Escherichia coli

J Am Acad Dermatol, 1983 Sep, 9(3), 428 - 34
Immunologic abnormalities in botryomycosis . A case report with review of the literature; Brunken RC et al.; Botryomycosis in an uncommon chronic bacterial infection that mimics fungal disease clinically and histologically . Microscopically the hallmark of the disease is the presence of fungus-like granules in which the causative organism is embedded . A patient with typical cutaneous botryomycosis is presented, along with the immunologic abnormalities discovered on laboratory examination . The botryomycosis literature is reviewed, with special emphasis on the immunologic status of the host . Additional studies of the causative organism and cell-mediated immunity in the host in future patients with botryomycosis may help to further elucidate the pathogenesis of this most interesting disease.

Chem Biol Interact, 1983 Sep 1, 46(2), 179 - 88
Template properties of DNA alkylated with N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea; Marushige K et al.; Alkylation of DNA with N-methyl-N-nitrosourea (MeNU) and N-ethyl-N-nitrosourea (EtNU) reduces its ability to support RNA synthesis catalyzed by exogenously added RNA polymerase . It is likely that 7-alkylguanine and alkyl phosphotriester in DNA are mainly responsible for the inhibition of RNA synthesis . The inhibitory effect of alkyl groups varies depending upon divalent metal ions and the type of RNA polymerase used as well as upon the presence of chromosomal proteins on DNA templates . Analyses of RNA products indicate that inhibition occurs primarily at the initiation step.

Biophys J, 1983 Sep, 43(3), 371 - 81
Simultaneous measurements of proton motive force, delta pH, membrane potential, and H+/O ratios in intact Escherichia coli; Setty OH et al.; An instrument is described that enables the simultaneous monitoring of proton motive force (PMF), membrane potential (delta psi), the delta pH across a membrane, oxidase activity, proton movements, and H+/O ratios . We have studied the relationship existing among these parameters of energy transduction as a critical condition is changed during an experiment . The major findings are: (a) In the pH range of 4.5 to 7.5, increasing the external pH causes an increase in delta psi, internal pH, and oxidase activity, a decrease in H+/O ratio, and a peak-plateau in PMF from pH 5.5 to 6.6 where delta pH is converted to delta psi . (b) An increase in {K+} from 1 to 100 mM, in the presence of 0.5 microM valinomycin, causes the conversion of delta psi to delta pH, a gradual decline in PMF and an increase in H+/O ratio, internal pH, and oxidase activity . (c) Increasing valinomycin concentration from 0 to 2.5 microM, in the presence of 50 mM {K+}, causes a decline in delta psi from 125 to 0 mV, and an increase in delta pH from 35 to 70 mV . From 2.5 to 10 microM, the delta pH and the PMF (which it solely represents), stay constant, H+/O ratio increases mainly from 0 to 0.5 microM and much more slowly from 2.5 to 10 microM . (d) Oxygen at only 10% of its concentration in air-saturated buffer can support the generation of 90% or more of the delta psi, delta pH, and PMF generated in an air-saturated solution . (e) The return of extruded protons to the cell (referred to here as "suck-back") represents a complicated process driven by delta psi and influenced by a variety of factors . (f) H+/O ratios measured by the kinetic technique used here are much higher than those measured by standard oxygen pulse techniques.

Am J Vet Res, 1983 Sep, 44(9), 1746 - 9
Gentamicin pharmacokinetics in horses given small doses of Escherichia coli endotoxin; Wilson RC et al.; The pharmacokinetics of gentamicin (3 mg/kg of body weight) were evaluated in 6 healthy horses and in 6 horses after they were given Escherichia coli endotoxin (0.113 microgram/kg) . In the horses given endotoxin, there were a maximum temperature increase of 1.97 +/- 0.44 degrees (C) and a fever index (between the 2 groups) of 8.754 units . Other mild signs of endotoxemia also occurred . Statistically significant changes were not observed in the rate constants for distribution (alpha) or elimination (beta) or in body clearance (ClB) of gentamicin in the 2 groups of horses . In the horses given endotoxin, significant (P less than 0.05) increases were found in the serum concentration data (A, B, and CoS), and significant decreases were found in the apparent volume of distribution {Vd(area)} and in the volume of the central compartment (Vc) . The alterations in gentamicin kinetics in the horses given endotoxin are believed to result from the decrease in Vc . This indicates that the extracellular fluid volume available for gentamicin distribution may be reduced by endotoxin.

Mutat Res, 1983 Sep, 121(3-4), 171 - 5
Further characterization of the expression of SOS functions in recA430 mutants of Escherichia coli; Barbe J et al.; recA-dependent inhibition of cell division and cessation of cell respiration are not expressed in recA430 (formerly lexB) mutants of Escherichia coli after ultraviolet irradiation . Our results suggest that, to be induced in UV-treated cells, inhibition of division as well as cessation of respiration require the protease activity of the RecA protein.

Genetics, 1983 Sep, 105(1), 1 - 18
Functional effects of PGI allozymes in Escherichia coli; Dykhuizen DE et al.; Five alleles representing three electromorphs of phosphoglucose isomerase (PGI) have been transferred from natural isolates of E . coli into the genetic background of E . coli K12 and examined for their effect on growth rate in chemostats limited for glucose or fructose . With glucose limitation, all alleles are selectively neutral or nearly neutral within the limit of resolution of the technique, whether the genetic background is nonmutant or whether it contains a deletion of the locus of glucose-6-phosphate dehydrogenase, the enzyme that provides an alternative metabolic pathway for the substrate of PGI . With fructose limitation, one of the naturally occurring alleles has a small but reproducible detrimental effect on growth rate . A kinetic difference in this detrimental allozyme, apparently relating to an inhibition constant, has been observed in some, but not all, lots of substrate, and a similar difference has also been noted in one of the rare electromorphs that could not be transferred into E . coli K12 . These results support a model of genetic variation in which the alleles are neutral or nearly neutral in the prevailing environment but have a potential for selection that can be expressed under the appropriate conditions of environment or genetic background . This hypothesis is discussed in the context of allozyme polymorphisms observed in other organisms.

Biochem Pharmacol, 1983 Sep 1, 32(17), 2505 - 9
Conversion of 2,6-diamino-9-(2-hydroxyethoxymethyl)purine to acyclovir as catalyzed by adenosine deaminase; Spector T et al.; Adenosine deaminase (ADA) was partially purified from several sources using affinity chromatography . These enzymes have the capacity to catalyze the deamination of 2,6-diamino-9-(2-hydroxyethoxymethyl)purine (A134U) to form the antiviral agent acyclovir {9-(2-hydroxyethoxymethyl)guanine} . Their relative substrate efficiencies (Vmax/Km) with A134U (standardized to adenosine = 100) were: dog ADA, 0.092; human ADA, 0.015-0.029; rat ADA, 0.025; calf ADA, 0.016; and Escherichia coli ADA, 0.0003 . In addition to having the lowest efficiency with A134U, the bacterial ADA was also distinguished by its lack of binding of the mammalian ADA inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine and by its weak binding to the 9-(p-aminobenzyl)adenine-agarose affinity column . Four minor metabolites of A134U and acyclovir have been reported to be produced in the rat . These compounds are oxidized on either the C-8 position of the ring or the terminal carbon of the side chain . Neither acyclovir nor any of these metabolites produced significant inhibition of calf intestine ADA . The oxidized metabolites containing an N-6 amino group were extremely slow substrates of this enzyme.

Surgery, 1983 Sep, 94(3), 487 - 93
Mechanisms of the adjuvant effect of hemoglobin in experimental peritonitis . VII . Hemoglobin does not inhibit clearance of Escherichia coli from the peritoneal cavity; Dunn DL et al.; Hemoglobin has been shown to be a potent adjuvant in experimental Escherichia coli peritonitis, although a satisfactory mechanistic rationale is still obscure . Hemoglobin has been thought to impair intraperitoneal neutrophil function, delay clearance of bacteria from the peritoneal cavity by the normal absorptive mechanisms, or directly enhance bacterial growth . Using highly purified stroma-free hemoglobin (SFHgb), we have largely discounted any direct effect of hemoglobin on peritoneal white blood cell function . In the present study, we confirmed that uncontrolled proliferation of bacteria takes place in the presence of hemoglobin in the peritoneal cavity . Nonviable 5-iododeoxyuridine 125I-labelled bacteria were then used to directly study peritoneal clearance kinetics, eliminating the problem of bacterial growth . SFHgb had no influence on the removal of intraperitoneal bacteria . The rate of bloodstream appearance of radiolabel was similar with or without intraperitoneal SFHgb . Thus, SFHgb does not prevent clearance of bacteria from the peritoneal cavity by interfering with normal host clearance mechanisms . SFHgb may act as a bacterial growth adjuvant, either by serving as a bacterial nutrient or by suitably modifying the environment so that extensive bacterial proliferation can occur . The latter hypothesis appears to be an area in which investigation concerning the adjuvant effect of hemoglobin may prove most fruitful.

Proc Soc Exp Biol Med, 1983 Sep, 173(4), 547 - 52
Blastogenic responsiveness of spleen cells from guinea pigs sensitized to Legionella pneumophila antigens; Widen R et al.; An in vitro leukocyte blastogenic assay was utilized to establish an in vitro correlate of cell-mediated immunity to Legionella pneumophila antigen with spleen cells from sensitized guinea pigs . Incubation of spleen cells from sensitized but not normal guinea pigs with graded amounts of killed whole cell Legionella bacteria or sonicate derived from the bacteria resulted in an antigen-induced blast cell proliferation as evidenced by an increased uptake of {3H}-thymidine into spleen cell cultures . Peak responses occurred approximately 4-6 days after incubation of the spleen cells with antigen . Sensitivity of spleen cells from animals immunized with Legionella vaccine in adjuvant persisted for at least 150 days, while responses after infection of guinea pigs with viable bacteria persisted about 4-6 weeks . The blastogenic responses of the spleen cells to Legionella antigen appeared to be a correlate of cell-mediated immunity.

Proc Natl Acad Sci U S A, 1983 Sep, 80(18), 5550 - 3
Isolation of Z-DNA-containing plasmids; Thomae R et al.; A purified, monoclonal antibody, specific for the left-handed Z-form of poly(dG-dC), was coupled covalently to Sephacryl S-1000 beads . Such an antibody column provides a convenient method to isolate and purify those plasmid DNAs that contain Z-DNA from a large excess of other DNAs, RNA, etc . From a library of Escherichia coli DNA, cloned into the vector plasmid pUC-8, several recombinant plasmids were isolated, which bind to this antibody . Thus, E . coli contains sequences, which in "natural" negatively supercoiled DNA, adopt a left-handed Z-DNA-like conformation.

Proc Natl Acad Sci U S A, 1983 Sep, 80(18), 5480 - 4
F sex factor encodes a single-stranded DNA binding protein (SSB) with extensive sequence homology to Escherichia coli SSB; Chase JW et al.; We have determined the sequence of the gene encoding a single-stranded DNA (ss DNA) binding protein (SSB) from the Escherichia coli F sex factor and the amino acid sequence of the protein it encodes . The protein has extensive homology with E . coli SSB, particularly within its NH2-terminal region, where 87 of the first 115 amino acid residues are identical to those of the E . coli protein . We have previously shown that this portion of E . coli SSB contains the DNA binding region . The sequences diverge extensively in their COOH-terminal regions, although small areas of homology exist in several places . Six of the last seven amino acid residues of the two proteins are identical, which may have implications in terms of the direct interactions of these proteins with other proteins required for DNA replication, recombination, and repair . The coding region of the F plasmid ssf gene is 537 base pairs . The protein encoded by the gene contains 178 amino acids (one more than E . coli SSB) and has a calculated molecular weight of 19,505 . Other than the presumptive Shine-Dalgarno sequence, the promoter and terminator regions of both genes are not similar . The most significant feature in this regard may be the lack of a region of dyad symmetry within the presumptive promoter of the F plasmid ssf gene as is found in the region of the presumptive E . coli ssb promoter . In this report the predicted secondary structures of both the F plasmid and E . coli SSB proteins are compared and the evolutionary significance of their sequence and structural similarities to the functional domains of the proteins are discussed.

Proc Natl Acad Sci U S A, 1983 Sep, 80(17), 5359 - 63
Repeated sequences and open reading frames in the 3' flanking region of the gene for the RNA subunit of Escherichia coli ribonuclease P; Reed RE et al.; We have determined the identity of about 700 nucleotides in the 3' flanking region of the gene for M1 RNA, the RNA component of RNase P (EC 3.1.26.5) . This region begins with a 113-base-pair segment of DNA, which is repeated approximately 3.5 times . The first repeating unit originates within the gene sequence for the 3' end of mature M1 RNA . The repeating units are highly conserved but diverge considerably after the partial fourth repeat . Segments of sequence homologous to the repeats have been identified both upstream in the M1 RNA transcription unit and downstream from the repeating units . Several overlapping open reading frames, with the potential to encode small basic proteins, have been identified in the repeated sequences . The structure of the M1 RNA gene 3' flanking region is very similar to the corresponding region at the Tyr T locus . In vitro and in vivo, transcription of the M1 RNA gene appears to terminate about 40 nucleotides downstream from the 3' terminus of mature M1 RNA . Therefore, an RNA processing event is involved in the biosynthesis of M1 RNA.

J Immunol, 1983 Sep, 131(3), 1189 - 94
The use of mutants of Escherichia coli for the identification of human lymphocyte subpopulations in blood smears; Bratescu A et al.; In previous work we have shown that some bacteria can bind to human lymphocytes and can be used to identify lymphocyte subpopulations in conventionally stained blood smears . These bacteria are of different species or genera, which makes it difficult to study the binding mechanism . Also, the main marker for B cells, Brucella melitensis, is of very small size and highly pathogenic . Here we show that B cells as well as some of the T cell subpopulations can be identified by different mutants obtained from a strain of an Escherichia coli . Two procedures were used to generate mutants . First, E . coli-YS57 (pro-his-trp-) was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and the binding to mouse spleen cells was used as a selective pressure . Second, phage-resistant mutants of E . coli-YS57 were obtained and tested for the ability to bind to lymphocytes . Out of 10 strains selected by the former procedure, 5 bound to a significant number of human lymphocytes . All four phage-resistant mutants bound to human lymphocytes . Out of the total of nine mutants that bound to lymphocytes, six bound consistently, i.e., they bound to similar percentages of peripheral blood lymphocytes from different normal donors . One phage-resistant mutant, E . coli USC-106, bound only to B cells . The subpopulations of lymphocytes identified by the mutants were essentially the same as those identified by different species or genera of bacteria . We concluded that E . coli mutants can be obtained that identify human lymphocyte subpopulations and that one of these mutants recognizes B cells; these mutants may be used to study the nature of the receptors for bacteria on lymphocytes, which appear to have a lectin-like nature.

J Bacteriol, 1983 Sep, 155(3), 983 - 8
Evidence for diffuse growth of the cylindrical portion of the Escherichia coli murein sacculus; Burman LG et al.; High-resolution autoradiography of thin sections of Escherichia coli cells whose murein was pulse-labeled with {3H}diaminopimelic acid after a period of diaminopimelic acid deprivation indicated that elongation of the murein sacculus occurs by a multisite (diffuse) process . Upon chasing, radioactivity in polar murein was stable, whereas radioactivity in cylindrical murein was reduced, indicating that diffuse intercalation of new murein occurred during cell elongation . Elongation and septation were shown to be overlapping processes.

J Bacteriol, 1983 Sep, 155(3), 1463 - 6
Chemotactic response of Escherichia coli to chemically synthesized amino acids; Hedblom ML et al.; In Escherichia coli, seven of the commonly occurring amino acids are strong attractants: L-aspartate, L-serine, L-glutamate, L-alanine, L-asparagine, glycine, and L-cysteine, in order of decreasing effectiveness . The chemotactic response to each amino acid attractant is mediated by either methyl-accepting chemotaxis protein I or II, but not by both . Seven of the commonly occurring amino acids are repellents . This work was carried out with chemically synthesized amino acids.

J Bacteriol, 1983 Sep, 155(3), 1450 - 4
Regulation of fatty acid transport in Escherichia coli: analysis by operon fusion; Sallus L et al.; The regulation of the fadL gene, which encodes a long-chain fatty acid (LCFA) transport component, was examined by constructing a strain of Escherichia coli K-12 that bears a phi (fadL-lacZ+) operon fusion plus a wild-type fadL gene . This merodiploid strain expressed LCFA transport and beta-galactosidase activity coordinately under noninducing, inducing, and catabolite-repressing conditions . Merodiploid strains which carried a defective fadR gene expressed LCFA transport and beta-galactosidase activity constitutively . These results suggest that expression of the fadL gene is regulated by the fadR gene and is inducible at the level of transcription by LCFA.

J Bacteriol, 1983 Sep, 155(3), 1426 - 8
Isolation of rel mutants of Escherichia coli B/r; Little R et al.; A method that relies on the biological effect of near-UV (340-nm) irradiation is described by which large numbers of independent rel mutants of Escherichia coli B/r may be rapidly isolated.

J Bacteriol, 1983 Sep, 155(3), 1306 - 15
The muc genes of pKM101 are induced by DNA damage; Elledge SJ et al.; A gene fusion was constructed in vitro that resulted in the synthesis of a hybrid protein consisting of the amino-terminal segment of the MucB protein of the mutagenesis-enhancing plasmid pKM101 joined to an enzymatically active carboxy-terminal segment of the beta-galactosidase protein . In strains bearing this fusion, beta-galactosidase activity was induced by UV radiation and other DNA-damaging agents . A genetic analysis of the regulation of expression of the phi (mucB'-lacZ') fusion was consistent with the LexA protein acting as the direct repressor of the mucB gene . Examination of the expression of the mucA and phi (mucB'-lacZ') gene products in maxicells in the presence and absence of a high-copy-number plasmid carrying the lexA+ gene demonstrated that lexA regulated both the mucA and mucB genes, thus supporting our conclusion that the two genes are organized in an operon with the mucA gene transcribed first . An analysis of the effects of the recA430(lexB30) mutation on muc expression led to the discovery of the differential ability of the recA430 gene product to induce expression of a dinB::Mu d1(Ap lac) fusion located on the chromosome and the same phi (dinB'-lacZ+) fusion cloned into plasmid pBR322 . Models to account for the role of the recA430 allele on the expression of damage-inducible genes and on mutagenesis are discussed.

J Bacteriol, 1983 Sep, 155(3), 1116 - 22
Isolation and preliminary characterization of Escherichia coli mutants deficient in exonuclease VII; Vales LD et al.; Strains of Escherichia coli containing reduced levels of exonuclease VII activity due to mutations in the xseB gene have been isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine . Seven mutants of independent origin deficient in exonuclease VII activity were obtained . Four of these contained defects in xseA, a locus which has been previously identified, and three others contained mutations in a gene distinct from xseA, which we have designated xseB . Genetic mapping studies place the xseB locus between proC and dnaZ . Exonuclease VII purified from KLC835 (xseA+ xseB3) is more heat labile than enzyme purified from the parent strain PA610 (xse+), showing that xseB is a structural gene for exonuclease VII . The isolation of lambda transducing phage carrying xseA is also described.

J Bacteriol, 1983 Sep, 155(3), 1110 - 5
Mutation causing overproduction of outer membrane protein OmpF and suppression of OmpC synthesis in Escherichia coli; Koga-Ban Y et al.; A novel mutation affecting the synthesis of major outer membrane proteins OmpF and OmpC in Escherichia coli K-12 is described . The mutation resulted in overproduction of the OmpF protein with concomitant suppression of OmpC synthesis . This mutation, designated as ompFp100, was mapped at 21 min on the E . coli chromosome map with the gene order aroA-aspC-ompF4-ompFp100-asnS-pyrD . This mutation was cis-dominant to the expression of the ompF gene . In addition, the direction of the mRNA transcription of the ompF gene was from asnS to aspC . These results strongly indicate that ompFp100 is a promoter mutation of the ompF gene . Introduction of an ompF mutation, which causes the disappearance of the OmpF protein, into strains carrying the ompFp100 mutation resulted in the reappearance of the OmpC protein in the outer membrane . Addition of a high concentration of sucrose to the medium, which suppresses the OmpF synthesis and stimulates the OmpC synthesis in the wild-type strain, resulted in the reappearance of the OmpC protein in the ompFp100 mutant with concomitant suppression of the overproduction of the OmpF protein . These results suggest that suppression of OmpC synthesis in the ompFp100 mutant is due to overproduction of the OmpF protein.

J Bacteriol, 1983 Sep, 155(3), 1027 - 32
Buoyant density constancy during the cell cycle of Escherichia coli; Kubitschek HE et al.; Cell buoyant densities were determined in exponentially growing cultures of Escherichia coli B/r NC32 and E . coli K-12 PAT84 by equilibrium centrifugation in Percoll gradients . Distributions within density bands were measured as viable cells or total numbers of cells . At all growth rates, buoyant densities had narrow normal distributions with essentially the same value for the coefficient of variation, 0.15% . When the density distributions were determined in Ficoll gradients, they were more than twice as broad, but this increased variability was associated with the binding of Ficoll to the bacteria . Mean cell volumes and cell lengths were independent of cell densities in Percoll bands, within experimental errors, both in slowly and in rapidly growing cultures . Buoyant densities of cells separated by size, and therefore by age, in sucrose gradients also were observed to be independent of age . The results make unlikely any stepwise change in mean buoyant density of 0.1% or more during the cycle . These results also make it unlikely that signaling functions for cell division or for other cell cycle events are provided by density variations.

Infect Immun, 1983 Sep, 41(3), 881 - 7
Hemolysis determinant common to Escherichia coli strains of different O serotypes and origins; de la Cruz F et al.; Ten Escherichia coli hemolytic strains, isolated from various types of human infection and belonging to at least six different O serotypes were analyzed . In all these strains, our results are consistent with the hly genetic determinant being located on the bacterial chromosome . An hly-specific probe (plasmid pAN215) was used to detect homologous fragments in the strains analyzed by the Southern blotting technique . It was found that all 10 strains contain sequences that hybridize against the probe (greater than 85% homology) and thus contain hly genetic determinants very similar to those found on Hly plasmids . These results strongly suggest that there is a unique ubiquitous genetic determinant responsible for the hemolytic phenotype in E . coli.

Infect Immun, 1983 Sep, 41(3), 1340 - 51
Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines; Moon HW et al.; Three strains of enteropathogenic Escherichia coli (EPEC), originally isolated from humans and previously shown to cause diarrhea in human volunteers by unknown mechanisms, and one rabbit EPEC strain were shown to attach intimately to and efface microvilli and cytoplasm from intestinal epithelial cells in both the pig and rabbit intestine . The attaching and effacing activities of these EPEC were demonstrable by light microscopic examination of routine histological sections and by transmission electron microscopy . It was suggested that intact colostrum-deprived newborn pigs and ligated intestinal loops in pigs and rabbits may be useful systems to detect EPEC that have attaching and effacing activities and for studying the pathogenesis of such infections . The lesions (attachment and effacement) produced by EPEC in these systems were multifocal, with considerable animal-to-animal variation in response to the same strain of EPEC . The EPEC strains also varied in the frequency and extent of lesion production . For example, three human EPEC strains usually caused extensive lesions in rabbit intestinal loops, whereas two other human EPEC strains usually did not produce lesions in this system.

Eur J Biochem, 1983 Sep 1, 135(1), 41 - 6
The location of redox-sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli; Poolman B et al.; Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli . One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane . Treatment of right-side-out membrane vesicles from E . coli ML 308-225 with the membrane-impermeable oxidant ferricyanide resulted in inhibition of L-proline uptake without having significant effect on the magnitude of the delta approximately mu H+ . Subsequent addition of reducing agents restored proline transport activity . The membrane-impermeable SH-reagent glutathione hexane maleimide inhibited proline transport in right-side-out membrane vesicles irreversibly . Pretreatment of the vesicles with ferricyanide protected the carrier against inactivation by glutathione hexane maleimide . Electron transfer in the respiratory chain of right-side-out vesicles led to the generation of a delta approximately mu H+, interior negative and alkaline, and the conversion of a disulphide to a dithiol in the proline carrier as is shown by the increased inhibition of proline transport by the membrane impermeable dithiol reagent 4-(2-arsonophenyl)azo-3-hydroxy-2,7-naphthalene disulphonic acid (thorin) . The inhibition exerted by thorin was completely reversed by dithiothreitol . Pretreatment of the vesicles with thorin protected against glutathione hexane maleimide inhibition, indicating that both reagents react with the same group . Treatment of inside-out membrane vesicles with ferricyanide inactivated the proline transport system reversibly . The oxidizing effect of ferricyanide in inside-out vesicles resulted in protection against inhibition by glutathione hexane maleimide . Imposition in these vesicles of a delta approximately mu H+, interior positive and acid, also protected the proline carrier against glutathione hexane maleimide inactivation, indicating that a dithiol is converted to a disulphide upon energization.

Clin Orthop, 1983 Sep, (178), 241 - 3
Septic dislocation of the hip with extension of emphysema; Lowery CE et al.; In a 58-year-old woman extension of emphysema was associated with septic arthritis and dislocation of the hip.

Radiology, 1983 Sep, 148(3), 823 - 6
Endotoxic shock and its effects on hepatobiliary scanning in dogs; Hughes KS et al.; Hepatobiliary scans were obtained with Tc-99m-disofenin in 15 dogs . Of these, 5 served as controls, 5 were infused with E . coli endotoxin for 4 hours (endotoxic shock group), and 5 were bled to a mean pressure similar to that of the endotoxic shock group (hemorrhagic shock group) . Scans of the controls and hemorrhagic shock group were identical . Scans of the endotoxic shock group were markedly abnormal, with a prolonged hepatic phase and little excretion of isotope into the biliary tract, a pattern characteristic of mechanical obstruction of the common bile duct . These results should alert the clinician to the potential danger of abnormal hepatobiliary scans in the septic patient.

Cancer Res, 1983 Sep, 43(9), 4172 - 5
Importance of treatment regimen of interferon as an antitumor agent; Lee SH et al.; A highly purified hybrid human leukocyte interferon, IFN-alpha AD, produced in Escherichia coli, has been used to define optimum treatment conditions for L1210 leukemia in mice . Treatments prior to tumor inoculation were ineffective . Treatments from the third day post-tumor inoculation were most effective, and treatments every third day were more effective than were regimens involving more frequent treatments . IFN-alpha AD was effective in vivo against tumors formed from a line of L1210 cells resistant to IFN-alpha AD in cell cultures . These and other results indicate the importance and nature of indirect mechanisms of action for efficacy of interferons against tumors.

Mol Biochem Parasitol, 1983 Sep, 9(1), 83 - 92
Isolation and characterization of an alpha-tubulin gene from Leishmania enriettii; Wirth DF et al.; An alpha-tubulin gene of Leishmania enriettii has been identified in genomic Southern blots by hybridization with a heterologous alpha-tubulin gene from Drosophila melanogaster . A clone containing this gene has been isolated from a plasmid library of size-selected L . enriettii DNA . It was identified by hybridization with the D . melanogaster tubulin gene . The cloned DNA fragment was characterized by restriction analysis and partial DNA sequence analysis . The cloned DNA fragment is 2 kb in length, bounded by Pst I sites, and appears to contain the entire coding region of the alpha-tubulin gene.

Sci Sin {B}, 1983 Sep, 26(9), 954 - 60
Cloning and restriction mapping of human HBV genome serotype adr; Wu XF et al.; A Bam HI cleaved 3.2 Kb fragment from the HBV adr genome was cloned in E . coli using pBR322 as vector . Sixteen restriction sites from the action of Bam HI, Hind III, Bgl I, Bgl II, Ava I, Hinc II, Sph I, Xba I, Xho I and Sst II were determined and mapped . No cleavage sites were found for Eco RI, Pst I, Sma I, Hpa I, Kpn I, Pvu II and Sst I . The restriction map for HBV adr is significantly different from those reported for the subtypes adw, ayw and adyw.

J Biochem (Tokyo), 1983 Sep, 94(3), 1021 - 4
Purification of dihydrofolate reductase amplified in Escherichia coli K-12; Iwakura M et al.; The Escherichia coli strain carrying pTP 6-10 which was constructed in our previous work (Iwakura, M., et al . (1983) J . Biochem . 93, 927-930) produces more than 400-fold dihydrofolate reductase as compared with the strain without the plasmid . Dihydrofolate reductase was highly purified from the cell-free extract of the plasmid strain simply by two steps; ammonium sulfate fractionation and ion-exchange chromatography . By 10-fold purification, the enzyme was essentially homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . The restriction map of pTP 6-10 was also determined and the plasmid was shown to have an Ava I, an EcoR I, a Pst I, a Pvu I, and a Pvu II site . Our results indicate that the plasmid strain is suitable as a source of the enzyme and that plasmid pTP 6-10 is promising as a versatile plasmid vector for efficiently yielding the product of the cloned gene.

Genetika, 1983 Sep, 19(9), 1419 - 25
{3 linkage groups of the genes of riboflavin biosynthesis in Escherichia coli}; Bandrin SV et al.; Using transposon Tn5 inactivation technology a collection of Escherichia coli mutants defective in riboflavine biosynthesis was obtained . All mutations were distributed within three linkage groups . With the help of P1-transduction mapping, group I mutations (ribA locus) were localized near cysB locus (28 min of the standard 100 min E . coli map) and mutations of group II (ribB locus) were mapped near tolC locus (66 min) . The location of group III mutations was approximately determined by the F' complementation analysis: this linkage group lies in the region of 56-60 min of the E . coli map.

Eur J Cell Biol, 1983 Sep, 31(2), 171 - 4
Immunocytochemical demonstration of human proinsulin chimeric polypeptide within cytoplasmic inclusion bodies of Escherichia coli; Paul DC et al.; Immunocytochemical techniques were used to identify human proinsulin chimeric protein in cytoplasmic inclusion bodies of genetically modified Escherichia coli . Antibodies to proinsulin chimeric protein (human proinsulin coupled at its amino-terminus to a portion of the E . coli tryptophan E gene product) were localized in E . coli using post-embedding staining with protein A-peroxidase labelling for transmission electron microscopy . The observable distribution of the labelled antibody was limited to that portion of the E . coli cytoplasm occupied by inclusion bodies . The localization of human peptides as insoluble masses within the bacterial cytoplasm has important implications in relation to the synthesis, recovery and purification of pharmacologically useful substances produced through the application of recombinant DNA technology.

Biosci Rep, 1983 Sep, 3(9), 857 - 61
Distinction between cofactor-dependent and -independent phosphoglycerate mutases by chromatography on Cibacron Blue-Sepharose; Price NC et al.; The binding of phosphoglycerate mutases from a variety of sources to Cibacron Blue-Sepharose has been examined . Those enzymes which are dependent on 2,3-bisphosphoglycerate (BPG) for activity bind to the immobilized dye and can be eluted by BPG . Those enzymes which are independent of BPG do not bind to the immobilized dye . The possible structural significance of this distinction is discussed.

Plasmid, 1983 Sep, 10(2), 175 - 83
Genetics of the replication and maintenance functions of the hemolytic plasmid pSU316 . Cloning of an IncFIII determinant; Rodriguez JC et al.; Two miniplasmids have been constructed from pSU306, a Tn802 insertion derivative of the IncFIII-IncFIV hemolytic plasmid pSU316 . One of these, pSU3027, is a low copy number plasmid expressing both IncFIII and IncFIV incompatibilities, but is rather unstable, and probably lacks a putative par gene . The other, pSU3025, is maintained in about 340 copies per genome equivalent and expresses only IncFIII incompatibility . Most of the PstI-generated fragments from pSU3027 have been cloned in pBR322 . One of the resulting plasmids, pSU3135, contains an insertion of 0.5 kb in the vector molecule, and expresses IncFIII, but not the IncFIV incompatibility . These results allowed us to identify and locate several genes involved in the control of pSU316 replication and stable plasmid maintenance.

Plasmid, 1983 Sep, 10(2), 111 - 8
Analysis of IS21-mediated mobilization of plasmid pACYC184 by R68.45 in Escherichia coli; Riess G et al.; Escherichia coli plasmids like pACYC184 or pBR325 can be mobilized by the P-type plasmid R68.45, which carries a tandem duplication of insertion element IS21, at a frequency of 10(-3)-10(-5) per donor cell . Analysis of exconjugant cells revealed that plasmid mobilization occurs via cointegrate formation involving transposition of IS21 . No resolution of cointegrates of pACYC184 and the P-type plasmid could be found in recA recipient cells . In the cointegrate, the E . coli plasmid is flanked by single copies of IS21 in direct orientation . After resolution of the cointegrate in recA+ recipients, the mobilizing plasmid R68.45 lost one copy of IS21 becoming indistinguishable from plasmid R68 . It was shown that during mobilization, insertion element IS21 transposes to the mobilized plasmid . Insertion sites and orientations of IS21 in 33 pACYC184::IS21 insertion mutants have been determined: IS21 was found to be integrated in plasmid pACYC184 in different regions but only in one orientation . The IS21 tandem structure of plasmid R68.45 and its role in the mobilization process is discussed.

Gene, 1983 Sep, 24(1), 93 - 8
Rapid preparation of vector-free hybridization probes suitable for screening recombinant libraries; Galau GA; A procedure is described to rapidly prepare radioactively labeled DNA inserts from crude recombinant plasmid DNA preparations . These probes can subsequently be used to identify homologous nucleotide sequences in bacteria containing recombinant plasmids by colony hybridization . In a single procedure, crude recombinant plasmid DNA is both 32P-labeled and fragmented by nick-translation in the presence of sufficient pancreatic DNase I to produce radioactive DNA of about 0.2-0.3-kb single-strand length . At this DNA fragment length the majority of the vector and insert sequences are on different DNA fragments . The insert DNA can then be separated from vector and contaminating Escherichia coli host chromosomal DNA by the following method . The DNA fragment population is first denatured and renatured under conditions such that the recombinant plasmid DNA reassociates but host DNA does not . The renatured plasmid DNA fragments are separated from the denatured host DNA by hydroxylapatite chromatography . The plasmid DNA fragments are then denatured and renatured with an excess of insert-free vector DNA . Conditions are chosen such that the insert DNA remains single-stranded while the vector DNA becomes double-stranded . The single-stranded insert DNA can be separated from the double-stranded vector DNA on hydroxylapatite and used directly for colony hybridization.

Arch Biochem Biophys, 1983 Sep, 225(2), 944 - 9
Fructose bisphosphatase from Escherichia coli . Purification and characterization; Babul J et al.; Escherichia coli fructose-1,6-bisphosphatase has been purified for the first time, using a clone containing an approximately 50-fold increased amount of the enzyme . The procedure includes chromatography in phosphocellulose followed by substrate elution and gel filtration . The enzyme has a subunit molecular weight of approximately 40,000 and in nondenaturing conditions is present in several aggregated forms in which the tetramer seems to predominate at low enzyme concentrations . Fructose bisphosphatase activity is specific for fructose 1,6-bisphosphate (Km of approximately 5 microM), shows inhibition by substrate above 0.05 mM, requires Mg2+ for catalysis, and has a maximum of activity around pH 7.5 . The enzyme is susceptible to strong inhibition by AMP (50% inhibition around 15 microM) . Phosphoenolpyruvate is a moderate inhibitor but was able to block the inhibition by AMP and may play an important role in the regulation of fructose bisphosphatase activity in vivo . Fructose 2,6-bisphosphate did not affect the rate of reaction.

J Virol, 1983 Sep, 47(3), 421 - 33
Molecular cloning and physical mapping of murine cytomegalovirus DNA; Ebeling A et al.; Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons . Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA) . The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI . In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184 . The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI . On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000 . All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen . We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions . Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.

Cell, 1983 Sep, 34(2), 641 - 6
The dnaK protein modulates the heat-shock response of Escherichia coli; Tilly K et al.; E . coli bacteria respond to a sudden upward shift in temperature by transiently overproducing a small subset of their proteins, one of which is the product of the dnaK gene . Mutations in dnaK have been previously shown to affect both DNA and RNA synthesis in E . coli . Bacteria carrying the dnaK756 mutation fail to turn off the heat-shock response at 43 degrees C . Instead, they continue to synthesize the heat-shock proteins in large amounts and underproduce other proteins . Both reversion and P1 transduction analyses have shown that the failure to turn off the heat-shock response is the result of the dnaK756 mutation . In addition, bacteria that overproduce the dnaK protein at all temperatures undergo a drastically reduced heat-shock response at high temperature . We conclude that the dnaK protein is an inhibitor of the heat-shock response in E . coli.

Proc Natl Acad Sci U S A, 1983 Sep, 80(17), 5235 - 9
DNA sequence of the Escherichia coli tonB gene; Postle K et al.; The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the tonB gene has been determined . Transcription initiation and termination sites for tonB RNA have been determined by S1 nuclease mapping . The tonB promoter and terminator resemble other E . coli promoters and terminators; the sequence of the tonB terminator region suggests that it may function bidirectionally . The DNA sequence specifies an open translation reading frame between the 5' and 3' RNA termini whose location is consistent with the position of previously isolated tonB::IS1 mutations . The DNA sequence predicts a proline-rich protein with a calculated size of 26.1-26.6 kilodaltons (239-244 amino acids), depending on which of three potential initiation codons is utilized . The predicted NH2 terminus of tonB protein resembles the proteolytically cleaved signal sequences of E . coli periplasmic and outer membrane proteins; the overall hydrophilic character of the protein sequence suggests that the bulk of the tonB protein is not embedded within the inner or outer membrane . A significant discrepancy exists between the calculated size of tonB protein and the apparent size of 36 kilodaltons determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis.

Proc Natl Acad Sci U S A, 1983 Sep, 80(17), 5225 - 9
Versatile cosmid vectors for the isolation, expression, and rescue of gene sequences: studies with the human alpha-globin gene cluster; Lau YF et al.; We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells . These cosmids were constructed by inserting one of the SV2-derived selectable gene markers--SV2-gpt, SV2-DHFR, and SV2-neo--in cosmid pJB8 . High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases . We isolated recombinant cosmids containing the human alpha-globin gene cluster from these genomic libraries . The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of alpha-globin genes in these cells . These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems . Both of the adult alpha-globin genes were more actively expressed than the embryonic zeta-globin genes in these transformed cell lines . Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA . Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

Proc Natl Acad Sci U S A, 1983 Sep, 80(17), 5198 - 202
Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells; Nielsen DA et al.; As an approach to the study of mammalian gene expression, the promoters and translation initiation regions of the rat preproinsulin II and the simian virus 40 early genes were fused to the structural gene of Escherichia coli beta-galactosidase, a sensitive probe for gene expression . These fusions were introduced into COS-7 cells, a simian virus 40 large tumor-antigen-producing monkey kidney cell line, where they directed the synthesis of enzymatically active hybrid beta-galactosidase proteins . Conditions for transfection were varied to optimize the expression of beta-galactosidase activity in the transfected cells . The pH optimum of this activity was found to be 7.0, the same as that of native E . coli beta-galactosidase and distinct from the major lysosomal "acid" beta-galactosidase . The fused preproinsulin-beta-galactosidase was further characterized by gel electrophoresis of nondenatured cell extracts stained by a fluorogenic substrate and by immunoprecipitation and gel electrophoresis of 3H-labeled cell proteins . These results all indicate that fully active tetrameric beta-galactosidase hybrids can be produced in mammalian cells . The expression of preproinsulin-beta-galactosidase activity was measured in the presence of high glucose, insulin, dexamethasone, or epidermal growth factor but no regulatory changes were observed.

J Bacteriol, 1983 Sep, 155(3), 973 - 82
Physical mapping of the exuT and uxaC operators by use of exu plasmids and generation of deletion mutants in vitro; Mata-Gilsinger M et al.; Operons uxaCA and exuT of the hexuronate system are very closely linked on the Escherichia coli genetic map . Using plasmid vectors constructed by Casadaban et al . (J . Bacteriol . 143:971-980, 1980), we formed exuT-lacZ and uxaA-lacZ fusions in vitro . The phenotypic properties of the new plasmids allowed us to confirm that the exuT and uxaCA operons are divergently transcribed . An analysis of these fusion plasmids and derivatives in the presence of multiple copies of the exuR regulatory gene demonstrated that the two operons possess separate control regions . The precise location of the operator site relative to endonuclease restriction sites was determined . In addition, deletions of different lengths were generated on exu plasmids by restriction enzymes and were recombined into the chromosome . The expression of the exu regulon genes in the resulting deletion mutants is in agreement with the postulated location of the exuT and uxaCA operators in the fusion plasmids.

J Bacteriol, 1983 Sep, 155(3), 966 - 72
A hybrid plasmid is a stable cloning vector for the cyanobacterium Anacystis nidulans R2; Golden SS et al.; Anacystis nidulans R2 is a highly transformable strain which is suitable as a recipient for molecular cloning in cyanobacteria . In an effort to produce an appropriate cloning vector, we constructed a hybrid plasmid molecule, pSG111, which contained pBR328 from Escherichia coli and the native pUH24 plasmid of A . nidulans . pSG111 replicated in and conferred ampicillin and chloramphenicol resistance to both hosts . It contained unique sites for the restriction enzymes EcoRI, SalI, SphI, and XhoI, which could be used for the insertion of exogenous DNA . To demonstrate that a molecule like pSG111 could serve as a shuttle vector for the cloning of A . nidulans genes, we constructed a hybrid plasmid, pRNA404, containing an A . nidulans rRNA operon . This recombinant molecule was genetically and structurally stable during passage through A . nidulans and E . coli . The stability of the hybrid plasmid and the inserted rRNA operon demonstrates the feasibility of cloning in A . nidulans with hybrid vectors, with the subsequent retrieval of cloned sequences.

J Bacteriol, 1983 Sep, 155(3), 1288 - 96
Structure and function of the internal promoter (hisBp) of the Escherichia coli K-12 histidine operon; Grisolia V et al.; The entire histidine operon of Escherichia coli K-12 was cloned in the vector plasmid pBR313, and a complete restriction map of the operon was determined . By using subclones, complementation tests, and enzyme assays, we were able to make a correlation between the physical map and the genetic map of the operon . We determined the sequence of a fragment of DNA 665 base pairs long, comprising the distal portion of the hisC gene, the proximal portion of the hisB gene, and the internal transcription initiation site hisBp . The efficiency of this promoter was assessed under different physiological conditions by cloning the DNA fragment in a recombinant vector system used to study transcriptional regulatory signals . The precise point at which transcription initiates was determined by S1 nuclease mapping.

J Bacteriol, 1983 Sep, 155(3), 1185 - 91
Nucleotide sequence of an incompatibility region of mini-Rts1 that contains five direct repeats; Kamio Y et al.; The plasmid mini-Rts1, consisting of an EcoRI/HindIII fragment of about 1.8 kilobases (kb), contains an incompatibility determinant in its EcoRI/AccI region (0.5 kb) . The nucleotide sequence of this incompatibility fragment was determined . A most striking feature of the sequence is the presence of five 24-base pair direct repeats . Four out of the five repeating units, which are contained in a 0.2-kb EcoRI/HincII fragment, were cloned en bloc in pACYC184 and found to express Rts1-specific incompatibility . In addition, the copy number of the mini-Rts1 plasmid appeared to be increased threefold upon removal of the 0.2-kb incompatibility region (incI) from the plasmid . This deletion derivative of mini-Rts1, as well as mini-Rts1, was maintained stably at 37 degrees C, but was cured at a high frequency at 42 degrees C . A possible role of the repeated nucleotide sequence was discussed . By subcloning the mini-Rts1 DNA, a second inc determinant (incII) was found on the AccI fragment, which is contiguous to the 0.5-kb EcoRI/AccI fragment.

J Bacteriol, 1983 Sep, 155(3), 1078 - 87
Cloning and mapping of the manganese superoxide dismutase gene (sodA) of Escherichia coli K-12; Touati D; An Escherichia coli gene bank composed of large DNA fragments (about 40 kilobases) was constructed by using the small cosmid pHC79 . From it, a clone was isolated for its ability to overproduce superoxide dismutase . The enzyme overproduced was manganese superoxide dismutase, as determined by electrophoresis and antibody precipitation . Maxicell analysis and two-dimensional O'Farrell polyacrylamide gel electrophoresis demonstrated that the structural gene, sodA, of manganese superoxide dismutase was cloned . Subcloning fragments from the original cosmid located the sodA gene within a 4.8-kilobase EcoRI-BamHI fragment . This fragment was inserted into a lambda phage which was deleted for the att region and consequently could only lysogenize by recombination between the cloned bacterial DNA insertion and the bacterial chromosome . Genetic mapping of the prophage in such lysogens indicated that the chromosomal sodA locus lies near 87 min on the E . coli map.

J Bacteriol, 1983 Sep, 155(3), 1071 - 7
Expression of a cloned K88ac adhesion antigen determinant: identification of a new adhesion cistron and role of a vector-encoded promoter; Kehoe M et al.; The determinant for the K88ac adherence antigen of porcine enterotoxigenic Escherichia coli has been cloned previously onto the vector plasmid pBR322 to form the K88ac-pBR322 hybrid plasmid pMK005 (M . Kehoe et al., Nature {London} 291:122-126) . Further studies on the expression of the K88ac antigen from pMK005 are presented in this paper . Expression was found to be dependent mainly on the P1 promoter of the pBR322 vector . The natural K88ac promoter was apparently not cloned from the original parental K88ac plasmid . The P1 promoter was deleted and replaced by a DNA sequence encoding the promoter-operator region of the E . coli tryptophan (Trp) operon . Cells harboring the Trp-pMK005 hybrid plasmid expressed high levels of K88ac antigen when the Trp promoter was repressed . If the promoter was derepressed either by growing the cells in low concentrations of tryptophan or in the presence of indole acrylic acid, growth of the cells harboring the Trp-pMK005 hybrid plasmid was inhibited . A quantitative assay was used to measure the levels of K88ac antigen expressed by cells harboring different pMK005::Tn5 plasmids . All cells were found to express a reduced level of K88ac antigen, providing evidence that a single transcription unit, initiating at promoter P1 of pBR322, may be involved in the expression of the K88ac antigen . By constructing specific deletion and insertion mutants of pMK005, a fifth adhesion cistron, tentatively named adhE, was identified and mapped at the proximal end of the K88ac determinant . Although the cistron is required for high-level expression of K88ac surface-associated fimbriae, as yet no gene product has been assigned to adhE.

J Bacteriol, 1983 Sep, 155(3), 1052 - 61
Genetic control of the hexose phosphate transport system of Escherichia coli: mapping of deletion and insertion mutations in the uhp region; Kadner RJ et al.; The Escherichia coli transport system responsible for the accumulation of a number of sugar phosphates is encoded by the uhp region and is induced by external, but not intracellular, glucose 6-phosphate . To delineate the genetic organization of the uhp region, a total of 225 independent point, deletion, and transposon Tn10 insertion mutations were collected . Mutations conferring the Uhp-phenotype were obtained on the basis of their resistance to fosfomycin and their inability to use sugar phosphates as carbon source . Deletions of uhp sequences were obtained as a consequence of imprecise excision of Tn10 insertions located on either side of uhp . Conjugal crosses between these deletions and the point of insertion mutations allowed determination of the relative order of the uhp alleles and of the deletion endpoints . Specialized lambda transducing phages carrying a uhpT-lac operon fusion and various amounts of adjacent uhp material were isolated and used as genetic donors . Results from these crosses corroborated those obtained in the conjugal crosses . The locations of the mutant alleles were compared with the regulatory properties of Uhp+ revertants of these alleles . This comparison suggested the existence of at least three genes in which mutation yields the Uhp-phenotype . Mapping experiments were consistent with the gene order pyrE-gltS-uhpTRA-ilvB, where uhpT encodes the transport system and uhpR and uhpA are regulatory genes whose products are necessary for proper uhp regulation.

Infect Immun, 1983 Sep, 41(3), 971 - 7
Stimulation of calcium uptake and cyclic GMP synthesis in rat basophilic leukemia cells by Escherichia coli heat-stable enterotoxin; Knoop FC et al.; The effect of Escherichia coli heat-stable (ST) enterotoxin on calcium and cyclic nucleotide metabolism in rat basophilic leukemia cell cultures was investigated . Addition of ST enterotoxin to rat basophilic leukemia cell cultures resulted in dose- and time-dependent stimulation of calcium uptake and elevation of the intracellular cyclic GMP (cGMP) concentration . The effect of ST enterotoxin on calcium uptake (P less than 0.02) and cGMP synthesis (P less than 0.02) was demonstrated after 5 and 30 min of incubation at 37 degrees C, respectively . In further studies ST enterotoxin did not enhance calcium release or the intracellular concentration of cyclic AMP . The stimulation of calcium uptake and cGMP synthesis by ST enterotoxin was inhibited by pharmacological and chemical agents which block cellular calcium entry and prostaglandin synthesis . These results demonstrate that ST enterotoxin induces calcium uptake and cGMP synthesis in rat basophilic leukemia cell cultures.

Yale J Biol Med, 1983 Sep-Dec, 56(5-6), 643 - 8
Interaction of mycoplasmas and phagocytes; Howard CJ et al.; Aspects of the interaction of certain mycoplasmas with macrophages and neutrophils in vivo and in vitro have been studied using two systems, one involving M . pulmonis in mice and the other involving M . bovis with bovine leucocytes . Studies with M . pulmonis indicated that the disappearance of viable organisms from the peritoneal cavity was not enhanced in SPF mice in which a peritoneal exudate rich in neutrophils had been induced . However, viable M . pulmonis organisms disappeared more rapidly from the peritoneal cavities with exudates containing increased numbers of macrophages . Experiments in vitro studied the opsonic effect of bovine IgG isotypes for bovine neutrophils and alveolar macrophages . Both IgG1 and IgG2 promoted killing of M . bovis by alveolar macrophages but IgG2 was more effective than IgG1 at promoting mycoplasma killing by neutrophils . Further studies in vitro indicated that certain bovine mycoplasma could inhibit killing of Escherichia coli by bovine neutrophils.

Chem Biol Interact, 1983 Sep 1, 46(2), 219 - 32
Platinum complexes with anticancer potential and their evaluation by a colorimetric lambda prophage induction assay; Das Sarma B et al.; A simple biochemical phage induction assay (BIA) showed significant activity with 90% of the antitumor platinum compounds tested and lack of activity for all Pd(II) compounds and Pt(II) cationic complexes, compounds that are expected to be inactive . Structure-activity relationships for a large number of chemicals can be studied simultaneously by this simple, rapid, inexpensive and quantitative biochemical assay . Fifty-three platinum complexes were tested, including a number of ethylenediamines synthesized for this work . The magnitude of inducing activity varied over a 25-fold range; differences among analogs reflected structural differences in a chemically consistent manner . Seven platinum complexes showed greater activity than that of cis-diamminedichloroplatinum(II) (cisplatin, cis-DDP), while other compounds appeared to be substantially less toxic . The assay was predictive for most compounds with very high or very low activity in vivo against L1210 . For compounds with intermediate levels of activity, no correlation between inducing and antitumor activity was observed.

Proc Natl Acad Sci U S A, 1983 Sep, 80(18), 5540 - 4
Termination sites of the in vitro nick-translation reaction on DNA that had photoreacted with psoralen; Piette JG et al.; A double-stranded circular DNA having a single nick at a specific site has been photochemically induced to react with 4'-hydroxymethyl-4,5', 8-trimethylpsoralen (HMT) and used as a substrate for nick-translation with Escherichia coli DNA polymerase I holoenzyme . By using the dideoxy chain-terminating sequencing procedure, it was possible to map the termination sites observed on the template that had photoreacted with HMT . These sites occur at nucleotides preceding potential psoralen crosslinking sites . Analysis of DNA products synthesized on templates that had photoreacted under conditions designed to maximize psoralen monoaddition revealed that DNA polymerase I is not stopped by this lesion . Psoralen monoadducts situated on the template strand act only as kinetic attenuators, whereas psoralen monoadducts localized on the nick-translated strand have no effect on the rate of synthesis . These data suggest that psoralen crosslinks are responsible for the lethal effects of psoralen photochemistry in E . coli . Mutagenesis may be associated, however, with the repair replication of psoralen monoadducts by E . coli DNA polymerase I.

J Bacteriol, 1983 Sep, 155(3), 1265 - 70
Cloning and expression of uncI, the first gene of the unc operon of Escherichia coli; Brusilow WS et al.; The unc operon of Escherichia coli consists of eight genes coding for the eight subunits of the proton-translocating ATPase . In vitro transcription-translation of DNA cloned from the beginning of the operon onto plasmids reveals that the reading frame uncI, which precedes the other genes of the operon, codes for a protein with a molecular weight of 14,500, called i . In minicells, the i protein is synthesized in amounts comparable to the amounts of the ATPase subunits, suggesting that it may be part of the ATPase complex . The presence of the unc promoter and uncI on a plasmid containing the other eight genes of the unc operon has little effect on the differential expression of the unc genes or the partitioning of the newly synthesized subunits into soluble or sedimentable fractions in the in vitro system . The i protein partitions into the sedimentable fraction.

Yale J Biol Med, 1983 Sep-Dec, 56(5-6), 357 - 66
Characterization of the mycoplasma genome; Razin S et al.; Recent advances on the properties of the mycoplasma genome, including size, base composition, replication, extrachromosomal DNA, and transfer of genetic material are briefly reviewed, with emphasis on their phylogenetic implications . The use of cleavage patterns of the mycoplasma genome by restriction endonucleases as "finger-prints" indicating genetic relatedness among strains is discussed . The data support the notion that strains of mycoplasma species of strict host and tissue specificity exhibit marked genetic homogeneity, suggesting a clonal origin for some species . The regions of the mycoplasma genome carrying the ribosomal RNA (rRNA) genes have been studied using restriction endonucleases, cloning, and hybridization procedures . The mycoplasmal rRNA cistrons cross-hybridized among themselves, and with the seven rRNA cistrons of Escherichia coli, demonstrating the marked conservation of structure during evolution of this part of the procaryotic genome . In most of the mollicutes tested so far the number of rRNA cistrons is two, but a few species appear to carry only one rRNA cistron in their genome.

Mol Biol (Mosk), 1983 Sep-Oct, 17(5), 958 - 64
{Stringent control of relA gene transcription in cells of Escherichia coli}; Bochkanov SS et al.; Regulation of E . coli K12 relA gene transcription was studied . The rate of relA-RNA synthesis was measured by RNA-DNA-hybridization technique using cloned fragment of relA gene . Under seryl-tRNA deficiency the rate of relA-RNA synthesis was reduced six times in the strain CP78 (relA+) and only two times in its' relA-variant CP79 . Chloramphenicol addition stimulated the rate of relA-RNA synthesis in starved relA+ cells . It was concluded that relA gene transcription is under "stringent control" . The rate of relA-RNA synthesis increases proportionally with bacteria growth rate . Grown in glucose-minimal media strain with several copies of relA gene exhibits elevated ppGpp level (50 pmol/A450) and reduced rate of relA-RNA synthesis per one copy of relA gene . Thus, relA gene transcription is under negative regulation of ppGpp.

Gene, 1983 Sep, 23(3), 255 - 65
Expression plasmid vectors containing Escherichia coli tryptophan promoter transcriptional units lacking the attenuator; Tacon WC et al.; Two DNA fragments which contain the Escherichia coli tryptophan promoter operator region but lack the attenuator have been used in the construction of a series of pAT153 based plasmids suitable for the regulated expression of foreign genes in E . coli . The first, a 139-bp HhaI fragment includes 59 bp of the trp leader sequence, ending within the "attenuator peptide" coding sequence, eleven codons from the N-terminus . A fusion-type expression plasmid incorporating this fragment has been constructed . The second, a 99-bp HaeIII-TaqI fragment contains no coding sequence but includes the "attenuator peptide" SD site situated 4 bp upstream of the TaqI site . This fragment has been incorporated in expression vectors which result in the direct expression of cloned gene sequences . To further maximise expression, plasmids with directly repeating trp promoter HaeIII-TaqI units have been constructed.

J Bacteriol, 1983 Sep, 155(3), 1382 - 92
Monoclonal antibodies reveal lamB antigenic determinants on both faces of the Escherichia coli outer membrane; Schenkman S et al.; LamB protein is involved in the transport of maltose across the outer membrane and constitutes the receptor for phage lambda . In this study we characterized six previously described anti-LamB monoclonal antibodies (mAbs) . Four of these, the E-mAbs, recognized determinants that were exposed at the cell surface, whereas the other two, the I-mAbs, recognized determinants which were not exposed . Competition experiments demonstrated that the domains recognized by these two classes of mAbs were completely distinct . In addition, the E-mAbs prevented LamB from neutralizing phage lambda in vitro and protected LamB against proteolytic degradation, whereas the I-mAbs had no such effects . The E-mAbs have been shown previously to constitute two classes: some E-mAbs inhibit maltose transport in vivo, and others do not . Immunoelectron microscopy demonstrated that the I-mAbs also define at least two types of determinants . One of these, which is accessible in membrane fragments from a mutant (lpp) devoid of lipoprotein but not in membrane fragments from an lpp+ strain, probably corresponds to a region of LamB that is involved in the interactions with peptidoglycan . The other determinant, which is fully accessible in LamB-peptidoglycan complexes and in LamB-containing phospholipid vesicles but only slightly accessible in membrane fragments from an lpp mutant, is probably located very close to the inner surface of the outer membrane . LamB also contains at least one additional determinant, which (i) is exposed at the inner surface of the membrane, (ii) is accessible to antibodies in membrane fragments from an lpp+ strain, and (iii) may be involved in the interaction of LamB with the periplasmic maltose-binding protein.

J Bacteriol, 1983 Sep, 155(3), 1279 - 87
In vivo 5' terminus and length of the mRNA for the proton-translocating ATPase (unc) operon of Escherichia coli; Jones HM et al.; The promoter for the proton-translocating ATPase (unc) operon of Escherichia coli was localized by using a plasmid promoter-screening vector system . S1 nuclease analysis, using the appropriate single-stranded DNA probe from this promoter region and in vivo mRNA, revealed that the 5' end of the in vivo unc mRNA initiates with a guanine residue 73 bases before the start of the proposed gene 1 or 474 bases before uncB . An in vivo unc mRNA species of approximately 7,000 nucleotides in length which initiates in the unc promoter region was shown to exist by RNA-DNA hybridization analysis . This unc mRNA species (based on DNA sequence analysis) is sufficient in length to contain all nine genes, gene 1 and uncBEFHAGDC . That gene 1 is cotranscribed with the unc genes was confirmed by using hybridization probes containing the promoter-proximal (gene 1) or -distal gene (uncC) . No strong internal promoters within the unc operon were revealed with either the promoter-screening vector system or the RNA-DNA hybridization analysis . The 5' terminus and the length of the unc mRNA were found to be identical in cells grown either aerobically or anaerobically . The level of unc operon expression, as assayed with the unc promoter plasmid, did not significantly differ when cells bearing the plasmid were grown either aerobically or anaerobically.

J Bacteriol, 1983 Sep, 155(3), 1271 - 8
Promoter for the unc operon of Escherichia coli; Porter AC et al.; Fragments of DNA carrying possible promoters for the unc operon of Escherichia coli were cloned into a promoter detection plasmid (pRZ5255) . Similar fragments were transcribed in vitro to produce transcripts whose sizes were used to determine the approximate start site for transcription . One strong promoter and at least two very much weaker ones were detected by these methods . The exact position of the strongest promoter, presumed to be the true unc promoter, was determined by S1 nuclease mapping and shown to lie 73 base pairs upstream from the open reading frame that precedes uncB . It therefore appears that this reading frame (uncI) is part of the unc operon . S1 mapping also revealed the presence of a third weak promoter 25 base pairs upstream of uncI . All of the weak promoters occur between the proposed unc promoter and uncB, but their role in vivo, if any, is unclear.

Res Vet Sci, 1983 Sep, 35(2), 234 - 9
Passive immunisation of neonatal lambs against infection with enteropathogenic Escherichia coli via colostrum of ewes immunised with crude and purified K99 pili; Altmann K et al.; Lambs sucking ewes immunised four to five weeks before parturition with crude preparations of K99 and purified K99 pili of single subunit composition were protected against challenge infection with heterologous enteropathogenic Escherichia coli strains . In contrast, the majority of lambs sucking sham-immunised ewes suffered severe diarrhoea and dehydration, followed by death in nearly half of the affected lambs . Protection was related to the presence of antibody in the colostral whey and lamb sera . K99-specific antibody activity in the colostral whey was found to be confined to IgM and IgG (IgG1 and IgG2) but not to the IgA class.

J Gen Microbiol, 1983 Sep, 129 (Pt 9), 2753 - 9
The production of F41 fimbriae by piglet strains of enterotoxigenic Escherichia coli that lack K88, K99 and 987P fimbriae; Morris JA et al.; The enterotoxigenic Escherichia coli strains 1676, 1706, 1751 and KEC96a, which do not produce fimbrial adhesive antigens of the K88, K99 or 987P antigen type reacted both in vitro and in vivo with antiserum to F41 fimbriae in an indirect immunofluorescent antibody technique . Antiserum used to demonstrate material B, an adhesive antigen thought to mediate the adhesive and mannose-resistant (MR) haemagglutinating properties of E . coli strains 1676, 1706 and 1751, reacted in vitro with an F41+ strain . The antiserum also inhibited the MR haemagglutinating activity of F41 antigen and gave an anionic precipitation line in immunoelectrophoresis experiments with an extract containing F41 antigen . The MR haemagglutinating properties of an antigen extract containing material B from E . coli strain 1706 was neutralized by antiserum to F41 fimbriae and by OK antisera to E . coli strains that produce both F41 and K99 fimbriae . These sera also gave an anionic precipitation line with the MR haemagglutinin from E . coli strain 1706 and the MR haemagglutinin gave a line of identity with F41 in gel diffusion experiments with antiserum to F41 fimbriae . OK antisera to K99+ F41- bacteria and OK antisera to K88+ bacteria and 987P+ bacteria did not react with this haemagglutinin . Transmission electron microscopy on the ileum of newborn gnotobiotic piglets infected with E . coli strain 1706 showed irregular, poorly defined filamentous material surrounding some,though not all, bacteria but regular fimbrial structures were not visible.(ABSTRACT TRUNCATED AT 250 WORDS)

Infect Immun, 1983 Sep, 41(3), 942 - 9
Genetics of digalactoside-binding adhesin from a uropathogenic Escherichia coli strain; Normark S et al.; The uropathogenic strain Escherichia coli J96 mediates mannose-resistant hemagglutination owing to production of a digalactoside-binding adhesin . A cosmid clone from this strain has been isolated that, when harbored in E . coli K-12, expressed Pap pili and this adhesin (R . Hull et al., Infect . Immun . 33:933-938, 1981) . By transposon mutagenesis and by the construction of a number of hybrid plasmid derivatives, we have demonstrated that about 8.5 kilobases of DNA is required to generate a mannose-resistant hemagglutination-positive phenotype in E . coli K-12 strain P678-54 . The structural gene for the Pap pili monomer, papA, has been identified and mapped close to the promotor-proximal end of the Pap operon . Although strain P678-54 that harbored a Tn5 insertion within papA showed a mannose-resistant hemagglutination-positive phenotype, it was negative in a competitive enzyme-linked immunosorbent assay with anti-Pap pilus serum . This could mean that a Pap adhesin is encoded by a region on the Pap operon that is distinct from papA.

Infect Immun, 1983 Sep, 41(3), 1368 - 9
Effect of sodium acetate on expression of K99 pili by Escherichia coli; Francis DH et al.; Sodium acetate suppressed K99 production in Escherichia coli strains cultured on a minimal medium, as determined by seroagglutination and enzyme-linked immunosorbent assay . The greatest suppression occurred when the medium contained both sodium acetate and glucose . Glucose alone did not suppress K99 production.

Infect Immun, 1983 Sep, 41(3), 1296 - 301
Monoclonal antibodies against colonization factor antigen I pili from enterotoxigenic Escherichia coli; Worobec EA et al.; Hybridomas secreting monoclonal antibodies directed against intact colonization factor antigen I pili have been produced by the fusion of spleen cells from immunized BALB/c mice with NS1/SP2 myeloma cells . The four monoclones with the highest antibody titer, as detected by enzyme-linked immunosorbant assay (ELISA), were chosen for antibody amplification by production of mouse ascitic fluid . These four were examined for antibody specificity by ELISA and immunoblot assays, using six different pilus types . Three of the four monoclonal isolates were specific for only colonization factor antigen I pili in both assays, whereas the remaining isolate showed a distinct cross-reactivity with K99 pili in the ELISA assay but not in immunoblot analysis . These results indicate that this monoclone may be recognizing a common structural element between the two adhesive pilus types.

Biochemistry, 1983 Aug 30, 22(18), 4192 - 7
Urea--DNA glycosylase in mammalian cells; Breimer LH; Urea-DNA glycosylase, an enzyme presumed to be active in the repair of DNA damage caused by oxidizing agents, has been identified previously in Escherichia coli . This enzyme has now been shown to be present in cell extracts of calf thymus and human fibroblasts . It catalyzes the release of free urea from a double-stranded polydeoxyribonucleotide containing thymine residues fragmented by KMnO4 and NaOH treatment . The calf thymus enzyme has been 400-fold purified and largely separated from previously identified mammalian DNA glycosylases . It has a molecular weight of about 25 000 and requires no cofactors . The identity of the enzymatically released product as unsubstituted urea has been verified by its susceptibility to urease.

Biochemistry, 1983 Aug 30, 22(18), 4310 - 5
Ribonucleic acid-protein cross-linking within the intact Escherichia coli ribosome, utilizing ethylene glycol bis{3-(2-ketobutyraldehyde) ether}, a reversible, bifunctional reagent: identification of 30S proteins; Brewer LA et al.; To obtain detailed topographical information concerning the spatial arrangement of the multitude of ribosomal proteins with respect to specific sequences in the three RNA chains of intact ribosomes, a reagent capable of covalently and reversibly joining RNA to protein has been synthesized {Brewer, L.A., Goelz, S., & Noller, H . F . (1983) Biochemistry (preceding paper in this issue)} . This compound, ethylene glycol bis{3-(2-ketobutyraldehyde) ether} which we term "bikethoxal", possesses two reactive ends similar to kethoxal . Accordingly, it reacts selectively with guanine in single-stranded regions of nucleic acid and with arginine in protein . The cross-linking is reversible in that the arginine- and guanine-bikethoxal linkage can be disrupted by treatment with mild base, allowing identification of the linked RNA and protein components by standard techniques . Further, since the sites of kethoxal modification within the RNA sequences of intact subunits are known, the task of identifying the components of individual ribonucleoprotein complexes should be considerably simplified . About 15% of the ribosomal protein was covalently cross-linked to 16S RNA by bikethoxal under our standard reaction conditions, as monitored by comigration of 35S-labeled protein with RNA on Sepharose 4B in urea . Cross-linked 30S proteins were subsequently removed from 16S RNA by treatment with T1 ribonuclease and/or mild base cleavage of the reagent and were identified by two-dimensional polyacrylamide gel electrophoresis . The major 30S proteins found in cross-linked complexes are S4, S5, S6, S7, S8, S9 (S11), S16, and S18 . The minor ones are S2, S3, S12, S13, S14, S15, and S17.

Biochemistry, 1983 Aug 30, 22(18), 4303 - 9
Ribonucleic acid-protein cross-linking within the intact Escherichia coli ribosome, utilizing ethylene glycol bis{3-(2-ketobutyraldehyde) ether}, a reversible, bifunctional reagent: synthesis and cross-linking within 30S and 50S subunits; Brewer LA et al.; We have used the reversible, bifunctional reagent ethylene glycol bis{3-(2-ketobutyraldehyde) ether} to cross-link RNA to protein within intact ribosomal subunits from Escherichia coli . Here we describe the synthesis of this compound (termed bikethoxal) and demonstrate its ability to form covalent attachments between RNA and protein in the 5S RNA-L18 complex and within 30S and 50S ribosomal subunits . The reagent is a symmetrical dicarbonyl compound and reacts with guanine in single-stranded RNA and with arginine in protein . RNA-protein cross-links generated with this reagent are stable, as demonstrated by the comigration of 35S-labeled ribosomal proteins with ribosomal RNA on neutrally buffered sodium dodecyl sulfate (SDS)-agarose gels . However, the cross-linked product is unstable in mildly basic conditions, allowing the identification of the linked macromolecules by conventional techniques . The reagent is potentially capable of cross-linking any combination of single-stranded RNA, single-stranded DNA, or protein; it should prove a useful probe of the RNA-protein proximities within the E . coli ribosome, since the SDS-agarose gel system we describe provides a rapid method of optimizing this RNA--protein cross-linking reaction.

Biochemistry, 1983 Aug 30, 22(18), 4272 - 5
A study of the quenching of the intrinsic fluorescence of succinyl-CoA synthetase from Escherichia coli by acrylamide, iodide, and coenzyme A; Prasad AR et al.; Escherichia coli succinyl-CoA synthetase (SCS) contains three tryptophan residues per mole of alpha beta dimer, and all of them are on the beta subunit . SCS shows an emission maximum at 335 nm which is shifted to 350 nm upon denaturation by urea or guanidine hydrochloride . Acrylamide is able to quench the tryptophan fluorescence in SCS by static and dynamic mechanisms . Substrates give protection against quenching by acrylamide . Binding of ATP to the alpha subunit which has no tryptophans gives as large an effect on the quenching by acrylamide as the binding of coenzyme A (CoA) to the beta subunit . Addition of CoA eliminates the curvature observed in Stern-Volmer plots for acrylamide quenching obtained by lifetime measurements . Potassium iodide does not quench the SCS fluorescence in the presence of CoA . These results suggest that there are heterogeneously emitting tryptophan residues in SCS that are located at the alpha beta subunit contact region close to the CoA binding site . Hence, the tryptophan residues can act as intrinsic reporters of events taking place at the active site of this enzyme . Further, the present results support models for SCS that put the active site at the alpha beta subunit contact region.

Biochem Biophys Res Commun, 1983 Aug 30, 115(1), 1 - 7
Spinach chloroplast thioredoxins in evolutionary drift; Tsugita A et al.; The amino acid sequences surrounding the active sites of spinach chloroplast thioredoxins m and f have been determined . Both types of thioredoxins share common ancestor genes with the E . coli one, demonstrated by invariant active site sequences . The m-type thioredoxins have closer homology with the E . coli one in the sequence analyzed as well as in enzymatic specificity, whereas the f-type is less homologous both in sequence and specificity . It suggests that the m-type gene represents a prototype conserved throughout evolutionary processes whereas the f-type has undergone mutations resulting in a modified specificity.

J Chromatogr, 1983 Aug 26, 266, 225 - 37
Reversed-phase high-performance liquid chromatography of Escherichia coli ribosomal proteins . Characteristics of the separation of a complex protein mixture; Kerlavage AR et al.; We have previously reported the application of reversed-phase high-performance liquid chromatography (RP-HPLC) to the separation of Escherichia coli ribosomal proteins (A . R . Kerlavage, L . Kahan and B . S . Cooperman, Anal . Biochem., 123 (1982) 342-348; A . R . Kerlavage, T . Hasan and B . S . Cooperman, J . Biol . Chem., in press) . In the present studies RP-HPLC is shown to yield much greater resolution of these proteins than does size-exclusion HPLC . In addition, we report on various aspects of RP-HPLC of ribosomal proteins including column capacity, resolution, reproducibility, recovery, separation of irreversibly denatured protein, and analysis of affinity-labeled ribosomal protein . The capacity of analytical columns was found to range from several micrograms to several milligrams with minimal loss in resolution and highly reproducible retention values . Recovery varied from protein to protein and ranged from 27% to 91%, with an average total protein recovery of 70% . The partitioning of several proteins between two peaks was shown to be due to irreversible denaturation of a small fraction . Finally, the utility of RP-HPLC in the study of the ribosome was demonstrated by analyses of {3H}puromycin-labeled ribosomal proteins, and the demonstration that labeling slightly alters protein elution.

Pharm Weekbl Sci, 1983 Aug 26, 5(4), 145 - 8
Determination of glutathione in biological material by high pressure liquid chromatography; Baars AJ et al.; A rapid and selective determination of reduced glutathione in biological material is described, based on its conjugation with 1-chloro-2,4-dinitrobenzene . The reaction product has a UV absorption maximum at 340 nm and is analysed by reversed phase high pressure liquid chromatography . Linear calibration graphs were obtained in the concentration range between 50 microM and 2 mM glutathione in standard solutions and in biological material (rat liver and bacterial homogenates) . The detection limit is about 2 microM glutathione when using 20 microliters injection samples.

J Chromatogr, 1983 Aug 26, 266, 385 - 94
New ion exchanger for the separation of proteins and nucleic acids; Kato Y et al.; The basic properties of the new weak anion exchanger TSK-GEL IEX-645 DEAE and applications to the separations of proteins and nucleic acids were investigated . IEX-645 DEAE was found to be very versatile for high-performance ion-exchange chromatography of biopolymers . It was especially superior in applications at high pH and to high-molecular-weight samples.

J Chromatogr, 1983 Aug 26, 266, 359 - 83
High-performance liquid chromatography of transfer RNAs . Separation of transfer RNAs from mammalian sources; Singhal RP; A survey of recent advances in high-performance liquid chromatography (HPLC) of tRNA is presented here . The polystyrene and reversed-phase anion exchangers are discussed for their ability to resolve tRNAs without loss of the aminoacyl-tRNA bond . The HPLC of a tRNA of choice, based on the affinity principle, is studied . Both chemical (boronate) and biological (plant lectins) affinity groups for the tRNA interaction are described . A comprehensive scheme is presented for the separation of four mammalian tRNAs . Scope of future research in this area is also discussed.

Nucleic Acids Res, 1983 Aug 25, 11(16), 5589 - 602
The binding of ribosomal protein S1 to S1-depleted 30S and 70S ribosomes . A fluorescence anisotropy study of the effects of Mg2+; Goss DJ et al.; We have determined the equilibrium constants for the binding of AEDANS-labelled S1 to S1-depleted 30S and 70S ribosomes . For "tight" ribosomes, the association of S1 increases with the sixth power of Mg2+ concentration, but for 30S subunits and "loose" ribosomes, there is virtually no dependence of the association on Mg2+ over the same concentration range, 2-10 mM in Mg2+ . The binding of S1 to 70S ribosomes at 10 mM Mg2+ is stabilized by 2 kcal/mol compared to the binding to 30S subunits . When intact S1 binds to tight ribosomes, the fluorescence anisotrophy is that for virtually complete rotational immobilization . The anisotropies vary considerably with the preparation and treatment of both S1 and ribosomes and these variations are detailed here . The results suggest the linkage of Mg2+-dependent conformational changes in the intact ribosomes, perhaps including rRNA, and the binding of S1.

J Mol Biol, 1983 Aug 25, 168(4), 729 - 45
Analysis of two purified mutants of Escherichia coli aspartate transcarbamylase with single amino acid substitutions; Silver RS et al.; Two mutant versions of Escherichia coli aspartate transcarbamylase have been purified and analyzed kinetically . Each of these mutant enzymes contains a single amino acid different from the wild-type enzyme, which was introduced by suppression of a nonsense codon within the E . coli pyrB gene . These enzymes exhibited alterations in both homotropic and heterotropic interactions with little change in specific activity . Depending upon the site of the substitution, the allosteric interactions have been either enhanced or diminished over the wild-type enzyme . Carbamyl phosphate saturation curves indicate that aspartate and carbamyl phosphate homotropic co-operativity are separable . Experiments employing the allosteric effectors indicate that the transmission of the regulatory effect is dependent upon the structure of the catalytic subunit, and that CTP inhibition can be partially decoupled from ATP activation . The kinetics of one of the mutants is unusually sensitive to dissociation at elevated temperatures . This sensitivity may be due to weakened interactions between the subunits of the enzyme.

J Biol Chem, 1983 Aug 25, 258(16), 9780 - 5
The large subunit of the fatty acid oxidation complex from Escherichia coli is a multifunctional polypeptide . Evidence for the existence of a fatty acid oxidation operon (fad AB) in Escherichia coli; Yang SY et al.; The subunit locations of the five enzymes associated with the fatty acid oxidation complex from Escherichia coli were studied by immunotitration and chemical modification . Antibodies raised against the purified complex caused the parallel inhibitions of enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase, while slightly stimulating 3-ketoacyl-CoA thiolase . All five component enzymes of the complex were inactivated by treatment with iodoacetamide . The inactivation of 3-ketoacyl-CoA thiolase was rapid, whereas the four other enzymes were inactivated at much slower, but almost equal rates . All enzymes except for 3-ketoacyl-CoA thiolase were protected against this inactivation by either NADH or crotonyl-CoA . The reaction of iodo{1-14C}acetamide with the complex in the presence and absence of NADH resulted in the differential labeling of the large subunit only . These observations together with published results (Pawar, S., and Schulz, H . (1981) J . Biol . Chem . 256, 3894-3899) lead to the suggestion that enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, cis-delta 3-trans-delta 2-enoyl-CoA isomerase, and 3-hydroxyacyl-CoA epimerase are located on the 78,000-Da subunit, whereas 3-ketoacyl-CoA thiolase is associated with the 42,000-Da subunit . Additionally, this study provides further evidence for the existence of a fatty acid oxidation (fad AB) operon that codes for the multienzyme complex of fatty acid oxidation and that is located at 85 min on the E . coli chromosome.

J Biol Chem, 1983 Aug 25, 258(16), 10098 - 103
Ribosomal protein L1 from Escherichia coli . Its role in the binding of tRNA to the ribosome and in elongation factor g-dependent gtp hydrolysis; Sander G; Two Escherichia coli mutants lacking ribosomal protein L1, previously shown to display 40 to 60% reduced capacity for in vitro protein synthesis (Subramanian, A . R., and Dabbs, E . R . (1980) Eur . J . Biochem . 112, 425-430), have been used to study partial reactions of protein biosynthesis . Both the binding of N-acetyl-Phe-tRNA to ribosomes and the 6 to 8-fold stimulation of the elongation factor G (EF-G)-dependent GTPase reaction by mRNA plus tRNA, assayed in the presence of wild type 30 S subunits, were low with L1-deficient 50 S subunits . Addition of pure protein L1 to the assay restored both reactions to 100% of the control . By contrast, the basic EF-G GTPase reaction in the absence of mRNA and tRNA was not at all affected (mRNA alone had no effect) . None of the following partial reactions were more than moderately modified by the lack of protein L1: binding to ribosomes of EF-G.GDP plus fusidic acid; the translocation reaction catalyzed by EF-G plus GTP; poly(U)-dependent binding to ribosomes of Phe-tRNAPhe (whether dependent on elongation factor Tu plus GTP or not); and the EF-Tu-dependent GTPase activity . It is concluded that protein L1 is involved in the interaction between ribosomes and peptidyl-tRNA (or tRNA) in the peptidyl site and consequently in the ribosomal GTPase activity depending on the simultaneous action of tRNA and EF-G.

Nucleic Acids Res, 1983 Aug 25, 11(16), 5775 - 91
Maintenance and incompatibility of plasmids carrying the replication origin of the Escherichia coli chromosome: evidence for a control region of replication between oriC and asnA; Stuitje AR et al.; Plasmids that replicate only by means of the cloned Escherichia coli replication origin (oriC) are called minichromosomes or oriC-plasmids . In this paper it is shown that sequences located between oriC and asnA are involved in maintenance and incompatibility of minichromosomes . These sequences include part of the 16kD and 17kD genes, previously allocated within this region (1,2) . Transcription towards oriC that is initiated at the 16kD promoter, specifically enhances the stability and copy-number of minichromosomes . Three regions are involved in minichromosome incompatibility . One region, incA, includes the minimal oriC sequence . A second, incB, maps within a 210 base pairs fragment that overlaps the 16kD promoter . The third, incC, encompasses the 17kD gene . Neither one of the regions expresses incompatibility on its own, but the additional presence of one of the others is required . The data presented indicate that sequences of the 16kD and 17kD genes are part of the replication control system of oriC-plasmids.

Nucleic Acids Res, 1983 Aug 25, 11(16), 5645 - 59
The nucleotide sequence of replication and maintenance functions encoded by plasmid pSC101; Churchward G et al.; The nucleotide sequence of 1100bp around the origin of replication of the pSC101 plasmid has been determined . This segment of DNA is capable of replication in the presence of a helper plasmid . The sequence data reveal similarities between pSC101 and several other replicons . The origin of replication contains three direct repeats of an 18bp sequence associated with a segment exceptionally rich in A-T base pairs . A promotor that probably directs transcription of a gene encoding an essential plasmid replication function is associated with a region of extensive potential secondary structure . The sequence presented here includes the sequence of the par region involved in partitioning of plasmids at cell division.

Nucleic Acids Res, 1983 Aug 25, 11(16), 5629 - 43
The nucleotide sequence of poliovirus type 3 leon 12 a1b: comparison with poliovirus type 1; Stanway G et al.; The complete nucleotide sequence of the genome of the Sabin vaccine strain of poliovirus type 3 (P3/Leon 12 a1 b) has been determined from cDNA cloned in E . coli . The genome comprises a 5' non-coding region of 742 nucleotides, a large open reading frame of 6618 nucleotides (89% of the sequence) and a 3' non-coding region of 72 nucleotides . There is 77.4% base-sequence homology and 89.6% predicted amino-acid homology between types 1 and 3 . Conservation of all glutamine-glycine and tyrosine-glycine cleavage sites suggests a mechanism of polyprotein processing similar to that established for poliovirus type 1.

J Biol Chem, 1983 Aug 25, 258(16), 9963 - 7
A trans-acting regulatory mutation that causes overproduction of phosphatidylserine synthase in Escherichia coli; Sparrow CP et al.; We have isolated three mutants of Escherichia coli which have elevated levels of the phospholipid synthetic enzyme phosphatidylserine synthase . One of these strains carries a mutation, designated pssR1, which maps near minute 84 of the chromosome, distinct from the synthase structural gene (pss) at minute 56 . The pssR1 mutation causes selective overproduction of phosphatidylserine synthase, since the levels of six other lipid synthetic enzymes are unaltered . The specific activity of the synthase in crude cell extracts of mutants harboring pssR1 is about five times greater than wild type . The synthase can also be overproduced 10-fold in wild type strains with hybrid ColE1 plasmids carrying the synthase structural gene (pss) . A pssR1 mutant harboring such a pss plasmid overproduces the synthase about 50-fold . This multiplicative interaction of pssR1 and cloned pss demonstrates that pssR1 is trans-acting . The synthase has been purified in parallel from pssR1 and pssR+ strains . The pssR1 mutant yields more total synthase protein than pssR+, but the pure enzyme has the same specific activity in both cases . Therefore, pssR1 acts by increasing the amount of the normal protein, not by activating the enzyme . The discovery of pssR shows that there are regulatory loci which control the production of enzymes involved in membrane lipid synthesis.

J Biol Chem, 1983 Aug 25, 258(16), 10136 - 43
Assembly of a functional F0 of the proton-translocating ATPase of Escherichia coli; Klionsky DJ et al.; We have investigated both structural and functional assembly of the F0 portion of the Escherichia coli proton-translocating ATPase in vivo . Fractionation of E . coli minicells containing plasmids which code for parts of the unc operon shows that each of the F0 peptides a, b, and c insert into the cytoplasmic membrane independent of each other and without the polypeptides which form the F1 portion of the complex alpha, beta, gamma, delta, and epsilon . Assays of membrane energization indicate that, while formation of a functional proton channel requires the presence of all three F0 polypeptides a, b and c, they are not sufficient . Synthesis of both the alpha and beta subunits of the F1 are required for formation of a functional proton channel.

Nucleic Acids Res, 1983 Aug 25, 11(16), 5497 - 520
Gene expression in rat brain; Milner RJ et al.; 191 randomly selected cDNA clones prepared from rat brain cytoplasmic poly (A)+ RNA were screened by Northern blot hybridization to rat brain, liver and kidney RNA to determine the tissue distribution, abundance and size of the corresponding brain mRNA . 18% hybridized to mRNAs each present equally in the three tissues, 26% to mRNAs differentially expressed in the tissues, and 30% to mRNAs present only in the brain . An additional 26% of the clones failed to detect mRNA in the three tissues at an abundance level of about 0.01%, but did contain rat cDNA as demonstrated by Southern blotting; this class probably represents rare mRNAs expressed in only some brain cells . Therefore, most mRNA expressed in brain is either specific to brain or otherwise displays regulation . Rarer mRNA species tend to be larger than the more abundant species, and tend to be brain specific; the rarest, specific mRNAs average 5000 nucleotides in length . Ten percent of the clones hybridize to multiple mRNAs, some of which are expressed from small multigenic families . From these data we estimate that there are probably at most 30,000 distinct mRNA species expressed in the rat brain, the majority of which are uniquely expressed in the brain.

J Mol Biol, 1983 Aug 25, 168(4), 809 - 30
Structure of partially denatured Escherichia coli 23 S ribosomal RNA determined by electron microscopy; Klein BK et al.; The secondary structure of 23 S ribosomal RNA was analyzed by electron microscopy after partial denaturation . A reproducible pattern of loops was seen when molecules were spread for electron microscopy in 50% formamide solutions containing various concentrations of Mg2+ and Na+ . Some loops were stabilized more than others by Na+ or by Mg2+; but in general, small amounts of Mg2+ (0.5 to 1.0 mM) markedly stabilized all the major loops, as did much greater amounts of Na+ (100 mM) . However, at all levels of Mg2+ examined, increasing levels of Na+ destabilized loop structures . These data are consistent with the known salt dependence of double-stranded DNA and transfer RNA structure . The four most frequently observed loops correspond, within the limits of measurement error, to the major loops in the secondary structure models of Noller et al . (1981) and Glotz et al . (1981) . These four loops are, in length and position of their midpoints along the 23 S rRNA molecule: 490 +/- 50 at 250 +/- 40; 350 +/- 50 at 1860 +/- 80; 400 +/- 70 at 2330 +/- 150; and 570 +/- 100 at 2350 +/- 100 . Three of the four have base-paired stems with delta G0 values among the lowest of all the loops in the two indirect models . At least two are also among the most stable loops found in computer searches of the 23 S rRNA sequence for dyad symmetry . These results demonstrate that partial denaturation mapping can both identify prominent features of secondary structure in rRNA and estimate their relative stability.

J Biol Chem, 1983 Aug 25, 258(16), 9793 - 800
Integration of F1 and the membrane sector of the proton-ATPase of Escherichia coli . Role of subunit "b" (uncF protein); Perlin DS et al.; Membranes derived from the Escherichia coli strain AN1460 which carries the multicopy plasmid pAN45 (unc+) (Downie, J . A., Langman, L., Cox, G . B., Yanofsky, C., and Gibson, (1980) J . Bacteriol . 143, 8-17) were enriched 5- to 10-fold in proton-ATPase activity . Incubation of F1-depleted AN1460 membranes with trypsin abolished F1-binding ability but did not inhibit proton transport through the membrane sector (F0) . Sodium dodecyl sulfate-gel electrophoresis indicated that subunit "b" (uncF protein) of F0 was cleaved by trypsin and prebound F1 protected against the trypsin effect . Subunits "a" (uncB protein) and "c" (uncE protein) were unaffected by the trypsin treatment . A water-soluble fragment (Mr = 14,800) was liberated after trypsin treatment and appeared to arise from subunit b . Studies of enzyme hybridization and of F1 binding to membranes derived from strains containing mutations in uncB, F, and E genes supported the suggestion that subunit b is involved in F1 binding to the F0 . Also, extraction of membranes with KSCN increased the relative proportion of subunit b in the membrane and this coincided with a parallel increase in trypsin-sensitive F1-binding ability . It is proposed that subunit b is involved in binding of F1 to the F0; this agrees with the presumed role of the protein as deduced from predictions of its secondary and tertiary structure (Walker, J . E., Saraste, M., and Gay, N . J . (1982) Nature (Lond.) 298, 867-869; Senior, A . E . (1983) Biochim . Biophys . Acta, in press).

FEBS Lett, 1983 Aug 22, 160(1-2), 296 - 300
Amino acid sequence around the active serine in the acyl transferase domain of rabbit mammary fatty acid synthase; McCarthy AD et al.; Rabbit mammary fatty acid synthase was labelled in the acyl transferase domain(s) by the formation of the O-ester intermediates after incubation with {14C}acetyl- or malonyl-CoA . Elastase peptides containing the labelled acyl groups were isolated using high performance liquid chromatography and sequenced by fast atom bombardment mass spectrometry . An identical peptide (acyl-Ser-Leu-Gly-Glu-Val-Ala) was obtained after labelling with acetyl- or malonyl-CoA . This confirms the hypothesis that, unlike Escherichia coli or yeast, a single transferase catalyses the transfer of both acetyl- and malonyl-groups in the mammalian complex . The sequence at this site is compared with that around the active serine in other acyl transferases and hydrolases.

FEBS Lett, 1983 Aug 22, 160(1-2), 129 - 33
Peptidyltransferase center of ribosomes . On the mechanism of action of alkaloid lycorine; Kukhanova M et al.; The molecular mechanism of action of the alkaloid lycorine has been revised . According to our results, lycorine inhibits the binding of CACCA-Leu comes from Ac to the donor site of the peptidyltransferase center of wheat-germ ribosomes, whereas the transpeptidation reaction in the system with the minimal model donor is not inhibited . The equilibrium constant of CACCA-Leu comes from Ac to the donor site of 80 S ribosomes is measured.

Carbohydr Res, 1983 Aug 16, 120, 131 - 41
Structure of the 3-deoxy-D-manno-octulosonic acid-(KDO)-containing capsular polysaccharide (K14 antigen) from Escherichia coli 06:K14:H31; Jann B et al.; The chemical structure of the K14-antigenic polysaccharide (K14 antigen) of Escherichia coli 06:K14:H31 was elucidated by determination of the composition, 1H- and 13C-n.m.r . spectroscopy, periodate oxidation, and study of the oligosaccharides obtained by partial hydrolysis . The polysaccharide consists of {O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl)-(1 leads to 5)-O-(3-deoxy-beta-D-manno-octulopyranosylonic acid)-(2 leads to 6)} repeating units, approximately 60% of the octonic acid units being O-acetylated and approximately 10% O-propionylated at O-8 . The sequence of acetylated and propionylated residues is not known . The serologically-specific part of the K14 antigen residues in the polysaccharide part.

Biochemistry, 1983 Aug 16, 22(17), 4159 - 64
Identification of sites of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen cross-linking in Escherichia coli 23S ribosomal ribonucleic acid; Turner S et al.; The reagent 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) was used to cross-link 23S rRNA from Escherichia coli under 50S ribosomal subunit reconstitution conditions . Following partial digestion of the RNA with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate fragments derived from the cross-linked sites . These fragments were analyzed by digestion with ribonucleases T1 and A and their positions in the 23S RNA sequence identified . Fragment a1 (positions 1325-1426) is cross-linked to a2 (positions 1574-1623); fragment b1 (positions 1700-1731) is cross-linked to b2 (positions 1732-1753); and a cross-link is formed within fragment c (or c') (positions 863-916) . In the latter case, the cross-link was located precisely, linking residues C867 and U913 . All three HMT-mediated cross-links are consistent with a proposed secondary structure model for 23S RNA {Noller, H . F., Kop, J., Wheaton, V., Brosius, J., Gutell, R . R., Kopylov, A . M., Dohme, F., Herr, W., Stahl, D . A., Gupta, R., & Woese, C . R . (1981) Nucleic Acids Res . 9, 6167-6189}.

Biochemistry, 1983 Aug 16, 22(17), 4071 - 81
Characterization of the Escherichia coli X-ray endonuclease, endonuclease III; Katcher HL et al.; The X-ray endonuclease endonuclease III of Escherichia coli has been purified to apparent homogeneity by using the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The most purified fraction shows endonucleolytic activity against apurinic and apyrimidinic (AP) sites and a dose-dependent response to DNA that has been X irradiated, UV irradiated, or treated with OsO4 . The endonuclease also nicks OsO4-treated DNA that has been subsequently treated with alkali to produce fragmented thymine residues and DNA treated with potassium permanganate . The enzyme does not incise the alkali-labile sites present in DNA X irradiated in vitro in the presence of hydroxyl radical scavengers . The most purified fractions exhibit two distinct activities, an AP endonuclease that cleaves on the 3' side of the damage leaving a 3'-OH and a 5'-PO4 and a DNA N-glycosylase that recognizes at least two substrates, thymine glycol residues and urea residues . The glycosylase activity is sensitive to N-ethylmaleimide while the AP endonuclease is not.

Biochim Biophys Acta, 1983 Aug 16, 746(3), 202 - 8
Co-operative effects in affinity labeling reveal the interaction of tRNA-recognition centers of phenylalanyl-tRNA synthetase; Gorshkova II et al.; A mathematical treatment of affinity labeling of the enzymes is presented . The model considered involves a dimeric enzyme with identical ligand binding sites . Equations are derived which describe the kinetics of modification; mutual influence of ligand molecules on association, on the rate of covalent attachment and the possibility of the existence of different sites of modification are taken into account . Experimental data on affinity labeling of phenylalanyl-tRNA synthetase (L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20) of Escherichia coli MRE-600 with N-bromoacetyl-{14C}phenylalanyl-tRNA are treated in terms of the model suggested . The affinity (association constant value) of the tRNAPhe analog molecule towards the enzyme is only slightly affected by another molecule, whereas the reaction rate constant of covalent attachment decreases significantly . The latter is assumed to be due to acceptor site change in the complex containing two molecules of the tRNAPhe analog.

Chem Biol Interact, 1983 Aug 15, 46(1), 101 - 8
Effects of bisulfite (sulfur dioxide) on DNA synthesis and fidelity by DNA polymerase I; Mallon RG et al.; In an attempt to explain the mechanism of comutagenesis by bisulfite, the extent and accuracy of DNA synthesis by E . coli DNA polymerase I was examined in the presence of sodium bisulfite . Bisulfite concentration of 100 mM caused nearly complete inhibition of dNTP incorporation into activated calf thymus DNA . Other salts (NaCL, Na2SO4) at the same concentration had no effect on enzyme activity . Preincubation of the various DNA synthesis assay components in 100 mM bisulfite showed that only preincubation of DNA polymerase I caused inhibition of DNA synthesis . Exonuclease functions of DNA polymerase I were unaffected by up to 100 mM bisulfite . Accuracy of DNA synthesis in the presence of bisulfite was determined using poly (dA-dT) as a template-primer . Concentrations of bisulfite greater than 50 mM caused a progressive decrease in enzyme accuracy . At 100 mM bisulfite there was an approximate 7.5-fold decrease in the fidelity of DNA synthesis, compared to control values, as measured by the ratio of noncomplementary (dGTP) to complementary (dTTP) nucleotide incorporated . Based on the known chemistry of bisulfite, it is hypothesized that sulfitolysis of the one disulfide group in DNA polymerase I by bisulfite might be responsible for the reduced polymerase activity and accuracy . The exonuclease functions of DNA polymerase I do not seem to require the disulfide linkage . These results suggest that the effects of bisulfite on mutation frequency might be mediated by effects on the fidelity of DNA repair systems.

J Mol Biol, 1983 Aug 15, 168(3), 489 - 503
Synthesis of yeast histone 3 in an Escherichia coli cell-free system; Mellado RP et al.; The gene for histone H3 from the yeast Saccharomyces cerevisiae was placed under the control of the lac promoter of Escherichia coli by fusing the H3 coding sequence to that of beta-galactosidase . The gene was shown to be transcribed in vivo, but its product was not detected in cell extracts . However, synthesis of the fused polypeptide was detected in an in vitro transcription-translation system derived from E . coli . Proteolytic degradation of the newly synthesized polypeptides may be the cause of their apparent absence in the in vivo experiment.

J Mol Biol, 1983 Aug 15, 168(3), 451 - 68
Nucleotide sequence of Escherichia coli pabA and its evolutionary relationship to trp(G)D; Kaplan JB et al.; We have determined the entire nucleotide sequence of Escherichia coli pabA . A comparison of the nucleotide and amino acid sequences of pabA and trp(G) D reveals extensive homology, suggesting that these two genes arose from a common ancestor . pabA and trp(G) D are 44% homologous at the amino acid level and 53% homologous at the nucleotide sequence level . The nucleotide sequences can be divided into regions of high homology, in which most nucleotide changes occur in the third position of codons and do not effect the amino acid sequence, and regions which show almost no DNA homology . Divergence in these non-homologous regions appears to have resulted from single-base substitutions as well as the rearrangement of small regions of DNA by inversion, deletion and duplication.

Eur J Biochem, 1983 Aug 15, 134(3), 429 - 38
500-MHz 1H-NMR studies of ribosomal proteins isolated from 70-S ribosomes of Escherichia coli; van de Ven FJ et al.; A method for the large-scale isolation of ribosomal proteins is described avoiding pre-separation of 30-S and 50-S subunits . Five proteins isolated in this way were studied with high-resolution 1H NMR at 500 MHz . These are S21, L18, L25, L30 and L33 . The results show that L18, L25 and L30 exhibit tertiary structure in solution and indications for secondary structure in S21 are found . Protein L33 appears to be a random coil . Several resonances in the 1H NMR spectra are assigned to particular protons of amino acid residues, e.g . the aromatic ring protons of tyrosines and histidines, and epsilon-protons of lysines.

Chem Biol Interact, 1983 Aug 15, 46(1), 67 - 84
Effect of 2-chloroethylnitrosoureas on plasmid DNA including formation of strand breaks and interstrand cross-links; Vadi HV et al.; Plasmid {3H}pBR 322 was incubated with various alkylating agents including chlorozotocin, N,N'-bis(2-chloroethyl)-N'-nitrosourea (BCNU), N-ethyl-N-nitrosourea (Enu) and dimethylsulfate (DMS) . Formation of DNA strand breaks was followed by separation of the various forms of DNA on agarose gels and liquid scintillation counting of the bands . All alkylating agents examined were capable of rapidly producing strand breaks in time and concentration dependent fashion . Bands migrating as relaxed circular and supercoiled forms of the plasmid disappeared, and extensive alkylation resulted in formation of a band that migrated faster than the linear form of DNA . Electron microscopy of this band showed that it consisted of relaxed circles . Prolonged storage of alkylated plasmid resulted in fragmentation of the DNA, possibly due to strand scission at apurinic sites . A new neutral denaturation technique was developed, which allowed for the detection of DNA interstrand cross-links with minimal effects on other potentially labile sites of the alkylated DNA . The level of alkylation was quantitated by incubating {3H}pBR 322 with {2-chloroethyl-U-14C}chlorozotocin and was shown to be independent of DNA concentration but have a linear relationship with drug concentration . Linear and relaxed circular forms of the plasmid were alkylated to a somewhat higher extent than supercoiled DNA . Alkylation of pBR 322 with defined superhelical densities showed no preferential loss in DNA with a specific superhelical density, indicating that alkylation-induced unwinding is independent of superhelicity under the experimental conditions used.

J Mol Biol, 1983 Aug 15, 168(3), 477 - 88
Regulation of the pho regulon in Escherichia coli K-12 . Genetic and physiological regulation of the positive regulatory gene phoB; Shinagawa H et al.; phoB is a positive regulatory gene for phoA, which codes for alkaline phosphatase, as well as for other genes belonging to the phosphate (pho) regulon whose expression is inducible by phosphate limitation in Escherichia coli . A hybrid plasmid that contains a phoB-lacZ fused gene was constructed in vitro . This plasmid enabled us to study phoB gene expression by measuring the beta-galactosidase level in the cells . The plasmid was introduced into various regulatory mutants related to the phosphate regulon, and phoB gene expression in these strains was studied under limited and excess phosphate conditions . It was found that the regulation of phoB expression was very similar to that of phoA expression . Expression of both genes was induced by phosphate starvation . Both genes were constitutively expressed in phoR, phoS, phoT and phoU mutants and were not expressed in a phoR-phoM double mutant . The implications of these findings for the regulatory mechanism of the pho regulon are discussed.

J Mol Biol, 1983 Aug 15, 168(3), 525 - 57
Initiation, processing and termination of ribosomal RNA from a hybrid 5 S ribosomal RNA gene in a plasmid; Szeberenyi J et al.; Transformation of an RNA-processing mutant (rne, RNase E-) of Escherichia coli with a recombinant plasmid containing the promoter region of the ribosomal cluster rrnA and portions from the 3' region of the rrnD cluster results in the accumulation of the precursors to 5 S ribosomal RNAs at the permissive as well as that of two full-length transcripts and a processing intermediate at the nonpermissive temperature . The two full-length transcripts start from the two rrnA promoters, which are about 120 nucleotides apart . This plasmid, pJR3 delta, contains an intact 5 S rRNA gene and portions from the 16 S and 23 S rRNA genes . Analysis of the major plasmid-specific RNA species revealed that RNA molecules initiated in vivo from the first promoter (P1) start with pppA, while transcripts from the second promoter (P2) contain either pppG or pppC at their 5' ends . Termination occurs mainly at the first available termination site . Full-length transcripts initiated from both promoters are processed to precursors of 5 S rRNAs in vivo at the permissive temperature, but only about 20% of these transcripts are processed to mature 5 S rRNA . RNA1 and RNA2 (the transcripts initiated from P1 and P2, respectively) and RNA3 (an RNA-processing intermediate containing the entire 5 S region and the 3' end of the transcripts) can be cleaved in vitro by cell extracts of wild type strains resulting in precursor and mature 5 S rRNAs in a reaction that is RNase E dependent but not ribosome dependent . The 5' end of the processed 5 S rRNA can correspond to the 5' end of mature 5 S rRNA or it can contain one to three additional nucleotides.

Nucleic Acids Res, 1983 Aug 11, 11(15), 5007 - 19
Molecular basis for modulated regulation of gene expression in the arginine regulon of Escherichia coli K-12; Cunin R et al.; We compare the nucleotide sequences of the regulatory regions of five genes or groups of genes of the arginine regulon of Escherichia coli K-12: argF, argI, argR, the bipolar argECBH operon and the carAB operon . All these regions harbour one or two copies of a conserved 18 bp sequence which appears to constitute the basic arginine operator sequence (ARG box) . We discuss the influence of ARG box copy number, degree of dyad symmetry, base composition, and position relative to the cognate promoter site on the derepression-repression ratios of the genes of the regulon . A novel hypothesis, based on structural considerations, is also put forward to account for the absence ot attenuation control.

Nucleic Acids Res, 1983 Aug 11, 11(15), 5299 - 313
Molecular structure of ilvIH and its evolutionary relationship to ilvG in Escherichia coli K12; Squires CH et al.; ilvIH of Escherichia coli K12 codes for a valine-sensitive acetohydroxy acid synthase (AHASIII) . The DNA sequence of ilvIH was determined . Open reading frames and appropriate translation signals exist for two polypeptides, one containing 565 amino acids (ilvI polypeptide) and the other 160 amino acids (ilvH polypeptide) . A graphic matrix analysis shows three clearcut regions of homology between ilvI and ilvG (codes for AHASII) . Within these three regions of homology, 50-60% of the amino acid sequences of AHASII and AHASIII are conserved . Inspection of the region between ilvG and ilvE (the K region) revealed that it can potentially code for an 86 amino acid polypeptide . A computer analysis shows small but significant homology between the predicted amino acid sequences of the N-terminal half of the ilvH polypeptide and the putative region K polypeptide . We conclude that ilvIH and ilvG-region K evolved from a common ancestor.

Nucleic Acids Res, 1983 Aug 11, 11(15), 5147 - 58
The nucleotide sequence of the trimethoprim-resistant dihydrofolate reductase gene harbored by Tn7; Fling ME et al.; The complete nucleotide sequence of the type I dihydrofolate reductase gene from Tn7 was determined . The structural gene coded for a polypeptide of 157 amino acid residues . The polypeptide deduced from the DNA sequence had a molecular weight of 17,577 which was in good agreement with that estimated by mobility in SDS-polyacrylamide gels . Sequences were identified proximal to the coding region which were similar to those found in the consensus E . coli promoter region and for the initiation of protein synthesis . Features consistent with the termination of RNA transcription were present distal to the structural gene . No homology was apparent when the DNA sequence of the type I gene was compared to the sequence of the type II plasmid DHFR genes, but sequence homology was evident when the type I and E . coli chromosomal enzymes were compared . Homology was greatest in the regions coding for amino acids which in the E . coli chromosomal enzyme are associated with substrate, cofactor and inhibitor binding.

Nucleic Acids Res, 1983 Aug 11, 11(15), 5103 - 12
Efficient site-directed mutagenesis by simultaneous use of two primers; Norris K et al.; A rapid and efficient procedure for site specific mutagenesis is described . A double primed synthesis with a 17-mer mismatch primer and a "universal" 15-mer M13 sequencing primer was used to introduce a T to A transversion into an ompF signal peptide gene cloned in the M13mp8 vector . The two primers were annealed to the circular single stranded M13 template . After a short extension and ligation reaction, a double stranded restriction fragment containing the mismatch (ompF*/EcoR1-SalI) was cut out of the partly single stranded circular DNA and inserted into pBR322 . 42% of the E.coli transformants harboured plasmid with the desired mutation, which could be detected by the appearance of a new restriction site (MboII) and by dot blot hybridization of plasmid DNA with the 32P-labeled 17-mer.

Nature, 1983 Aug 11-17, 304(5926), 559 - 60
Determination of the absolute handedness of knots and catenanes of DNA; Krasnow MA et al.; DNA winds about itself in a right-handed or left-handed fashion at several structural levels . The double helix is generally right-handed and is given a (+) sign by convention, whereas supercoiling of the helix axis is always (-) in the cell . The winding in higher -order forms such as knots and catenanes is unknown, and this has impeded elucidation of the mechanisms of their formation and resolution by replication, recombination and topoisomerase action . We introduce here a procedure for determining the handedness of DNA winding by inspection of electron micrographs of DNA molecules coated with Escherichia coli RecA protein . We demonstrate the validity of the method and show that DNA topoisomerase I of E . coli generates an equal mixture of (+) and (-) duplex DNA knots, and that one product of recombination by resolvase of transposon Tn3 (refs 8, 9) is a catenane of uniquely (+) sign.

J Biol Chem, 1983 Aug 10, 258(15), 9208 - 12
A complementary DNA oligomer releases a transcription pause complex; Fisher R et al.; The formation of alternative secondary structures in the transcript of the tryptophan (trp) operon leader region regulates expression of the trp operons of Escherichia coli and other bacterial species . During in vitro transcription RNA polymerase pauses near base pair 90 after the first hairpin secondary structure in E . coli trp leader mRNA is formed . The E . coli L-factor enhances transcription pausing at this site (Farnham, P . J., Greenblatt, J., and Platt, T . (1982) Cell 29, 945-951); presumably it does so by facilitating recognition of the RNA hairpin by polymerase . We show that addition of a DNA oligomer complementary to the proximal segment of the RNA hairpin relieves transcription pausing in vitro both in the presence and absence of L-factor . The oligomer apparently interferes with formation of the RNA hairpin which we believe is recognized by polymerase as the pause signal . The oligomer also relieves pausing in L-factor-induced paused complexes, suggesting that the oligomer can disrupt a preformed secondary structure in the transcript.

J Biol Chem, 1983 Aug 10, 258(15), 9237 - 44
Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli . In vitro and in vivo incorporation into phospholipids; Orr GA et al.; sn-Glycerol 3-phosphorothioate, a bacteriocidal analog of sn-glycerol 3-phosphate in strains of Escherichia coli with a functioning glycerol phosphate transport system, was investigated for its ability to be incorporated into phospholipid under in vitro and in vivo conditions . A cell-free particulate fraction from E . coli strain 8 catalyzes the transfer of sn-{3H}glycerol 3-phosphoro{35S}thioate to chloroform-soluble material in the presence of either CDP-diglyceride or palmitoyl coenzyme A . With CDP-diglyceride as the co-substrate, the product of the reaction was tentatively identified as phosphatidylglycerol phosphorothioate . No formation of phosphatidylglycerol was observed, suggesting that the specific phosphatase required for the synthesis of phosphatidylglycerol does not catalyze, or else at a greatly reduced rate, the hydrolysis of the phosphorothioate monoester linkage . The kinetics of incorporation of sn-{3H}glycerol 3-phosphate and phosphorothioate into chloroform-soluble material in the presence of CDP-diglyceride are almost identical . In the presence of palmitoyl coenzyme A, sn-{3H}glycerol 3-phosphoro{35S}thioate was converted to the phosphorothioate analog of phosphatidic acid . Kinetic analysis showed that the apparent Km values for the incorporation of the phosphate and the phosphorothioate derivatives into phospholipid were 0.4 and 0.8 mM, respectively . The Vmax for the phosphorothioate analog was approximately half that for the phosphate derivative . Chemically synthesized thiophosphatidic acid was not a substrate for CTP:phosphatidic acid cytidylyltransferase . sn-{3H}Glycerol 3-phosphoro{35S}thioate was incorporated into phospholipid by cultures of E . coli strain 8 . The major phosphorothioate-containing phospholipid synthesized in vivo was identified as 1,2-diacyl-sn-{3H}glycerol 3-phosphoro{35S}thioate . The phosphorothioate analog of phosphatidylglycerol phosphate was not observed despite our observations that this analog can be synthesized in vitro . Our results indicate that the phosphorothioate analog is an effective sn-glycerol 3-phosphate surrogate and suggest that a major reason for its toxicity toward E . coli strain 8 may be due to a total blockade of endogenous phospholipid biosynthesis.

J Biol Chem, 1983 Aug 10, 258(15), 9159 - 65
The purification and characterization of the cytochrome d terminal oxidase complex of the Escherichia coli aerobic respiratory chain; Miller MJ et al.; The aerobic respiratory chain of Escherichia coli is branched . In aerobically grown cells harvested in midexponential phase, a respiratory chain containing only b-type cytochromes is predominant . This chain contains a terminal oxidase which is a b-type cytochrome, referred to as cytochrome o . However, when the bacteria are grown under conditions of oxygen limitation, additional components of the respiratory chain are induced, as evidenced by the appearance of new spectroscopic species . These include a new b-type cytochrome, cytochrome b558, as well as cytochrome a1 and cytochrome d . In this paper, a purification protocol and the initial characterization of the terminal oxidase complex containing cytochrome d are reported . Solubilization of the membrane is effected by Zwittergent 3-12, and purification is accomplished by chromatography with DEAE-Sepharose CL-6B and hydroxyapatite . The complex contains cytochrome b558, a1, and d . Analysis by sodium dodecyl sulfate-polyacrylamide gels indicates that the complex contains only two types of polypeptides with the molecular weights estimated to be 57,000 and 43,000 . The purified complex has oxidase activity in the presence of detergents, utilizing substrates including ubinquinol-1, N,N,N',N'-tetramethyl-p-phenylenediamine, and 2,3,5,6-tetramethyl-p-phenylenediamine . The cytochrome d complex contains protoheme IX and iron, but does not contain nonheme iron or copper . Approximately half of the cytochromes which are thought to participate in E . coli aerobic respiration are accounted for by this single complex . These results suggest that the E . coli aerobic respiratory chain is organized around a relatively small number of cytochrome-containing complexes.

J Biol Chem, 1983 Aug 10, 258(15), 9046 - 9
Different binding of human interferon alpha 1 and alpha 2 to common receptors on human and bovine cells . Studies with recombination interferons produced in Escherichia coli; Yonehara S et al.; Minicells from Escherichia coli DS410 harboring cDNA for human interferon (IFN) alpha 1 or alpha 2 were metabolically labeled with {3H}leucine and the radioactive IFN was purified to homogeneity by immune precipitation with anti-IFN-alpha serum . These preparations of radioactive IFN-alpha 1 and -alpha 2 were used to study the binding on two human (FL and Daudi) and one bovine (MDBK) cell lines . IFN-alpha 2 specifically bound well to both human and bovine cells, while IFN-alpha 1 bound very poorly to human cells but well to bovine cells . Specific binding of radioactive IFN-alpha 2 to these cell lines was completely inhibited by not only nonradioactive IFN-alpha 2 but also IFN-alpha 1, and binding of IFN-alpha 1 to bovine cell was also competed by IFN-alpha 2 as well as IFN-alpha 1, indicating that the receptors for both IFNs are identical . However, 50-100-fold (on human cells) or 4-fold (on bovine cell) more nonradioactive IFN-alpha 1 than -alpha 2 was required to inhibit the binding of radioactive IFN-alpha 2 to the receptors . Scatchard analysis showed that IFN-alpha 1 and -alpha 2 bind to the receptors on human cells with an apparent Kd of greater than 6 X 10(-10) and 3 X 10(-11) M, respectively, while on bovine cells with a Kd of 4.2 X 10(-11) and 1.6 X 10(-11) M, respectively . These results show that the different target cell specificity of IFN-alpha 1 and -alpha 2 in regard to antiviral activity (Streuli, M., Hall, A., Boll, W., Stewart, W . E., II, Nagata, S., and Weissmann, C . (1981) Proc . Natl . Acad . Sci . U . S . A . 78, 2848-2852) is due to the different binding activity of IFN-alpha molecules to their common receptors.

FEBS Lett, 1983 Aug 8, 159(1-2), 102 - 6
Specific DNA binding of the cyclic AMP receptor protein to a synthetic oligodeoxyribonucleotide . A circular dichroism study; Martin SR et al.; The interaction of the cAMP receptor protein (CRP) of Escherichia coli with a synthetic DNA undecamer (11 mer) comprising a portion of the specific target site in the gal operon and containing 8 basepairs out of the 10 basepair concensus making up specific CRP sites, has been studied by circular dichroism spectroscopy . The binding constants for the interaction of CRP with the 11 mer in the presence and absence of cAMP have been determined, and it is shown that CRP, both in the presence and absence of cAMP, induces a B-C transition in the conformation of the 11 mer.

J Mol Biol, 1983 Aug 5, 168(2), 307 - 20
Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli . I . Identification of a lysR gene encoding an activator of the lysA gene; Stragier P et al.; The synthesis of diaminopimelate decarboxylase, which catalyzes the decarboxylation of diaminopimelate into lysine, is known to be repressed by lysine and induced by diaminopimelate in Escherichia coli K12 . Until now only mutations in lysA, the structural gene for diaminopimelate decarboxylase, have been described that lead to a Lys- phenotype . A set of plasmids carrying adjacent inserts of the lysA region was constructed and employed to transform different Lys- mutants . The complementation pattern observed and the corresponding expression of the lysA gene show that in fact the Lys- phenotype can be obtained by mutations in two different and closely linked loci: one being the lysA structural gene, and the other called lysR . We propose that the lysR gene encodes a positive effector required for the full expression of the lysA gene . The synthesis of a hybrid lysA-lacZ protein constructed in vitro was observed to be decreased dramatically in lysR mutants . Moreover, all the regulatory features were lost, indicating that the LysR activator is necessary for the regulation of lysA expression . The gene order is thyA lysA lysR clockwise around 61 minutes on the chromosome, lysA being transcribed counter-clockwise.

Eur J Pharmacol, 1983 Aug 5, 91(4), 493 - 9
A lipopolysaccharide and concanavalin A induce variations of serotonin levels in mouse tissues; Endo Y; The administration of an Escherichia coli lipopolysaccharide (LPS), or an endotoxin into mice produced a variation in tissue serotonin (5HT) levels within 4.5 h . 5HT levels in the kidney and lung were decreased by the higher doses of LPS, but those in the liver and spleen were increased even by lower doses of the agent . The increase in liver 5HT was most marked . Such variations in 5HT levels were also produced by the administration of concanavalin A . In vitro experiments using extracts from livers of LPS-treated and non-treated mice indicated that there was no difference in the 5HT formation from 5-hydroxytryptophan between the two groups, but that 5HT formation from tryptophan was higher in the LPS-treated mice . The LPS-induced 5HT increase in liver was suppressed by p-chlorophenylalanine (an inhibitor of tryptophan hydroxylase), actinomycin D, cycloheximide and dexamethasone, but not by Ro 4-4602 (an inhibitor of aromatic amino acid decarboxylase), pargyline (an inhibitor of monoamine oxidase) and indomethacin . A possible mechanism of the 5HT increase in the liver is discussed on the basis of these results.

J Mol Biol, 1983 Aug 5, 168(2), 333 - 50
Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli . III . Nucleotide sequence and regulation of the lysR gene; Stragier P et al.; The complete nucleotide sequence of the lysR gene, which encodes the activatory protein required for lysA expression, has been determined . Bal31 deletions and translational fusions were used to localize the promoter region and the initiator ATG of the lysR gene which encodes a 311 amino acid polypeptide . Both lysA and lysR coding sequences were found to be divergent and separated by a very short intergenic region consisting of 121 base-pairs between the postulated ATGs of the two proteins . Transfer of the whole lysR gene on a plasmid carrying a lysR-lacZ fusion shows that lysR expression is autoregulated by a factor of 7 . The same binding site (73 base-pairs fragment) could be involved in both effects of the LysR product, acting simultaneously as an operator for lysR expression and an initiator for lysA expression . The genetic organization of the whole region (4127 base-pairs) is given . A strikingly symmetrical pattern is observed with the four tightly packed galR, lysA, lysR and orfX (an unidentified open reading frame) genes, in a very unusual arrangement of both divergent and convergent overlapping transcription units.

J Mol Biol, 1983 Aug 5, 168(2), 321 - 31
Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli . II . Nucleotide sequence of the lysA gene and its regulatory region; Stragier P et al.; The complete nucleotide sequence of the lysA gene and its regulatory region was determined . At the 3' end of the lysA gene an open reading frame was revealed in the opposite direction and was identified as the galR coding region . Only six base-pairs are present between the two translational stops and thus both transcription units are overlapping in vivo . Translational gene fusions constructed in vitro with the beta-galactosidase gene were used to identify the lysA initiating ATG . The sequence encodes a 420 amino acid long peptide for a predicted molecular weight of 46,099 . No attenuation-like sequence was found at the beginning of the lysA gene . A target of the LysR activator protein was localized on a 73 base-pair fragment found 48 base-pairs upstream from the lysA coding region . The presence of this DNA sequence on a multicopy plasmid led to a net decrease of lysA expression, indicating limiting amounts of active LysR protein in the cytoplasm.

J Mol Biol, 1983 Aug 5, 168(2), 285 - 305
Molecular cloning of the gene for phosphofructokinase-2 of Escherichia coli and the nature of a mutation, pfkB1, causing a high level of the enzyme; Daldal F; The pfkB gene of Escherichia coli is known to specify a minor phosphofructokinase, Pfk-2, in the wild-type strain; the pfkB1 mutation causes a 25-fold increase in the amount of Pfk-2 so that it adequately substitutes for mutational loss of the major phosphofructokinase, Pfk-1 (specified by pfkA); and another closely linked mutation, pfkB10, affects the structure of Pfk-2 . This paper is about the pfkB1 mutation . pfkB+, pfkB1 and pfkB1 pfkB10 were cloned and subcloned on plasmid pBR322; their functions were carried, in all three cases, by a 2.1 X 10(3) base-pair fragment with a similar or identical restriction pattern . Experiments with "maxicells" confirmed that the cloned fragments included the structural gene as well as the determinants of its level of expression . Results of N-terminal sequencing of the enzyme matched with the DNA sequence and established the position and direction of the gene . A HindIII-SmaI fragment of 408 base-pairs, which included 294 base-pairs of the non-coding 5' region and 114 base-pairs of the protein coding sequence, was fused to galK on the promoter cloning vector pK01; in the fusion pfkB1 caused a high level expression of galK . Transcription in vitro from pfkB+ and pfkB1 allowed the determination of the +1 position in both cases, at about 19 base-pairs before the initiating methionine codon; the level of transcription was much higher from pfkB1 than from pfkB+ . The DNA sequence of the 408 base-pair fragments from pfkB+ and pfkB1 were found to differ only in a single residue, the pfkB1 mutation thus proving to be a C to T change at position about -12 from the initiation of transcription.

Science, 1983 Aug 5, 221(4610), 551 - 3
High efficiency DNA-mediated transformation of primate cells; Gorman C et al.; Tissue culture cells from several mammalian species, including three primate lines, were transfected with recombinant vectors carrying Escherichia coli xanthine-guanine phosphoribosyltransferase or Tn5 aminoglycoside phosphotransferase dominant selectable markers . Human HeLa and SV40-transformed xeroderma pigmentosum cells exhibited stable transformation frequencies of at least 10(-3) (0.1 percent) . CV-1, an African green monkey kidney cell line, could be stably transformed with the exceptionally high frequency of 6 X 10(-2) (6 percent).

Biochemistry, 1983 Aug 2, 22(16), 3913 - 20
Cross-links between ribosomal proteins of 30S subunits in 70S tight couples and in 30S subunits; Lambert JM et al.; Ribosome 70S tight couples and 30S subunits derived from them were modified with 2-iminothiolane under conditions where about two sulfhydryl groups per protein were added to the ribosomal particles . The 70S and 30S particles were not treated with elevated concentrations of NH4Cl, in contrast to those used in earlier studies . The modified particles were oxidized to promote disulfide bond formation . Proteins were extracted from the cross-linked particles by using conditions to preclude disulfide interchange . Disulfide-linked protein complexes were fractionated on the basis of charge by electrophoresis in polyacrylamide/urea gels at pH 5.5 . The proteins from sequential slices of the urea gels were analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis . Final identification of proteins in cross-linked complexes was made by radioiodination of the proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis . Attention was focused on cross-links between 30S proteins . We report the identification of 27 cross-linked dimers and 2 trimers of 30S proteins, all but one of which were found in both 70S ribosomes and free 30S subunits in similar yield . Seven of the cross-links, S3-S13, S13-S21, S14-S19, S7-S12, S9-S13, S11-S21, and S6-S18-S21, have not been reported previously when 2-iminothiolane was used . Cross-links S3-S13, S13-S21, S7-S12, S11-S21, and S6-S18-S21 are reported for the first time . The identification of the seven new cross-links is illustrated and discussed in detail . Ten of the dimers reported in the earlier studies of Sommer & Traut (1976) {Sommer, A., & Traut, R . R . (1976) J . Mol . Biol . 106, 995-1015}, using 30S subunits treated with high salt concentrations, were not found in the experiments reported here.

Biochim Biophys Acta, 1983 Aug 2, 740(3), 282 - 90
7 S RNA: a single site substrate for the RNA processing enzyme ribonuclease E of Escherichia coli; Szeberenyi J et al.; 7 S RNA accumulates at non-permissive temperatures in an RNAase E strain containing the recombinant plasmid pJR3 delta which carries a single 5 S rRNA gene and expression sequences . 7 S RNA is a processing intermediate that contains the complete sequence of 5 S rRNA as well as a stem-and-loop structure encoded by the terminator of rrnD . 7 S RNA can be processed in vitro by RNAase E . Structural analysis of the products (5 S rRNA and the stem) of in vitro processing of 7 S RNA revealed that the cleavage site of RNAase E in 7 S RNA is 3 nucleotides downstream from the 3' end of the mature 5 S rRNA . The cleavage generates 3'-hydroxyl and 5'-phosphate termini.

J Gen Virol, 1983 Aug, 64 (Pt 8), 1815 - 8
Characterization and properties of a modified human interferon-alpha containing an additional 18 amino acids at the N-terminus; King RM et al.; A modified human interferon-alpha 2 was produced in Escherichia coli cells infected with phage M13 mp7 containing an interferon-alpha gene . After purification by immunochromatography with the monoclonal antibody NK2, the N-terminal amino acid sequence was determined . The N-terminal methionine was absent but an additional sequence of 18 amino acids at the N-terminus was retained . The modified interferon-alpha 2 was indistinguishable from authentic interferon-alpha 2 in its ability to activate natural killer cells, to slow the growth of Daudi cells, and to confer virus resistance on heterologous cells.

Mol Cell Biol, 1983 Aug, 3(8), 1501 - 10
Location of the initial cleavage sites in mouse pre-rRNA; Bowman LH et al.; The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques . These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA . Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream . This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini . Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA . Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms . Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W . E . Goldman, G . Goldberg, L . H . Bowman, D . Steinmetz, and D . Schlessinger, Mol . Cell . Biol . 3:1488-1500, 1983).

Mol Cell Biol, 1983 Aug, 3(8), 1488 - 500
Mouse rDNA: sequences and evolutionary analysis of spacer and mature RNA regions; Goldman WE et al.; Two regions of mouse rDNA were sequenced . One contained the last 323 nucleotides of the external transcribed spacer and the first 595 nucleotides of 18S rRNA; the other spanned the entire internal transcribed spacer and included the 3' end of 18S rRNA, 5.8S rRNA, and the 5' end of 28S rRNA . The mature rRNA sequences are very highly conserved from yeast to mouse (unit evolutionary period, the time required for a 1% divergence of sequence, was 30 X 10(6) to 100 X 10(6) years) . In 18S rRNA, at least some of the evolutionary expansion and increase in G + C content is due to a progressive accretion of discrete G + C-rich insertions . Spacer sequence comparisons between mouse and rat rRNA reveal much more extensive and frequent insertions and substitutions of G + C-rich segments . As a result, spacers conserve overall G + C richness but not sequence (UEP, 0.3 X 10(6) years) or specific base-paired stems . Although no stems analogous to those bracketing 16S and 23S rRNA in Escherichia coli pre-rRNA are evident, certain features of the spacer regions flanking eucaryotic mature rRNAs are conserved and could be involved in rRNA processing or ribosome formation . These conserved regions include some short homologous sequence patterns and closely spaced direct repeats.

J Gen Microbiol, 1983 Aug, 129 (Pt 8), 2359 - 66
Isolation of polysomes from Nostoc sp . MAC and translation of messenger RNA in a heterologous cell-free system; Gupta M et al.; A method has been established which isolated polysomes from the lysozyme/EDTA-shocked cyanobacterium, Nostoc sp . MAC . In a typical preparation the total recovery of RNA as polysomes was 83%, in which 77% of the polysome fraction was present at sizes greater than 5-mers and 23% as 2-4-mers . Messenger RNA isolated from such a preparation of polysomes produced a 10-fold stimulation in the incorporation of {35S}methionine into polypeptides by a cell-free system of Escherichia coli . The in vitro-synthesized polypeptides were analysed on an SDS-polyacrylamide gradient gel together with in vivo-labelled proteins of Nostoc sp . MAC: seven polypeptides co-migrated with the in vivo-synthesized products . This is the first report of the expression of cyanobacterial messenger RNA in a heterologous cell-free system from E . coli; the efficiency of the system is discussed.

J Antibiot (Tokyo), 1983 Aug, 36(8), 1001 - 6
Total synthesis of edeine D; Czerwinski A et al.; Syntheses of the peptides with sequences postulated for active and inactive isomer of edeine D were carried out . The peptides obtained were identical with natural product in regard to chromatographic and electrophoretic properties . Biological data for synthesized compounds confirmed that in active and inactive isomer isoserine is linked with the alpha- or beta-amino group of alpha, beta-diaminopropionic acid, respectively.

Biochem J, 1983 Aug 1, 213(2), 473 - 8
Dog and human acid beta-D-galactosidases are structurally similar; Hubert JJ et al.; The purification of dog liver acid beta-galactosidase is described . The dog enzyme migrated as a single major band on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, with a molecular weight of 60000 . Antiserum raised against purified human liver acid beta-galactosidase cross-reacted with beta-galactosidase from dog liver, but not with those from cat liver or Escherichia coli . Tryptic peptide maps of the dog and human acid beta-galactosidases indicate that 21 of the 24 peptides observed were homologous; a similar result was obtained after chymotryptic peptide mapping . We conclude that dog and human acid beta-galactosidases are structurally similar, and that canine GM1 gangliosidosis (acid beta-galactosidase deficiency) is an excellent model for the same disease in man.

Proc Natl Acad Sci U S A, 1983 Aug, 80(15), 4619 - 23
Alpha-pyridine nucleotides as substrates for a plasmid-specified dihydrofolate reductase; Smith SL et al.; The alpha epimers of pyridine nucleotides are almost totally inactive as reductants in dehydrogenase reactions . In contrast, the R plasmid R67-specified dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) isolated from trimethoprim-resistant Escherichia coli utilized alpha-NADPH and alpha-NADH in addition to the "normal" beta-epimers . The enzymes from bacterial and mammalian sources used only beta-NADPH and beta-NADH . THe Km value for alpha-NADPH (16 microM) was 4-fold greater than that for beta-NADPH (4 microM), while the maximal velocity of the alpha-NADPH-catalyzed reaction was 70% of that seen with the beta-NADPH . beta-NADP+ and alpha-NADP+ were competitive inhibitors of the R67 enzyme . Pyridine nucleotide analogues such as deamino- and acetyl-NADPH were used readily by bacterial, plasmid, and mammalian enzymes, whereas thio-NADPH was used only by the plasmid enzyme . These data suggest that the enzyme from R plasmid R67 possesses a pyridine nucleotide binding site different from that of other dihydrofolate reductases and dehydrogenases.

J Med Chem, 1983 Aug, 26(8), 1193 - 6
Novel fluorinated antifolates . Enzyme inhibition and cytotoxicity studies on 2'- and 3'-fluoroaminopterin; Henkin J et al.; Two novel analogues of aminopterin with a single fluorine substitution in the 2' (compound 8) or in the 3' (compound 9) position of the p-aminobenzoyl group were synthesized and evaluated as inhibitors of dihydrofolate reductase from two bacterial species and from human HeLa cells . The 2'-fluoro compound was bound essentially the same as aminopterin itself, while the 3'-fluoro derivative bound two- to threefold more tightly in all cases . UV spectral shifts indicated normal binding of the pteridine . Cytotoxicity studies against mouse leukemia L1210 cells and the human stomach cancer line HuTu80 indicated equivalent toxicity of the parent drug with the 2'-fluoro analogue . 3'-Fluoroaminopterin was, however, twice as toxic as aminopterin to both cell lines.

Carcinogenesis, 1983 Aug, 4(8), 997 - 1000
Excision of aflatoxin B1-imidazole ring opened guanine adducts from DNA by formamidopyrimidine-DNA glycosylase; Chetsanga CJ et al.; This investigation has confirmed the earlier reports that when aflatoxin B1-DNA adducts are stored under physiological conditions some aflatoxin B1-guanine adducts are converted to a secondary product in which fission of the imidazole ring of the adduct guanine has occurred . Incubation of DNA containing aflatoxin B1-guanine adducts for an increasing number of hours under physiological conditions resulted in a progressive increase in the number of adducts in which the imidazole rings of guanines underwent fission . It was shown that the Escherichia coli enzyme, formamidopyrimidine-DNA glycosylase exercises from the 6-day incubated DNA, an amount of imidazole ring opened guanines equivalent to 40% of the aflatoxin B1-guanine adducts present in the DNA . The enzymatic excision of imidazole ring opened aflatoxin B1-guanine adducts is inhibited by Cibacron Blue F3GA a strong inhibitor of formamidopyrimidine-DNA glycosylase . Treatment of aflatoxin B1-DNA with mild alkali (pH 9.6), resulted in a 2-fold increase in the amount of aflatoxin B1-guanines with opened imidazole rings; this was revealed by enzyme assays using this alkaline treated DNA substrate as well as by analysis of acid hydrolysates of the alkaline treated DNA.

Clin Pediatr (Phila), 1983 Aug, 22(8), 582 - 4
Neonatal lead poisoning . An unusual clinical manifestation; Sensirivatana R et al.; A case of lead encephalopathy in a 2-month-old child is reported . Modes of poisoning are discussed and the unusual clinical manifestations of metallic brownish discoloration of nails and subdural effusion are presented . The possibility of lead poisoning as a cause of convulsions in neonates should be considered by doctors caring for these patients . Detailed history of lead exposure in prenatal and postnatal periods aids in early diagnosis and treatment which, thus, prevent severe neurological sequelae.

Anal Biochem, 1983 Aug, 133(1), 58 - 61
A method for concentration of nucleoside triphosphates by coprecipitation with calcium fluoride; Kukko EI et al.; A method for concentration of nucleoside triphosphates (NTP) is described . NTPs are quantitatively coprecipitated from the solution with calcium fluoride . The precipitate is separated by filtration through a membrane filter and NTPs are dissolved from the filter by immersing it in 0.5 N H2SO4 . With this method also nucleoside diphosphates can be efficiently concentrated, but the method does not work with nucleoside monophosphates or cyclic AMP.

Anal Biochem, 1983 Aug, 133(1), 153 - 6
A one-step procedure for the purification of uridine phosphorylase from Escherichia coli; Vita A et al.; Adsorption to Matrex Gel Green A and successive elution with uridine provides a one-step procedure for the purification of uridine phosphorylase from Escherichia coli B . The homogeneous preparation of the enzyme can be obtained within a single day with an activity recovery above 80%.

J Gen Microbiol, 1983 Aug, 129 (Pt 8), 2509 - 19
The separation of proteins based on their age, for the study of protein degradation in Escherichia coli; Cocucci M et al.; Density labelling with 2H2O has been used in association with isopycnic centrifugation to isolate proteins of known age from cultures of Escherichia coli . The physical properties of protein samples of known age have been examined to detect a correlation between specific properties and susceptibility to degradation . No evidence of a correlation between size, charge, thermodynamic properties or amino acid composition, on one hand, and susceptibility to degradation, on the other hand, was observed following step-down conditions of growth . However, the SH content of proteins in E . coli does appear to be correlated with their susceptibility to degradation.

J Biochem (Tokyo), 1983 Aug, 94(2), 415 - 20
Inhibitory effect of long chain fatty acyl CoAs on RNA polymerase from Escherichia coli; Yokokawa M et al.; RNA polymerase from Escherichia coli was inhibited by long chain fatty acyl CoAs, such as myristoyl CoA (Ki = 17.2 microM), palmitoyl CoA (Ki = 8.9 microM), oleoyl CoA (Ki = 5.5 microM), and stearoyl CoA (Ki = 0.94 microM) . The inhibition by these CoA thioesters was non-competitive against nucleoside triphosphates . Short chain fatty acyl CoAs, such as acetyl CoA, propionyl CoA, acetoacetyl CoA, butyryl CoA, and decanoyl CoA, failed to inhibit RNA polymerase . CoA, Na-myristate, Na-palmitate, Na-oleate, Na-stearate, palmitoyl carnitine, and carnitine did not inhibit the enzyme . The inhibition of RNA polymerase by long chain fatty acyl CoAs was competitive against template DNA.

J Biochem (Tokyo), 1983 Aug, 94(2), 409 - 13
Studies on the metabolism of unsaturated fatty acids . XII . Reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli; Mizugaki M et al.; Incorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli (E . coli) was investigated . When trans-2,cis-4-decadienoyl-CoA was incubated with 4R- or 4S-{4-2H1}NADPH in the presence of purified 2,4-dienoyl-CoA reductase, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to its pyrrolidine amide . On the other hand, when the dienoyl-CoA was incubated in the presence of NADPH and the reductase in 2H2O, two deuterium atoms were incorporated: One deuterium atom was located at the C-4 position of trans-2-decenoate, and the other at the C-5 position . The UV and shorter wavelengths of the visible spectrum of the reductase solution revealed that the reductase contained flavin as a prosthetic group . Therefore it is considered that a hydrogen atom of NADPH was first transferred to the flavin moiety of the reductase, and then the hydrogen atom was rapidly exchanged for one in the medium before its direct transfer to the substrate.

Genetika, 1983 Aug, 19(8), 1221 - 6
{Action of plasmid ColIb-P9 on the survival after ultraviolet irradiation and on the mutagenesis of the imiC, uvm, recL, uvrE and tif1 sfiA lexA spr mutants of Escherichia coli K-12 cells}; Kopylov VM et al.; To clarify the mechanisms whereby the ColIb-P9 plasmid affects DNA repair processes, its effect was studied in mutant Escherichia coli K-12 cells with altered mutagenesis and DNA repair . The plasmid was shown to protect umuC, uvm, recL and uvrE mutants after UV irradiation . The frequency of UV-induced his+ revertants increased in the presence of the plasmid in umuC, uvm and recL mutant cells . The ColIb-P9 plasmid completely restored the UV mutability and survival of umuC mutants . These results suggest that the ColIb-P9 plasmid may encode a product similar to that of the umuC gene . In the tif1 sfiA lexA spr mutant cells where SOS functions are constitutively expressed, the ColIb-P9 plasmid increased the number of his+ revertants several times . This suggests that the action of ColIb-P9 is probably brought about not via the derepression of the recA gene but at the subsequent stages of the recA+lexA+-dependent DNA error-prone repair.

Mol Cell Endocrinol, 1983 Aug, 31(2-3), 199 - 214
Developmental aspects of androgen-dependent mRNA from rat ventral prostate using cloned cDNA; Carter DB et al.; The androgen-dependence of two mRNAs from rat ventral prostate coding for a 20 000 and an 11 000 dalton translation product has been investigated using complementary DNA cloned in the bacterial plasmid PBR322 . One of the cloned insert DNAs from a recombinant plasmid, C-27, arrests the in vitro translation of C2 (Peeters et al . (1980) J . Biol . Chem . 255, 7017-7023) . The other cloned insert DNA arrests the translation of a glycoprotein 20 000 daltons in size, with unknown function . The quantities of mRNA coding for the 20 000 and 11 000 dalton translation product were determined by hybridization of 32P-labeled inserts to filter-bound total RNA or poly(A+)-mRNA . Castration caused a decline in both mRNAs of 250-fold over 8 days . Stimulation with androgen of 5-week castrates restored the mRNA levels to 17% of intact for the 20 000 dalton translation product and 31% of intact for the 11 000 dalton translation product . The quantity of the two mRNAs found in the lateral poly(A+)-mRNA was about 1/10 that of the ventral level and the mRNAs were not detectable in the dorsal prostate, seminal vesicle or human prostate poly(A+)-mRNA populations . RNA from the ventral prostates of animals 10-21 days old contained mature levels of complementary sequences, suggesting a form of developmental posttranscriptional regulation for synthesis of the polypeptides which are not synthesized in mature quantities at this stage of development (Heyns et al . (1978) Endocrinology 103, 1090-1095; Kistler et al . (1981) Proc . Natl . Acad . Sci . (U.S.A.) 78, 737-741).

Am J Vet Res, 1983 Aug, 44(8), 1497 - 500
Endotoxin-induced hemodynamic changes in dogs: role of thromboxane and prostaglandin I2; Bottoms GD et al.; Plasma concentrations of thromboxane and prostaglandin I2 (PGI2) before and after IV injection of endotoxin and resulting hemodynamic changes were evaluated . Effects of flunixin meglumine on plasma concentrations of these prostaglandins and the related hemodynamic changes were also determined . Shock was induced in 2 groups of anesthetized dogs . Four dogs were given endotoxin only and 4 dogs were given endotoxin and then were treated with flunixin meglumine . Arterial blood pressure (BP), cardiac output (CO), and heart rate were measured, and blood samples were collected at postendotoxin hours (PEH) 0, 0.1, 0.25, 0.5, 1, 2, 3, and 4 . Plasma thromboxane and PGI2 concentrations were increased in canine endotoxic shock . Thromboxane concentration was highest early in shock, and appeared to be associated with an initial decrease in BP and CO . The increased concentration of PGI2 was associated with systemic hypotension at PEH 1 to 2 . Treatment of dogs with flunixin meglumine at PEH 0.07 prevented further increase of thromboxane and blocked the release of PGI2, resulting in an increased CO, BP, and tissue aerobic metabolism.

Am J Vet Res, 1983 Aug, 44(8), 1442 - 5
Hemolytic complement titers and complement C3 levels in endotoxin-induced mastitis; Mueller R et al.; Escherichia coli lipopolysaccharide B was instilled through the lactiferous duct of cows to induce acute mastitis . Hemolytic complement (C) activity and C3 concentrations were determined in blood serum and in renninprecipitated whey before, and at certain times after, mastitis was induced . Hemolytic complement activity was detected in the whey only during the first 36 hours after endotoxin was instilled, whereas activity was not seen before and 48 or more hours after the endotoxin was given . The maximum titer as measured with the guinea pig RBC/bovine natural antibody system was 1:64 . The C3 concentrations in normal whey (before installation of endotoxin), measured by radial immunodiffusion, were between 1% and 4% of the base-line blood serum values (pool from healthy cows) . The whey concentration of C3 increased (to 5% to 18%) during the first 8 hours of mastitis . However, at 72 hours, the whey values were back to preinstillation concentrations in all quarters.

J Virol, 1983 Aug, 47(2), 367 - 9
Measurement of repair patch size by quantitation of nucleotides excised during DNA repair in vivo; Radany EH et al.; Escherichia coli uvr- cells, prelabeled in their DNA, were infected with phage T4 denV+ or T4 denV- under conditions that preclude phage-mediated degradation of the bacterial chromosome . Measurement of the distribution of acid-soluble radioactivity between pyrimidine dimers and nondimer nucleotides in cell extracts yielded calculated estimates of the average size of excision repair tracts that are in good agreement with the size of repair patches determined by others using direct measurement of repair synthesis.

Gene, 1983 Aug, 23(2), 195 - 8
Rapid isolation of Escherichia coli minicells by glass-fiber filtration: study of plasmid-coded polypeptides; Christen AA et al.; A filtration technique is described to purify Escherichia coli chi 1488 minicells much more rapidly than the usual method involving sucrose gradient centrifugation, and to produce minicells that have not been subjected to osmotic stress . The minicells so prepared are metabolically active as indicated by the in vivo incorporation of {35S}methionine into plasmid-coded polypeptides.

Biochem J, 1983 Aug 1, 213(2), 495 - 502
Comparison of inhibitors of S-adenosylmethionine decarboxylase from different species; Pegg AE et al.; S-Adenosyl-L-methionine decarboxylases were purified from rat ventral prostate, yeast (Saccharomyces cerevisiae), slime mould (Physarum polycephalum) and bacteria (Escherichia coli) and tested for inhibition by a variety of nucleosides related to S-adenosylmethionine and by methyl- and ethyl-glyoxal bis(guanylhydrazone) . Although the enzymes from these different sources are markedly different with respect to activation by cations, the inhibition by nucleosides was quite similar . Very little inhibition was seen when analogues of S-adenosylmethionine with a different base were tested or when the ribose ring was opened or the positive charge on the sulphur atom was not present . Some derivatives in which the amino acid portion of the molecule was altered were more potent inhibitors, but again there was little difference between the enzymes from different sources . 5'-(Dimethylsulphonio)-5'-deoxyadenosine and S-adenosyl-3-methylthiopropylamine were the most inhibitory substances and had similar Ki values, suggesting that the aminopropyl group does not contribute significantly to the binding . All of the S-adenosylmethionine decarboxylases were strongly competitively inhibited by methylglyoxal bis(guanylhydrazone) and even more powerfully by its ethyl analogue, although the putrescine-activated enzymes from prostate and yeast were more sensitive than the bacterial and slime-mould enzymes . All of the S-adenosylmethionine decarboxylases tested bound to a column of methylglyoxal bis(guanylhydrazone) linked to Sepharose and were not eluted by 0.5 M-NaCl, but could be released by 1 mM concentrations of the drug, providing a rapid and efficient method for their purification.

Biull Eksp Biol Med, 1983 Aug, 96(8), 76 - 7
{Conjugativity and incompatibility of derepressed pAP11-2 plasmid}; Buianova NI et al.; It has been demonstrated during investigation of Colplasmid pAP11-2 and its varieties labeled with transpozone (Tn1 and Tn9) that this plasmid is a derepressed one in terms of transfer functions in E . coli strain K-12 cells as well as in some of serologically typed strains of this type . The plasmid under study is incompatible with reference plasmids belonging to two different groups (FI and FIV) and is marked by a number of the properties common to the system of genetic control over Tra-functions.

Am Rev Respir Dis, 1983 Aug, 128(2), 282 - 7
Procoagulant activity of rabbit alveolar macrophages; Sitrin RG et al.; Alveolar macrophages are thought to participate in the clearance of fibrin from the injured lung, but their ability to facilitate the conversion of fibrinogen to fibrin (procoagulant activity) has not been described . In order to characterize their procoagulant properties, unstimulated alveolar macrophages obtained from normal rabbits were tested for their ability to accelerate the coagulation of plasma in a one-stage clotting assay . Compared with control assays containing no macrophages (coagulation times greater than 500 s), intact cells (10(6)/ml) were shown to display procoagulant activity (coagulation time, 153.6 +/- 11.3 s mean +/- SEM) . Cell lysis caused further procoagulant activity to be expressed (125.6 +/- 11.8 s) . Alveolar macrophages that were stimulated in vitro with bacterial lipopolysaccharide (LPS) or the purified complement fragments C5a and C5a des Arg caused further significant (p less than 0.002) reductions in coagulation times (intact cells, 71 to 76 s; lysed cells, 27 to 32 s), representing 5- to 6-fold and 30- to 40-fold increases in the procoagulant activity of intact and lysed cells, respectively . The generation of this material was independent of the presence of lymphocytes . The procoagulant material was identified as a cell-associated tissue thromboplastin, acting via the extrinsic coagulation pathway . These findings show that alveolar macrophages have procoagulant activity that is markedly augmented by LPS and complement fragments . This suggests that alveolar macrophages may contribute to intra-alveolar fibrin deposition in vivo.

Urology, 1983 Aug, 22(2), 212 - 4
Mycotic aneurysm of renal graft artery: diagnosis by ultrasonography; Squifflet JP et al.; Mycotic aneurysm of the renal graft artery is rarely detected before its rupture . Diagnosis up to now relied on arteriography . We report on a patient whose preoperative diagnosis was made by ultrasonography . Recent progress with this noninvasive procedure should allow earlier recognition of this life-threatening complication.

Proc Natl Acad Sci U S A, 1983 Aug, 80(16), 4936 - 9
Anticodon shift in tRNA: a novel mechanism in missense and nonsense suppression; Murgola EJ et al.; In a previous publication, an unusual UGG-reading missense suppressor caused by insertion of an extra adenylate residue in the anticodon loop of an Escherichia coli glycine tRNA was described . In this study, we provide in vivo evidence that the additional nucleotide causes an "anticodon shift" by one nucleotide in the 3' direction and that the "new" anticodon can explain the unanticipated coding properties of the suppressor . We converted the UGG suppressor with ethyl methanesulfonate, a base-substitution mutagen, to suppressors that read codons related to UGG by a single base change . Sequence analysis of each mutant tRNA revealed that its mutational alteration was an anticipated base change in one of the three nucleotides of the "new" anticodon . Although the new suppressors read codons beginning with A or U, the mutant tRNAs lack the customary hypermodified nucleosides on the 3' side of the anticodon . As determined on the basis of their in vivo coding specificities, the new mutant tRNAs do not continue to utilize the original anticodon triplet for decoding . Furthermore, the failure of the UGG suppressor to correct frameshift mutations throughout each of three genes of the trp operon suggests that the addition of a nucleotide to the anticodon loop of a tRNA does not necessarily result in out-of-frame decoding by the tRNA . Therefore, a "frameshift" mutation in a tRNA has principally changed the triplet codon recognition properties of the molecule.

Proc Natl Acad Sci U S A, 1983 Aug, 80(16), 4904 - 8
Molecular cloning and sequence of partial cDNA for interferon-induced (2'-5')oligo(A) synthetase mRNA from human cells; Merlin G et al.; By using a translation assay in oocytes, a 17S RNA fraction coding for the interferon-induced (2'-5')oligo(A) synthetase was purified from human cells . A cDNA library was prepared by cloning in Escherichia coli plasmid pBR322 and screened by positive hybridization-translation in oocytes . A cDNA clone corresponding to the (2'-5')oligo(A) synthetase mRNA was identified . In SV80 cells, this E cDNA recognizes three RNAs of 1.65, 1.85, and 3.6 kilobases, which are present only after interferon treatment of the cells . In Namalva cells, mainly one RNA of 1.8 kilobases is seen.

Proc Natl Acad Sci U S A, 1983 Aug, 80(16), 4884 - 8
Escherichia coli polymerase I can use O2-methyldeoxythymidine or O4-methyldeoxythymidine in place of deoxythymidine in primed poly(dA-dT).poly(dA-dT) synthesis; Singer B et al.; O2-and O4-alkyldeoxythymidine are among the four O-alkyl base-modified derivatives produced by the reaction of N-nitroso alkylating agents with nucleic acids in vitro and in vivo . We find that both O2- and O4-methyl-dTTP can substitute for dTTP in alternating poly(dA-dT)-primed DNA synthesis . Up to 22% of the pyrimidines in the newly synthesized polymer were found by HPLC analysis to be O-methyldeoxythymidine . Little polymer synthesis was observed in the absence of dTTP . However, the O-methyl-dTTPs did not inhibit polymerization of dATP and dTTP . Polymers containing O2- or O4-methyldeoxythymidine were obtained in good yield, retaining the secondary structure of alternating poly(dA-dT) . This was shown by the data for thermal transition under different conditions . In contrast, poly(dA-dT).poly(dA-dT) methylated or ethylated to less than 4% total modification by alkylnitrosoureas had a distinctly less stable structure . Neither O2- nor O4-methyldeoxythymidine can form more than one hydrogen bond with adenosine . The unchanged secondary structure of polymers containing these modified thymidines indicates that stacking interactions must play a major role in helix stabilization . O-Alkyldeoxythymidine may be formed by N-nitroso carcinogens that react intracellularly . We have shown that the triphosphates can be utilized by Escherichia coli DNA polymerase I as dTTP . The incorporated O4-methyl-dT causes misincorporation of G, both in transcription and synthesis . When O2-methyl-dT is present, less, but definite, misincorporation results.

Proc Natl Acad Sci U S A, 1983 Aug, 80(16), 4875 - 8
Association of 16S and 23S ribosomal RNAs to form a bimolecular complex; Burma DP et al.; Association of the 30S and 50S subunits to generate the 70S ribosomes of Escherichia coli has long been known but the mechanism of this interaction remains obscure . Light-scattering studies indicate that naked 16S and 23S RNAs can also associate under conditions similar to those required for the assembly of ribosomes from the constituent RNAs and proteins . The RNA-RNA association also takes place in the presence of ethanol, which promotes folding of 16S and 23S RNAs into specific compact structures with the morphological features of 30S and 50S ribosomes, respectively . Equimolar amounts of the two RNAs are involved in the association . The formation of a stoichiometric complex was shown by light scattering, sucrose density gradient centrifugation, and composite polyacrylamide/agarose gel electrophoresis . The presence of the two species of RNA in the complex was also shown by gel electrophoresis . The association of naked 16S and 23S RNAs suggests that RNA-RNA interaction may play an important role in the association of 30S and 50S subunits.

Proc Natl Acad Sci U S A, 1983 Aug, 80(15), 4624 - 8
Molecular requirements for B-lymphocyte activation by Escherichia coli lipopolysaccharide; Raetz CR et al.; Certain Escherichia coli mutants altered in phosphatidylglycerol metabolism accumulate fatty acyl derivatives of glucosamine 1-phosphate . Especially prominent is 2,3-diacylglucosamine 1-phosphate (previously designated lipid X), which may be an early precursor of lipid A . We have examined the activity of lipid X (Mr = 711.9) and several related compounds as mitogens towards mouse lymphocytes . As judged by labeling with {methyl-3H}thymidine, lipid X is mitogenic, and it mimics the properties of lipopolysaccharide and lipid A . The following evidence suggests that lipid X exerts its effects by a route similar to that of lipopolysaccharide: (i) lymphocytes from C3H/HeJ mice, which are unresponsive to lipopolysaccharide, are also not stimulated by lipid X; (ii) polymyxin B abrogates lymphocyte stimulation by lipid X; and (iii) lipid X induces the proliferation and maturation of lymphocytes to antibody-producing plaque-forming cells . Selective removal of the ester-linked hydroxymyristate moiety at position 3 totally abolishes mitogenic activity . Other phospholipids, such as phosphatidic acid, CDP-diglyceride, phosphatidylcholine, and lysophosphatidylcholine, have no activity as mitogens . If lipid X and lipid A induce by common mechanism(s) B-lymphocyte proliferation, then it follows from structural comparison that the reducing-end subunit of lipid A is the minimal structural requirement for this activity . Because the structure of lipid X is completely defined, biochemical and pharmacological dissection of B-cell activation by lipopolysaccharide should now be possible.

J Bacteriol, 1983 Aug, 155(2), 922 - 5
Alternative explanation for excision repair deficiency caused by the polAex1 mutation; Wahl AF et al.; An investigation of the mechanism of the polAex1 mutation in vitro suggested that the excision repair deficiency observed in vivo does not result from an inability of the enzyme to nick translate . The defect appears to reside in the inability of the enzyme to effectively generate a nick structure to serve as a substrate for DNA ligase.

J Bacteriol, 1983 Aug, 155(2), 854 - 9
Identification of the rodA gene product of Escherichia coli; Stoker NG et al.; Plasmids that carry the Escherichia coli cell shape gene rodA directed the synthesis of a cytoplasmic membrane protein (Mr, 31,000 {31K protein} ) in minicells, maxicells, and an in vitro-coupled transcription-translation system . The 31K protein was identified as the rodA gene product, because it was not synthesized from the vector plasmids or from a plasmid in which the rodA gene was inactivated by insertion of Tn1000 . Furthermore, a purified 1.6-kilobase KpnI-BamHI DNA fragment that contained the intact rodA gene directed the synthesis of only the 31K protein in an in vitro system . The apparent molecular weight of the protein was identical whether synthesized in vivo or in vitro, indicating that the rodA gene product is not made as a preprotein . The direction of transcription of rodA was from the KpnI site towards the BamHI site . The 31K protein was unusual in that it could only be detected when cell membranes were solubilized at low temperature (e.g., 37 degrees C) before sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Apparently the rodA gene product aggregates after being boiled in sodium dodecyl sulfate and fails to enter a polyacrylamide gel.

J Bacteriol, 1983 Aug, 155(2), 768 - 75
hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis; Moyed HS et al.; Except for a small fraction of persisters, 10(-6) to 10(-5), Escherichia coli K-12 is killed by prolonged inhibition of murein synthesis . The progeny of persisters are neither more resistant to inhibition of murein synthesis nor more likely to persist than normal cells . Mutants have been isolated in which a larger fraction, 10(-2), persists . The persistent response of the mutants, Hip (high persistence), is to inhibition of murein synthesis at early or late steps by antibiotics (phosphomycin, cycloserine, and ampicillin) or by metabolic block (starvation for diaminopimelic acid) . Killing of the parent strain by each of the four inhibitors has two phases: The first is rapid and lasts about 30 min; the second is slower, but still substantial, and lasts 3 to 4 h . The first phase also occurs in the Hip mutants, but then viability of the mutants remains constant after about 30 min . Neither tolerance, resistance, impaired growth, nor reversion of spheroplasts accounts for high-frequency persistence . Two of the mutations map at 33.8 min in a region containing few other recognized functions . This position and the phenotypes define hipA as a newly recognized gene . Transposons Tn5 and Tn10 have been inserted close to hipA making it possible to explore the molecular genetics of persistence, a long recognized but poorly understood phenomenon.

J Bacteriol, 1983 Aug, 155(2), 728 - 33
Sequence of heat-labile enterotoxin of Escherichia coli pathogenic for humans; Yamamoto T et al.; We determined the complete nucleotide sequence of the toxB gene (375 base pairs in length), which encodes the B subunit of heat-labile enterotoxin produced from Escherichia coli pathogenic for humans (hLT) . The amino acid sequence of the B subunit of hLT was deduced from the nucleotide sequence . Consequently, it has become possible to study the homology between the B subunits of three similar toxins: hLT, LT produced from E . coli pathogenic for piglets (pLT), and cholera toxin (the latter two sequences have been reported by others) . The three B subunits are all 103 amino acids in length . A comparison of the toxB gene and the eltB gene, which encodes the B subunit of pLT, showed a 98% homology at the nucleotide level and a 95% homology at the amino acid (of a precursor) level, indicating the possibility that the two genes share a common ancestor . With respect to the B-subunit sequences, the homologies between hLT and pLT, between hLT and cholera toxin, and between pLT and cholera toxin were 96, 81, and 79%, respectively . Several large common sequences are conserved by the three peptides . In contrast, no sequences are present in both pLT and cholera toxin but missing in hLT.

J Bacteriol, 1983 Aug, 155(2), 664 - 80
Escherichia coli RecBC pseudorevertants lacking chi recombinational hotspot activity; Schultz DW et al.; Pseudorevertants of an Escherichia coli exonuclease V (RecBC enzyme)-negative mutant have been isolated after ethyl methane sulfonate mutagenesis of a recC73 (presumed missense) mutant . The remedial mutations in each of the four pseudorevertants studied in detail map and complement as recC mutations . By several criteria, such as recombination proficiency, support of phage growth, RecBC nuclease activity, and cell viability, the pseudorevertants appear to have regained partially or completely various aspects of RecBC activity . However, chi recombinational hotspots, which stimulate exclusively the RecBC pathway of recombination, have no detectable activity in lambda vegetative crosses in the pseudorevertants . The properties of these mutants, in which the RecBC pathway of recombination is active yet in which chi is not active, are consistent with the hypothesis that wild-type RecBC enzyme directly interacts with chi sites; alternatively, the mutants may block or bypass the productive interaction of another recombinational enzyme with chi.

J Bacteriol, 1983 Aug, 155(2), 447 - 53
Changes in the composition of Escherichia coli murein as it ages during exponential growth; Burman LG et al.; Escherichia coli murein was specifically labeled with {14C}diaminopimelic acid in the mutant strains W7 (dap lysA) and BUG6 . Pulse-labeled heat-denatured E . coli cells were digested with 2 mg of egg-white lysozyme per ml to degrade the murein completely and free any lipoprotein-bound muropeptide trimers, dimers, and monomers . Pulse-chase experiments showed that the relative percentage of trimers and dimers found in the newly synthesized murein increased somewhat with time at the expense of monomers . The increase in cross-links indicated that the radioactive monomers served as acceptors in multisite transpeptidations occurring after the labeling period . The content of nonreducing monomers (C7 and C8) remained unaltered, indicating that the oligosaccharide chain length did not change with time . A gradual conversion of the reducing disaccharide tetrapeptide monomer to its tripeptide analog occurred during chasing . Braun lipoprotein was linked to about 2% of the murein subunits within 30 s of the incorporation of subunits into insoluble murein, and after one-half a generation of chase, lipoprotein-associated muropeptides had approached the maximum (16% of the total murein subunits) . The distribution of muropeptides was similar in lipoprotein-linked and lipoprotein-free murein, showing that the enzyme that links Braun lipoprotein to murein does not discriminate between monomers, dimers, and trimers . No evidence for a chasable, soluble polymer of murein was found in our experiments . Hence, our data support the idea that new murein is incorporated directly into the sacculus without first existing as a soluble intermediate.

Infect Immun, 1983 Aug, 41(2), 709 - 21
Identification and preliminary characterization of Treponema pallidum protein antigens expressed in Escherichia coli; Stamm LV et al.; We have previously described the construction in Escherichia coli K-12 of a hybrid plasmid colony bank of Treponema pallidum (Nichols strain) genomic DNA . By screening a portion of this bank with an in situ immunoassay, we identified six E . coli clones that express T . pallidum antigens . In this study, the recombinant plasmids from each of these clones have been analyzed in E . coli maxicells and have been found to encode a number of proteins that are not of vector pBR322 origin and are, therefore, of treponemal origin . In each case, several of these proteins can be specifically precipitated from solubilized maxicell extracts by high-titer experimental rabbit syphilitic serum . Certain of these proteins are also precipitated by high-titer latent human syphilitic sera (HSS) . The T . pallidum DNA inserts in these plasmids range in size from 6.2 to 14 kilobase pairs, and from the restriction patterns of the inserts and the protein profiles generated by each plasmid in maxicells, it is apparent that we have recovered a total of four unique clones from our colony bank . Recombinant plasmids pLVS3 and pLVS5 were of particular interest . Plasmid pLVS3 encodes three major protein antigens with molecular weights of 39,000, 35,000, and 25,000 . These three proteins, which were not recognized by pooled normal human sera, were efficiently precipitated by most secondary HSS, latent HSS, and late HSS tested . These proteins were also precipitated, although somewhat inefficiently, by most primary HSS tested . Plasmid pLVS5 encodes a major protein antigen with a molecular weight of 32,000 and several minor protein antigens that, although efficiently precipitated by experimental rabbit syphilitic serum, were generally not recognized by the various HSS tested . Evidence is presented indicating that the protein antigens encoded by plasmids pLVS3 and pLVS5 are specific for pathogenic treponemal species . We have also demonstrated that immunoglobulin G antibodies directed against these protein antigens can be detected in rabbits experimentally infected with T . pallidum Nichols as early as 11 days postinfection.

Am J Vet Res, 1983 Aug, 44(8), 1446 - 50
Influence of intramammary infusion of polymyxin B on the clinicopathologic course of endotoxin-induced mastitis; Ziv G et al.; The influence of giving 1 dose of polymyxin B (1.6 X 10(6) U/quarter) by the intramammary route to dairy cows to modify the clinicopathologic course of mastitis induced by intramammary infusion of Escherichia coli endotoxin (1 mg/quarter) was examined . Pretreatment with polymyxin B or its simultaneous administration reduced and delayed the typical febrile and leukopenic responses to endotoxin infusion, prevented an increase in plasma lactate dehydrogenase activity and a reduction in plasma zinc concentration, but only marginally influenced the degree of udder inflammation and had no effect on leukocytosis in the milk . Intramammary infusion of polymyxin B at 30 or 60 minutes after endotoxin was infused prevented the increase in plasma lactate dehydrogenase activity and moderated the decrease in plasma zinc concentration, but otherwise failed to alter the clinicopathologic course of endotoxin-induced acute mastitis.

Mol Cell Biol, 1983 Aug, 3(8), 1421 - 9
Amplification and hormone-regulated expression of a mouse mammary tumor virus-Eco gpt fusion plasmid in mouse 3T6 cells; Chapman AB et al.; Mouse 3T6 cells were transformed with a chimeric DNA plasmid, pSVMgpt, in which the mouse mammary tumor virus (MMTV) promoter was fused to the Escherichia coli gene encoding xanthine-guanine phosphoribosyl transferase (Eco gpt) . The transformants exhibited glucocorticoid-inducible expression of Eco gpt . With limiting xanthine concentrations, conditions were established in which cell growth became hormone dependent . Cells selected for their ability to grow in limiting concentrations of both xanthine and glucocorticoids contained amplified levels of Eco gpt DNA, and expression of Eco gpt remained glucocorticoid inducible in these amplified cells . Thus, amplification of the MMTV promoter region in itself did not abolish hormonal responsiveness of a gene . In addition to increased levels of Eco gpt DNA, some of the selected cells also exhibited increased levels (two- to threefold) of glucocorticoid receptors . Lastly, we found that excessive expression of Eco gpt is toxic to 3T6 cells; by maintaining low hormone levels and, therefore, low levels of expression, we were able to select cells with amplified Eco gpt . Thus, the MMTV promoter may be of general utility in expressing genes whose products may be lethal if they are produced in excessive quantities.

Gene, 1983 Aug, 23(2), 167 - 74
Cloning and characterization of the umu operon responsible for inducible mutagenesis in Escherichia coli; Shinagawa H et al.; In Escherichia coli, radiation and chemically inducible mutagenesis requires a functional umuC gene product . The umuC mutants are defective in mutagenesis and slightly sensitive to DNA damaging agents . A chromosomal fragment that complemented the umuC mutations for UV mutability and UV resistance was cloned into miniF vector plasmid pMF3 by a shotgun method . A restriction map of the hybrid plasmid was constructed . Further subcloning, Tn1000 insertion inactivation, and complementation tests revealed that there are two genes, umuD and umuC in the former umuC region . The gene products of umuD and umuC were identified by the maxicell method to be proteins with Mr of 18 000 and 46 000, respectively . The two genes comprise an operon, and the transcriptional direction is from umuD to umuC . A plasmid carrying an umuC'-lac'Z gene fusion was constructed in vitro to study the regulation of the umu operon . It was shown that the umu operon is inducible by UV and chemical mutagens, and is regulated by the recA and lexA genes.

Gene, 1983 Aug, 23(2), 149 - 56
Overlapping divergent promoters control expression of Tn10 tetracycline resistance; Bertrand KP et al.; We have previously examined the genetic organization and regulation of the Tn10 tetracycline-resistance determinant in Escherichia coli K-12 . The structural genes for tetA, the Tn10 tetracycline-resistance function, and for tetR, the Tn10 tet repressor, are transcribed in opposite directions from promoters in a regulatory region located between the two structural genes . Expression of both tetA and tetR is induced by tetracycline . Here we report the DNA sequence of the Tn10 tet regulatory region . The locations of the tetA and tetR promoters within this region were defined by S1 nuclease mapping of the 5' ends of in vivo tet RNA . The tetA and tetR promoters overlap; the transcription start points are separated by 36 bp . We propose that two similar regions of dyad symmetry within the Tn10 tet regulatory region are operator sites at which tet repressor binds to tet DNA, thereby inhibiting transcription initiation at the tetA and tetR promoters . The Tn10 tet regulatory region and the pBR322 tet regulatory region show significant DNA sequence homology (53%).

Proc Natl Acad Sci U S A, 1983 Aug, 80(16), 4889 - 93
Reconstitution of active transport in proteoliposomes containing cytochrome o oxidase and lac carrier protein purified from Escherichia coli; Matsushita K et al.; Most active transport across the bacterial cell membrane is driven by a proton electrochemical gradient (delta-muH+, interior negative and alkaline) generated via electron transfer through a membrane-bound respiratory chain . This phenomenon is now reproduced in vitro with proteoliposomes containing only two proteins purified from the membrane of Escherichia coli . An o-type cytochrome oxidase was extracted from membranes of a cytochrome d terminal oxidase mutant with octyl beta-D-glucopyranoside after sequential treatment with urea and cholate and was purified to homogeneity by ion-exchange chromatography . The purified oxidase contains four polypeptides (MrS 66,000, 35,000, 22,000, and 17,000), two b-type cytochromes (b558 and b563), and 16-17 nmol of heme b per mg of protein, and it catalyzes the oxidation of ubiquinol and other electron donors with specific activities 20- to 30-fold higher than crude membranes . The lac carrier protein was purified as described . Proteoliposomes were formed in the presence of the oxidase and lac carrier protein by detergent dilution, followed by freeze-thaw/sonication . The system generates a delta-muH+ (interior negative and alkaline) with ubiquinol as electron donor and the magnitude of delta-muH+ is dependent on the concentration of cytochrome o in the proteoliposomes . Furthermore, the proteoliposomes transport lactose against a concentration gradient to an extent that is commensurate with the magnitude of delta-muH+ generated . The results provide powerful additional support for the "chemiosmotic hypothesis" and demonstrate that purified lac carrier protein retains the ability to function in a physiological manner.

Proc Natl Acad Sci U S A, 1983 Aug, 80(15), 4784 - 8
Mini-F plasmid genes that couple host cell division to plasmid proliferation; Ogura T et al.; A mechanism for stable maintenance of plasmids, besides the replication and partition mechanisms, has been found to be specified by genes of a mini-F plasmid . An oriC plasmid carrying both a mini-F segment necessary for partition {coordinates 46.4-49.4 kilobase pairs (kb) on the F map} and another segment (42.9-43.6 kb), designated ccd (coupled cell division), is more stably maintained than are oriC plasmids carrying only the partition segment; the stability is comparable to that of the parental mini-F plasmid . When replication of a plasmid carrying ccd is prevented and the plasmid copy number decreases, to as few as one per cell, host cell division is inhibited, but not increase of turbidity or chromosome replication . Appearance of plasmid-free segregants is therefore effectively prevented under such conditions . Experimental results suggest that reduction of the copy number of plasmids carrying the ccd region causes an inhibition of cell division and that the ccd region can be dissected into two functional regions; one (ccdB) inhibits cell division and the other (ccdA) releases the inhibition . The interplay of the ccdA and ccdB genes promotes stable plasmid maintenance by coupling host cell division to plasmid proliferation.

Proc Natl Acad Sci U S A, 1983 Aug, 80(15), 4775 - 9
Cyclic AMP-dependent constitutive expression of gal operon: use of repressor titration to isolate operator mutations; Irani M et al.; When the gal operator region is present in a multicopy plasmid it binds to all ("titrates") the gal repressor and "induces" the chromosomal gal operon . To make operator mutations (Oa) with reduced affinity toward the repressor, plasmid DNA was irradiated with UV light and mutant derivatives were isolated that were unable to release the chromosomal gal genes from repression . Then with such an Oa plasmid operator revertants were isolated that had reacquired the ability to release repression . Both sets of mutations have been localized by DNA sequence analysis . When the Oa mutations were transferred from the plasmid to the chromosome by recombination these mutant operators were found to make gal expression constitutive (independent of repressor) but still dependent on cAMP, whereas the previously reported gal operator mutants (Oc) are constitutive both in the presence and in the absence of cAMP . The titration method of isolating mutants enables the isolation of strains with operator mutations that also affect normal promoter activity, and it provides an easy way to isolate revertants of operator mutations.

Proc Natl Acad Sci U S A, 1983 Aug, 80(15), 4659 - 63
Identification of Escherichia coli DNA helicase I as the traI gene product of the F sex factor; Abdel-Monem M et al.; Active DNA helicase I (Mr 180,000) can be isolated from Escherichia coli F+ strains but not F- strains . The transfer of the F sex factor to F- strains by conjugation permits the purification of the enzyme from the transconjugant strains . We conclude from this that helicase I is coded for by a portion of the F factor . Results also obtained by using recombinant plasmids carrying different DNA fragments of the F factor transfer region suggest that DNA helicase I is identical to the product of traI, one of the transfer genes of the F factor.

Proc Natl Acad Sci U S A, 1983 Aug, 80(15), 4639 - 43
Methyl-directed repair of DNA base-pair mismatches in vitro; Lu AL et al.; An assay has been developed that permits analysis of DNA mismatch repair in cell-free extracts of Escherichia coli . The method relies on repair of heteroduplex molecules of f1 R229 DNA, which contain a base-pair mismatch within the single EcoRI site of the molecule . As observed with mismatch heteroduplexes of lambda DNA {Pukkila, P . J., Peterson, J., Herman, G., Modrich, P . & Meselson, M . (1983) Genetics, in press}, in vivo mismatch correction of f1 heteroduplexes is directed by the state of dam methylation of d(G-A-T-C) sequences within the DNA duplex . Thus, the heteroduplex (formula: see book) is repaired in vivo to an EcoRI-sensitive form if the strand bearing the wild-type EcoRI sequence carries the dam modification and the other does not . Such molecules are also subject to mismatch repair by E . coli extracts . The in vitro activity is also dependent on ATP, the state of dam methylation of mismatch heteroduplexes, and products of mutH, mutL, mutS, and uvrE loci . However, crude fractions deficient in these gene products do complement in the cell-free system, thus providing assays for their isolation . The in vitro reaction is accompanied by repair synthesis on the unmethylated DNA strand.

Proc Natl Acad Sci U S A, 1983 Aug, 80(15), 4629 - 33
The carB gene of Escherichia coli: a duplicated gene coding for the large subunit of carbamoyl-phosphate synthetase; Nyunoya H et al.; Previous genetic and biochemical studies indicate that the carB gene of Escherichia coli codes for the large subunit of carbamoyl-phosphate synthetase (EC 6.3.5.5) . We have determined the nucleotide sequence of a 4-kilobase-pair cloned fragment of E . coli DNA with genetic determinants for carB . The DNA sequence is a 3,219-nucleotide-long reading frame . The polypeptide encoded by this reading frame has been verified to be the large subunit of carbamoyl-phosphate synthetase . The gene product is similar to the large subunit in its molecular weight, amino acid composition and amino-terminal residue, and carboxyl-terminal sequence . The amino acid sequence derived from the nucleotide sequence shows a highly significant homology between the amino- and carboxyl-terminal halves of the protein . We propose that the carB gene was formed by an internal duplication of a smaller ancestral gene.

J Bacteriol, 1983 Aug, 155(2), 943 - 6
Inversion in the lactose region of Escherichia coli K-12: inversion termini map within IS3 elements alpha 3 beta 3 and beta 5 alpha 5; Savic DJ et al.; In this work, the previously described inversion in the lactose region of the Escherichia coli K-12 chromosome (D . J . Savic, J . Bacteriol . 140:311-319, 1979) is analyzed in greater detail . The results presented indicate that the inversion most likely occurred by a homologous recombination between alpha 3 beta 3 and beta 5 alpha 5 IS3 elements.

J Bacteriol, 1983 Aug, 155(2), 910 - 4
Conservation of capR (lon) DNA of Escherichia coli K-12 between distantly related species; Rupprecht KR et al.; Mutations in the capR gene of Escherichia coli K-12 are responsible for a wide variety of phenotypic changes, including defects in cell division . Since this gene plays a critical role in cell division, it might be evolutionarily conserved . Of the DNAs examined by Southern analysis, capR probe sequences were found not only in other enterics but also in Caulobacter crescentus CB13 and the distantly related archebacterium Halobacterium halobium R1.

J Bacteriol, 1983 Aug, 155(2), 793 - 801
Molybdenum cofactor in chlorate-resistant and nitrate reductase-deficient insertion mutants of Escherichia coli; Miller JB et al.; We examined molybdenum cofactor activity in chlorate-resistant (chl) and nitrate reductase-deficient (nar) insertion mutants and wild-type strains of Escherichia coli K-12 . The bacterial molybdenum cofactor was assayed by its ability to restore activity to the cofactor-deficient nitrate reductase found in the nit-1 strain of Neurospora crassa . In the wild-type E . coli strains, molybdenum cofactor was synthesized constitutively and found in both cytoplasmic and membrane fractions . Cofactor was found in two forms: the demolybdo form required additional molybdate in the assay mix for detection, whereas the molybdenum-containing form was active without additional molybdate . The chlA and chlE mutants had no detectable cofactor . The chlB and the narG, narI, narK, and narL (previously designated chlC) strains had cofactor levels similar to those of the wild-type strains, except the chlB strains had two to threefold more membrane-bound cofactor . Cofactor levels in the chlD and chlG strains were sensitive to molybdate . When grown in 1 microM molybdate, the chlD strains had only 15 to 20% of the wild-type levels of the demolybdo and molybdenum-containing forms of the cofactor . In contrast, the chlG strains had near wild-type levels of demolybdo cofactor when grown in 1 microM molybdate, but none of the molybdenum-containing form of the cofactor . Near wild-type levels of both forms of the cofactor were restored to the chlD and chlG strains by growth in 1 mM molybdate.

J Bacteriol, 1983 Aug, 155(2), 593 - 600
In vivo regulation of the Escherichia coli araC promoter; Hahn S et al.; The ara pC promoter is known to be derepressed about fivefold for 20 to 30 min after the addition of arabinose . This transient derepression was studied by using araC::Mu lac insertions and araC-lacZ gene fusions . In strains containing increased levels of araC protein, the pC promoter became progressively less derepressible, but the ara pBAD promoter remained normally inducible . Repression of pC was reestablished 20 min after induction in araB mutants, but did not occur in arabinose-transport-deficient mutants . Finally, mutant araCc proteins which normally do not repress pC did so in the presence of arabinose.

J Bacteriol, 1983 Aug, 155(2), 565 - 77
Genetics of methyl-accepting chemotaxis proteins in Escherichia coli: organization of the tar region; Slocum MK et al.; The tar locus of Escherichia coli specifies one of the major species of methyl-accepting proteins involved in the chemotactic behavior of this organism . The physical and genetic organization of the tar region was investigated with a series of specialized lambda transducing phages and plasmid clones . The tar gene was mapped at the promoter-proximal end of an operon containing five other chemotaxis-related loci . Four of those genes (cheR, cheB, cheY and cheZ) are required for all chemotactic responses; consequently, polar mutations in the tar gene resulted in a generally nonchemotactic phenotype . The fifth gene, tap, was mapped between the tar and cheR loci and specified the production of a 65-kilodalton methyl-accepting protein . Unlike the tar locus, which is required for chemotaxis to aspartate and maltose, mutants lacking only the tap function had no obvious defects in chemotactic ability . Genetic and physical maps of the tar-tap region were constructed with Mu d1 (Apr lac) insertion mutations, whose polar properties conferred a phenotype suitable for deletion mapping studies . Restriction endonuclease analyses of phage and plasmid clones indicated that all of the genetic coding capacity in the tar region is now accounted for.

J Bacteriol, 1983 Aug, 155(2), 557 - 64
Mutations in multicopy Tn10 tet plasmids that confer resistance to inhibitory effects of inducers of tet gene expression; Moyed HS et al.; Escherichia coli K-12 strains that carry the Tn10 tetracycline resistance determinant (tet) on multicopy plasmids are hypersensitive to 5a,6-anhydrotetracycline and heated chlortetracycline, two tetracycline derivatives that are relatively more effective as inducers of tet gene expression than as inhibitors of bacterial growth . Twenty spontaneous mutations that confer resistance to anhydrotetracycline (Atr) and resistance to heated chlortetracycline (Ctr) were isolated and characterized . All of these Atr mutations are located in the Tn10 tet region; the majority (18 of 20) have no effect on tetR repressor function . Atr mutations can increase, reduce, or eliminate the phenotypic expression of plasmid tetracycline resistance (Tcr) . IS insertions that result in an Atr Tcs phenotype are clustered in a 150-base-pair promoter-proximal region of the tetA resistance gene . Some Atr mutations reduce expression of the tetA gene by altering either the tetR repressor or the tetA promoter . In addition, it appears that E . coli cannot tolerate constitutive expression of the wild-type tetA gene from a multicopy plasmid containing a tetR deletion . These observations support the proposal that high level expression of the 36-kilodalton tetA gene product inhibits the growth of E . coli . We speculate that this inhibition is related to the interaction of the tetA gene product with the cytoplasmic membrane.

J Bacteriol, 1983 Aug, 155(2), 549 - 56
Multicopy Tn10 tet plasmids confer sensitivity to induction of tet gene expression; Moyed HS et al.; We inserted the Tn10 tetracycline resistance determinant (tet) into the multicopy plasmid pACYC177, and we examined the phenotype of Escherichia coli K-12 strains harboring these plasmids . In agreement with others, we find that Tn10 tet exhibits a negative gene dosage effect . Strains carrying multicopy Tn10 tet plasmids are 4- to 12-fold less resistant to tetracycline than are strains with a single copy of Tn10 in the bacterial chromosome . In addition, we find that multicopy tet strains are 30- to 100-fold less resistant to the tetracycline derivative 5a,6-anhydrotetracycline than are single-copy tet strains . Multicopy tet strains are, in fact, 10- to 25-fold more sensitive to anhydrotetracycline than are strains that lack tet altogether . The hypersensitivity of multi-copy strains to anhydrotetracycline is correlated with the effectiveness of anhydrotetracycline as an inducer of tet gene expression, rather than its effectiveness as an inhibitor of protein synthesis . Anhydrotetracycline is 50- to 100-fold more effective than tetracycline as an inducer of tetracycline resistance and as an inducer of beta-galactosidase in strains that harbor tet-lac gene fusions . In contrast, anhydrotetracycline appears to be two- to fourfold less effective than tetracycline as an inhibitor of protein synthesis . Both anhydrotetracycline and tetracycline induce synthesis of tet polypeptides in minicells harboring multicopy tet plasmids . Differences between E . coli K-12 backgrounds influence the tetracycline and anhydrotetracycline sensitivity of multicopy strains; ZnCl2 enhances the tetracycline and anhydrotetracycline sensitivity of these strains two- to threefold . We propose that the overexpression of one or more Tn10 tet gene products inhibits the growth of multicopy tet strains and accounts for their relative sensitivity to inducers of tet gene expression.

J Bacteriol, 1983 Aug, 155(2), 459 - 66
Amplified DNA in Streptomyces fradiae; Fishman SE et al.; A spontaneous mutant of Streptomyces fradiae contained an amplifiable unit of DNA with a sequence length of approximately 10.5 kilobases that was amplified to approximately 500 copies per chromosome . The amplified DNA appears to be cryptic . SalI fragments of the amplified DNA were cloned into Escherichia coli to construct a restriction map and characterize the amplified DNA . The amplified DNA contained tandem repeats of the amplifiable unit of DNA . The unit had an average base composition of 71% guanine plus cytosine, similar to the chromosomal DNA of Streptomyces species . At least a portion of the amplifiable unit of DNA was present at a low copy number in the wild-type strain . The phenotype of amplified DNA was designated Ads1SF for amplified DNA sequence 1 in S . fradiae.

Eur J Biochem, 1983 Aug 1, 134(2), 355 - 64
Kinetic evidence for an activation step following binding of human interferon alpha 2 to the membrane receptors of Daudi cells; Mogensen KE et al.; A single species of human interferon alpha (IFN alpha) was labelled with 125I to high incorporation for binding studies on the B-lymphoblastoid cell line, Daudi, whose growth is inhibited by low doses of IFN, the effect being saturated at about 100 U/ml (25 pM) . The radiolabelled IFN was shown to be fully active and the binding affinity to cellular sites was shown to be unchanged by iodination . Experimental conditions were standardized such that binding and cell growth experiments could be performed on the same initial culture of cells . 125I-labelled IFN alpha 2 (IFN alpha prepared from Escherichia coli carrying human alpha 2 gene) was added to exponentially growing cultures (mean specific growth rate 0.77 +/- 0.07 days-1) at a mean concentration of 235000 +/- 20000 cells ml-1 . Two types of binding could be discerned on growing cultures: the first with a transient peak followed by a loss or discharge of available sites, the second reaching equilibrium some 3 h after the addition of IFN . Large differences in the apparent dissociation constants were evident . The affinity of binding at the 'steady-state', appeared to be much higher . An analysis of the displacement rates for bound IFN suggested that the two reactions were occurring consecutively over the whole of the dose range studied (1-100 U/ml; 0.25-25 pM IFN) . In this dose range we found that Daudi cells would eventually stop growing at all doses and that the rates of deceleration of cellular growth were linearly proportional to the dose of IFN in a double-reciprocal plot (i.e . in analogy to Michaelis-Menten kinetics) . A good congruence was found between the equilibrium constants for binding and for growth inhibition (2.65 pM and 2.39 pM, respectively) . The amount of IFN bound at steady state thus determines the rate at which growth is inhibited . We propose that the first reaction represents binding of IFN to surface receptors, and the second transfer of IFN to an activation complex on the cell membrane . Appropriate models and their general applicability to IFN action are discussed.

J Lab Clin Med, 1983 Aug, 102(2), 240 - 9
Effects of endotoxemia on cyclic nucleotides in the unanesthetized sheep; Snapper JR et al.; The effects of endotoxemia on hemodynamics, pulmonary fluid, and solute exchange and cGMP and cAMP concentrations in lung lymph and pulmonary artery and left atrial blood were studied in six unanesthetized sheep . Cyclic AMP levels increased early in the endotoxin reaction, reaching peak concentration 1 hr after endotoxemia (during the period of pulmonary hypertension) . Cyclic GMP levels increased gradually during the endotoxin reaction, reaching peak concentrations 5 hr after endotoxemia (during the period of "increased pulmonary vascular permeability") . The changes observed in cyclic nucleotide levels in lung lymph and pulmonary artery and left atrial blood suggested pulmonary production of cGMP but not cAMP . Cyclic AMP concentrations correlated with PPA, Qlymph, and a drop in the L/P ratio, whereas lung lymph cGMP correlated with an increased clearance of protein in the lymph . These results further characterize the sheep endotoxin reaction and suggest a possible role for cyclic nucleotides in the pathogenesis of the changes in lung vascular function that follow endotoxin infusion in sheep.

J Biochem (Tokyo), 1983 Aug, 94(2), 443 - 50
Construction of a lambda packageable ColE1 vector which permits cloning of large DNA fragments: cloning of thyA gene of Escherichia coli; Fujiyoshi T et al.; A small packageable plasmid depending on the ColE1 replicon was constructed from ColE1-gal-cos lambda:: Tn3, pKY2113 (T . Nakamura et al . (1981) J . Biochem . 90, 1013), and named pKY2662 . This plasmid carries ampicillin resistance and colicin E1 immunity genes as selective markers, and has neither mobilization function nor movable transposon . The molecular size of pKY2662 is 8.7 kb, and it has a single cleavage site each for BamHI, Bg/II, ClaI, EcoRI, HpaI, PstI, and Tth111I . By using pKY2662 as a vector, a 32 kb Escherichia coli DNA fragment covering thyA, recC, recB, and argA genes was cloned . This new small cosmid is among the most efficient vectors hitherto found for in vitro cloning of large DNA fragments.

Gene, 1983 Aug, 23(2), 199 - 209
Nucleotide sequence of the promoter and amino-terminal coding region of the glutamate dehydrogenase structural gene of Escherichia coli; Valle F et al.; A 610-bp DNA fragment carrying the promoter and amino-terminal coding regions of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli has been sequenced . The amino-terminal sequence of the enzyme was also determined to help localize the transcriptional and translational signals for this gene . Three possible promoters and a CRP binding site were identified by concensus criteria . The sequence of 102 amino acids at the amino terminus of the enzyme is compared with the amino acid sequence from other GDH enzymes.

Proc Natl Acad Sci U S A, 1983 Aug, 80(15), 4599 - 603
Importance of secondary structure in the signal sequence for protein secretion; Emr SD et al.; Mutant Escherichia coli strains in which export of the LamB protein (coded for by the lamB gene) to the outer membrane of the cell is prevented have been described previously . One of these mutant strains contains a small (12-base pair) deletion mutation within the region of the lamB gene that codes for the NH2-terminal signal sequence . In this mutant strain, export but not synthesis of the LamB protein is blocked . We have isolated pseudorevertants that restore export of functional LamB protein to the outer membrane . DNA sequence analysis showed that two of the revertants contain a point mutation in addition to the original deletion . These point mutations lead to amino acid substitutions within the signal sequence . Our results indicate that these secondary mutations efficiently suppress the export defect caused by the deletion mutation . Analysis of the secondary structure of the wild-type, mutant, and pseudorevertant LamB signal sequences suggests that the secondary mutations restore export by allowing the formation of a stable alpha-helical conformation in the central, hydrophobic region of the signal sequence.

J Bacteriol, 1983 Aug, 155(2), 473 - 80
SOS induction by P1 Km miniplasmids; Capage MA et al.; We have constructed (in vitro) a set of P1 miniplasmids . The smallest of these that could function as an independent replicon contained the right side of EcoRI-5 plus all of EcoRI-8 . Those miniplasmids that lack EcoRI-6 induce the SOS pathway of the cell as shown by (i) increased expression of the recA operon, (ii) excision of the cryptic genetic element e14, (iii) spontaneous induction of lambda, and (iv) dependence of e14 excision on recA+ function . This induction was contingent upon the replication of the P1 Km miniplasmids from their P1 origin and, thus, was apparently caused by an aberrant initiation of DNA replication . When P1 EcoRI-6 was present in cis or trans with a P1 Km miniplasmid, neither e14 nor lambda was excised, but the expression of the recA operon was still induced . These results suggest that P1 EcoRI fragments 5 and 8 are insufficient for normal replication, and thus our P1 Km miniplasmids induced SOS functions . A product of EcoRI-6 may partially restore normal replication.

Jpn J Exp Med, 1983 Aug, 53(4), 181 - 5
Highly sensitive-beta-D-galactosidase-linked immunosorbent assay for anti-myelin basic protein antibody; Akiyama K; A highly sensitive enzyme-linked immunosorbent assay (ELISA) procedure has been devised for the determination of anti-myelin basic protein antibodies . First, rabbit anti-myelin basic protein (MBP) sera to be assayed were incubated with MBP immobilized on small pieces of silicone rubber . Next, anti-MBP bound specifically to the solid phase was allowed to react either with Fab' of guinea pig anti-rabbit IgG conjugated with beta-D-galactosidase from Escherichia coli or with protein A conjugated with beta-D-galactosidase . The amount of fixed anti-MBP antibodies was assayed by fluorometric determination of the enzyme activity using 4-methylumbelliferyl-beta-D-galactoside as a substrate . The sensitivity of the present assay was at least an order of magnitude higher than the hitherto available radioimmunoassay and ELISA.

Biochem J, 1983 Aug 1, 213(2), 451 - 8
The F1F0-ATPase of Escherichia coli . Substitution of proline by leucine at position 64 in the c-subunit causes loss of oxidative phosphorylation; Fimmel AL et al.; The uncE410 allele differs from the normal uncE gene in that C leads to T base changes occur at nucleotides 190 and 191, resulting in proline at position 64 in the c-subunit of the F1F0-ATPase being replaced by leucine . Two partial-revertant strains were isolated in which alanine-20 of the c-subunit was replaced by proline, owing to a G leads to C base change at nucleotide 58 . These c-subunits, coded for by the uncE501 and uncE502 alleles, therefore contained two amino acid changes, namely proline-64 leads to leucine, and alanine-20 leads to proline . Membranes prepared from the partial-revertant strains lacked ATP-dependent atebrin-fluorescence-quenching activity but were able to carry out oxidative phosphorylation . The ATPase activity of the F1-ATPase was inhibited when bound to membranes from strains carrying the uncE410, uncE501 and uncE502 alleles . It is concluded that a bend in the helix axis in one of the arms of the c-subunit hairpin structure is required for integration of the c-subunit into a functional F1F0-ATPase.

J Immunol, 1983 Aug, 131(2), 554 - 60
Single cell studies on hapten-specific B lymphocytes: differential cloning efficiency of cells of various sizes; Pike BL et al.; Three separate forms of in vitro stimulation were assessed for their capacity to activate hapten-gelatin fractionated, fluorescein- (Flu) specific murine splenic B lymphocytes . They were: a) Flu-polymerized flagellin (Flu-POL) acting on a single Flu-specific B cell in microculture in the absence of "filler" or feeder cells, but in the presence of T cell-derived B cell growth and differentiation factor(s) (BGDF); b) a mixture of mitogens, E . coli lipopolysaccharide (LPS) and dextran sulfate, acting on a single Flu-specific B cell in the absence of added BGDF; and c) Flu-POL plus BGDF acting on single Flu-specific B cells as in a but with thymus filler cells also present . System c was markedly superior in causing antibody formation, 15 to 22% of cells forming a clone of Flu-specific antibody-forming cells (AFC), in contrast to 6% for system b and 3 to 6% for system a . Each stimulus was applied to single cells that had been size fractionated into samples of increasing size by using the forward light-scattering parameter of the fluorescence-activated cell sorter . Surprisingly, the smaller sized fractions proliferated poorly in system a and contributed less than 10% of the antibody-forming potential of the total population . The smaller cells proliferated better in system b, but only 10 to 15% of proliferating clones generated Flu-specific AFC, whereas the larger cells contributed 86% of the total AFC response . Even in system c, only 6% of the small cells formed AFC clones compared with 41% of the larger cells . It thus appears that the smaller half of murine B lymphocytes is relatively resistant to activation into proliferation and differentiation by "T-independent" antigens; when activated by mitogens, they clone less efficiently than larger cells . Despite these limitations, system c could generate a total of up to four hapten-specific AFC for every B cell placed into culture, making it the most efficient system of specific antibody formation yet described.

J Biochem (Tokyo), 1983 Aug, 94(2), 487 - 92
An enzymic cycling method for the determination of free fatty acids with acyl-CoA synthetase and acyl-CoA hydrolase; Kawaguchi A et al.; A method for measuring free fatty acids by enzymic cycling is described . Free fatty acids are converted to acyl-CoAs by acyl-CoA synthetase, then the acyl-CoAs are hydrolyzed back to the free fatty acids by acyl-CoA hydrolase in a cyclic fashion . The amounts of AMP produced during this cyclic reaction are determined from the absorbance at 340 nm in the presence of AMP deaminase and glutamate dehydrogenase . This method is sensitive to as low as 0.1 nmol of free fatty acids, and the standard curve is linear up to 1.0 nmol . This method shows a broad specificity for long-chain fatty acids (C12--C20) and the recoveries of fatty acids added to bacterial cell-free extracts are more than 90%.

Virology, 1983 Jul 30, 128(2), 265 - 70
Studies on polylysogens containing lambda N-cI- prophages . II . Role of high multiplicities in lysogen formation by lambda N-cI- phage; Chattopadhyay DJ et al.; Results of the experiments presented in this paper show that lambda N-cI- phage can lysogenize a nonpermissive host Escherichia coli when it infects at very high multiplicities (around 100), and lambda N-cI-cII- and lambda cIII-N-cI- lysogenize poorly at similar high multiplicities . The latter two phages lysogenize with appreciable frequency when either lambda N-cI- or lambda int-cN-cI-cII- is used as helper . The phages, lambda N-cI-, lambda N-cI-cII-, and lambda cIII-N-cI- can lysogenize also at relatively low m.o.i . of 20 in presence of the above lambda int-c helper, and the lambda int-cN-cI-cII- phage alone forms converted lysogens at an m.o.i . as low as 12 . All these results suggest that the establishment of prophage integration by lambda N-cI- is positively regulated, like lambda N+cI+ phage, by the cII/cIII-promoted expression of the int gene of lambda, and under the N- condition, high multiplicities are needed to provide optimum levels of cII and cIII products, especially the latter.

Biochem Biophys Res Commun, 1983 Jul 29, 114(2), 882 - 8
Putrescine and spermidine sensitivity of lysine decarboxylase in Escherichia coli: evidence for a constitutive enzyme and its mode of regulation; Wertheimer SJ et al.; Cells of Escherichia coli grown under physiological (noninducing) conditions have a low level of lysine decarboxylase activity . This activity differs from the enzyme found in induced cells in its sensitivity to putrescine (33% of control in the presence of 20 mM putrescine) . It is also sensitive to spermidine (20% of control in the presence of 6 mM spermidine) . A mixture of putrescine and spermidine completely eliminated lysine decarboxylase activity . This provides evidence for the existence of a biosynthetic enzyme and suggests a mechanism to explain the appearance of cadaverine in polyamine-depleted cells.

Biochem Biophys Res Commun, 1983 Jul 29, 114(2), 684 - 9
A kinetic and binding study of the reactivity of Escherichia coli ATPase to N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; Satre M et al.; Escherichia coli H+-ATPase (ECF1) was inactivated in a time- and concentration-dependent manner by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a selective carboxyl group reagent . Among the subunits of ECF1, only the beta subunit was modified by EEDQ . The reaction of 1 mol of EEDQ per mol of ECF1 resulted in total inactivation, in spite of the fact that the enzyme possesses three beta subunits.

Biochem Biophys Res Commun, 1983 Jul 29, 114(2), 670 - 6
The majority of cDNA clones with strong positive signals for the interferon-induction-specific sequences resemble mitochondrial ribosomal RNA genes; Tsuzuki T et al.; A complementary DNA library prepared from the 12S polyadenylated RNAs extracted from interferon-induced KG-1 cells, a human myeloblast cell line, was screened for the presence of induction-specific sequences . Clones that exhibited strong positive signals were separated by hybridization criteria into nine classes . Clones from classes I through IV consisted of about 78% of the total and unexpectedly were found to resemble human mitochondrial ribosomal RNA genes.

Nucleic Acids Res, 1983 Jul 25, 11(14), 4923 - 32
Chemical crosslinking of elongation factor G to the 23S RNA in 70S ribosomes from Escherichia coli; Skold SE; Elongation factor G was crosslinked to the 23S RNA of 70S Escherichia coli ribosomes with the bifunctional, cleavable reagent diepoxybutane (DEB) . The EF-G-23S RNA complex was isolated and digested with ribonuclease A . After digestion, an RNA fragment, protected by EF-G was cleaved from the complex and isolated . The nucleotide sequence of this RNA fragment was determined by partial ribonuclease digestion . It proved to be 27 nucleotides long and it could be identified with residues 1055 to 1081 of the nucleotide sequence of E . coli 23S RNA . In the presence of thiostrepton, which prevents binding of EF-G to the ribosome, there was a dramatic decrease in the yield of this complex.

Nucleic Acids Res, 1983 Jul 25, 11(14), 4703 - 12
Complete nucleotide sequence of the influenza B/Singapore/222/79 virus hemagglutinin gene and comparison with the B/Lee/40 hemagglutinin; Verhoeyen M et al.; The complete nucleotide sequence of the hemagglutinin (HA) gene of the human type B influenza virus B/Singapore/222/79 is presented . Comparison with the only other known sequence of a B hemagglutinin (B/Lee/40) shows that antigenic drift in type B HA genes is essentially the same as already observed within the influenza A H3 subtype, i.e., an accumulation of point mutations . The main difference is that the apparent evolution is significantly slower, most likely due to the cumulative effect of a lower occurrence in the population (slower evolution) and/or less immunological pressure . There is a striking cluster of changes at positions 127 until 137 of the HA1 subunit which may represent one of the antigenic sites of the molecule.

FEBS Lett, 1983 Jul 25, 158(2), 301 - 4
Cloning and expression of a gene coding for the prolipoprotein signal peptidase of Escherichia coli; Yamagata H et al.; An Escherichia coli mutant, Y815, has a temperature-sensitive prolipoprotein signal peptidase . IPTG-induced synthesis of the major outer membrane prolipoprotein (PLP) results in the inhibition of cell growth because of accumulation of PLP in its envelope {J . Bacteriol . (1982) 152, 1163-1168} . The 2000 E . coli strains of Clarke and Carbon's collection were screened for the presence of a plasmid complementing the IPTG-sensitivity of the growth of Y815 . One plasmid, pLC3-13, complemented the IPTG-sensitivity . The envelope fraction prepared from Y815 transformed by pLC3-13 showed high activity of the PLP signal peptidase in vitro at high temperature . A 4 kb AccI fragment subcloned onto plasmid pHY001 was shown to carry the gene for the PLP signal peptidase.

J Biol Chem, 1983 Jul 25, 258(14), 8637 - 41
Specific incorporation of selenium into lysine- and glutamate- accepting tRNAs from Escherichia coli; Wittwer AJ; Amino acid transfer nucleic acids (tRNAs) that contain selenium-modified bases are synthesized by Escherichia coli in the presence of low levels (0.1-0.5 microM) of {75Se}selenite or {75Se}selenate . The amount of selenium incorporated (1-2 g atoms/100 mol of tRNA) was unchanged by 10-20-fold variations in selenium or sulfate concentrations or by the addition of 1 mM cysteine, sulfide, or sulfite . Specific incorporation of selenium (as opposed to nonspecific substitution for sulfur) was further indicated by the different reversed phase chromatographic elution patterns of 35S- and 75Se-labeled tRNAs isolated from cells labeled with 35SO2-4 and 75SeO2-4 . Also, E . coli mutants unable to synthesize an abundant sulfur-modified base, 4-thiouracil, nevertheless produced normal levels of selenium-modified tRNAs . Two different methods of distinguishing between aminoacylated and nonaminoacylated tRNA, one which examined mobility during reversed phase chromatography and another which employed anti-AMP antibodies, indicated that over 50% of the selenium-containing tRNA had lysine or glutamate acceptor activity.

Nucleic Acids Res, 1983 Jul 25, 11(14), 4809 - 21
Isolation and characterization of genomic mouse DNA clones containing sequences homologous to tRNAs and 5S rRNA; Hu JC et al.; We have cloned and characterized three fragments of Balb/c mouse DNA which hybridize to mouse cell tRNAs . Fractionation of the tRNAs which hybridize to these clones reveals that two of the clones, lambda Mt-4A and lambda Mt-6A hybridize to only one or two tRNAs, while one clone, lambda Mt-4B, hybridizes to at least seven tRNAs . Two of the tRNAs were identified as tRNAProCCG and tRNAGlyGGA, and others have been identified as tRNAs which are selectively encapsidated into virions of murine leukemia virus and avian reticuloendotheliosis virus . The DNA sequences of putative genes for tRNAProCCG and tRNAGlyGGA, plus flanking regions, were determined . A clone of Balb/c mouse DNA which selectively hybridized to 5S rRNA was also isolated and partially characterized.

Nucleic Acids Res, 1983 Jul 25, 11(14), 4677 - 88
Inducible high level synthesis of mature human fibroblast interferon in Escherichia coli; Remaut E et al.; We have obtained high level synthesis in Escherichia coli of mature human fibroblast interferon using a plasmid vector that was designed to allow easy coupling of a DNA coding region to the initiator AUG of the replicase gene of the RNA phage MS2 cloned downstream of phage lambda's leftward promoter . The activity of the promoter can be regulated by temperature . Induced cells accumulated the interferon up to 4% of the total cellular protein . The biological activity of the product amounted to 4 X 10(9) international units per litre of culture . The synthesis of human fibroblast interferon was shown to drastically inhibit the growth rate of the bacterial host.

J Biol Chem, 1983 Jul 25, 258(14), 8984 - 92
Differential template recognition by the Caulobacter crescentus and the escherichia coli RNA polymerases; Amemiya K et al.; Transcription of Escherichia coli and Caulobacter crescentus phage DNAs by their respective host RNA polymerase was examined to determine their ability to recognize specific transcription signals on the heterologous template . Analysis of coliphage T7 in vitro transcripts showed that, like the E . coli enzyme, the C . crescentus RNA polymerase initiated transcription from the three major T7 early promoters and recognized the terminator at the end of the early region . On the other hand, several differences were found between the C . crescentus and E . coli RNA polymerases with respect to their interaction with Caulobacter phage phiCdl DNA . The rates of open complex formation and RNA elongation were slower when phiCdl DNA was transcribed by the E . coli RNA polymerase . In addition, transcription of phiCdl DNA by the E . coli enzyme produced a subset of transcripts not synthesized by the C . crescentus enzyme . The production of these different transcripts by the E . coli enzyme was dependent on salt concentration and, in at least one case, appeared to be the result of differential termination . Although both enzymes protected the same sites on phiCdl DNA from cleavage with HincII, the E . coli enzyme was unable to form stable complexes with some phiCdl restriction fragments that formed stable complexes with the C . crescentus RNA polymerase . These results indicate that although the C . crescentus RNA polymerase can accurately recognize transcription signals on a heterologous phage template, the E . coli enzyme exhibits altered specificity with a heterologous phage template of higher G + C content.

Nucleic Acids Res, 1983 Jul 25, 11(14), 4853 - 65
Cloning of cDNA for pyruvate, Pi dikinase from maize leaves; Hague DR et al.; To obtain molecular probes for studies of gene regulation in photosynthetic tissues of maize, we have cloned DNA complementary to poly(A)+RNA extracted from green leaves by insertion into plasmid pBR322 and transformation of E . coli, strain RR1 . Colonies were screened by sequential hybridization with 32P-labeled single stranded cDNA synthesized from pooled aliquots of poly(A)+RNA fractionated by sucrose density centrifugation . Among the clones bearing cDNA homologous to high molecular weight poly(A)+RNA, we identified one with an insert of 440 base pairs homologous to mRNA for pyruvate, Pi dikinase, a C-4 carbon cycle protein localized in mesophyll cells of the leaf . Our work indicates that the dikinase subunits are synthesized in the cytoplasm as precursors approximately 13,000 daltons larger than the mature peptide subunits . Leaves of seedlings illuminated during growth have higher levels of pyruvate, Pi dikinase mRNA than leaves of dark-grown plants.

Science, 1983 Jul 22, 221(4608), 378 - 80
Clustered third-base substitutions among wild strains of Escherichia coli; Milkman R et al.; Nucleotide sequences of translated regions of the trp operon in 12 wild strains of Escherichia coli reveal striking uniformity among eight strains (suggesting recent common ancestry and supporting the importance of periodic selection in natural populations) and clustered substitutions in four strains (implicating events affecting runs of nucleotides).

J Theor Biol, 1983 Jul 21, 103(2), 313 - 28
Periodic enzyme synthesis and oscillatory repression: why is the period of oscillation close to the cell cycle time?
Tyson JJ.
During exponential growth of a cell culture, some enzymes are synthesized periodically . In a synchronous culture, in which all cells undergo DNA synthesis and division more-or-less synchronously, the burst of enzyme synthesis also occurs synchronously in each cell once per division cycle . However, there are a number of interesting cases in which periodic enzyme synthesis continues in the absence of synchronous DNA replication or cell division . In all cases of periodic enzyme synthesis in asynchronous cultures, the time between bursts of enzyme synthesis, though no longer identical to the cell cycle time, is still close to the interdivision time of the growing, replicating cells . The theory of oscillatory repression looks for an explanation of this phenomenon in the periodic repression of gene transcription caused by periodic fluctuations in the concentration of the endproduct of the metabolic pathway of which the enzyme is a part . A major difficulty with this theory is that there is no obvious relationship between the periodicity of the negative feedback loop, which is determined by the kinetics of synthesis and degradation of the individual components of the feedback loop, and the periodicity of the cell cycle, which is determined by overall net synthetic rates of cellular macromolecules . Why should the period of oscillation of a repressible gene transcription system be close to the interdivision time of a population of growing cells? In this paper, I show that the relationship may be coincidental: the two fundamental periods are close to each other because they are both close to the mass-doubling time of the cell culture . That the mean interdivision time must be close to the mass-doubling time is a consequence of "balanced" growth: there is a stable size distribution of cells in a growing culture . That the period of oscillation of the negative feedback loop is also close to the mass-doubling time is shown to be a consequence of the large, nearly constant demand for endproduct and the assumed stability of the enzyme . The period of oscillation is largely attributable to the slow dilution of the stable enzyme by cell growth . For reasonable values of the parameters describing the gene-control system, I show that the enzyme must be diluted by a factor of two (approximately), that is, by the growth accomplished by one mass-doubling (nearly).

Biochemistry, 1983 Jul 19, 22(15), 3664 - 71
Microtubule assembly with the guanosine 5'-diphosphate analogue 2',3'-dideoxyguanosine 5'-diphosphate; Hamel E et al.; The GDP analogue 2',2'-dideoxyguanosine 5'-diphosphate (ddGDP) supports efficient tubulin polymerization . Microtubule-associated protein (MAP) dependent microtubule assembly occurs in 0.1 M 2-(N-morpholino)-ethanesulfonate, and sheets of parallel protofilaments are formed in 1.0 M glutamate without MAPs . The nucleotide is bound to tubulin in the course of polymerization, presumably in the exchangeable GTP site . The ddGDP is not hydrolyzed, however, and is completely stable in the reaction mixture . Nor was the nonexchangeable GTP bound to tubulin hydrolyzed in ddGDP-supported polymerization: equivalent amounts of GTP remained associated with polymerized tubulin after polymerization with either ddGDP or GTP . Higher concentrations of ddGDP than GTP were required under all conditions . Nevertheless, under optimum conditions for the ddGDP-supported reaction, polymerization began with a shorter lag period and both the rate and extent of polymerization were greater with ddGDP than with GTP . The MAP-dependent reaction with ddGDP is temperature dependent, cold reversible, and inhibited by calcium and antimitotic drugs . It differs from the GTP-supported reaction in being most vigorous at minimal Mg2+ concentrations and exquisitely sensitive to GDP inhibition.

Biochemistry, 1983 Jul 19, 22(15), 3563 - 70
Nonspecific interaction of the lac repressor headpiece with deoxyribonucleic acid: fluorescence and circular dichroism studies; Schnarr M et al.; The nonspecific interaction of the short headpiece, the NH2-terminal domain of the lac repressor, with natural DNA and alternating polydeoxynucleotides has been studied by means of fluorescence and circular dichroism . The important quenching of the intrinsic tyrosine fluorescence of the headpiece upon complexation has been used for the determination of the binding isotherms under various environmental conditions . By comparison with theoretical binding curves, we have determined a physical site size of three base pairs . The "perturbated" site size as determined from circular dichroism measurements (about four base pairs) is slightly greater . As in the case of the entire lac repressor, the interaction is strongly ionic strength dependent . The plots log Kobsd as a function of log {NaCl} are linear for salt concentrations greater than 50 mM for the interaction with DNA and greater than 25 mM for the interaction with poly{d(G-C)} . From the slopes of the linear parts of these plots, we determine a number of three electrostatic interactions, assuming no anion release from the protein upon complexation . This value is independent of pH . On the contrary, the association constant Kobsd depends on pH . Complexation requires the protonation of one titrating group of the headpiece with a pK value of 6.7 +/- 0.3, probably the residue histidine-29 . The finding is in favor of the idea that the lac repressor interacts with DNA via two headpieces as earlier work has shown that the interaction of the lac repressor with DNA requires the protonation of two groups of the protein.

Biochemistry, 1983 Jul 19, 22(15), 3537 - 46
Elementary steps in the DNA polymerase I reaction pathway; Bryant FR et al.; The polymerization reaction catalyzed by Escherichia coli DNA polymerase I (Pol I) has been studied by using the homopolymer template-primer system poly(dA).oligo(dT) . Isotope-partitioning experiments indicate that the reaction follows an ordered mechanism in which Pol I first combines with template-primer to form an E.poly complex followed by addition of MgTTP and catalysis . The parameters governing the binding of Pol I to the template-primer are kon = 1.2 X 10(6) M-1 s-1, koff = 0.25 s-1, and KD = 2 X 10(-7) M . Efforts to demonstrate the catalytic competence of the binary E.MgTTP complex were unsuccessful . Following initiation of the catalytic cycle, Pol I catalyzes the incorporation of an average of 40-50 TTP molecules into polymer before dissociating from the template-primer . The processive nature of the polymerization reaction as reflected by the isotope-trapping time dependence can be accounted for by a model in which processive synthesis is treated as a simple partitioning between continued polymerization (kcat = 3.8 s-1, 22 degrees C) and dissociation of the enzyme from the template-primer under steady-state conditions (koffss = 0.1 s-1) . The rapid quench time course of the polymerization reaction (kcat = 2.5 s-1, 20 degrees C) exhibited a pre-steady-state burst consistent with two partially rate-determining steps, one of which precedes the actual chemical phosphodiester bond-forming step (k = 4.6 s-1) and the other which follows this step (k = 4.0 s-1) . Binding of MgTTP to the E.poly complex was shown to be a rapid equilibrium step by steady-state isotope-partitioning experiments . This suggested that the first rate-determining step may be a first-order isomerization which follows the binding of substrates and precedes bond formation.

Biochem Biophys Res Commun, 1983 Jul 18, 114(1), 222 - 9
Stimulatory effect of UTP on peptide chain initiation in Streptomyces aureofaciens; Mikulik K et al.; The formation of the 30S and 70S initiation complex in Streptomyces aureofaciens differs from that in E.coli and B.stearothermophilus with respect to the requirement for nucleotide triphosphates for the maximum activity . In the presence of GTP and initiation factors from S.aureofaciens the codon specific binding of fMet-tRNA to ribosomes of S.aureofaciens was stimulated by ATP or UTP . UTP exhibited the most significant effect increasing the binding about 3.5 times, whereas CTP had no effect on the reaction . The stimulatory effect of UTP is GTP-dependent and was not observed in experiments with E.coli ribosomes.

Biochem Biophys Res Commun, 1983 Jul 18, 114(1), 348 - 54
Incorporation of 5S RNA into 16S-23S RNA complex; Tewari DS et al.; 5S RNA as such is not incorporated into 16S-23S RNA complex formed under reconstitution condition . However, the addition of 50S ribosomal proteins, L5, L18 and L25/L15 results in its incorporation in stoichiometric amount . None of the proteins added individually is capable of incorporating 5S RNA into the complex . Of the different combinations in pairs that are possible out of the four proteins, the pairs L5, L18 and L15, L18 stimulate the incorporation to some extent . Of the four possible triplets, L5, L18, L25 or L5, L15, L18 is the most efficient for maximum incorporation of 5S RNA . The presence of all the four proteins is no more effective than the combinations of the three.

Biochem Biophys Res Commun, 1983 Jul 18, 114(1), 1 - 7
Necessity of the sulfoxide moiety for the biochemical and biological properties of an analog of sparsomycin; Flynn GA et al.; An analog of the peptidyl transferase inhibitor sparsomycin was a competitive inhibitor (Ki = 1.8 microM) of peptidyl-puromycin synthesis on E . coli polysomes . Preincubation of polysomes with the compound enhanced the degree of inhibition of peptide bond formation . A model for the involvement of a histidine residue in peptidyl transferase activity is presented as a result of our observations which include direct association of {3H} labelled analog with 70S ribosomes . The correct oxidation state of sulfur in the compound was necessary for the "preincubation effect" and entry of the compound into bacterial cells.

J Mol Biol, 1983 Jul 15, 167(4), 791 - 808
The SOS regulatory system: control of its state by the level of RecA protease; Little JW; Our current understanding of the SOS regulatory system suggests that it can exist in two extreme states: in the repressed state, LexA protein is active, and it represses a particular set of genes called SOS genes . In the induced state, which results from various impairments to DNA replication, LexA repressor is cleaved by the specific protease activity of the RecA protein; in consequence, the SOS genes are derepressed and they express various functions that are believed to aid cell survival in induced cells . Since high levels of RecA protease activity turn on this system, it seems plausible that the level of protease activity will also control the transitions between the two states of the system . In order to assess the in vivo level of protease activity, antibody techniques were used to study the stability of LexA repressor during various phases of the SOS regulatory cycle . Repressor was reasonably stable in the repressed state, but it was degraded within a few minutes after an inducing treatment . Cleavage depended upon the RecA protease activity and resulted in the same products as seen in vitro . Cleavage preceded, and did not depend upon, derepression of any SOS gene . During the transition to the repressed state, LexA repressor became increasingly stable with time, suggesting that as DNA damage was repaired the level of protease declined . This decline depended upon derepression of the regulatory system, consistent with the belief that an inducing signal, resulting from DNA damage, reversibly activates the RecA protease and is removed by the action of one or more SOS functions . At low levels of DNA damage, a subinduced state was observed in which repressor level was reduced by a low level of cleavage . These data indicate that the level of RecA protease activity controls the state of the system and the transitions between its two states.

Anal Biochem, 1983 Jul 15, 132(2), 259 - 64
New assay for enzymatic phosphate release: application to aspartate transcarbamylase and other enzymes; Bencini DA et al.; The colorimetric method for phosphate determination described in the preceding paper is adapted for the assay of orthophosphate liberated in the aspartate transcarbamylase reaction . The method provides for simple, accurate, and sensitive measurement of enzyme activity . The assay uses ammonium molybdate and zinc acetate to form a colored complex with the enzymatically released phosphate; mild conditions which minimize the nonenzymatic background degradation of the substrate, carbamoyl phosphate, are used . Since the assay procedure is relatively rapid, it is especially attractive in situations where results are desired immediately . The method can be used for the assay of any enzyme which releases inorganic phosphate, even in the presence of labile organophosphate compounds.

Anal Biochem, 1983 Jul 15, 132(2), 236 - 42
Determination of nanogram quantities of osmium-labeled nucleic acids by stripping (inverse) voltammetry; Palecek E et al.; Modification of nucleic acids with OSO4 in the presence of pyridine results in a formation of a covalently bound electroactive center in a polynucleotide chain detectable by polarographic (voltammetric) methods . It has been shown that DNA modified with osmium (DNA-Os) accumulates at the hanging mercury-drop electrode during a waiting time in a wide range of potentials between 0 and -1.0 V (against the saturated calomel electrode) and produce at neutral pH a well-developed reduction peak at about -1.2 V due to scanning in the cathodic direction . Using the differential-pulse stripping (inverse) voltammetry, nanogram quantities of single-stranded DNA-Os can be determined at relatively short waiting times (1-3 min) . Double-stranded DNA is modified with osmium to a much lesser extent as compared to single-stranded polynucleotides . The degree of modification of double-helical DNA is influenced by the presence of single-stranded and distorted double-stranded regions in the DNA molecules and by the environmental conditions which influence the DNA conformation . Osmium can thus be used as a probe of the DNA structure, and a few micrograms of double-helical DNA sample suffice for the voltammetric analysis.

Science, 1983 Jul 15, 221(4607), 295 - 6
Immunochemical localization of NADP-specific isocitrate dehydrogenase in Escherichia coli; Swafford JR et al.; The intracellular localization of isocitrate dehydrogenase was determined by immunochemical techniques with ultrathin sections of Escherichia coli . The thin sections, which were obtained by ultracryomicrotomy, were incubated first with antiserum specific for the enzyme and then with a protein A-gold complex . Transmission electron microscopy showed that the gold label was dispersed mainly in the cytoplasm.

J Mol Biol, 1983 Jul 15, 167(4), 895 - 9
On the origin of selectivity in recognition by cyclic adenosine 3',5'-monophosphate receptor protein of its specific binding site of the lactose promoter region; Takahashi M et al.; From fluorescence measurements we could analyse the binding of cyclic adenosine 3',5'-monophosphate receptor protein (CRP) from Escherichia coli to its specific site on a 301 base-pair long DNA fragment containing the control region of the lactose operon . At physiological ionic strength selection of the specific site is strictly dependent on the allosteric effector cAMP, and binding of the cAMP . CRP complex to its specific site is favoured over the non-specific binding by 5 kcal/mol with Kass (specific) = 10(8) M-1 at 37 degrees C.

Eur J Biochem, 1983 Jul 15, 134(1), 77 - 82
Proline dehydrogenase from Escherichia coli K12 . Properties of the membrane-associated enzyme; Abrahamson JL et al.; We have examined the oxidative activities of inverted cytoplasmic membrane preparations from Escherichia coli bearing proline dehydrogenase . Our measurements include both direct substrate:2,6-dichloroindophenol and substrate:O2 oxidoreductase assays and the 9-aminoacridine fluorescence assay for proton translocation, employing succinate and NADH dehydrogenases as comparative standards . Our data show the following . (a) Membranes prepared in a new buffer system bear proline dehydrogenase that is stable in both activity and membrane association . This membrane-associated enzyme shows an apparent Km for proline 20-fold lower than that estimated from the solubilized and purified enzyme . (b) Electrons are transferred from proline to O2 via the respiratory chain since proline utilization requires porphyrin synthesis and it is coupled to trans-membrane proton translocation . (c) Patterns of inhibition by 5-ethyl-5-isopentyl barbituric acid (Amytal) and 2-heptyl-4-hydroxyquinoline-N-oxide (HpHOQnO) suggest that parallel pathways of electron flux from NADH and proline converge at a cyanide-sensitive terminal oxidase . Succinate:O2 and succinate:DCIP oxidoreductase activities are stimulated by HpHOQnO and Amytal, and the former is inhibited by cyanide in this system . (d) Amytal is a non-competitive inhibitor of proline dehydrogenase . (e) Analysis of our fluorescence data suggests that Amytal and HpHOQnO dissipate delta pH at concentrations as low as 5 mM and 8.5 microM, respectively, in this system.

Virology, 1983 Jul 15, 128(1), 166 - 75
Evidence of cell fragility caused by gene kil following lambda induction; Volpi L et al.; Escherichia coli cells carrying lambda cI857 prophage lyse 40 min after lambda thermoinduction; the lysis depends on the lambda genes Q, R, and S . If chloramphenicol (CAP) is added within 20 min after lambda cI857 induction, an early, unproductive lysis occurs . This lysis is independent of the genes int, rex, O, P, Q, and all late genes . Instead, early lysis depends upon the kil gene . The early lysis is under the positive control of lambda gene N and the negative control of gene cro . One or more events specifically connected with lambda induction appear to be necessary for the occurrence of early lysis, since early lysis cannot be observed after lambda infection . Induced lambda kil+ lysogens are more sensitive to osmotic shock than induced lambda kil- lysogens . CAP-induced early lysis can be prevented in a hypertonic medium . These results suggest that induction of lambda causes an osmotic fragility due to a damage of the cell envelope which requires repair; in the absence of protein synthesis the cell envelope is not repaired and cell lysis ensues.

J Mol Biol, 1983 Jul 15, 167(4), 757 - 71
A dominant (mutD5) and a recessive (dnaQ49) mutator of Escherichia coli; Maruyama M et al.; The two known strong mutators of Escherichia coli K12, mutD5 (Degnen & Cox, 1974) and dnaQ49 (Horiuchi et al., 1978), are located at almost the same position, at five minutes on the linkage map . To clarify the genetical and functional relationships between these two mutators, we have constructed hybrid plasmids and phages carrying dnaQ+ or mutD5 by using in vivo and in vitro recombination techniques and examined their effect on the phenotype of wild-type or mutant bacteria . The results indicated that the mutD5 mutator is dominant over the wild-type allele whereas dnaQ49 is recessive . Thus, mutD5 plasmid or mutD5 transducing lambda phage can be used to convert a wild-type strain to a highly mutable strain . Both dnaQ+ and mutD5 plasmids carried a 1.5 X 10(3) base DNA fragment derived from the E . coli chromosome and they were indistinguishable from each other by restriction enzyme analysis . Moreover, specific labeling of the plasmid-encoded proteins by the maxicell method revealed that the mutD5 plasmid codes for two proteins, one whose molecular weight is 25,000 and the other whose molecular weight is 21,000, which correspond to the dnaQ protein and RNase H, respectively . Insertion of the gamma delta sequence into the mutD gene of the plasmid resulted in disappearance of the 25,000 Mr protein . These results suggested that the dnaQ49 and mutD5 mutator are mutations that have arisen in a single gene, though they differ in many respects.

Fortschr Med, 1983 Jul 14, 101(26), 1237 - 40
{Long-term results after endoscopic papillotomy}; Brandstatter G et al.; A long-term control study after endoscopic papillotomy was performed in 102 patients at least 12 months after the procedure . 14 patients were no longer alive, from those only one patient died due to biliary problems . Complications occurred in 10 patients (9,8%) . There was no cholangitis, but 2 cases of an acute cholecystitis some time after papillotomy . Therefore a cholecystectomy after papillotomy is suggested in the interval.

Biochim Biophys Acta, 1983 Jul 13, 732(1), 204 - 9
Reconstitution of the GalP galactose transport activity of Escherichia coli into liposomes made from soybean phospholipids; Henderson PJ et al.; Galactose transport activity from Escherichia coli was solubilized with octyl glucoside, and reconstituted into liposomes made from soybean or E . coli lipid . Galactose counterflow in the proteoliposomes was inhibited by glucose, talose, 2-deoxygalactose and 6-deoxygalactose, confirming that it was due to GalP and not one of the other E . coli galactose transport systems.

Nucleic Acids Res, 1983 Jul 11, 11(13), 4611 - 27
A DNA recombinant database management system; Tolstoshev CM et al.; A set of computer programs is described which constitutes a clone database management system . Maintenance of the database and the stocks of material is designed to be under the control of one person or group of people, who may insert, delete or modify data entries, and who may interrogate the database as to which stocks are in need of checking . The system is organised in such a way that information is freely and speedily available to all users . Database entries may be accessed by name or key word.

Nucleic Acids Res, 1983 Jul 11, 11(13), 4379 - 89
Molecular cloning and sequencing of pheU, a gene for Escherichia coli tRNAPhe; Schwartz I et al.; A recombinant plasmid (designated pID2) carrying the E . coli gene for tRNAPhe has been isolated from a plasmid bank constructed by the ligation of a total EcoRI digest of E . coli K12 DNA into the EcoRI site of pACYC184 DNA . The plasmid was selected by virtue of its ability to complement a temperature-sensitive lesion in the gene (PheS) for the alpha-subunit of phenylalanyl-tRNA synthetase . Crude tRNA isolated from such transformants exhibited elevated levels of phenylalanine acceptor activity . The tRNAPhe gene has been localized within the first 300 base pairs of a 3.6 kb SalI fragment of pID2 . The sequence of the gene and its flanking regions is presented.

Nucleic Acids Res, 1983 Jul 11, 11(13), 4325 - 33
Common terminal repeats of the macronuclear DNA are absent from the micronuclear DNA in hypotrichous ciliate, Stylonychia pustulata; Oka Y et al.; Comparison of nucleotide sequences of a macronuclear DNA and its micronuclear counterpart of a hypotrichous ciliate, Stylonychia pustulata, demonstrates that common terminal repeats (C4A4) of the macronuclear DNA are not present at the corresponding region in the micronuclear genome . The results indicate that the common terminal C4A4 repeat is added or translocated during or after the rearrangement of the micronuclear DNA to the macronuclear DNA.

FEBS Lett, 1983 Jul 11, 158(1), 113 - 5
Monoclonal antibodies inhibiting RNA polymerase from Escherichia coli; Nikiforov VG et al.; Monoclonal hybridoma antibodies directed against RNA polymerase from E . coli have been obtained . Only a few have been found to inhibit the enzyme activity . Antibodies produced by two clones, PYN-1 and PYN-2, inhibit RNA polymerase at the stage of RNA chain elongation . The PYN-1 antibodies react with the beta'-subunit of the enzyme . The PYN-2 antibodies react with the beta-subunit and with its 130 kDa amber fragment.

J Biol Chem, 1983 Jul 10, 258(13), 8146 - 50
Mutations of the beta subunit of RNA polymerase alter both transcription pausing and transcription termination in the trp operon leader region in vitro; Fisher RF et al.; RNA polymerase was purified from rifampicin-resistant mutants of Escherichia coli which exhibit altered transcription termination at the trp operon attenuator in vivo . These mutant polymerases were used to investigate transcription pausing at the trp leader pause site and transcription termination at the trp attenuator . The mutant polymerases examined in vitro mimic their in vivo termination responses; i.e . RNA polymerase isolated from a mutant which displays high transcriptional read-through of the trp operon in vivo allows greater transcriptional read-through in vitro, while RNA polymerase prepared from a mutant which has reduced read-through in vivo exhibits greater termination of transcription in vitro . The observed differences are not due to the presence of--or response to--alternate secondary structures in the trp leader transcript since deletion of the DNA segment corresponding to some of these alternate structures does not affect termination efficiency . The mutant polymerases also have comparable effects on the kinetics of transcription pausing at the trp leader pause site; the termination-deficient polymerase exhibits diminished pausing while the termination-proficient polymerase displays enhanced pausing . We suggest that this correlation reflects polymerase recognition of similar features of RNA secondary structures in the pause and termination events . In addition, since single mutational changes in RNA polymerase affect two activities, pausing and termination, it is likely that a single site or region of the polymerase is involved in both events.

J Biol Chem, 1983 Jul 10, 258(13), 8074 - 80
Purification and characterization of coliphage N4 RNA polymerase II activity from infected cell extracts; Zehring WA et al.; The soluble components of the RNA polymerase activity (N4 RNA polymerase II) required for coliphage N4 middle RNA synthesis have been purified to homogeneity using a complementation assay described elsewhere . These soluble components are found to exhibit the properties of a DNA-dependent RNA polymerase which is resistant to both rifampicin and streptolydigin and transcribes denatured N4 DNA with marked preference but with little selectivity for the middle region of the N4 genome . In its native form, the activity is composed of one Mr = 40,000 polypeptide (p4, the product of N4 cistron 4) and one Mr = 30,000 polypeptide (p7, the product of N4 cistron 3) . Its physical properties and the mechanism of transcription selectivity are discussed.

J Biol Chem, 1983 Jul 10, 258(13), 7981 - 90
Amino acid sequence of the N alpha-terminal 201 residues of human erythrocyte membrane band 3; Kaul RK et al.; We have determined the amino acid sequence of the N alpha-terminal portion of band 3, the anion transport protein of the human erythrocyte membrane . The material analyzed was a 201-residue, 23,053-Da fragment cleaved from the cytoplasmic end of band 3 by S-cyanylation . The sequence had these notable features . 1) The N alpha-terminal region was extraordinarily acidic, second only to a segment of similar size from the sigma factor of Escherichia coli RNA polymerase . The first 33 residues contained 6 aspartic acid and 12 glutamic acid residues, no basic residue, and a blocked N alpha-amino group . 2) The first 11 residues of the protein had a striking resemblance to the following 11 residues . 3) In contrast to the acidic N alpha-terminal third, the COOH-terminal two-thirds of the 23,053-Da fragment had a predominantly basic character . The highly acidic character of the N alpha-terminal portion of band 3 accounts for the capacity of this part of the protein to bind glycolytic enzymes in a highly electrostatic fashion, presumably through interaction with their cationic substrate-binding sites.

J Biol Chem, 1983 Jul 10, 258(13), 7918 - 20
Primary structure analysis of the mutant recA 441 and recA 430 proteins; Stachelek C et al.; The mutant recA 441 and recA 430 proteins from Escherichia coli have single amino acid substitutions which distinguish each of them from the wild type recA protein . recA 441 mutants express SOS functions at 42 degrees C even without DNA damage whereas recA 430 mutants are totally unable to express SOS functions at any temperature . Both mutant recA proteins have altered protease functions . However, the amino acid substitutions in each protein are located at a considerable linear distance from one another in their respective polypeptide chains, the recA 441 protein having a lysine in place of a glutamic acid residue at position 68, while the recA 430 protein has a serine in place of a glycine at position 204.

J Biol Chem, 1983 Jul 10, 258(13), 8456 - 61
Introduction and characterization of amber mutations in the bacteriorhodopsin gene; McCoy JM et al.; A fusion between the genes for bacteriorhodopsin and beta-galactosidase was constructed on a multicopy plasmid, pXB/Gal 101 . The fusion gene, containing the bacteriorhodopsin gene fused upstream from the beta-galactosidase gene, was under the control of tandem lipoprotein and lac gene promoters . When expressed in Escherichia coli the fusion protein retained beta-galactosidase activity . Mutations in the fusion gene were produced by passage of pXB/Gal 101 through the E . coli mutator strain mut D5 . Amber mutations were then selected by examining the loss of the lac+ phenotype imparted by the fusion protein to lac- E . coli cells . Amber mutations occurring within the bacteriorhodopsin gene were localized by replacing the beta-galactosidase region of each mutant plasmid with a beta-galactosidase region which was known to be unmutated . Precise localization of the mutations was achieved first by sizing the prematurely terminated peptides produced by the mutant plasmids in in vitro coupled transcription-translation reactions, and secondly by DNA sequence analysis . Six amber mutants in the gene for bacteriorhodopsin were characterized in this way . One of these was a transversion mutation at a lysine codon; the other five were all transition mutations at tryptophan codons, codons 10, 12, 80, 86, and 137 of the bacteriorhodopsin sequence.

J Biol Chem, 1983 Jul 10, 258(13), 8421 - 8
Double strand DNA cleavage by type II DNA topoisomerase from Drosophila melanogaster; Sander M et al.; The purified type II DNA topoisomerase from the embryos of Drosophila melanogaster exists in its native form as a dimer of 170,000-dalton polypeptides . In addition to the 170,000-dalton polypeptides, 3 polypeptides with molecular weights of 151,000, 141,000, and 132,000 were resolved when the enzyme was analyzed by electrophoresis under denaturing conditions . All four polypeptides can participate in the topoisomerase cleavage reaction and form covalent complexes with the cleaved DNA . Furthermore, immunochemical and biochemical data showed that they are structurally related and, therefore, the smaller polypeptides are likely generated from the 170,000-dalton polypeptide by proteolysis . The double strand DNA cleavage reaction of Drosophila topoisomerase has different site specificity from the Escherichia coli DNA gyrase-effected reaction . However, they result in an identical DNA structure at the cleavage site, which is a staggered double strand break with 4-nucleotide long 5'-protruding ends . The 3'-ends at the site of cleavage by Drosophila topoisomerase II have free hydroxyl groups and can be extended by exactly 4 nucleotides with T4 DNA polymerase, while the 5'-ends are covalently linked to the topoisomerase molecules . This similarity in cleavage site structure for Drosophila topoisomerase II and E . coli DNA gyrase suggests that they share some fundamental features in their mechanism of action.

J Biol Chem, 1983 Jul 10, 258(13), 8068 - 73
Appearance of monoglyceride and triglyceride in the cell envelope of Escherichia coli mutants defective in diglyceride kinase; Rotering H et al.; Diglyceride kinase mutants of Escherichia coli contain about 50- to 100-fold more 1,2-diglyceride than wild type cells . We now report that monoglyceride and triglyceride also accumulate in these strains . In mutant RZ60 (dgk-6) these compounds represent about 1 and 0.2%, respectively, of the total lipid fraction, while diglyceride represents 5-8% under most conditions . Monoglyceride accumulates predominantly in the outer membrane, while triglyceride builds up together with diglyceride in the cytoplasmic membrane . Under typical growth conditions about two-thirds of the diglyceride in E . coli arises in conjunction with synthesis of the membrane-derived oligosaccharides (Raetz, C.R.H., and Newman, K.F . (1979) J . Bacteriol . 137, 860-868) . Inhibition of membrane-derived oligosaccharides (MDO) synthesis also curtails the accumulation of monoglyceride and triglyceride . However, there appears to be at least one other MDO-independent source of diglyceride and related metabolites . Since MDO synthesis is suppressed by high osmolarity (Kennedy, E.P . (1982) Proc . Natl . Acad . Sci . U.S . A . 79, 1092-1095), we have examined the effects of osmolarity on diglyceride accumulation in RZ60 (dgk-6) . As expected, if MDO synthesis and diglyceride formation are coupled, the diglyceride level in RZ60 is higher at low osmolarity, while at high osmolarity the level of diglyceride is reduced to that observed in double mutants defective both in MDO synthesis and diglyceride kinase . Since dgk mutants do not grow at very low osmolarity, we have isolated several spontaneous phenotypic revertants that do . One class regains diglyceride kinase and has low diglyceride levels under all conditions . The other class remains defective in diglyceride kinase but tolerates higher diglyceride levels which amount to 13% of the total lipid during maximal induction of MDO synthesis at low osmolarity.

J Biol Chem, 1983 Jul 10, 258(13), 7890 - 3
Initiation in vivo at the internal trp p2 promoter of Escherichia coli; Horowitz H et al.; We have identified an RNA transcript initiated in vivo at the internal promoter of the Escherichia coli trp operon . The 5' end of this message overlaps the distal portion of the trpD structural gene, and the startpoint of transcription is the same as that previously determined in vitro . The relative abundance of the primary and secondary promoter transcripts in cells grown under varying conditions confirms previous genetic data suggesting that the p2 promoter is expressed at a low level, but constitutively . Comparison of p2 with a number of other recently identified internal promoters suggests that the primary function of these elements may be to provide a differentially regulated source of transcription for a subset of genes within the operon.

J Mol Biol, 1983 Jul 5, 167(3), 751 - 6
n' Protein activator sites of plasmid pBR322 are not essential for its DNA replication; Van der Ende A et al.; The lagging strand DNA synthesis of the Escherichia coli bacterial chromosome and plasmids is thought to be initiated by the mobile promotor, the primosome . This primosome is assembled at a specific site on single-stranded DNA . This process is initiated by the interaction of one of the at least seven components, the n' protein, with this site . Indeed n' protein activator sites are found in the plasmids Col E1 and pBR322 . To investigate the in vivo function of these n' protein sites, deletion derivates of pBR322 were constructed in which the n' protein sites are removed . The deletion plasmids show no change in stability and only threefold reduction in copy number compared to pBR322 . Using a transduction system for single-stranded plasmid DNA it was shown that no other specific initiation signals for lagging strand DNA synthesis were present in the deletion plasmids . It was concluded that the n' protein activator sites in pBR322 are not essential for its DNA replication in vivo.

J Submicrosc Cytol, 1983 Jul, 15(3), 705 - 12
Endotoxin effect on the isolated perfused rat liver: functional and ultrastructural observations; Gaeta GB et al.; The effect of E . coli endotoxin on bile flow and hepatic ultrastructure was studied in the isolated perfused rat liver . Within 30 min after its addition to the perfusate, endotoxin caused bile flow reduction . Despite this cholestatic effect, parenchymal cell structure did not show impressive changes on electron microscope examination . This feature contrasts with the in vivo findings by others, and suggests that the activation of extrahepatic mechanisms may contribute to the endotoxin toxicity for parenchymal cells in vivo.

Exp Cell Res, 1983 Jul, 146(2), 339 - 47
Fractionation and characterization of DNA at sites of replication from rat liver nuclei; Kaufman DG et al.; Regenerating rat liver nuclei when sonicated and centrifuged in a Cs2SO4 gradient were fractionated into three distinct bands . These bands were designated as light band (LB), middle band (MB), and heavy band (HB) according to their density . LB and MB were shown to consist of large granular particles with varying electron densities, but LB also contained remnants of nuclear membrane . When analysed by gel electrophoresis, LB and MB displayed more than 35 bands of proteins . The third fraction, HB, consisted largely of small chromatin fibers and its proteins were predominantly the four histones of the nucleosomal core particle . Following short pulses with {3H}thymidine in vivo, the specific activity of DNA in LB and MB was significantly higher than that of bulk DNA contained in HB . DNA in all three fractions became equally labelled as the duration of the labelling interval increased beyond 30 min . Newly synthesized DNA was characterized by electrophoresis on analytical 1.7% acrylamide -0.5% agarose composite gels . After a 1-min labelling interval in vivo, 17% of the rapidly labelled DNA from LB and MB was stationary at the gel origin like replication forks from E . coli, but only 3% of HB DNA had zero mobility . Electron microscopy confirmed the presence of DNA replication forks in LB, MB, and HB . With increasing time of synthesis the proportion of labelled DNA exhibiting zero mobility decreased in all three fractions . Denaturation of DNA or digestion of single-stranded DNA with S1 nuclease released newly synthesized DNA from the gel origin . Ribonuclease was without effect . DNA recovered from LB and MB also had a higher molecular weight than the HB DNA . Together these results indicate (1) that LB and MB are enriched in newly replicated DNA; (2) that an increased proportion of newly replicated DNA in LB and MB is associated with DNA replication forks; and (3) that the replicating DNA recovered in LB and MB may be associated with other nuclear constituents in situ because this DNA appears to be protected from the more frequent chain breaks introduced into the bulk of chromatin (HB) by sonication.

Cell, 1983 Jul, 33(3), 887 - 97
Initiation of transcription at phage T4 late promoters with purified RNA polymerase; Kassavetis GA et al.; We have previously identified T4 late promoters governing the in vivo expression of T4 late genes 23 and 24 (P23 and P24) . T4 late transcription in vivo is known to involve the binding of at least five phage-coded proteins to the bacterial RNA polymerase and normally requires concurrent DNA replication for DNA template activation . We show here that in vitro transcription, primarily of plasmids carrying T4 genes 23 and 24, by RNA polymerase purified from Escherichia coli at late times after T4 infection allows specific initiation at P23 and P24 in the absence of DNA replication . These promoters are not utilized by E . coli RNA polymerase holoenzyme, by RNA polymerase core, or by T4-modified RNA polymerase purified from cells infected with a T4 gene 55 mutant (gene 55 codes for an RNA polymerase binding protein required for late transcription) . The utilization of P23 and P24 in vitro is sharply inhibited by NaCl concentrations greater than 100 mM, and this inhibition is partly reversed by the addition of 10% DMSO . Relaxation of plasmid DNA containing P23 (with topoisomerase I) reduces P23 utilization at low salt (50 mM Na+) and nearly abolishes it at high salt (250 mM Na+) . P23 utilization is discernible in linear, glucosylated hydroxymethylcytosine-containing T4 virion DNA.

Yale J Biol Med, 1983 Jul-Aug, 56(4), 293 - 301
The effect of endotoxin and endotoxin tolerance on inflammation induced by mycobacterial adjuvant; Rosenbaum JT et al.; Peptidoglycan, the substance in mycobacteria thought to be responsible for inducing adjuvant arthritis, and endotoxin (lipopolysaccharide or LPS) share many inflammatory properties . Since repeated administration of LPS produces tolerance, i.e., resistance to the toxic and inflammatory effects of LPS, we tested whether LPS and/or LPS tolerance might influence inflammation due to mycobacterial adjuvant . Male Sprague-Dawley rats were injected with Escherichia coli LPS or saline intraperitoneally and then challenged with 100 micrograms killed Mycobacteria butyricum (adjuvant) in the footpad . A single dose of 100 micrograms LPS three or 24 hours before adjuvant markedly, but transiently, reduced the local footpad swelling that begins within hours of the adjuvant injection and histologically resembles a sterile abscess . Animals that received multiple doses of LPS and were therefore tolerant or animals that received LPS 72 hours before adjuvant demonstrated adjuvant-induced footpad swelling nearly equal to controls . The anti-inflammatory effect of LPS was transient since footpad swelling in all groups was nearly comparable six days after the adjuvant injection and LPS failed to inhibit consistently the arthritis that develops two or more weeks after adjuvant injection . These studies establish that LPS can markedly inhibit the prodrome of adjuvant arthritis (footpad swelling due to M . butyricum), that inhibition of this prodrome does not prevent the subsequent development of arthritis, and that LPS tolerance diminishes this anti-inflammatory effect of LPS.

Mikrobiologiia, 1983 Jul-Aug, 52(4), 634 - 7
{Sensitivity of Escherichia coli phages to the action of the physicochemical factors accompanying the process of cryopreservation}; Tsutsaeva AA et al.; A temperature shock, a change in the pH of the medium for conservation within the range of 4.0 to 10.0, and an increase of NaCl concentration up to 5 M do not inactivate Escherichia coli phages T3, T4 and phi X174 . The hydrostatic pressure of 2 X 10(3) atm inactivates phages T4 and phi X174 . The sensitivity of the phages to the pressure correlates with their survival rate after freezing.

Hematol Oncol, 1983 Jul-Sep, 1(3), 243 - 50
Chromosome studies in stimulated lymphocytes of B-cell chronic lymphocytic leukemias; Sadamori N et al.; Using a sister chromatid differentiation technique, cell cycle study of stimulated lymphocytes of B-cell chronic lymphocytic leukemia (B-CLL) revealed their cell cycle progression to be similar to that of normal lymphocytes stimulated by T-cell and various polyclonal B-cell activators (PBA) . The chromosome constitutions of stimulated lymphocytes in 62 patients with B-CLL were examined using PBA such as Epstein-Barr virus (EBV) and lipopolysaccharide W from E . coli 055:B5 (LPS) . Of the 20 patients with abnormal clones, 11 patients had trisomy 12; other less common abnormalities were trisomy 1, 6q-, i(7q), 14q+, trisomy 16, trisomy 18, reciprocal translocations, and marker chromosomes of unknown origin . These findings indicate that trisomy 12 may be a unique and common karyotypic change in B-CLL . The fact that 3 out of 4 patients with marker chromosomes showed stage IV disease may indicate that a clone with a marker is a predictor of an unfavourable prognosis . The near correlation between trisomy 12 and kappa chains existed (0.05 less than p less than 0.10) . Trisomy 12 was seen in all 5 patients with monoclonal paraprotein.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4258 - 62
Molecular cloning of the cDNA coding for rat ornithine transcarbamoylase; Horwich AL et al.; Ornithine transcarbamoylase is a mitochondrial matrix enzyme composed of three identical subunits encoded on the X chromosome . The subunit is synthesized on cytoplasmic polysomes as a precursor that is cleaved during transport into mitochondria . We report here the isolation and characterization of cDNA clones containing sequences corresponding to the mRNA encoding the ornithine transcarbamoylase subunit . cDNA was synthesized using rat liver mRNA enriched by polysome immunoadsorption for the low-abundance messenger species encoding the enzyme subunit . After insertion of cDNA into plasmid pBR322 and cloning in Escherichia coli, identification of the desired plasmids was accomplished by (i) differential colony hybridization using cDNA probes synthesized from mRNA of various tissues; (ii) differential blot hybridization using cDNA probes synthesized from mRNA enriched for or depleted of the ornithine transcarbamoylase message; (iii) hybrid-selected translation assays; and (iv) most definitively, structural analysis, which matched 25 consecutive amino acid residues determined by sequential Edman analysis of the carboxyl-terminal portion of the purified enzyme subunit with coding sequence present in the insert of one of the plasmids.

J Biochem (Tokyo), 1983 Jul, 94(1), 163 - 9
Adsorption chromatography of nucleic acids on silicone-coated porous glass; Mizutani T; Bovine liver tRNA was adsorbed on silicone-coated porous glass in 5 M NaCl, 10 mM Tris-HCl (pH 7.6) and fractionated by elution with decreasing NaCl concentrations . tRNAPro, tRNAVal, tRNAIle, tRNAThr, tRNASer, and tRNAPhe were eluted in this order . tRNA which had been digested with ribonuclease A was not adsorbed . Q beta RNA (adsorbed onto the glass in 5 M NaCl) was eluted with 1.5 M NaCl . RNA species in a crude rRNA fraction from Escherichia coli were separated into tRNA, 5S rRNA, and high molecular weight rRNA on siliconized porous glass . A half of calf thymus DNA was adsorbed on the glass in 5 M NaCl and the residual part passed through the column . The CD spectra showed that DNA and tRNA took the C-form and the A-form in 5 M NaCl, respectively . Therefore, the discrepancies of behavior of the DNA and RNA on siliconized porous glass may be related to the occurrence of these forms . The recovery of these nucleic acids from the column was 83-100% . Adsorption chromatography on siliconized porous glass may be a useful method for the separation of tRNA, rRNA, and mRNA.

J Appl Physiol, 1983 Jul, 55(1 Pt 1), 92 - 9
Effect of granulocyte depletion on altered lung mechanics after endotoxemia in sheep; Hinson JM Jr et al.; To examine the role of circulating granulocytes in the airway changes caused by endotoxemia, we measured the response of chronically instrumented unanesthetized sheep to endotoxemia before and after granulocyte depletion with hydroxyurea . Granulocyte depletion did not affect the increases in mean pulmonary arterial pressure caused by endotoxin {peak pressure 59 +/- 8 cmH2O +/- (SE) control, 51 +/- 8 cmH2O granulocyte depleted} . However, the early (30-60 min after endotoxin) airway response to endotoxemia was markedly attenuated . Without granulocyte depletion, endotoxin caused dynamic compliance (Cdyn) to decrease to 41 +/- 10% of the base-line value and total lung resistance (RL) to increase to 283 +/- 61% of base line . When animals were granulocyte depleted, endotoxin decreased Cdyn to 69 +/- 6% (P less than 0.05) of base line and increased RL to 141 +/- 20% of base line (P less than 0.05) . Granulocyte depletion also attenuated the effect of endotoxin on arterial oxygenation . During the maximum airway response to endotoxin, the alveolar-to-arterial oxygen gradient was 47 +/- 5 Torr in control studies and 32 +/- 2 Torr in granulocyte depleted studies (P less than 0.05) . We conclude that interaction of granulocytes with the lung contributes to the changes in lung mechanics observed following endotoxemia and that the early pulmonary hypertension and the early alterations in lung mechanics caused by endotoxemia are caused by separate processes.

Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4281 - 5
Cloning of human purine-nucleoside phosphorylase cDNA sequences by complementation in Escherichia coli; Goddard JM et al.; We have obtained cDNA clones that contain the entire coding region of the human purine-nucleoside phosphorylase (PNP; EC 2.4.2.1) mRNA . The cDNA sequences were generated by reverse transcription of PNP-enriched mRNA obtained by immunoadsorption of HeLa cell polyribosomes with monospecific antibody to human PNP . cDNA molecules that were close in length to PNP mRNA were separated by agarose gel electrophoresis and inserted into the Pst I site of the plasmid pBR322 . Plasmid DNA from the pooled clones was used to transform PNP-deficient Escherichia coli cells, and those transformants that phenotypically expressed PNP were isolated on selective media . The presence of human PNP in the selected bacterial cells was detected by immunoprecipitation with human PNP antibody.

J Trauma, 1983 Jul, 23(7), 605 - 9
Enteral and parenteral feeding influences mortality after hemoglobin-E . coli peritonitis in normal rats; Kudsk KA et al.; Enteral feeding with 25% dextrose-4.25% Freamine II (TPN) improves the survival of malnourished animals to normal levels after hemoglobin-E . coli adjuvant peritonitis, whereas intravenous feeding does not . To determine whether intravenous feeding maintained a high survival rate in previously well-nourished animals, 81 rats received TPN via gastrostomy or intravenous infusion for 12 days . They were then fasted for 24 hours and given a septic challenge . Gastrostomy-fed animals survived the challenge significantly better than intravenously fed animals . Enteral feeding appears to be important in producing a high survival rate after hemoglobin-E . coli adjuvant peritonitis.

J Clin Invest, 1983 Jul, 72(1), 63 - 76
Effects of cyclooxygenase inhibitors on the alterations in lung mechanics caused by endotoxemia in the unanesthetized sheep; Snapper JR et al.; The effects of Escherichia coli endotoxin on lung mechanics, hemodynamics, gas exchange, and lung fluid and solute exchange were studied in 12 chronically instrumented unanesthetized sheep . A possible role for cyclooxygenase products of arachidonate metabolism as mediators of the endotoxin-induced alterations in lung mechanics was investigated by studying sheep before and after cyclooxygenase inhibition with sodium meclofenamate and ibuprofen . Sheep were studied three times in random order: (a) sodium meclofenamate (or ibuprofen) infusion alone; (b) E . coli endotoxin alone; and (c) meclofenamate (or ibuprofen) and endotoxin . Meclofenamate alone had no effect on any of the variables measured . Endotoxin alone caused early marked changes in lung mechanics: resistance to airflow across the lungs (RL) increased 10-fold, dynamic lung compliance (Cdyn) decreased 80% and functional residual capacity (FRC) decreased by greater than 30% . The alveolar-to-arterial oxygen difference (delta AaPO2) increased markedly following endotoxemia . In the presence of sufficient meclofenamate to inhibit accumulation of thromboxane-B2 and 6-keto-prostaglandin F1 alpha in lung lymph, endotoxin caused no increase in RL, Cdyn decreased by less than 40%, and FRC decreased by only 6% . Meclofenamate significantly attenuated the hypoxemia and early pulmonary hypertension caused by endotoxemia but had no effect on the late increases in lung fluid and solute exchange . Ibuprofen had similar effects to those observed with meclofenamate . We conclude that both the pulmonary hypertension and changes in lung mechanics observed after endotoxemia may be mediated, at least in part, by constrictor prostaglandins or thromboxanes and that gas exchange may be improved by preventing endogenous synthesis of these mediators.

J Bacteriol, 1983 Jul, 155(1), 49 - 55
Evidence that repression mechanisms can exert control over the thr, leu, and ilv operons of Escherichia coli K-12; Johnson DI et al.; Mutants of Escherichia coli K-12 resistant to either the threonine analog DL-alpha-amino-beta-hydroxyvaleric acid or the leucine analog 5',5',5'-trifluoro-DL-leucine were isolated . One DL-alpha-amino-beta-hydroxyvaleric acid-resistant mutant strain, designated SP572, constitutively expressed the thr and ilv operons . The mutant allele, avr-16, was localized between trpR and the thr operon at min 0 . The wildtype allele of avr-16, designated ileR, is trans dominant . One 5',5',5'-trifluoro-DL-leucine-resistant mutant strain, designated FLR9, expressed the leu and ilv operons constitutively . The mutant allele, flr-9, is linked to entA at min 13 . The constitutive expression of the thr, leu, and ilv operons in mutants avr-16 and flr-9 was partly reversed in cells harboring a plasmid, which leads to elevated levels of the trpR gene product, the Trp aporepressor protein . Operator-like sequences situated upstream from the transcription startpoints of the thr, leu, and ilv operons are plausible candidates for targets of systems of repressor-operator control functioning in parallel with attenuation.

J Bacteriol, 1983 Jul, 155(1), 129 - 37
Regulation of hydrogenase activity in vegetative cells of Anabaena variabilis; Spiller H et al.; Heterocyst-free (NH4+-grown) cultures of the cyanobacterium Anabaena variabilis produce a hydrogenase which is reversibly inhibited by light and O2 . White or red light at an intensity of 5,000 lx inhibited greater than 95% of the activity . Oxygen at concentrations as low as 0.5% inhibited more than 85% of the hydrogenase in the vegetative cells of CO2-NH4+-grown cultures . The vegatative cell hydrogenase is also sensitive to strong oxidants like ferricyanide . In the presence of strong reductants like S2O4(2-), hydrogenase activity was not inhibited by light . However, hydrogenase activity in the heterocysts was insensitive to both light (greater than 5,000 lx) and O2 (10%) . Heterocysts and light-insensitive hydrogenase activity appear simultaneously during differentiation of the vegetative cells into heterocysts (an NH4+-grown culture transferred to NH4+-free, N2-containing medium) . This light-insensitive hydrogenase activity was detected several hours before the induction of nitrogenase activity . These results suggest a mode of regulation of hydrogenase in the vegetative cells of A . variabilis that is similar to "redox control" of hydrogenase and other "anaerobic" proteins in enteric bacteria like Escherichia coli.

Mikrobiologiia, 1983 Jul-Aug, 52(4), 615 - 9
{Action of sulfochlorantine on cell-free systems of Escherichia coli}; Viktorova LS et al.; Sulfochlorantine containing active chlorine was shown to produce a general toxic action inactivating a number of Escherichia coli enzyme systems involved in protein biosynthesis . DNA synthesis catalysed by calf thymus DNA polymerase alpha was repressed by 50% and the synthesis of aminoacyl-tRNA catalysed by aminoacyl-tRNA synthetases was repressed by 70% in the cell-free systems at the lethal concentration of the bactericide (0.01% and higher) . Sulfochlorantine produced the strongest inhibiting action on the ribosomal step of protein biosynthesis, inhibiting the poly(U)-directed polyphenylalanine formation by 95% at the sublethal concentration of the bactericide (0.005%).

J Pharmacobiodyn, 1983 Jul, 6(7), 496 - 504
Effect of inorganic arsenics on cytotoxicity and mutagenicity of ultraviolet light on Escherichia coli and the mechanism involved; Okada S et al.; The genetic effect of arsenite (AsO-2) and arsenate (HAsO4(2-)) on the cells of E . coliB (argF-) tester strains was investigated . Both the arsenics, which did not show mutagenic effect on the tester strains, reduced UV-induced mutation frequency of H/r30R (wild-type; Exc+Rec+) cells, while they did not change that of Hs30R (uvrA-; Exc-Rec+) cells . To elucidate the mechanism of this antimutagenicity, modifying effect of the arsenics on UV-induced cytotoxicity for Hs30R and NG30 (recA-; Exc+Rec-) cells was examined . In a nutrient medium containing the arsenics, the survived cell fraction of NG30 after ultraviolet (UV) irradiation was markedly increased, whereas that of Hs30R was not altered . When the UV-exposed NG30 cells were subjected to recovery incubation in a nutrient medium containing the arsenics, the survived cell fraction was remarkably increased before the cell proliferation in a similar manner as liquid-holding recovery observed in an arsenic-free and non-nutrient medium in which DNA replication was suppressed . The arsenics were found to cause a delay of DNA replication in UV-irradiated NG30 cells . These results indicate that arsenite and arsenate enhance the error-free excision repair of UV-damaged DNA by retarding the DNA replication and thus by prolonging the period for excision repair . This may lead to the reduction in UV-induced mutation.

Mech Ageing Dev, 1983 Jul-Aug, 22(3-4), 253 - 63
Age-correlated changes in the DNA template in the nematode Caenorhabditis elegans; Klass M et al.; Analysis of DNA from the nematode Caenorhabditis elegans demonstrated a number of significant age-correlated changes . The number of single-strand breaks as assayed by an in vitro assay procedure using Escherichia coli DNA polymerase I increased significantly with age . There was also an exponential increase in the amount of 5-methylcytosine in C . elegans DNA as the worm matured and aged . Furthermore, DNA isolated from older worms exhibited reduced transcriptional capacity when assayed in a HeLa cell in vitro transcription system . Finally, a biological assay to determine age-correlated changes in the DNA of aging sperm demonstrated a significant reduction in the capacity of the sperm to support zygotic development as the age of the male increased . These findings demonstrated significant age-correlated alterations and modifications occurring in the DNA template of the nematode, and their implications to the aging process are discussed.

Acta Paediatr Scand, 1983 Jul, 72(4), 577 - 82
Antibodies to Escherichia coli O antigens and the in-vitro bacteriostatic properties of human milk and its IgA; Dolby JM et al.; Only a very small part of the iron-reversed bacteriostatic activity of milk against Escherichia coli, demonstrable in vitro, is due to its anti-O antibody . Most of its growth-inhibitory activity is due to another lactoferrin-dependent, non-specific system . IgA prepared from milk is bacteriostatic for E . coli in the presence of lactoferrin, if it contains O-antibody for the indicator strain and if the strain is susceptible . Susceptibility depends to some extent on virulence, since those inhibited by IgA antibody to their own O-antigens were enteropathogenic or enterotoxigenic, whereas the growth of commensal strains was inhibited only slightly or not at all.

Plasmid, 1983 Jul, 10(1), 82 - 95
Conditional lethal mutants of the kilB determinant of broad host range plasmid RK2; Pohlman RF et al.; We have examined the relationship of kilB to the other known determinants which map in the 14'-22' region of RK2 . These are trfA, which encodes a diffusible replication function, and tra3, which specifies a function required for plasmid transmissibility . We found that, in addition to kilB, both tra3 and trfA functions are expressed by the cloned 14'-22' region of RK2 . Four temperature-sensitive mutants of kilB were isolated by in vitro mutagenesis of the cloned segment . At 42 degrees C these mutant plasmids can be maintained in Escherichia coli cells which lack a korB+ helper plasmid . At 30 degrees C the helper plasmid is required . Our analysis of these mutants revealed that kilB function is distinct from those of trfA and tra3 . One mutant plasmid was temperature-sensitive for maintenance of an RK2 ori plasmid, but this phenotype was shown to be independent of the KilB(ts) phenotype . Thus, kilB appears to be a separate new locus in this portion of the RK2 genome . In addition, these mutants allowed us to test for the existence of an essential replication determinant (trfB) in the 50.4'-56.4' region of RK2 . Our results demonstrate that this region is non-essential for replication from the RK2 ori in E . coli . We propose an alternative hypothesis to explain the role of the RK2 trfB region for plasmid maintenance in E . coli.

Plasmid, 1983 Jul, 10(1), 73 - 81
Two mini-F-encoded proteins are essential for equipartition; Austin S et al.; The par region of mini-F is both necessary and sufficient to promote equipartition of plasmid copies to daughter cells . It is approximately 2.5 kb long and contains the coding sequences for two proteins, F1 (41 kDa) and F2 (37 kDa) . We isolated 13 mutants of a phage lambda-mini-F hybrid that form unstable plasmids . Two of these putative Par- mutants are fully suppressible nonsense (amber) mutants . One of the amber mutants, par-41, eliminates the synthesis of F1, generating a large nonsense fragment of the protein . The other mutant, par-36, eliminates the synthesis of F2 . Thus both proteins appear to be essential for plasmid equipartition.

Plasmid, 1983 Jul, 10(1), 45 - 54
The structure of cloned hemolysin DNA from plasmid pHly185; Stark JM et al.; The complete DNA segment coding for hemolysis on Escherichia coli plasmid, pHly185, on to the vector pBR322 has been cloned . The recombinant plasmid, pJS204, coded the production of externalized hemolysin and contained four contiguous EcoRI fragments from the original plasmid . A restriction map of pJS204 was generated with six endonucleases . Deletion mutations were constructed from the recombinant plasmid by excision with either BamHI and PstI and religation . The BamHI deletion removed a 3680-bp portion from the recombinant plasmid and prevented the synthesis of active hemolytic activity . The PstI-generated deletion removed 7800 bp from the opposite end of the Hly sequence and resulted in a plasmid coding for the production of active intracellular hemolytic activity but contained no information for the export of hemolysin . We examined the ability of the deletion mutants of pJS204 to complement each other, and Tn5 insertion mutations in the hly region of the parent plasmid, pHly185 . The complementation studies indicated the presence of three separate cistrons able to complement in trans in a recA E . coli host . Two cistrons were found to be required for synthesis and one cistron for export of the hemolysin . The presence of two promoters and the directions of transcription for the synthesis of the hly gene products was also inferred from the complementation studies.

J Antimicrob Chemother, 1983 Jul, 12 Suppl A, 377 - 81
Clinical studies on ceftazidime in surgery in Japan; Nakayama I; Ceftazidime was evaluated in the treatment of post surgical infections in 280 patients at 19 institutions . The patients were mainly elderly and clinical assessment was possible in 232 cases . The overall clinical efficacy rate ('excellent' and 'improved') was 81% . The success rate in the treatment of cases which had failed to respond to other antibiotics was high (68%) . Bacteria were eradicated in 76% of all cases . No severe adverse reactions were observed.

Gene, 1983 Jul, 23(1), 1 - 13
The 19-kDal protein encoded by early region 1b of adenovirus type 12 is synthesized efficiently in Escherichia coli only as a fused protein; Fukui Y et al.; We have constructed recombinant plasmids that direct the synthesis of the Mr 19 000 protein encoded by the adenovirus type 12 (Ad12) E1b region as either a native protein or a protein fused to the amino-terminal portion of the elongation factor EF-TuB in Escherichia coli cells . Using these recombinants, we could synthesize a large amount of the fused protein, while only a small amount of the native Mr 19 000 protein was produced . The failure to synthesize the native Mr 19 000 protein in E . coli cells was ascribed to inefficient translation.

Genetika, 1983 Jul, 19(7), 1070 - 4
{Mutations of resistance to rifampicin leading to increased activity of the uridine phosphorylase gene in Escherichia coli}; Astvatsaturian MZ et al.; A number of rifampicin resistance mutations which have been mapped in the region of the rpoB gene, cause an increase in the activity level of a catabolite sensitive uridine phosphorylase (udp) gene . This effect is observed in bacterial cells deficient for the active protein repressor cytR, controlling expression of the udp gene . In cytR mutant cells grown on the medium containing glucose, the level of uridine phosphorylase is further increased by a factor of 1,5 to 2 under the influence of rif-r mutations . Concomitantly, the activity of some other catabolite sensitive genes controlled by the cytR protein is also increased on the medium with glucose . The data obtained suggest that the RNA polymerase beta-subunit participates in regulation of some catabolite sensitive genes.

Biofizika, 1983 Jul-Aug, 28(4), 570 - 2
{Circular dichroism of the complexes between poly-L-lysine and DNA with different GC contents}; Novoseler MA; Studies were carried out of circular dichroism spectra of the complexes between poly-l-lysine (PL) and calf thymus DNA, E . coli DNA, T2--and T7--phage DNA, Modiolus sp . DNA . The results indicate that PL more strongly changes AT--DNA conformation as compared to GC DNA conformation . This change correlates with the size of minimal PL clusters on DNA investigated . Sequence of DNA bases produces almost no effect on conformational changes caused by the complex-formation with PL.

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