WA802/p alpha NE2, was used for alpha-neo-endorphin purification . From 10.9 g of wet cells, we isolated 4 mg of chemically pure and biologically active alpha-neo-endorphin. J Biol Chem, 1982 Mar 10, 257(5), 2657 - 63 Purification and characterization of DNA polymerase III' . Identification of tau as a subunit of the DNA polymerase III holoenzyme; McHenry CS; DNA polymerase III', a new form of DNA polymerase III, has been purified 15,000-fold to 90% homogeneity from an Escherichia coli K12 strain . DNA polymerase III's is a subassembly of four subunits of the DNA polymerase III holoenzyme; it has functional and physical properties intermediate between the core DNA polymerase III and holoenzyme . Polyacrylamide gel electrophoresis performed under denaturing conditions indicates DNA polymerase III' to be a complex of the alpha, epsilon, and theta subunits of DNA polymerase III and a newly assigned subunit of the DNA polymerase III holoenzyme, tau (Mr = 83,000) . Both gel filtration and phosphocellulose chromatography separate DNA polymerase III from DNA polymerase III' . All enzyme forms can utilize a duplex template containing short gaps . DNA polymerase III', like the DNA polymerase III holoenzyme, can synthesize DNA on a long single-stranded template in the presence of 5 mM spermidine; DNA polymerase III cannot . Alone, DNA polymerase III' is inert in the G4 natural replicative system in which the DNA polymerase III holoenzyme is active . Molecular weight and subunit stoichiometry determinations suggest that DNA polymerase III' contains two units of core DNA polymerase III and two tau subunits. J Biol Chem, 1982 Mar 10, 257(5), 2649 - 56 Immunoelectron microscopic localization of puromycin binding on the large subunit of the Escherichia coli ribosome; Olson HM et al.; Ribosomes from Escherichia coli strain Q13 have been photoaffinity labeled with {3H}puromycin in the presence of tetracycline . Puromycin-modified 50 S subunits appear to be identical with untreated subunits in electron micrographs and are precipitated by antibodies to the N6,N6'dimethyladenosine moiety of puromycin . Electron micrographs of subunit-antibody complexes show ribosomal subunits to which an individual antibody molecule is bound and pairs of subunits linked by an IgG molecule . Two regions of puromycin binding have been identified . The primary area, seen in 76% of the ribosome monomer complexes and 93% of the antibody-linked dimers, is beside (or on) the small central protuberance and on the side opposite the L7/L12 arm . A secondary area, maximally distant from the central protuberance, is seen in 22% of the monomeric complexes but only 7% of the antibody-linked dimers . In conjunction with our earlier localization of puromycin binding on the 30 S subunit (Olson, H . M., Grant, P . G., Glitz, D . G., and Cooperman, B . S . (1980) Proc . Natl . Acad . Sci . U . S . A . 77, 890-894), we now define a puromycin-binding neighborhood of the 70 S ribosome . In addition to providing evidence for the localization of the peptidyl transferase center within the 50 S subunit, our results contribute to the formulation of a model for tRNA binding to both 30 S subunits and 70 S ribosomes. J Biol Chem, 1982 Mar 10, 257(5), 2641 - 8 A unified mechanism for the nuclease and unwinding activities of the recBC enzyme of Escherichia coli; Muskavitch KM et al.; Using a gentle method to prepare complexes of duplex DNA and the recBC enzyme for electron microscopy, structures not seen previously were observed to be associated with the double strand DNA exonuclease activity of the enzyme . These were terminal forms and loop + tail(s) structures . Both individual terminal single-stranded tails and single-stranded regions within duplexes were also observed . Observation of the terminal single-stranded structures present after various reaction conditions and after various potentially disruptive treatments helped to identify those structures which might represent true reaction intermediates . Based on these results and those of previous studies, a modified model for the mechanism of recBC enzyme action on double-stranded, linear DNA is proposed. Mol Cell Biochem, 1982 Mar 5, 43(1), 43 - 7 Reversible modification of 50S ribosomal subunits with dimethylmaleic anhydride: protein-deficient particles; Pintor-Toro JA et al.; The reversible modification of protein amino groups with dimethylmaleic anhydride, which had already been used to dissociate proteins from the 70S ribosomes of Escherichia coli (Pintor-Toro, J . A., et al . (1979) Biochemistry 18, 3219) was applied to the preparation of protein-deficient particles from the 50S subunits . Three successive cycles of treatment with dimethylmaleic anhydride, separation of dissociated proteins and regeneration of the modified amino groups produce partially inactivated ribosomal 'cores' lacking proteins L7, L11 and L12, and having very small amounts of L1, L6 and L10 . Incubation of these 'cores' with the corresponding split proteins is accompanied by complete reactivation of the polypeptide synthesizing activity as compared with control 50S subunits. Biochemistry, 1982 Mar 2, 21(5), 830 - 4 Zinc-sulfur bonds of aspartate transcarbamylase studied by x-ray absorption spectroscopy; Phillips JC et al.; X-ray absorption spectra have been recorded for aspartate transcarbamylase {unligated and ligated with the transition-state analogue N-(phosphonoacetyl)-L-aspartate} and for the model compound zinc dimethyldithiocarbamate . The spectra confirm that, in the enzyme, the zinc atom is ligated to four sulfur atoms, with a mean distance of 2.34 +/- 0.03 A . A spread in bond lengths of 0.1 +/- 0.03 A is possible, due to thermal and/or static disorder . No significant difference was found between the spectra of the ligated and unligated enzymes. Biochemistry, 1982 Mar 2, 21(5), 1068 - 75 Phosphorus-31 nuclear magnetic resonance studies of bioenergetics in wild-type and adenosinetriphosphatase(1-) Escherichia coli cells; Ugurbil K et al.; By use of 31P NMR, the transmembrane pH gradient (delta pH) and the intracellular levels of phosphorylated metabolites were measured in aerobic suspensions of wild-type Escherichia coli cells in the presence and absence of the adenosinetriphosphatase (ATPase) inhibitor dicyclohexylcarbodiimide (DCCD); the same parameters were also determined in E . coli mutants deficient in ATPase activity under both anaerobic and aerobic conditions . A method is described by which dense suspensions of E . coli cells (approximately 3 X 10(11) cells/mL) were oxygenated so that steady-state O2 levels in the suspensions were far greater than the Km for O2 consumption . Under these conditions, in wild-type MRE600 cells, the intracellular concentrations of PI, NTP, and NDP were measured to be 3.0 +/- 1.5, 8 +/- 1, and 1.2 +/- 1 mM, respectively, while the intracellular pH was approximately 7.5 over the external pH range studied (6 to approximately 7.0) . Upon treatment with DCCD, the intracellular NTP level was drastically reduced and intracellular Pi concentration increased in respiring wild-type cells; in the same cells, however, DCCD did not affect the intracellular pH and the delta pH . During respiration in the presence of lactate, ATPase- cells established a delta pH but failed to synthesize any detectable levels of NTP . Conversely, ATPase- cells accumulated high levels of NTP but did not generate a delta pH during glycolysis under anaerobic conditions . These results are in complete agreement with the generally accepted chemiosmotic hypothesis . 31P NMR data on intact ATPase- NR70 cells were in agreement with the previously proposed {Rosen, B . P., Brey, R., & Hasan, S . (1978) J . Bacteriol . 134, 1030} existence of a proton leak in this strain which is sealed by DCCD or by spontaneous mutation into strain NR71 . However, the NMR data also indicated that other major differences exist between NR71 and NR70 cells. Scand J Immunol, 1982 Mar, 15(3), 311 - 8 Soluble cytostatic factor(s) released from human monocytes . I . Production and effect on normal and transformed human target cells; Hammestrom J; Human monocytes activated with lymphokines after 3-7 days of in vitro maturation released stable cytostatic factor(s) (CF), as determined by reduction of {methyl-3H}thymidine incorporation and cell counts in target cell cultures . Lipopolysaccharide from Escherichia coli slightly enhanced the release of CF . The CF release was greatest at an intermediate stage of the in vitro monocyte maturation to large, macrophage-like cells . Three transformed human cell lines (NHIK 3025, K-562, and U-20S) and normal skin fibroblasts were consistently inhibited by CF . Normal human mesothelial cells were variably affected, while normal human lymphocytes proliferating in response to allogeneic cells in mixed lymphocyte culture were not inhibited . Further analysis of CF may disclose one of the mechanisms whereby human monocytes and macrophages inhibit growth of human tumour cells. Biokhimiia, 1982 Mar, 47(3), 398 - 404 {Characterization of DNAse from rat brain}; Ivanov VA et al.; Using gel filtration and ion exchange chromatography, a DNAse specific to a single-stranded DNA was isolated from the soluble fraction of rat brain nuclear proteins . The nuclear nuclease corresponds to alkaline DNAse obtained earlier from the intact brain . The enzyme is a Mg2+- or Mn2+-dependent exodeoxyribonuclease and hydrolyzes at the same rates the ss-DNAse from rat brain, calf thymus, E . coli and poly (dA) . The major products of hydrolysis are nucleoside-5'-monophosphates . A possible role of the enzyme in reparation of neuronal DNA is discussed. Cancer Res, 1982 Mar, 42(3), 1088 - 93 Cloning and screening of sequences expressed in a mouse colon tumor; Augenlicht LH et al.; Polyadenylic acid-containing cytoplasmic RNA was isolated from a dimethylhydrazine-induced mouse transplantable colon carcinoma . Double-stranded complementary DNA synthesized using these molecules was inserted into the HindIII site of pBR322 using HindIII linkers, and the recombinant molecules were cloned in Escherichia coli . Clones were screened with labeled complementary DNA synthesized from the polyadenylic acid-containing cytoplasmic RNA of the tumor or normal mouse colon, liver, or kidney . Of 378 clones screened with the normal colon and tumor probes, seven showed major increases in abundance in the tumor tissue as compared to normal, and one showed a major decrease . Twenty-five other sequences were found which showed smaller increases and 22 showed smaller decreases . Of 373 clones screened with tumor, colon, liver, and kidney probes, 79% exhibited little evidence of tissue specificity in expression . The data for those sequences which do show tissue-specific regulation of expression suggest that the tumor continues to express sequences characteristic of the colon but also shows a loss, or decrease, in other gene products, most of which (19 of 25) are characteristic of the colon . Finally, nine of 10 sequences which increase from low abundance in the normal colon to high abundance in the tumor are found at a moderate to high level in the liver and kidney . On the other hand, 23 of 36 sequences which show more modest increases in the tumor as compared to normal are scarce or absent in the liver and kidney. Ann Immunol (Paris), 1982 Mar-Apr, 133C(2), 189 - 97 {Cloning of cDNA for C3, the third component of complement (author's transl)}; Fey G et al.; A collection of bacterial plasmids has been constructed carrying cDNA inserts corresponding to mouse liver C3 . Eight overlapping cDNA fragments cover 4 700 out of the 5,100 +/- 200 nucleotides of the mRNA including its 3'-end . By partial DNA sequencing it has been found that the beta-chain is encoded in the 5'-half of the mRNA and must thus be located in the amino-terminal portion of the precursor polypeptide pro C3 . From DNA sequences it is predicted that the amino-terminus of mouse C3-beta coincides in 12/15 residues with the known amino-acid sequence of guinea-pig C3 beta and in 9/10 residues with that of human C3 beta . A gene bank has been constructed from mouse DNA, from which four C3 genomic clones have been isolated . Two of them carry direct neighbouring fragments of 14- and 18-kilobase pairs of DNA, and contain one entire C3 gene (24-kilobase pairs) . The 3'- and 5'-ends of the gene have been mapped . The DNA sequence at the 5'-end predicts that the initial translation product of the C3-mRNA is a prepro C3 molecule which carries at its amino-terminus a signal peptide of 24 amino-acids of typical composition . Human C3 genomic clones have been isolated and used to map the human C3 gene on a chromosome. Rev Infect Dis, 1982 Mar-Apr, 4(2), 255 - 60 Synergism in the folate pathway; Harvey RJ; Synergism between inhibitors acting on different enzymes of a metabolic pathway can occur as a simple consequence of the nonlinear nature of normal enzyme kinetics . However, whether synergism does or does not occur is then determined entirely by the configuration of the pathway . Synergism between trimethoprim and sulfamethoxazole results from the cyclic configuration of the folate pathway. Rev Infect Dis, 1982 Mar-Apr, 4(2), 246 - 54 Molecular mechanisms of resistance to trimethoprim; Burchall JJ et al.; Resistance to inhibitors of dihydrofolate reductase arises from a variety of mechanisms involving enzyme alteration, cellular impermeability, enzyme overproduction, inhibitor modification, and loss of binding capacity . The mechanism of greatest clinical importance is the production of plasmid-encoded, trimethoprim-resistant forms of dihydrofolate reductase . At least two different types of these enzymes have been documented . The trimethoprim-resistant reductases differ from all other dihydrofolate reductases in molecular weight, subunit structure, kinetic properties, and binding of inhibitors . Colony hybridization techniques, developed for the detection of plasmid DNA coding for trimethoprim-resistant reductases, enable researchers to evaluate the prevalence and distribution of plasmid-borne resistance . Preliminary results obtained with a series of enzymatically characterized clinical isolates suggest that the colony hybridization technique may provide a convenient epidemiological tool for monitoring the dissemination of plasmid-borne resistance to trimethoprim. Int J Radiat Oncol Biol Phys, 1982 Mar-Apr, 8(3-4), 791 - 3 Structure-cytotoxicity relationships of nitroimidazoles in an in vitro system; Edwards DI et al.; Correlations of cytotoxicity of nitroimidazoles and their electron affinity depend upon the degree of reduction of the drugs . If the end-point chosen to assess cytotoxicity is one at which incomplete reduction has occurred the slope of the correlation is positive, with respect to E71, but if measurements are taken after complete reduction of the drugs the slope is a negative one . The production of nitrite ion by 5-nitroimidazoles depends on the base composition of DNA suggesting that the active agent responsible for cytotoxicity is the one-electron radical anion, R--NO2-. Int J Radiat Oncol Biol Phys, 1982 Mar-Apr, 8(3-4), 777 - 80 Reduction of nitroimidazoles in model chemical and biological systems; Wardman P et al.; Some chemical and biological properties of intermediates obtained during reduction of nitroimidazoles are discussed . These include: rate data for the decay of the nitro radical-anion, stoichiometry and absorption spectra for reduction via the radical-anion or using dithionite, stoichiometry with other reducing agents, and rate of reduction by xanthine/xanthine oxidase . Increased radiosensitization by misonidazole is seen upon prolonged pre-irradiation incubation using E . coli, enabling demonstration that a freely-diffusable metabolite is responsible for this effect . Preliminary experiments designed to extend studies of the radiobiological properties of extracellularly-added metabolites to mammalian cells and the use of liver perfusion to generate metabolites are described. Int J Radiat Oncol Biol Phys, 1982 Mar-Apr, 8(3-4), 399 - 401 Radiosensitization by non-nitro compounds; Wardman P et al.; The effects of 23 non-nitro compounds on the radiosensitivity of hypoxic Chinese hamster V79-379A or E . coli AB 1157 cells in vitro are outlined . Imidazole derivatives substituted with several alternative electron-withdrawing groups are described; the dicyanovinyl function conferred considerable radiosensitizing activity . 2,4,5-Tribromoimidazole and 2,4-dinitrophenol may show unusual radiosensitizing activity because of interference with oxidative phosphorylation . Attempts to influence radiosensitivity by compounds potentially capable of depleting intracellular sulphydryls are also described. Gene, 1982 Mar, 17(3), 317 - 21 Physical map of pColA-CA31, an amplifiable plasmid, and location of colicin A structural gene; Morlon J et al.; Evidence showing that the plasmic ColA, derived from strain CA31{pColA} can be amplified in the presence of chloramphenicol is presented . This plasmid has been purified and its Mr-value has been found to be 4.6 X 10(6) or 7 kb . Twelve cleavage sites have been mapped in pColA by using single and double restriction endonuclease digestions . These sites were ordered in relation to the single HindIII site . The other restriction endonucleases used were, respectively, SmaI, AvaI, PstI and HincII . Establishment of the map was helped by hybridization of pColA endonuclease digest products with 32P-labeled colicin A-mRNA . The structural gene for colicin A was contained in a 2.17-kb HincII fragment. Gene, 1982 Mar, 17(3), 311 - 6 Nucleotide sequences of cloned cDNA fragments specific for six Xenopus laevis ribosomal proteins; Amaldi F et al.; We have previously constructed and selected six recombinant plasmids containing cDNA sequences specific for different ribosomal proteins of Xenopus laevis (Bozzoni et al., 1981) . DNA cloned in these plasmids have been isolated and sequenced . Amino acid sequences of the corresponding portions of the proteins have been derived from DNA sequences; they are arginine- and lysine-rich as expected for ribosomal proteins . One of the cDNA sequences has an open reading frame also on the strand complementary to the one coding for the ribosomal protein; this fragment has inverted repeats twenty nucleotides lone at the two ends . The codon usage for the six sequences appears to be non-random with some differences among the ribosomal proteins analysed. Gene, 1982 Mar, 17(3), 279 - 89 Synthesis of a human insulin gene . V . Enzymatic assembly, cloning and characterization of the human proinsulin DNA; Brousseau R et al.; To form a 258-bp sequence coding for human proinsulin, 41 synthetic deoxyribo-oligonucleotide fragments of 11 to 15 nucleotides in length were assembled by enzymatic methods . The coding sequence is preceded by ATG and following by TGA for translation start and stop signals, and terminated in an EcoRI and a BamHI recognition sequence . The complete synthetic sequence was ligated to a plasmid and cloned in Escherichia coli . The cloned DNA was shown to have the correct human proinsulin coding sequence. Gene, 1982 Mar, 17(3), 271 - 7 The making of strand-specific M13 probes; Hu N et al.; A novel approach has been developed for the preparation of highly radioactive, strand-specific M13 probes . A universal primer, complementary to the region 5' to the multiple cloning sites of M13mp7, was used to initiate the DNA synthesis of the complementary strand of the M13 sequence downstream from the inserted sequence . The synthesis of the (-) strand, which was labeled with a radioactively labeled precursor, did not proceed to completion so that the inserted sequence was kept single-stranded . Thus, a partially double-stranded probe that had the specificity of this inserted sequence was obtained . As an example for the application of single-stranded specific hybridization probes, an M13mp7 subclone of a zein cDNA clone of maize (A30) was labeled and used in a dot hybridization test to select from the hundreds of M13mp7 subclones of the zein genomic clone, Z4, the sequences complementary to the probe . The specificity of the probe was confirmed by dideoxy chain terminator sequencing experiments. Cell Calcium, 1982 Mar, 3(1), 43 - 54 Phospholipase A2 activity in pancreatic islets is calcium-dependent and stimulated by glucose; Laychock SG; Phospholipase A2 activity in islet cell homogenates and dispersed islet cells of the rat was determined using an exogenous radiolabeled phospholipid substrate from E . coli membranes . Phospholipase A2 activity in islet homogenates was found to have two pH optima in acid or neutral/alkaline pH ranges . The enzyme activity at pH 7.5 was calcium dependent and responded to increasing calcium concentrations with graded increases in phospholipid hydrolysis . Preincubation of islets with a concentration of glucose known to elicit maximum rates of insulin secretion resulted in a stable activation of phospholipase A2 activity which was assayable in islet homogenates . Glucose stimulated phospholipase A2 in these preparations by as much as 220% above control . 2-Deoxy-D-glucose, a nonsecretory analogue of glucose, did not elicit a significant increase in islet phospholipase A2 activity . The glucose sensitive enzyme was associated with a membrane-enriched subcellular fraction in which the glucose-stimulated activity was greater than 2-fold higher than control activity . Glucose stimulation potentiated the phospholipase A2 activity measured in the presence of high calcium concentrations . Phospholipase A2 activity was also found in dispersed islet cell preparations where glucose stimulation of what may be a partly externalized membrane enzyme was most apparent at low calcium concentrations . These data indicate that islet cells possess phospholipase A2 activity which may be in part localized to the plasma membrane as well as other membrane systems, and which exhibits the characteristic properties of pH and calcium dependency, and sensitivity to secretagogue stimulation reported for the enzyme in other secretory systems. Ann Microbiol (Paris), 1982 Mar-Apr, 133(2), 269 - 73 Mechanism of regulation of the lactose permease by the phosphotransferase system in Escherichia coli: evidence for protein-protein interaction; Osumi T et al.; Binding of enzyme IIIglc to membranes was demonstrated in vitro, using membrane fragments from an E . coli strain which produces elevated levels of the lactose permease . Lactose and other substrates of the lactose permease enhanced the binding, but phosphoenolpyruvate decreased it in the presence of enzyme I and HPr . HPr also bound to the membranes under some conditions . The results support a model of permease regulation involving allosteric protein-protein interactions. Ann Microbiol (Paris), 1982 Mar-Apr, 133(2), 235 - 41 Superposition of genetic sites in the regulatory region of the bipolar argECBH operon of Escherichia coli; Cunin R et al.; The nucleotide sequence of mutants in the regulatory region of the argECBH divergent operon of Escherichia coli explains the dual effect of these mutants on the rate of expression of both wings of the gene cluster and emphasizes the high degree of integration of the genetic sites at the argE-argC boundary. Ann Microbiol (Paris), 1982 Mar-Apr, 133(2), 219 - 24 Systems for generating and detecting mutations in the galactose operon promoter region; Busby S et al.; We discuss the use of plasmid systems which enable us to generate and detect mutations in the Escherichia coli galactose operon promoter region . We describe the isolation of mutants which map the galactose promoters and which pinpoint the sites for the binding of the cyclic AMP-binding protein (CRP) and the galactose repressor . Finally, we describe how such mutations can be transferred from the plasmid systems to the intact galactose operon on the host chromosome. Ann Microbiol (Paris), 1982 Mar-Apr, 133(2), 205 - 7 Lipopolysaccharide requirement of phage T6 inactivation in Escherichia coli K12; Yamato I et al.; T6-phage inactivation by homogeneous, lipopolysaccharide-free Tsx protein showed a clear dependence on added lipopolysaccharide, when assayed in the presence of a non-ionic detergent, octyl-polyoxyethylene (octyl-POE) . Under these conditions, the protein exists in a monomeric state. Proc Natl Acad Sci U S A, 1982 Mar, 79(6), 1945 - 9 Carcinogenic epoxides of benzo{a}pyrene and cyclopenta{cd}pyrene induce base substitutions via specific transversions; Eisenstadt E et al.; We have determined the spectrum of base-pair substitution mutations induced in the lacI gene of a uvrB- strain of Escherichia coli by two polycyclic aromatic hydrocarbons--(+/-)7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10 tetrahydrobenzo{a}pyrene (BPDE), and 3,4-epoxycylopenta{cd}pyrene (CPPE) . Approximately 10% of all lacI mutations induced by either BPDE or CPPE are nonsense mutations, suggesting that base-pair substitutions are a large fraction of the mutational events induced by these agents in the uvrB- bacteria . Both carcinogens specifically induced the G . C leads to T . A and, to a lesser extent, the A . T leads to T . A transversions . One possible mechanism for transversion induction at G . C sites by BPDE might involve carcinogen binding to the exocyclic amino group of guanine in the template strand followed by a rotation of the modified base around its glycosylic bond from the anti to the syn conformation . This could allow specific pairing of modified bases with an imino tautomer of adenine. Proc Natl Acad Sci U S A, 1982 Mar, 79(6), 1935 - 9 Broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells; Figurski DH et al.; Cloning of specific regions of RK2, a broad host range incompatibility group P plasmid, has revealed three genes: kilA, kilB, and kilC . Each of these genes can cause loss of viability of an Escherichia coli host . This effect on the host is normally prevented by the functions of three additional RK2 genes: korA, korB, and korC . Each kor gene is specific for a particular kil gene . The kil and kor genes are located in four distinct regions of the RK2 genome . The three kil genes are not clustered and, with the possible exception of kilA, they are also well separated from their corresponding kor genes . We have found that the korA and korB determinants are not peculiar to RK2 but instead are highly conserved throughout the incompatibility group P plasmids. Proc Natl Acad Sci U S A, 1982 Mar, 79(6), 1830 - 3 Cellular location affects protein stability in Escherichia coli; Talmadge K et al.; We study the biosynthesis of preproinsulin and proinsulin in Escherichia coli by pulse-chase experiments . When E . coli is transformed with plasmids bearing a gene consisting of a fusion of preproinsulin DNA to part or to all of the DNA encoding a bacterial signal sequence, a variety of hybrid preproinsulin molecules can be made . Molecules with largely complete hybrid signal sequences are secreted into the periplasm . Molecules with defective signal sequences, lacking the hydrophobic core, are not secreted and remain in the cytoplasm . Such molecules are degraded with a 2-min half-life, whereas the molecules transported to the periplasm are at least 10 times more stable . A full-length preproinsulin precursor appears transiently before the signal sequence is cleaved off by the bacterial signal peptidase, and we propose a model to account for this. Proc Natl Acad Sci U S A, 1982 Mar, 79(6), 1805 - 9 Identification and cloning of a yeast nuclear gene (CBP1) involved in expression of mitochondrial cytochrome b; Dieckmann CL et al.; Nuclear pet mutants of Saccharomyces cerevisiae deficient in mitochondrial respiration have been studied genetically and biochemically . Seven noncomplementing mutations leading to a deficiency of mitochondrial cytochrome b have been assigned to a single complementation group (group 60) . Examination of mitochondrial RNA by blot hybridization on diazobenzyloxymethyl-paper has revealed that group 60 mutants produce a large number of novel apocytochrome b transcripts not detected in wild-type yeast . The product of the gene affected in the mutants, therefore, appears to be required either for correct transcription or for processing of apocytochrome b premessenger RNA . The gene has been designated CBP1 . A representative mutant from complementation group 60 (N5-26) has been transformed to respiratory competency with a recombinant plasmid pool consisting of random fragments of wild-type yeast nuclear DNA inserted into a vector capable of replicating in yeast and Escherichia coli . The complementation of the N5-26 mutation has been shown for a number of independent transformants to be due to the presence of plasmid DNA . The plasmid pG60/T10 was further characterized to have a nuclear DNA insert of 6.7 kilobase pairs . This plasmid complements the mutations of all group 60 mutants, thus confirming that it contains the CBP1 gene. Proc Natl Acad Sci U S A, 1982 Mar, 79(6), 1766 - 70 Amplification of the uvrA gene product of Escherichia coli to 7% of cellular protein by linkage to the pL promoter of pKC30; Yoakum GH et al.; We have constructed a hybrid pKC30-uvrA plasmid (pGHY5003) in which transcription of the uvrA gene can be induced under pL control to amplify the uvrA gene product to 7% of cellular protein . To construct pGHY5003, we developed a genetic selection using the basal level of expression (30 degrees C) from pL in thermosensitive cI857 lysogens to isolate appropriately tailored repair genes inserted at the Hpa I site of pKC30 from recombinant DNA mixtures with a variety of products . In addition, a post-UV-irradiation radiolabeling method was adapted to screen inserts for temperature-inducible polypeptide synthesis directed by transcription under pL control rapidly . This should prove generally useful for isolating genes inserted at the Hpa I site of plasmid pKC30 with the following characteristics: (i) genetically functional hybrid plasmids selected from a large population of exonucleolytically tailored fragments ligated into Hpa I of pKC30 and (ii) production of high-level amplification for the gene product of interest by screening for post-UV-irradiation temperature inducibility of polypeptides synthesized from hybrid plasmids . The level of amplification obtained for the uvrA gene product from pGHY5003 is approximately 10,000-fold higher than estimates of the level of uvrA protein in logarithmic phase Escherichia coli. Prikl Biokhim Mikrobiol, 1982 Mar-Apr, 18(2), 225 - 30 {Influence of various factors in polyacrylamide gel immobilization on the viability of Escherichia coli B cells}; Starostina NG et al.; The entrapment of an E . coli cell population into polyacrylamide gel (PAAG) is associated with a drastic decline of its viability . The effect of immobilization factors (the reagents used for PAAG preparation, their mixtures and the elevated temperature) upon the viability of the E . coli cell population was investigated by the method of microcultural analysis . It was found that all the components of the polymerization mixture, except for acrylamide, as well as the washed-off granules of polymerized del did not appreciably influence that viability of the cell population . Acrylamide at a concentration of 10% appeared very toxic . The decrease of its concentration to 5% and of the initial temperature of the polymerization mixture markedly lowered the toxic effect and, consequently, increased the viability of the population of immobilized E . coli cells. J Gen Microbiol, 1982 Mar, 128(Pt 3), 579 - 84 Purification and properties of histidinol dehydrogenase from Escherichia coli B; Andorn N et al.; Histidinol dehydrogenase has been purified from a derepressed mutant of