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 incubat