Mutagenesis, 1989 May, 4(3), 230 - 4 The dissimilar mutational consequences of SN1 and SN2 DNA alkylation pathways: clues from the mutational specificity of dimethylsulphate in the lacI gene of Escherichia coli; Zielenska M et al.; The mutational specificity of the monofunctional alkylating agent dimethylsulphate has been determined through the DNA sequence characterization of 121 lacI-d mutations of Escherichia coli . The predominant mutation induced was the G:C----A:T transition (75%) . Transversions constituted 20% of all mutation with the greatest contribution being that of G:C----T:A events (12%) . Runs of G:C base pairs were the preferred sites of frameshift mutation . One 6-bp sequence (5'-CCCGCG-3') appeared to be highly susceptible to all classes of mutation and events within this sequence accounted for 33% of all mutations characterized . Although the distribution of G:C----A:T mutations appeared non-random, the site-specificity observed was quite different from that reported for SN1 alkylating agents . The results of this study highlight the differences between the consequences of SN1 and SN2 alkylation pathways. Am J Trop Med Hyg, 1989 May, 40(5), 455 - 64 Immunization of Saimiri sciureus boliviensis with recombinant vaccines based on the circumsporozoite protein of Plasmodium vivax; Collins WE et al.; We report a trial in squirrel monkeys of 2 recombinant Plasmodium vivax malaria vaccine candidates based on the circumsporozoite (CS) protein . One recombinant (NSl81V20), produced in Escherichia coli, contains the repeat region of the CS protein . The other (VIVAX-1) recombinant is yeast-derived and contains the entire repeat domain and part of the surrounding N-terminal and C-terminal regions . Both antigens were administered with alum and muramyl tripeptide as adjuvants . No formulations caused toxic side effects . Both antigens, when administered with alum, induced high levels of sporozoite antibodies in all animals . Another group of animals was immunized with irradiated sporozoites alone . Upon challenge, a few immunized animals did not develop detectable parasitemia and others developed parasitemia only after a prolonged prepatent period . Monkeys immunized with irradiated sporozoites had higher levels of antibody but no increased protection . There was no correlation between protection and either antibody level or the in vitro proliferation of lymphocytes in response to the antigens . This is the first time P . vivax sporozoite vaccines have been tested in monkeys with a subsequent sporozoite challenge. Proc Natl Acad Sci U S A, 1989 May, 86(10), 3699 - 703 Nonrandom utilization of codon pairs in Escherichia coli; Gutman GA et al.; We have analyzed protein-coding sequences of Escherichia coli and find that codon-pair utilization is highly biased, reflecting overrepresentation or underrepresentation of many pairs compared with their random expectations . This effect is over and above that contributed by nonrandomness in the use of amino acid pairs, which itself is highly evident; it is much weaker when nonadjacent codon pairs are examined and virtually disappears when pairs separated by two or three intervening codons are evaluated . There appears to be a high degree of directionality in this bias: any codon that participates in many nonrandom pairs tends to make both over- and underrepresented pairs, but preferentially as a left- or right-hand member . We show a relationship between codon-pair utilization patterns and levels of gene expression: genes encoding proteins expressed at high levels tend to contain more abundant, but more highly underrepresented, codon pairs, relative to genes expressed at low levels . The nonrandom utilization of codon pairs may be a consequence of their effects on translational efficiency, which in turn may be related to the compatibility of adjacent aminoacyl-tRNA isoacceptors at the A and P sites of a translating ribosome. Proc Natl Acad Sci U S A, 1989 May, 86(10), 3689 - 93 Genetic selection for genes encoding sequence-specific DNA-binding proteins; Elledge SJ et al.; We describe a genetic selection method designed to facilitate the cloning of genes encoding sequence-specific DNA-binding proteins . The strategy selects for clones expressing particular sequence-specific DNA-binding activities from a library of clones encoding other, nonspecific proteins . Specific DNA-binding sites have been placed near the start of transcription of the strong synthetic conII promoter to create promoters that can be repressed by the corresponding sequence-specific DNA-binding proteins . Transcription from the conII derivatives in the absence of repression interferes with the phenotypic expression of an adjacent drug-resistance gene, aadA . Sequence-specific DNA-binding proteins are shown to repress these promoters and alleviate transcriptional interference of aadA, resulting in drug resistance in cells expressing the appropriate DNA-binding protein. Proc Natl Acad Sci U S A, 1989 May, 86(10), 3579 - 83 Determinants of EcoRI endonuclease sequence discrimination; Needels MC et al.; The arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the guanine of the sequence GAATTC and thus be an important determinant of sequence discrimination . Arg-200 was replaced by each of the other 19 naturally occurring amino acids, and the mutant endonucleases were assessed for activities in vivo and in vitro . The mutant endonuclease with lysine at position 200 exhibits the most in vivo activity of all the position 200 mutants, although the in vitro activity is less than 1/100th of wild-type activity . Five other mutants show more drastically reduced levels of in vivo activity (Cys, Pro, Val, Ser, and Trp) . The Cys, Val, and Ser mutant enzymes appear to have in vivo activity which is specific for the wild-type canonical site despite the loss of hydrogen bonding potential at position 200 . The Pro and Trp mutants retain in vivo activity which is independent of the presence of the EcoRI methylase . In crude cell lysates, only the Cys mutant shows a very low level of in vitro activity . None of the mutant enzymes show a preference for alternative sites in assays in vitro . The implications of these results are discussed. Biophys J, 1989 May, 55(5), 927 - 36 Triplet state properties of tryptophan residues in complexes of mutated Escherichia coli single-stranded DNA binding proteins with single-stranded polynucleotides; Tsao DH et al.; Complexes of point-mutated E . coli single-stranded DNA-binding protein (Eco SSB) with homopolynucleotides have been investigated by optical detection of magnetic resonance (ODMR) of the triplet state of tryptophan (Trp) residues . Investigation of the individual sublevel kinetics of the lowest triplet state of Trp residues 40 and 54 in the poly (dT) complex of Eco SSB-W88F,W135F (a mutant protein whose Trp residues at positions 88 and 135 have been substituted by Phe) shows that Trp 54 is the most affected residue upon stacking with thymine bases, confirming previous results based on SSB mutants having single Trp----Phe substitutions . (Zang, L . H., A . H . Maki, J . B . Murphy, and J . W . Chase . 1987 . Biophys . J . 52:867-872) . The Tx sublevel of Trp 54 shows a fourfold increase in the decay rate constant, as well as an increase in its populating rate constant by selective spin-orbit coupling . The two nonradiative sublevels show no change in lifetime, relative to unstacked Trp . For Trp 40, a weaker perturbation of Tx by thymine results in a sublevel lifetime about one-half that of normal Trp . Trp54 displays a 2{E}transition of negative polarity in the double mutant SSB complex with Poly (dT), but gives a vanishingly weak {D} - {E} signal, thus implying that the steady-state sublevel populations of Tx and Tz are nearly equal in this residue . Poly (5-BrU) induces the largest red-shift of the Eco SSB-W88F,W135F Trp phosphorescence 0,0-band of all polynucleotides investigated . Its phosphorescence decay fits well to two exponential components of 1.02 and 0.12 s, with no contribution from long-lived Trp residues . This behavior provides convincing evidence that both Trp 40 and 54 are perturbed by stacking with brominated uridine . The observed decrease in the Trp {D} values further confirms the stacking of the Trp residues with 5-BrU . Wave-length-selected ODMR experiments conducted on the {D{ + {E} transition of Eco SSB-W88F,W135F complexed with poly(5HgU) indicate the presence of multiple heavy atom-perturbed sites . Measurements made on poly (5-HgU) which each of its 4 Trp residues has been replaced in turn by Phe demonstrate that Trp 40 and 54 are the only Trp residues undergoing stacking with nucleotide bases, as previously proposed. Proc Natl Acad Sci U S A, 1989 May, 86(9), 3237 - 41 A plant manganese superoxide dismutase is efficiently imported and correctly processed by yeast mitochondria; Bowler C et al.; In the plant Nicotiana plumbaginifolia, manganese superoxide dismutase (MnSOD) is synthesized in the cytoplasm as a preprotein and is subsequently translocated to the mitochondrial matrix with corresponding cleavage of an NH2-terminal leader sequence . To determine whether the plant enzyme could replace the endogenous SOD activities of Escherichia coli and yeast, constructions have been made in appropriate vectors for expression of the preprotein and the mature MnSOD . These were introduced into SOD-deficient strains for complementation studies . In E . coli, both forms of the protein were shown to be active and able to complement SOD deficiency to different degrees . Expression of the preprotein in a yeast strain lacking a mitochondrial MnSOD resulted in a restoration of wild-type growth, only possible if the plant protein was being targeted to the mitochondria . Subsequent studies revealed that the protein was processed and that the leader sequence was cleaved at the identical position as recognized by the mitochondrial peptidase of plants . The components mediating mitochondrial import thus appear to be highly conserved between plants and yeast. Proc Natl Acad Sci U S A, 1989 May, 86(9), 3119 - 22 Evidence for several higher order structural elements in ribosomal RNA; Woese CR et al.; Comparative analysis of small subunit ribosomal RNA sequences suggests the existence of two new higher order interactions: (i) a double-helical structure involving positions 505-507 and 524-526 (Escherichia coli numbering) and (ii) an interaction between the region of position 130 and the helix located approximately between positions 180 and 195 . In the first of these, one of the strands of the helix exists in the bulge loop, and the other strand exists in the terminal loop of a previously recognized compound helix involving positions 500-545 . Therefore, the new structure formally represents a pseudoknot . In the second, the insertion/deletion of a nucleotide in the vicinity of position 130 correlates with the length of the helix in the 180-195 region, the latter having a 3-base-pair stalk when the base in question is deleted and a stalk of approximately 10 pairs when it is inserted. Proc Natl Acad Sci U S A, 1989 May, 86(9), 3060 - 4 Protein fragments as probes in the study of protein folding mechanisms: differential effects of dihydrofolate reductase fragments on the refolding of the intact protein; Hall JG et al.; We describe an approach for investigating the protein folding process, using protein fragments as inhibitory probes of the refolding protein . The refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3), reversibly unfolded in 7 M urea, was monitored by the reappearance of enzyme activity after diluting the unfolded enzyme into low urea concentrations (less than or equal to 2 M) in the presence of substrates . Of eight protein fragments produced by limited proteolysis of the 159-residue enzyme, three isolated peptides--Ser-49/Glu-90, Ile-91/Glu-154, and Gln-102/Glu-154--were evaluated for their effects on the recovery of the refolding protein's enzymatic activity . By this criterion, 13 microM peptide Gln-102/Glu-154 inhibits the refolding of 0.015 microM enzyme by approximately 80%, while the related peptide, Ile-91/Glu-154, and peptide Ser-49/Glu-90 at the same concentration inhibit the recoverable activity of the refolding enzyme by less than or equal to 20% . None of these three peptides has any significant effect on the activity of the folded enzyme . Our results indicate that peptides may inhibit refolding differentially and that these effects may be extremely sensitive to fragment sequence and composition . We suggest that peptide specificity in the inhibition of protein folding may be exploited as a structural probe of protein folding mechanisms. Proc Natl Acad Sci U S A, 1989 May, 86(9), 3026 - 30 A host-encoded DNA-binding protein promotes termination of plasmid replication at a sequence-specific replication terminus; Sista PR et al.; We have purified approximately 6600-fold an approximately 40-kDa protein (Ter protein) encoded by Escherichia coli that specifically binds to two sites at the 216-base-pair replication terminus (tau) of the plasmid R6K . Chemical footprinting experiments have shown that the Ter protein binds to two 14- to 16-base-pair sequences that exist as inverted repeats in the tau fragment . Site-directed mutagenesis of one of the terminus sequences (tau R) resulted in a mutant tau R that failed to bind to the Ter protein . The same mutant terminus also failed to terminate DNA replication in vivo . These experiments strongly suggest that the interaction of the Ter protein with tau sequences plays an essential role in the termination of DNA replication, specifically at tau. J Cell Biol, 1989 May, 108(5), 1637 - 46 In vitro membrane assembly of a polytopic, transmembrane protein results in an enzymatically active conformation; Ahrem B et al.; In vitro integration of the polytopic, transmembrane lactose permease into membrane vesicles from Escherichia coli is demonstrated . To this end the enzyme was synthesized in a homologous, cell-free transcription-translation system . In this system, synthesis occurred in an essentially membrane-free environment leading to the formation of lactose permease aggregates, which were resistant to protease digestion and detergent solubilization . However, if inverted membrane vesicles from E . coli were included in the synthesis reaction, most de novo-synthesized lactose permease could be recovered from a membrane-containing subfraction (enriched in leader {signal} peptidase activity) . This membrane association of lactose permease was Na2CO3 resistant, detergent sensitive, and yielded a distinct pattern of proteolytic cleavage peptides . Moreover, membrane vesicles when present cotranslationally during synthesis of lactose permease, acquired the capability to accumulate lactose, strongly suggesting a correct in vitro assembly of the enzyme . Because of the extensive aggregation of lactose permease synthesized in the absence of membranes, only low amounts originating from the soluble enzyme pool integrated posttranslationally into the membrane vesicles . Unlike the translocation of the outer membrane protein LamB into membrane vesicles, integration of lactose permease was found to be independent of the H+-motive force. Eur J Biochem, 1989 May 1, 181(2), 363 - 70 Purification and characterization of ribonuclease M and mRNA degradation in Escherichia coli; Cannistraro VJ et al.; A previously unreported endoribonuclease has been identified in Escherichia coli, which has a preference for hydrolysis of pyrimidine-adenosine (Pyd-Ado) bonds in RNA . It was purified about 7000-fold to give a single band after SDS/polyacrylamide gel electrophoresis; the eluted protein gave the same RNase specificity . The sizes of the native and denatured enzymes agreed suggesting that the enzyme exists as a monomer of approximately 26 kDa . It is called RNase M . The only other reported broadly specific endoribonuclease in E . coli is RNase I, a periplasmic enzyme . Based on differences in charge, heat stability and substrate specificity, it was clear that RNase M is not RNase I . The specificity of RNase M was remarkably similar to that of pancreatic RNase A even though the two enzymes differ in charge characteristics and size . Earlier studies had shown that mRNA from the lactose operon of E . coli is hydrolyzed in vivo primarily between Pyd-Ado bonds {Cannistraro et al . (1986) J . Mol . Biol . 192, 257-274} We propose that this major RNase activity accounts for these cleavages observed in vivo and that it is the endonuclease for mRNA degradation in E . coli. Eur J Biochem, 1989 May 1, 181(2), 351 - 6 Immunoblotting analysis of protein-protein crosslinks within the 50S ribosomal subunit of Escherichia coli . A study using dimethylsuberimidate as crosslinking reagent; Redl B et al.; 50S ribosomal subunits of Escherichia coli have been crosslinked with the bifunctional imidoester dimethyl-suberimidate and the protein-protein crosslinks have been analyzed by immunoblotting, using antisera specific for the individual ribosomal proteins of the large ribosomal subunit . Crosslinked protein pairs which occurred in yields higher than 5% have been unambiguously identified . Thus 13 crosslinks have been identified, namely L1-L33, L5-L7/12, L6-L19, L7/12-L10, L7/12-L11, L9-L28, L10-L11, L13-L20, L16-L27, L17-L32, L18-L22, L19-L25 and L27-L33 . These data, together with the results which we will be presenting elsewhere, contribute considerably to our knowledge of the protein topography of the 50S ribosomal proteins as determined by immunoelectron microscopy . We can now propose the approximate locations of ten proteins that have not previously been localized. Am Rev Respir Dis, 1989 May, 139(5), 1118 - 24 Relationship between lung injury and lung lipid peroxidation caused by recurrent endotoxemia; Demling R et al.; We compared the relationship between lung lipid peroxidation and the histologic and physiologic changes seen after repeated doses of low dose endotoxin in unanesthetized sheep . Thirty-two sheep with lung lymph fistula were given from 1 to 10 doses of 1 micrograms/kg Escherichia coli endotoxin, 12 h apart . Animals were killed 5 h after 1, 3, 5, or 9 doses of endotoxin or 3 to 5 days after the tenth dose of endotoxin . The lipid peroxidation process was monitored by circulating conjugated dienes and lung tissue malondialdehyde (MDA) content . We found that conjugated dienes and MDA were increased after one dose of endotoxin corresponding in time with the increased prostanoid production and increased permeability . Acute lung inflammation was also evident histologically . Lipid peroxidation was not increased, however, when 3 to 7 doses were given . The permeability change was also markedly attenuated whereas severe lung inflammation was still present on histologic examination . After 9 doses, we noted a fourfold increase in lung tissue MDA that corresponded histologically with a marked mononuclear cell infiltration . Physiologic changes included a sustained 50% increase in oxygen consumption . However, lung lymph flow was not increased, again, reflecting lung inflammation with no change in lung vascular permeability . The MDA remained increased 5 days after the last dose of endotoxin along with a marked lung mononuclear cell infiltration . The lung MDA content corresponded with the level of increase in VO2, but not with changes in pulmonary vascular permeability . Conjugated dienes were increased only after the first injection of endotoxin . The lung lipid peroxidation process does not appear to correspond to physiologic or histologic lung changes after recurrent exposures to endotoxin. J Neurochem, 1989 May, 52(5), 1425 - 32 Human brain-type glycogen phosphorylase: quantitative localization in human tissues determined with an immunoassay system; Kato K et al.; Glycogen phosphorylase (EC 2.4.1.1) from human brain tissue was purified to homogeneity . Antisera were developed in rabbits with purified phosphorylase as the immunogen . Antibodies were first affinity-purified with a column of brain phosphorylase-coupled Sepharose, and then the antibody fraction was adsorbed with a column of muscle phosphorylase-coupled Sepharose to remove antibodies reactive also with muscle phosphorylase . By using the specific antibodies, a sandwich-type immunoassay system for measurement of brain phosphorylase was prepared . The assay system consisted of polystyrene balls with immobilized antibrain phosphorylase F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli . The assay was sensitive and specific to the brain phosphorylase . The minimum detection limit of the assay was 0.1 ng/assay tube, and the cross-reactivity of the assay with muscle phosphorylase was less than 1% . Tissue concentrations of immunoreactive brain-type phosphorylase were estimated . The phosphorylase was present in the heart at as high a level as in the brain . The immunoreactivity for brain phosphorylase was distributed widely at a significant concentration in various peripheral tissues, such as the digestive tract, bladder, aorta, liver, and testis . Immunohistochemical localization of brain phosphorylase in the CNS revealed that the enzyme is present in most astrocytes and amyloid bodies, as well as in some neurons in the cerebral cortex and Golgi cells in the cerebellar cortex. J Bacteriol, 1989 May, 171(5), 2783 - 8 Synthesis, inactivation, and localization of extracellular and intracellular Escherichia coli hemolysins; Oropeza-Wekerle RL et al.; Extra- and intracellular Escherichia coli hemolysin expressed by two cloned hly determinants, both under the control of the activator element hlyR, were analyzed . One determinant carried all four hly genes (hlyC, hlyA, hlyB, and hlyD), whereas the other carried only the two genes (hlyC and hlyA) required for synthesis of active hemolysin but not those essential for its secretion . It was shown that the total amounts of HlyA protein and of hemolytic activity are similar in both cases in logarithmically growing cultures . The E . coli strain carrying the complete hly determinant released most hemolysin into the media and accumulated very little HlyA intracellularly . The active extracellular hemolysin (HlyA*) was inactivated in the stationary phase without degradation of the HlyA protein . In contrast, the hemolysin which accumulated intracellularly in the E . coli strain carrying hlyA and hlyC only was proteolytically degraded at the end of the logarithmic growth phase . Immunogold labeling indicates that active intracellular HlyA bound preferentially to the inner membrane, whereas that part of the extracellular HlyA which remained cell-bound was located exclusively at the cell surface . It was shown by fluorescence-activated cell sorter analysis that active extra- and intracellular HlyA* bound with similar efficiency to erythrocytes, whereas hemolytically inactive HlyA protein did not bind to these target cells. J Bacteriol, 1989 May, 171(5), 2728 - 34 DNA sequence analysis, gene product identification, and localization of flagellar motor components of Escherichia coli; Malakooti J et al.; The Escherichia coli operon designated flaA contains seven flagellar genes; among them are two switch protein genes whose products are believed to interface with the motility and chemotaxis machinery of the cell . Complementation analysis using several plasmids carrying different portions of the flaA operon and analysis of expression of these plasmids in minicells allowed the identification of two flagellar gene products . The MotD (now called FliN) protein, a flagellar switch protein, was determined to have an apparent molecular weight of 16,000, and the FlaAI (FliL) protein, encoded by a previously unidentified gene, had an apparent molecular weight of 17,000 . DNA sequence analysis of the motD gene revealed an open reading frame of 414 base pairs . There were two possible initiation codons (ATG) for motD translation, the first of which overlapped with the termination codon of the upstream gene, flaAII (fliN) . The wild-type flaAI gene on the chromosome was replaced with a flaAI gene mutated in vitro . Loss of the flaAI gene product resulted in a nonmotile and nonflagellated phenotype . The subcellular location for both the MotD and FlaAI proteins was determined; the FlaAI protein partitioned exclusively in the insoluble fraction of a whole minicell sonic extract, whereas the MotD protein remained in both the soluble and insoluble fractions . In addition, we subcloned a 2.2-kilobase-pair DNA fragment capable of complementing the remaining four genes of the flaA operon (flbD {fliO}, flaR {fliP}, flaQ {fliQ}, and flaP {fliR}). J Bacteriol, 1989 May, 171(5), 2697 - 707 Nucleotide sequence and replication characteristics of RepFIB, a basic replicon of IncF plasmids; Saul D et al.; A second autonomous replicon of P307, RepFIB, has been isolated that has significant homology with other replicons in IncFI group plasmids . Eleven homologous repeats of 21 base pairs are present on the sequence and flank an open reading frame capable of coding for a protein of about Mr = 40,000 . This protein was identified by maxicell analysis of cloned RepFIB . A series of deletion mutations of RepFIB were inserted into a DNA polymerase I-dependent vector and examined for their replication proficiency in a polA1 strain . These experiments defined a minimal replication region of 1.6 kilobases which includes the three repeats immediately upstream and downstream of the open reading frame . Deletion of a second set of repeats further downstream doubled the copy number of a chimeric plasmid replicating under RepFIB control . It was concluded that these repeats control the copy number of the replicon . Incompatibility tests showed that all three sets of repeats could express incompatibility with a resident RepFIB plasmid. J Bacteriol, 1989 May, 171(5), 2673 - 9 pCloDF13-encoded bacteriocin release proteins with shortened carboxyl-terminal segments are lipid modified and processed and function in release of cloacin DF13 and apparent host cell lysis; Luirink J et al.; By oligonucleotide-directed mutagenesis, stop codon mutations were introduced at various sites in the pCloDF13-derived bacteriocin release protein (BRP) structural gene . The expression, lipid modification (incorporation of {3H}palmitate), and processing (in the presence and absence of globomycin) of the various carboxyl-terminal shortened BRPs were analyzed by a special electrophoresis system and immunoblotting with an antiserum raised against a synthetic BRP peptide, and their functioning with respect to release of cloacin DF13, lethality, and apparent host cell lysis were studied in Sup-, supF, and supP strains of Escherichia coli . All mutant BRPs were stably expressed, lipid modified, and processed by signal peptidase II, albeit with different efficiencies . The BRP signal peptide appeared to be extremely stable and accumulated in induced cells . Full induction of the mutant BRPs, including the shortest containing only 4 amino acid residues of the mature polypeptide, resulted in phospholipase A-dependent and Mg2+-suppressible apparent cell lysis . The extent of this lysis varied with the mutant BRP used . Induction of all mutant BRPs also prevented colony formation, which appeared to be phospholipase A independent . One shortened BRP, containing 20 amino acid residues of the mature polypeptide, was still able to bring about the release of cloacin DF13 . The results indicated that the 8-amino-acid carboxyl-terminal segment of the BRP contains a strong antigenic determinant and that a small segment between amino acid residues 17 and 21, located in the carboxyl-terminal half of the BRP, is important for release of cloacin DF13 . Either the stable signal peptide or the acylated amino-terminal BRP fragments (or both) are involved in host cell lysis and lethality. J Bacteriol, 1989 May, 171(5), 2634 - 8 Regulation of isocitrate dehydrogenase by phosphorylation in Escherichia coli K-12 and a simple method for determining the amount of inactive phosphoenzyme; Edlin JD et al.; In several Escherichia coli K-12 strains grown on a limiting concentration of glucose, isocitrate dehydrogenase (IDH) was inactivated about 90% after cessation of growth upon exhaustion of the glucose . Such inactivation has been previously observed in several E . coli strains but not in E . coli K-12 (unless acetate was added to the bacterial culture when growth ceased) . IDH was inactivated 75 to 80% in all E . coli K-12 strains we examined during growth on acetate . The inactivation involved phosphorylation of the enzyme and is considered to be a regulatory mechanism facilitating metabolite flow along the glyoxylate shunt . Phospho-IDH interacted with antibodies to enzymatically active IDH . We have devised a method, based on this immunological cross-reaction, for determining the proportions of active and inactive (phospho-) IDH in cell extracts. J Bacteriol, 1989 May, 171(5), 2626 - 33 Nucleotide sequences of the fecBCDE genes and locations of the proteins suggest a periplasmic-binding-protein-dependent transport mechanism for iron(III) dicitrate in Escherichia coli; Staudenmaier H et al.; The fec region of the Escherichia coli chromosome determines a citrate-dependent iron(III) transport system . The nucleotide sequence of fec revealed five genes, fecABCDE, which are transcribed from fecA to fecE . The fecA gene encodes a previously described outer membrane receptor protein . The fecB gene product is formed as a precursor protein with a signal peptide of 21 amino acids; the mature form, with a molecular weight of 30,815, was previously found in the periplasm . The fecB genes of E . coli B and E . coli K-12 differed in 3 nucleotides, of which 2 gave rise to conservative amino acid exchanges . The fecC and fecD genes were found to encode very hydrophobic polypeptides with molecular weights of 35,367 and 34,148, respectively, both of which are localized in the cytoplasmic membrane . The fecE product was a rather hydrophilic but cytoplasmic membrane-bound protein of Mr 28,189 and contained regions of extensive homology to ATP-binding proteins . The number, structural characteristics, and locations of the FecBCDE proteins were typical for a periplasmic-binding-protein-dependent transport system . It is proposed that after FecA- and TonB-dependent transport of iron(III) dicitrate across the outer membrane, uptake through the cytoplasmic membrane follows the binding-protein-dependent transport mechanism . FecC and FecD exhibited homologies to each other, to the N- and C-terminal halves of FhuB of the iron(III) hydroxamate transport system, and to BtuC of the vitamin B12 transport system . FecB showed some homology to FhuD, suggesting that the latter may function in the same manner as a binding protein in iron(III) hydroxamate transport . The close homology between the proteins of the two iron transport systems and of the vitamin B12 transport system indicates a common evolution for all three systems. J Bacteriol, 1989 May, 171(5), 2609 - 13 Chromosomal transformation of Escherichia coli recD strains with linearized plasmids; Russell CB et al.; Wild-type Escherichia coli are resistant to genetic transformation by purified linear DNA, probably in part because of exonuclease activity . We demonstrate that E . coli containing a recD mutation could be easily transformed by linearized plasmids containing a selectable marker . The marker was transferred to the chromosome by homologous recombination, whereas plasmid markers not in the region of homology were lost. J Bacteriol, 1989 May, 171(5), 2547 - 52 5-Aminolevulinic acid synthesis in Escherichia coli; Li JM et al.; A hemA mutant of Escherichia coli containing a multicopy plasmid which complemented the mutation excreted 5-aminolevulinic acid (ALA) into the medium . {1-14C}glutamate was substantially incorporated into ALA by this strain, whereas {2-14C}glycine was not . Periodate degradation of labeled ALA showed that C-5 of ALA was derived from C-1 of glutamate . The synthesis of ALA by two sonicate fractions which had been processed by gel filtration and dialysis, respectively, was dependent on glutamate, ATP, NADPH, tRNA(Glu), and pyridoxal phosphate . tRNA(Glu) stimulated ALA synthesis in a concentration-dependent manner . Pretreatment with RNase reduced this stimulation . The amino acid sequence of the cloned insert, derived from the nucleotide sequence (J.-M . Li, C . S . Russell, and S . D . Cosloy, J . Cell Biol . 107:617a, 1988), showed no homology with any ALA synthase sequenced to date . These results suggest that E . coli synthesizes ALA by the C5 pathway from the intact five-carbon chain of glutamate. J Bacteriol, 1989 May, 171(5), 2533 - 41 Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage; Ennis DG et al.; Evidence is presented that recA functions which promote the SOS functions of mutagenesis, LexA protein proteolysis, and lambda cI repressor proteolysis are each genetically separable from the others . This separation was observed in recombination-proficient recA mutants and rec+ (F' recA56) heterodiploids . recA430, recA433, and recA435 mutants and recA+ (F' recA56) heterodiploids were inducible for only one or two of the three functions and defective for mutagenesis . recA80 and recA432 mutants were constitutively activated for two of the three functions in that these mutants did not have to be induced to express the functions . We propose that binding of RecA protein to damaged DNA and subsequent interaction with small inducer molecules gives rise to conformational changes in RecA protein . These changes promote surface-surface interactions with other target proteins, such as cI and LexA proteins . By this model, the recA mutants are likely to have incorrect amino acids substituted as sites in the RecA protein structure which affect surface regions required for protein-protein interactions . The constitutively activated mutants could likewise insert altered amino acids at sites in RecA which are involved in the activation of RecA protein by binding small molecules or polynucleotides which metabolically regulate RecA protein. J Bacteriol, 1989 May, 171(5), 2485 - 98 Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression; Sawers G et al.; The sequence of the 5' regulatory region of the gene encoding pyruvate formate-lyase is presented together with a detailed analysis of the transcriptional signals required for its expression . The sequence data revealed that a gene coding for an open reading frame (orf) of unknown function is situated just upstream of the pfl gene . Analysis of RNA transcripts by Northern blot hybridization demonstrated that the genes for orf and pfl were cotranscribed as an operon but that the pfl gene was also transcribed alone . S1 nuclease protection analysis, primer extension, and construction of lacZ fusions with sequential deletions in the pfl 5' regulatory sequence revealed that transcription initiated from at least six promoters which spanned 1.2 kilobases of DNA . Three of these lay within the orf structural gene and were responsible for the high expression of pfl . All transcripts originating from these promoters terminated in the 3' untranslated region of the pfl gene at a strong rho-independent transcription terminator . All of the promoters were coordinately regulated by anaerobiosis, pyruvate, nitrate, and the fnr gene product, and the sequences thought to be responsible for this regulation lay 0.8 to 1.3 kilobases upstream of the translational initiation codon of the pfl gene . There were two sequences within this region which showed strong homology with that proposed to be required for recognition by the Fnr protein. J Bacteriol, 1989 May, 171(5), 2480 - 4 Spontaneous and UV-induced mutations in Escherichia coli K-12 strains with altered or absent DNA polymerase I; Bates H et al.; The induction of mutations to valine resistance and to rifampin resistance occurs after UV irradiation in bacteria carrying a deletion through the polA gene (delta polA), showing that DNA polymerase I (PolI) is not an essential enzyme for this process . The PolI deletion strain showed a 7- to 10-fold-higher spontaneous mutation frequency than the wild type . The presence in the deletion strain of the 5'----3' exonuclease fragment on an F' episome caused an additional 10-fold increase in spontaneous mutation frequency, resulting in mutation frequencies on the order of 50- to 100-fold greater than wild type . The mutator effect associated with the 5'----3' exonuclease gene fragment together with much of the effect attributable to the polA deletion was blocked in bacteria carrying a umuC mutation . The mutator activity therefore appears to reflect constitutive SOS induction . Excision-proficient polA deletion strains exhibited increased sensitivity to the lethal effect of UV light which was only partially ameliorated by the presence of polA+ on an F' episome . The UV-induced mutation rate to rifampin resistance was marginally lower in delta polA bacteria than in bacteria carrying the polA+ allele . This effect is unlikely to be caused by the existence of a PolI-dependent mutagenic pathway and is probably an indirect effect caused by an alteration in the pattern of excision repair, since it did not occur in excision-deficient (uvrA) bacteria . An excision-deficient polA deletion strain possessed UV sensitivity similar to that of an isogenic strain carrying polA+ on an F' episome, showing that none of the functions of PolI are needed for postreplication repair in the absence of excision repair . Our data provide no evidence for a pathway of UV mutagenesis dependent on PolI, although it remains an open question whether PolI is able to participate when it is present. J Bacteriol, 1989 May, 171(5), 2466 - 73 The ssb gene of plasmid ColIb-P9; Howland CJ et al.; The IncI1 plasmid ColIb-P9 was found to carry a single-stranded DNA-binding (SSB) protein gene (ssb) that maps about 11 kilobase pairs from the origin of transfer in the region transferred early during bacterial conjugation . The cloned gene was able to suppress the UV and temperature sensitivity of an ssb-1 strain of Escherichia coli K-12 . The nucleotide sequence of the ColIb ssb gene was determined, giving a predicted molecular weight of 19,110 for the SSB protein . Sequence data show that ColIb ssb is very similar to the ssb gene on plasmid F, which is also known to map in the leader region . High-level expression of ssb on ColIb required derepression of the transfer (tra) genes and the activity of the positive regulatory system controlling these genes, suggesting that the SSB protein contributes to the conjugative processing of DNA . A mutant of ColIbdrd-1 carrying a Tn903-derived insertion in ssb was constructed, but it was unaffected in the ability to generate plasmid transconjugants and it was maintained apparently stably in donor cells both following mating and during vegetative growth . Hence, no biological role of ColIb SSB protein was detected . However, unlike the parental plasmid, such ColIb ssb mutants conferred a marked Psi+ (plasmid-mediated SOS inhibition) phenotype on recA441 and recA730 strains, implying a functional relationship between SSB and Psi proteins. J Bacteriol, 1989 May, 171(5), 2458 - 65 Localization and assembly into the Escherichia coli envelope of a protein required for entry of colicin A; Bourdineaud JP et al.; Mutations in tolQ, previously designated fii, render cells tolerant to high concentrations of colicin A . In addition, a short deletion in the amino-terminal region of colicin A (amino acid residues 16 to 29) prevents its lethal action, although this protein can still bind the receptor and forms channels in planar lipid bilayers in vitro . These defects in translocation across the outer membrane in the tolQ cells or the colicin A mutant cannot be bypassed by osmotic shock . The TolQ protein, which is constitutively expressed at a low level, was studied in recombinant plasmid constructs allowing the expression of various TolQ fusion proteins under the control of the inducible caa promoter . The TolQ protein was thus "tagged" with an epitope from the colicin A protein for which a monoclonal antibody is available . A fusion protein containing the entire TolQ protein plus the 30 N-terminal residues of colicin A was shown to complement the tolQ mutation . Pulse-chase labeling followed by gradient fractionation indicated that the bulk of the overproduced fusion protein was rapidly incorporated into the inner membrane, with small amounts localized to regions corresponding to the attachment sites between inner and outer membranes and to the outer membrane itself . However, most of the protein was rapidly degraded, leaving only that localized to the attachment sites and the outer membrane remaining at very late times of chase. J Bacteriol, 1989 May, 171(5), 2415 - 23 New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis; Dutreix M et al.; To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication . By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations . A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination . The mutation rendered the host not mutable by UV, even in a lexA(Def) background . Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD . Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein . Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination . The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination . Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage . Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates . The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+ . With respect to other RecA functions, recA1730 was recessive to recA+ . This demonstrates that RecA protein has an additional role in mutagenesis beside mediating the cleavage of LexA and UmuD proteins. J Bacteriol, 1989 May, 171(5), 2353 - 60 Control of the ccd operon in plasmid F; Tam JE et al.; The F sex factor plasmid of Escherichia coli contains a pair of genes, ccdA and ccdB, whose protein gene products are involved in an unusual feature of plasmid maintenance . The CcdB protein is a cytotoxin that becomes activated when the F plasmid is lost, thereby killing the F- segregant cells . In F+ cells, the CcdA protein protects against the lethal effects of CcdB . In the present study we show that ccdA and ccdB expressions are negatively autoregulated at the level of transcription . Genetic studies showed that repression required at least ccdB; ccdA alone was without effect, and ccdB alone was not examined because it is lethal . Ccd-operator complexes were purified and contained a mixture of both CcdA and CcdB proteins; however, we could not conclude from our results whether CcdA was necessary for DNA binding or autorepression . By using restriction fragments of the promoter-operator region, we obtained results indicating that at least two DNA-binding sites existed for the Ccd protein(s) . Subsequent footprinting of the binding sites showed protection over about a 113-base-pair region encompassing the putative promoter-operator and the beginning of the ccdA gene. J Bacteriol, 1989 May, 171(5), 2337 - 46 Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism; Bukau B et al.; DnaK is a major heat shock protein of Escherichia coli and has been previously reported to be essential for growth at high temperatures . We systematically investigated the role of DnaK in cellular metabolism at a wide range of growth temperatures by analyzing cellular defects caused by deletion of the dnaK gene (delta dnaK52) . At intermediate temperatures (30 degrees C), introduction of the delta dnaK52 allele into wild-type cells caused severe defects in cell division, slow growth, and poor viability of the cells . delta dnaK52 mutants were genetically unstable at 30 degrees C and frequently acquired secondary mutations . At high (42 degrees C) and low (11 and 16 degrees C) temperatures the delta dnaK52 allele could only be introduced into the subpopulation of wild-type cells that had duplicated the dnaK region of their chromosome . delta dnaK52 mutants isolated at 30 degrees C were cold sensitive as well as temperature sensitive for growth . Cell division defects of delta dnaK52 mutants at 30 degrees C were largely suppressed by overproduction of the FtsZ protein, which is normally required for septation during cell division; however, slow growth and poor viability at 30 degrees C and cold sensitivity and temperature sensitivity of growth were not suppressed, indicating that delta dnaK52 mutants had additional defective cellular functions besides cell division. J Bacteriol, 1989 May, 171(5), 2303 - 11 Analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide; Puziss JW et al.; Oligonucleotide-directed mutagenesis was employed to investigate the role of the hydrophilic segment of the Escherichia coli maltose-binding protein (MBP) signal peptide in the protein export process . The three basic residues residing at the amino terminus of the signal peptide were systematically substituted with neutral or acidic residues, decreasing the net charge in a stepwise fashion from +3 to -3 . It was found that a net positive charge was not absolutely required for MBP export to the periplasm . However, export was most rapid and efficient when the signal peptide retained at least a single basic residue and a net charge of +1 . The nature of the adjacent hydrophobic core helped to determine the effect of charge changes in the hydrophilic segment on MBP export, which suggested that these two regions of the signal peptide do not have totally distinct functions . Although the stepwise decrease in net charge of the signal peptide also resulted in a progressive decrease in the level of MBP synthesis, the data do not readily support a model in which MBP synthesis and export are obligately coupled events . The export defect resulting from alterations in the hydrophilic segment was partially suppressed in strains harboring certain prl alleles but not in strains harboring prlA alleles that are highly efficient suppressors of signal sequence mutations that alter the hydrophobic core. Health Phys, 1989 May, 56(5), 691 - 704 Photobiology of low-power laser effects; Karu T; Quantitative studies have been performed to determine the action of low-intensity visible monochromatic light on various cells (E . coli, yeasts, HeLa, Chinese hamster fibroblasts and human lymphocytes); also irradiation conditions (wavelength, dose and intensity) conducive to vital activity stimulation have been examined . Respiratory chain components are discussed as primary photoacceptors . The possible ways for photosignal transduction and amplification are discussed . It is proposed that enhanced wound healing due to irradiation with low-intensity visible laser light (He-Cd, He-Ne and semiconductor lasers) is due to the increasing proliferation of cells. Carcinogenesis, 1989 May, 10(5), 949 - 52 The complexity of nitrosoguanidine mutagenesis increases with size: observations of the mutational specificity of N-propyl-N'-nitro-N-nitrosoguanidine; van der Vliet GM et al.; The mutational specificity of the monofunctional alkylating agent N-propyl-N'-nitro-N-nitrosoguanidine (PNNG) has been determined through the DNA sequence characterization of 109 LacI- mutations of Escherichia coli . The predominant mutation induced was the G:C----A:T transition (73%), presumably the result of O6-propylguanine damage . Transversions constituted 18% of the mutants, almost entirely due to G:C----T:A (9%) and A:T----C:G (8%) events . Two identical deletions, one single base pair frameshift and a tandem double base change were also recovered . In contrast, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was previously found to induce only transitions, again predominantly G:C----A:T events (98%) . Moreover, the site specificity observed for PNNG-induced G:C----A:T transitions is quite distinct from that induced by MNNG . G:C----A:T transitions recovered following PNNG treatment do not appear to be influenced by neighbouring base sequence to the extent seen for MNNG. Carcinogenesis, 1989 May, 10(5), 827 - 32 Detection of DNA adducts by high-performance liquid chromatography with electrochemical detection; Park JW et al.; A method for the detection of rare adducts in DNA has been developed by combining the resolution of high-performance liquid chromatography with the specificity and sensitivity of electrochemical detection . Many adducts are electrochemically active, while the normal bases, except for guanine, are not . Enzymatic hydrolysis of DNA is used to obtain the deoxynucleosides for analysis, or where appropriate, acid hydrolysis or thermal depurination of DNA is used to free the adduct base for analysis . Various types of DNA damage have been induced by in vitro exposure of DNA to acrolein, dimethyl sulfate, sodium nitrite, ascorbate/Cu2+ and gamma-irradiation . Several adducts are detected at a level of one adduct in 10(5)-10(6) normal bases in micrograms of DNA . The method is also useful for measuring O6-methylguanine (O6MeGua) in DNA from rats treated with N-nitrosodimethylamine and 8-hydroxydeoxyguanosine (oh8dG), and O6-MeGua in DNA from bacteria treated with hydrogen peroxide and dimethyl sulfate . oh8dG, which appears to be the most suitable marker for measuring the steady-state level of oxidative DNA damage, can be measured at fmol levels in DNA from normal rat tissues . The method is applicable to the analysis of DNA base damage caused by major endogenous processes relevant to aging, such as deamination, methylation and oxidation . The analysis of DNA adducts with this simple assay also may be potentially useful for studies on carcinogenesis and as a tool in studies on the epidemiology of cancer. Carcinogenesis, 1989 May, 10(5), 817 - 22 Mutational specificities of environmental carcinogens in the lacI gene of Escherichia coli . I . The direct-acting analogue N-nitroso-N-methyl-N-alpha-acetoxymethylamine; Horsfall MJ et al.; The mutational specificity of N-nitroso-N-methyl-N-alpha-acetoxymethylamine in Escherichia coli has been determined through the DNA sequence characterization of 190 forward mutations in the lacI gene . Consistent with the methylating ability of this compound and the predicted mutagenic potential of O6-methylguanine damage, the predominant mutation was the G:C----A:T transition . An analysis of the neighbouring template revealed a similar 5' flanking sequence influence on G:C----A:T site mutability reported for other direct-acting SN1 alkylating agents . However, a dose-dependency was observed . The preference for transition at guanine residues flanked (5') by a purine over those preceded by a pyrimidine decreased from a ratio of 19:1 (upon 1 mM treatment) to 6:1 (upon 10 mM treatment) and 4:1 (upon 30 mM treatment) . Two double G:C----A:T transition mutants were characterized . In addition, this nitrosamine appears to be relatively more efficient at producing other kinds of mutations . In total 19 non-G:C----A:T mutations were identified . These included: A:T----G:C transitions, transversions and frameshifts . The relative contribution of these events was found also to decrease with increasing dose . These results may help explain why the parent carcinogen N-nitroso-N,N-dimethylamine is hepatotropic while other methylating carcinogens, similarly metabolized in the liver, are not. Pol Tyg Lek, 1989 May 1-8, 44(18-19), 430 - 2 {Ability of granulocytes to reduce nitroblue tetrazolium in uremic patients treated conservatively}; Golec K et al.; NTB reduction test, both spontaneous and stimulated with E . coli endotoxin, was performed in peripheral blood granulocytes of 40 individuals of both sexes aged between 18 and 64 years treated conservatively at the nephrologic outpatient clinic . Serum creatinine, urea and uric acid were assayed at the same time . A control group included 40 healthy individuals of both sexes aged between 20 and 56 years . Statistically significant increase in spontaneous reduction of NTB was achieved in the group of the uremic patients in comparison with the control group . Moderately positive correlation between creatinine level and percentage of NTB-positive cells in the spontaneous test was shown . Possibility of granulocyte stimulation by uremic toxins is being considered. J Gen Microbiol, 1989 May, 135 ( Pt 5), 1135 - 44 Characterization of a putative colonization factor (PCFO166) of enterotoxigenic Escherichia coli of serogroup O166; McConnell MM et al.; Enterotoxigenic Escherichia coli (ETEC) of serogroup O166 gave mannose-resistant haemagglutination (MRHA) with bovine and human erythrocytes . The strains did not react with antisera prepared against the known colonization factors CFA/I, CFA/II, CFA/III, CFA/IV and PCFO159:H4 . Strain E7476 of serotype O166:H27, which produced heat-stable enterotoxin (ST), was examined initially . It produced fimbriae about 7 nm in diameter . On SDS-PAGE two possible fimbrial polypeptides of molecular mass 15.5 and 17.0 kDa were seen . When variants of strain E7476 were isolated, loss of ST and MRHA together was associated with loss of a 98 MDa plasmid, while loss of ST alone correlated with plasmid deletion . An absorbed anti-strain E7476 antiserum reacted specifically with the 15.5 and 17.0 kDa polypeptides in Western immunoblotting and bound to the intact fimbriae by immuno-electron microscopy . When this antiserum was used in an ELISA to examine other strains of serogroup O166, a positive reaction was obtained with all the ST- and MRHA-positive strains . One strain of serotype O71:H27 and two strains of serotype O98:H- also reacted with the absorbed anti-strain E7476 antiserum . The antiserum did not react with ETEC carrying known colonization factors . E . coli K12 and a number of E . coli of different serotypes carrying a plasmid coding for ST transferred from strain E7476, all gave MRHA and reacted with the absorbed anti-strain E7476 antiserum . The term putative colonization factor O166 (PCFO166) is proposed to describe the adhesive factor(s) on ETEC of serogroup O166 because of the similarity of properties with those of known colonization factors. J Gen Microbiol, 1989 May, 135 ( Pt 5), 1123 - 34 Properties of adherence factor plasmids of enteropathogenic Escherichia coli and the effect of host strain on expression of adherence to HEp-2 cells; McConnell MM et al.; EPEC adherence factor (EAF) plasmids from three strains of enteropathogenic Escherichia coli (EPEC) - E2347/69 (O127:H6), E20517 (O111:H2) and E24582 (O142:H6) - were examined . The EAF plasmids were all marked with ampicillin resistance by transposition of Tn801 to give pDEP1, pDEP2 and pDEP11, respectively . All three plasmids showed incompatibility with an FIme and an FIV plasmid and had some similarity in restriction enzyme digest patterns . Plasmid pDEP1 differed from pDEP2 and pDEP11 in being autotransferring and fertility-inhibition positive . An EAF probe consisting of a 1 kb BamHI-SalI restriction endonuclease fragment of the prototype EAF-associated plasmid pMAR2 hybridized to similar-sized SalI-BamHI fragments of pDEP1 and pDEP11 but to a different-sized fragment of plasmid pDEP2 . Loss of the EAF plasmids from EPEC strains resulted in a marked reduction in the ability of these strains to adhere to HEp-2 cells . The EAF-plasmid-negative variants did not express a 94 kDa outer-membrane protein (OMP) . When these EAF plasmids were reintroduced into EAF-plasmid-negative EPEC strains a high level of adherence equivalent to that of the parent EPEC strains was restored and a 94 kDa OMP was usually expressed . However, when EAF plasmids were transferred into E . coli K12 or non-EPEC E . coli the host strains either did not adhere or adhered poorly to the HEp-2 cells . These transconjugants did not express a 94 kDa OMP. FEMS Microbiol Lett, 1989 May, 50(1-2), 87 - 91 Some strains of Escherichia coli of putative enteroadherent-aggregative serotypes produce an unusual fibrillar haemagglutinin; Old DC et al.; Two strains of Escherichia coli that formed on unusual kind of mannose-resistant and eluting haemagglutinin (MREHA) reacting with the red blood cells of rat and mouse, when cultured at 37 degrees C but not at 18 degrees C, were examined by electron microscopy . Production of this rare rodent-positive MREHA was correlated with the presence of fine fibrillae of estimated diameter 2.5 nm that were demonstrated by negative staining and immuno-gold labelling with MREHA-specific anti-serum . These two strains belonged to serotypes 078:H- and 078:H33; thus, it would be useful to know whether enteroadherent-aggregative strains of E . coli of these and other serotypes also possess this unusual MREHA. FEMS Microbiol Lett, 1989 May, 50(1-2), 21 - 5 Cloning and expression of Thiobacillus versutus aspartate-semialdehyde dehydrogenase gene in Escherichia coli; Jagusztyn-Krynicka EK et al.; The Thiobacillus versutus asd gene coding for aspartate-semialdehyde dehydrogenase was cloned in Escherichia coli cells using pBR322 as a vector . The gene was expressed independently of its orientation, suggesting that E . coli RNA polymerase recognized T . versutus promoter sequence . The T . versutus DNA coded protein, of the molecular weight 44,000, was identified by the analysis of the proteins produced by minicells. APMIS, 1989 May, 97(5), 436 - 40 Mechanism of spontaneous loss of heat-stable toxin (STa) production in enterotoxigenic Escherichia coli; Sommerfelt H et al.; Five strains of enterotoxigenic Escherichia coli (ETEC) showing spontaneous loss of heat-stable enterotoxin (STa) production were studied to elucidate the underlying genetic mechanisms . Southern blot analysis revealed that loss of STa production, and the corresponding lack of hybridization with the STa gene probes, were associated with deletions of DNA fragments harboring the relevant toxin genes rather than with loss of plasmids. Proc Natl Acad Sci U S A, 1989 May, 86(9), 3094 - 8 Changes in stability and allosteric properties of aspartate transcarbamoylase resulting from amino acid substitutions in the zinc-binding domain of the regulatory chains; Eisenstein E et al.; Changes in subunit interaction energies linked to the allosteric transition of the regulatory enzyme aspartate transcarbamoylase (ATCase; EC 2.1.3.2) from Escherichia coli are localized in part at interfaces between the six catalytic (c) and six regulatory (r) polypeptide chains . Site-directed mutagenesis has been used to construct enzymes with amino acid substitutions in a limited region of the zinc-binding domain of the r chains . Substitution of Ser or His for r114 Cys, one of four cysteines binding the structural zinc ion in the regulatory chain, leads to incorrectly folded chains as shown by the inability to detect stable assembled holoenzyme in cell extracts . Replacement of r111 Asn by Ala at the interface between an r chain and a c chain in the apposing catalytic trimer causes a complete loss of the homotropic and heterotropic effects characteristic of wild-type ATCase . Moreover, sedimentation velocity experiments demonstrated that this mutant enzyme exists in the R ("relaxed") conformation in the absence of active site ligands due to preferential destabilization of the T ("taut") conformation relative to the R state . In contrast, replacement of r113 Asn by Ala at the interface between adjacent r and c chains leads to an increase in the cooperativity of the enzyme . When r139 Lys is replaced by Met, Vmax is reduced by 50% compared to wild-type ATCase, whereas it is increased about 2-fold when r142 Glu is replaced by Asp . Amino acid substitutions in this domain significantly affect subunit interaction energy as measured by rate of subunit exchange when holoenzymes are incubated with isolated catalytic subunits, thus permitting measurements of the effect of the bisubstrate analog N-(phosphonacetyl)-L-asparatate in weakening intersubunit interactions . Subunit exchange increased about 9-fold for the r142 Glu----Asp mutant and almost 20-fold for the r142 Glu----Ala mutant in the presence of the ligand. Int J Radiat Biol, 1989 May, 55(5), 739 - 45 Post-ultraviolet DNA synthesis in the absence of repair: role of the single-strand DNA-binding protein; Trgovcevic Z et al.; Post-ultraviolet DNA synthesis kinetics were investigated in the Escherichia coli uvrA recA strain and its isogenic counterpart, overproducing single-strand DNA-binding protein (SSB) . It was demonstrated that large quantities of SSB enhance the capacity of the unmodified replisome to use the UV-damaged template for DNA synthesis . DNA thus synthesized is of low molecular weight, as shown by sedimentation in alkaline sucrose gradients . It is therefore suggested that SSB actively participates in the replisome translocation past dimers and/or the initiation of new DNA chains downstream of these lesions. J Bacteriol, 1989 May, 171(5), 2889 - 93 Differentiation of arcA, arcB, and cpxA mutant phenotypes of Escherichia coli by sex pilus formation and enzyme regulation; Iuchi S et al.; In Escherichia coli, mutations in arcA (dye) or arcB anaerobically derepress the synthesis of a multitude of enzymes of aerobic function, and mutations in arcA or cpxA impair F-pilus formation . It is thought that arcA encodes a promoter-recognizing protein, whereas arcB and cpxA encode sensor proteins which interact with the arcA product . In this study we found that anaerobic growth of a wild-type F' strain decreased the synthesis of both the enzymes and the pilus . Although the two arcA mutants examined were both anaerobically derepressed in the enzymes and impaired in aerobic pilus formation as expected, one mutant hyperproduced the pilus anaerobically . The two arcB mutants examined showed normal pilus formation when grown aerobically . When grown anaerobically they developed more pili than the wild-type strain did when grown aerobically . When a cpxA mutant was examined for synthesis of two aerobic enzymes, normal regulation was found . The available data suggest the following . The arcA product anaerobically represses certain genes of aerobic function and activates certain genes related to F function . It appears that the arcB product senses the redox or energy state; absence of the gene function shifts the arcA product to the nonrepressive form for enzyme synthesis for aerobic pathways . The cpxA product, on the other hand, senses the sexual state; absence of the gene function shifts the arcA product to the inactive form for F-pilus synthesis. Infect Immun, 1989 May, 57(5), 1604 - 11 Distribution of the P-associated-pilus (pap) region among Escherichia coli from natural sources: evidence for horizontal gene transfer; Plos K et al.; Variation in chromosomal DNA in Escherichia coli was studied with probes specific for the P-associated-pilus (pap) region . The presence of DNA homologous to pap was determined by dot blots . Variation in the number of copies of pap and in the organization of internal and flanking sequences was determined by Southern blot hybridization . The 229 strains studied were also classified by O:K:H serotyping and multilocus enzyme electrophoresis . There was considerable heterogeneity in the presence of pap and distribution of pap-homologous DNA in these E . coli strains from natural sources . In general, there was less variation in pap among strains of the same specific O:K:H serotype and enzyme electrophoretic type than among random isolates . There were, however, E . coli strains identified as members of the same clone by O:K:H serotyping and enzyme electrophoresis that were pap positive and pap negative or had different Southern blot patterns for the pap probes (pap type) . There were also isolates of the same pap type that differed in two of three O:K:H serotype antigens and the majority of enzymes that determined their enzyme electrophoretic type . These latter two observations were interpreted as evidence for the horizontal (infectious) transfer of the pap-homologous sequences among clones of E . coli. Infect Immun, 1989 May, 57(5), 1506 - 11 Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli; Benz I et al.; The adherence of enteropathogenic Escherichia coli (EPEC) to the small bowel mucosa is an important step in the pathogenesis of diarrheal diseases . It has been shown that many EPEC strains adhere to HEp-2 and especially HeLa cells in characteristic patterns termed localized adherence (LA) and diffuse adherence (DA) . A plasmid-derived DNA fragment encoding a factor specific for LA hybridized only to EPEC strains expressing LA, which demonstrated that LA and DA are mediated by two genetically distinct adhesins . EPEC strain 2787 (O127:H27), isolated from a case of infantile diarrhea, exhibited three major properties: (i) it showed DA to HeLa cells, (ii) it carried two large (ca . 100-kilobase {kb}) plasmids and one small plasmid of about 3 kb, and (iii) no fimbriae could be detected by electron microscopy in organisms grown on agar plates or in liquid cultures . Whole isolated plasmid DNA was partially digested with EcoRI and cloned into the vector pBR322 . One recombinant clone (pIB6) was found to exhibit the same DA pattern on HeLa cells as did the parent strain . This clone contained an 11-kb DNA fragment derived from the largest of the three plasmids, as shown by Southern hybridization . By deletion analysis, a 6.0-kb DNA fragment was shown to be sufficient for expression of the DA phenotype . This insert encoded the production of a 100,000-dalton protein mediating adhesion to HeLa cells. J Gen Microbiol, 1989 May, 135 ( Pt 5), 1263 - 73 Role of guanosine kinase in the utilization of guanosine for nucleotide synthesis in Escherichia coli; Hove-Jensen B et al.; Using purine auxotrophic strains of Escherichia coli with additional genetic lesions in the pathways of interconversion and salvage of purine compounds, we demonstrated the in vivo function of guanosine kinase and inosine kinase . Mutants with increased ability to utilize guanosine were isolated by plating cells on medium with guanosine as the sole purine source . These mutants had altered guanosine kinase activity and the mutations were mapped in the gene encoding guanosine kinase, gsk . Some of the mutants had acquired an additional genetic lesion in the purine de novo biosynthetic pathway, namely a purF, a purL or a purM mutation . A revised map location of the gsk gene is presented and the gene order established as proC-acrA-apt-adk-gsk-purE. Plasmid, 1989 May, 21(3), 195 - 204 The EcoDXX1 restriction and modification system: cloning the genes and homology to type I restriction and modification systems; Skrzypek E et al.; The Escherichia coli plasmid pDXX1 codes for a type I restriction and modification system, EcoDXX1 . A 15.5-kb BamHI fragment from pDXX1 has been cloned and contains the hsdR, hsdM, and hsdS genes that encode the EcoDXX1 system . The EcoDXX1 hsd genes can complement the gene products of the EcoR124 and EcoR124/3 hsd systems, but not those of EcoK and EcoB . Hybridization experiments using EcoDXX1 hsd genes as a probe demonstrate homology between EcoDXX1 and EcoR124 and EcoR124/3 restriction-modification systems, but weak or no homology between EcoDXX1 and EcoK or EcoB systems. Mol Biol (Mosk), 1989 May-Jun, 23(3), 862 - 71 {Analogs of pyrophosphate in a pyrophosphorolysis reaction catalyzed by DNA polymerases}; Rozovskaia TA et al.; The reaction of pyrophosphorolysis catalyzed by Escherichia coli DNA polymerase I Klenov fragment, calf thymus DNA polymerase alpha, rat liver DNA polymerase beta and AMV reverse transcriptase was studied . Some pyrophosphate (PPi) analogs were taken as low molecular weight substrates . It was shown that only imidodiphosphonic acid acted as the PPi substrate analog for the reactions catalyzed by DNA polymerases I and alpha, both imidodiphosphonic acid and methylenediphosphonic acid were active in the case of DNA polymerase beta and reverse transcriptase . Other analogs tested were neither nucleotide residue acceptors, nor inhibitors of the pyrophosphorolysis reaction with PPi . The abilities of some PPi analogs to inhibit the DNA elongation catalyzed by reverse transcriptase were investigated . The principles of specificity of low molecular substrates recognition by DNA polymerases and some problems concerning the mechanisms of DNA synthesis inhibition by PPi analogues are discussed. Mol Gen Genet, 1989 May, 217(1), 85 - 96 The Escherichia coli dam gene is expressed as a distal gene of a new operon; Jonczyk P et al.; DNA containing the Escherichia coli dam gene and sequences upstream from this gene were cloned from the Clarke-Carbon plasmids pLC29-47 and pLC13-42 . Promoter activity was localized using pKO expression vectors and galactokinase assays to two regions, one 1650-2100 bp and the other beyond 2400 bp upstream of the dam gene . No promoter activity was detected immediately in front of this gene; plasmid pDam118, from which the nucleotide sequence of the dam gene was determined, is shown to contain the pBR322 promoter for the primer RNA from the pBR322 rep region present on a 76 bp Sau3A fragment inserted upstream of the dam gene in the correct orientation for dam expression . The nucleotide sequence upstream of dam has been determined . An open reading frame (ORF) is present between the nearest promoter region and the dam gene . Codon usage and base frequency analysis indicate that this is expressed as a protein of predicted size 46 kDa . A protein of size close to 46 kDa is expressed from this region, detected using minicell analysis . No function has been determined for this protein, and no significant homology exist between it and sequences in the PIR protein or GenBank DNA databases . This unidentified reading frame (URF) is termed urf-74.3, since it is an URF located at 74.3 min on the E . coli chromosome . Sequence comparisons between the regions upstream of urf-74.3 and the aroB gene show that the aroB gene is located immediately upstream of urf-74.3, and that the promoter activity nearest to dam is found within the aroB structural gene . This activity is relatively weak (about 15% of that of the E . coli gal operon promoter) . The promoter activity detected beyond 2400 bp upstream of dam is likely to be that of the aroB gene, and is 3 to 4 times stronger than that found within the aroB gene . Three potential DnaA binding sites, each with homology of 8 of 9 bp, are present, two in the aroB promoter region and one just upstream of the dam gene . Expression through the site adjacent to the dam gene is enhanced 2- to 4-fold in dnaA mutants at 38 degrees C . Restriction site comparisons map these regions precisely on the Clarke-Carbon plasmids pLC13-42 and pLC29-47, and show that the E . coli ponA (mrcA) gene resides about 6 kb upstream of aroB. Mol Gen Genet, 1989 May, 217(1), 26 - 30 Cloning of a gene involved in cellulose biosynthesis in Acetobacter xylinum: complementation of cellulose-negative mutants by the UDPG pyrophosphorylase structural gene; Valla S et al.; Three cellulose-negative (Cel-) mutants of Acetobacter xylinum strain ATCC 23768 were complemented by a cloned 2.8 kb DNA fragment from the wild type . Biochemical analysis of the mutants showed that they were deficient in the enzyme uridine 5'-diphosphoglucose (UDPG) pyrophosphorylase . The analysis also showed that the mutants could synthesize beta(1-4)-glucan in vitro from UDPG, but not in vivo from glucose . This result was expected, since UDPG is known to be the precursor for cellulose synthesis in A . xylinum . In order to analyze the function of the cloned gene in more detail, its biological activity in Escherichia coli was studied . These experiments showed that the cloned fragment could be used to complement an E . coli mutant deficient in the structural gene for UDPG pyrophosphorylase . It is therefore clear that the cloned fragment must contain this gene from A . xylinum . This is to our knowledge the first example of the cloning of a gene with a known function in cellulose biosynthesis from any organism, and we suggest the gene be designated celA. Mol Gen Genet, 1989 May, 217(1), 178 - 81 Escherichia coli parA is an allele of the gyrB gene; Kato J et al.; A thermosensitive (ts) parA mutant, MFT110, of Escherichia coli carried at least two ts mutations . The major ts defect, resulting from a mutation mapped originally at 95 min and complemented by pLC8-47, was most probably due to psd . A plasmid carrying the 1.6 kb BamHI-PvuII fragment recloned from pLC8-47 complemented the major ts mutation in MFT110 and psd(ts) in two mutants, but did not correct the Par phenotype of MFT110 . The second ts mutation was salt-repairable and mapped at 83 min close to recF and tnaA . This mutation was linked with the Par phenotype as shown unambiguously by 4',6-diamidino-2-phenylindole stained nucleoids in parA mutant cells with the W3110 genetic background . Both salt-repairable ts and Par traits were corrected concomitantly by a plasmid carrying the chromosomal region solely for the gyrB gene . This strongly suggests that parA is an allele of gyrB. Mol Gen Genet, 1989 May, 217(1), 126 - 31 Isolation and characterization of the phosphoglucose isomerase gene from Escherichia coli; Froman BE et al.; The nucleotide sequence of the gene encoding the glycolytic enzyme phosphoglucose isomerase (PGI) from Escherichia coli is presented . The gene encodes a polypeptide of 549 amino acids . The transcriptional start point of the gene was determined and found to lie within a consensus promoter region . The amino acid sequence derived from the E . coli PGI gene can be aligned without insertions or deletions to the predicted amino acid sequence of a nuclear-encoded chloroplast isozyme of PGI from a higher plant, and the two sequences have a similarity of 87.6% . The amino acid sequence similarity between E . coli and that predicted from cDNA sequences for mouse and pig PGI is approximately 65%. EMBO J, 1989 May, 8(5), 1609 - 13 Directionality of DnaA protein/DNA interaction . Active orientation of the DnaA protein/dnaA box complex in transcription termination; Schaefer C et al.; The complex of DnaA protein with its 9 bp consensus binding site, the dnaA box 5'-TT(A/T)T(A/C)CA(A/C)A, blocks transcribing RNA polymerase . In a model system, the rate of transcription was monitored distal to the dnaA box 5'-TTTTCCACA by the expression of a reporter gene . DnaA-dependent transcription termination occurred irrespective of whether the dnaA box region was or was not translated . Only the dnaA box orientation 5'-TTTTCCACA on the non-coding strand, but not the reverse orientation, was active in termination . This suggests that DnaA protein contacts only one strand of the DNA duplex . Oligonucleotide-directed mutation of a dnaA box present within the dnaA coding region resulted in increased expression of dnaA . This demonstrates that DnaA protein-directed transcription termination is an element of the autoregulation of the dnaA gene. Electrophoresis, 1989 May-Jun, 10(5-6), 360 - 5 Studies of DNA-protein interactions by gel electrophoresis; Ceglarek JA et al.; The use of gel electrophoresis in studies of nucleic acid-protein (especially DNA-protein) interactions has yielded much qualitative and quantitative information about a variety of such systems . The reduction in mobility of complexes relative to free DNA allows isolation and characterization of the complexes as well as determination of thermodynamic and kinetic properties of the interactions . This article begins with a review of recent applications of the "gel retardation" assay, by way of introduction to experiments in two areas . In the first, a hypothesis is tested regarding whether a DNA molecule with sizable proteins bound very near to each end migrates through a polyacrylamide gel differently than does the corresponding complex having the proteins in the middle of the DNA fragment . The data show little mobility differences for these types of complexes, implying that both may move in a linear, "snakelike", manner through the gel . The experiments also provide results pertaining to questions of DNA bending caused by the binding of the E . coli catabolite activator protein (CAP) and RNA polymerase to the lactose promoter region . It appears that DNA bending by CAP at its wild type lac binding site is retained in complexes where RNA polymerase is bound simultaneously at the lac UV5 promoter. Appl Environ Microbiol, 1989 May, 55(5), 1106 - 11 Solubilization and renaturation of overexpressed aggregates of mutant tryptophan synthase alpha-subunits; Lim WK et al.; Certain Escherichia coli tryptophan synthase mutant alpha-subunits encoded from mutagenized trpA-containing plasmids were overexpressed as insoluble aggregates which were seen as large, intracellular inclusion bodies . The insoluble aggregates were solubilized to various degrees by several neutral, chaotropic salts . The order of effectiveness of these salts (KSCN, NaI greater than NaNO3, LiBr greater than CaCl2) followed that for the Hofmeister series . Optimum conditions for the use of KSCN resulted in a maximum 70 to 75% solubilization of the aggregate forms for all mutant alpha-subunits examined . Removal of KSCN by dialysis resulted in the recovery of biological activity and of certain characteristic structural properties . Such salts may be a useful alternative for other recombinant protein aggregates which resist complete renaturation by commonly used treatments with guanidine or urea. Antimicrob Agents Chemother, 1989 May, 33(5), 746 - 50 Organization of complex transposon Tn2610 carrying two copies of tnpA and tnpR; Yamamoto T; Transposon Tn2610 has two elements of 3.5 kilobase pairs as inverted repeats, one set at each end . This unique terminal element contained the transposition genes tnpA and tnpR . Only the tnpA gene in the left element was functional for transposition, whereas both tnpR genes were active . Possible evolutionary relationships among class II transposable elements are proposed on the basis of the genetic and structural organization of Tn2610. Genetika, 1989 May, 25(5), 784 - 98 {Interaction of products of su(Hw) and su(f) genes with MDG4 region regulating its transcription suppresses mutations in Drosophila caused by insertion of this gene}; Mizrokhi LIu et al.; The mdg4 (gypsy) mobile element of Drosophila contains two closely situated regions binding to proteins from nuclear extracts . One of these is an imperfect palindrome having homology with the lac operator of Escherichia coli, the other contains a reiterated sequence (5'PyPuTCTGCATACTPyPy) homologous to the octamer which is the core of many enhancers and upstream promoter elements . The transient expression of deletion mutants has shown that these DNA regions are negative and positive regulators, respectively, of mdg4 transcription . As was demonstrated earlier, mutations induced by the presence of mdg4 at different loci are suppressed, owing to either repression or activation of mdg4 transcription in Drosophila lines carrying unlinked mutations in su(Hw) or su(f) genes . We have shown that binding to a negative regulator (silencer) is weakened in nuclear extracts isolated from cell lines carrying su(f) mutations which activate mdg4 transcription; therefore, the su(f) gene codes for a protein capable of mdg4 repression . Furthermore, binding to a positive regulator is weakened in nuclear extracts isolated from cell lines carrying su(Hw) gene mutations which decrease the level of mdg4 transcription; hence, the su(Hw) gene encodes a protein which activates mdg4 transcription. Biochem J, 1989 May 1, 259(3), 693 - 700 X-ray-absorption and electron-paramagnetic-resonance spectroscopic studies of the environment of molybdenum in high-pH and low-pH forms of Escherichia coli nitrate reductase; George GN et al.; Previous e.p.r . work {George, Bray, Morpeth & Boxer (1985) Biochem . J . 227, 925-931} has provided evidence for a pH- and anion-dependent transition in the structure of the Mo(V) centre of Escherichia coli nitrate reductase, with the low-pH form bearing both an anion and probably a hydroxy-group ligand . Initial e.x.a.f.s . measurements {Cramer, Solomonson, Adams & Mortenson (1984) J . Am . Chem . Soc . 106, 1467-1471} demonstrated the presence of sulphur (or chloride) ligands in the Mo(IV) and Mo(VI) oxidation states, as well as a variable number of terminal oxo (Mo = O) groups . To synthesize the e.p.r . and e.x.a.f.s . results better, we have conducted new e.p.r . experiments and complementary e.x.a.f.s . measurements under redox and buffer conditions designed to give homogeneous molybdenum species . In contrast with results on other molybdoenzymes, attempts to substitute the enzyme with 17O by dissolving in isotopically enriched water revealed only very weak hyperfine coupling to 17O . The significance of this finding is discussed . Experiments with different buffers indicated that buffer ions (e.g . Hepes) could replace the Cl- ligand in the low-pH Mo(V) enzyme form, with only a small change in e.p.r . parameters . E.x.a.f.s . studies of the oxidized and the fully reduced enzyme were consistent with the e.p.r . work in indicating a pH- and anion-dependent change in structure . However, in certain cases non-stoichiometric numbers of Mo = O interactions were determined, complicating the interpretation of the e.x.a.f.s . Uniquely for a molybdenum cofactor enzyme, a substantial proportion of the molecules in a number of enzyme samples appeared to contain no oxo groups . No evidence was found in our samples for the distant 'heavy' ligand atom reported in the previous e.x.a.f.s . study . The nature of the high-pH-low-pH transition is briefly discussed. Am J Vet Res, 1989 May, 50(5), 655 - 61 DNA homology of Brucella abortus strains 19 and 2308; Muzny DM et al.; The restriction endonuclease digestion DNA patterns from Brucella abortus strains 19 and 2308 were examined with 11 restriction enzymes (AvaI, BamHI, BglII, BstEII, DdeI, EcoRI, HindIII, KpnI, PstI, XbaI, and SalI) . The DNA electrophoretic banding patterns between the 2 strains were highly similar, using this restriction enzyme analysis . Differences were not discernable between B abortus strains 19 and 2308 in any of the restriction banding patterns examined . Methylation at CCGG or GATC sites was not detectable on the basis of digestion with isoschizomers (HpaII and MspI, and DpnI, Sau3AI and MboI) . Homology between B abortus strains 19 and 2308 was assessed, using solution-hybridization techniques followed by S1 nuclease assays . Results of these reassociation experiments indicated 98.6 to 99.3% homology between B abortus strains 19 and 2308 with 13.5 to 18.6% homology between B abortus (strains 19 and 2308) and the E coli HB101 control . We concluded that any DNA differences between the 2 B abortus strains are small and will require analysis at the DNA sequence level. Proc Natl Acad Sci U S A, 1989 May, 86(10), 3554 - 8 Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis; Charles IG et al.; Protein P.69 is localized on the outer membrane of Bordetella pertussis and is one of the virulence factors believed to contribute to the disease state of whooping cough . We demonstrate that protein synthesis of P.69 is under genetic control of the vir locus . Using oligonucleotide probes derived from the protein sequence of a cyanogen bromide fragment, we have cloned the gene for P.69 from B . pertussis CN2992 . Analysis of the DNA sequence reveals a G + C-rich gene capable of encoding a protein of 910 amino acids with a Mr of 93,478, suggesting that P.69 is a processed form of a larger precursor . In common with some of the genes in the pertussis toxin operon, the sequence CCTGG was found 5' to the ATG initiation codon . At the 3' end, 29 bases after the TAA stop codon, the sequence GTTTTTCCT was found and may have some function in transcription termination . A full-length clone of the gene for P.69 carried by the cosmid pBPI69 was unable to direct the expression of P.69 protein in an Escherichia coli host . The generation of P.69-fusion products allowed the detection of P.69-specific protein products synthesized in E . coli. Proc Natl Acad Sci U S A, 1989 May, 86(10), 3489 - 93 Vaccinia DNA topoisomerase I promotes illegitimate recombination in Escherichia coli; Shuman S; Vaccinia virus encapsidates a Mr 32,000 type IDNA topoisomerase . Although the vaccinia gene encoding the topoisomerase is essential for virus growth, the role of the enzyme in vivo remains unclear . In the present study, the physiologic consequences of vaccinia topoisomerase action have been examined in a heterologous system, Escherichia coli . The vaccinia topoisomerase gene was inducibly expressed in an int-lambda lysogen BL21(DE3) using a T7 RNA polymerase-based transcription system . Expression of active topoisomerase in this context resulted in recA-dependent lysogenic induction as well as cell lysis . Surprisingly, topoisomerase expression also effected a 200-fold increase in the titer of infectious lambda phage, apparently by promoting int-independent prophage excision . This effect was not observed during lysogenic induction with nalidixic acid . Restriction analysis of genomic DNA from plaque-purified excisants revealed (in 10 of 10 cases) gross alterations of the DNA structure around the att site relative to the structure of the parental phage DE3 . It is construed therefore that vaccinia DNA topoisomerase I acts to promote illegitimate recombination in E . coli. Virology, 1989 May, 170(1), 311 - 5 Human papillomavirus type 16 E7 protein expressed in Escherichia coli and monkey COS-1 cells: immunofluorescence detection of the nuclear E7 protein; Sato H et al.; Human papillomavirus type 16 E7 protein, expressed in Escherichia coli as fusion protein lac alpha peptide-E7 (lac-E7), was purified by electrophoresis and used to immunize rabbits to raise antisera . The anti-lac-E7 sera recognized another bacterially expressed fusion protein trpE-E7 by the Western blot method . The antisera were used to identify E7 protein transiently expressed in monkey COS-1 cells from an SV40-derived expression plasmid containing E7 gene . Like the E7 protein from CaSki cells, the majority of 19K E7 protein in the transfected COS-1 cells was found by immunoprecipitation in the soluble cytoplasmic fraction . By immunofluorescence staining, however, the E7 protein was detectable in the nuclei of the transfected COS-1 cells . The results suggest that the E7 protein expressed in monkey cells is nuclear, although it is readily released from nuclei when the cell structure is broken. Virology, 1989 May, 170(1), 302 - 6 Insertional mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase: evidence that the gene is essential for virus growth; Shuman S et al.; Vaccinia virus encodes a type I DNA topoisomerase whose function in virus replication is not known . To determine whether topoisomerase is required for growth of vaccinia in cell culture, we attempted to isolate null mutations in the topoisomerase gene through insertional mutagenesis . Plasmids containing mutant topoisomerase alleles were constructed by intragenic insertion of the Escherichia coli gpt gene . Recombinant viruses containing the gpt insertion were isolated by selection for growth in the presence of mycophenolic acid . Analysis of the genome structures of drug-resistant viruses revealed that in every case (n = 22) both the wild-type and the gpt-inserted allele were present in viral DNA . We interpret the retention of the wild-type allele as indicative of the essential nature of the topoisomerase gene for vaccinia virus growth. Proc Natl Acad Sci U S A, 1989 May, 86(9), 3271 - 5 Exonuclease III and the catalase hydroperoxidase II in Escherichia coli are both regulated by the katF gene product; Sak BD et al.; The levels of both exonuclease III (exo III, product of xthA) and hydroperoxidase II (HP-II, product of katE) activity in Escherichia coli were influenced by a functional katF gene . The katF gene product is also necessary for synthesis of HP-II . Mutations in either katF or xthA, but not katE, result in sensitivity to H2O2 and near-UV (300-400 nm) radiation . Exo III, encoded by the xthA locus, recognizes and removes nucleoside 5'-monophosphates near apurinic and apyrimidinic sites in damaged DNA . Extracts of katF mutant strains had little detectable exo III activity . When a katF+ plasmid was introduced into the katF mutant, exo III activity exceeded wild-type levels . We propose that the katF gene is a trans-acting positive regulator of exo III and HP-II enzymes, both of which are involved in cellular recovery from oxidative damage. J Bacteriol, 1989 May, 171(5), 2909 - 12 Control mechanism of the Escherichia coli K-12 cell cycle is triggered by the cyclic AMP-cyclic AMP receptor protein complex; Utsumi R et al.; The role of cyclic AMP (cAMP) in the cell cycle of Escherichia coli K-12 was studied in three mutant strains . One was KI1812, in which the cya promoter is replaced by the lacUV5 promoter . In KI1812, isopropyl-beta-D-thiogalactopyranoside induced the synthesis of cya mRNA, and at the same time cell division was inhibited and short filaments containing multiple nuclei were formed . The other strains were constructed as double mutants (NC6707 cya sulB {ftsZ(Ts)} and TR3318 crp sulB {ftsZ(Ts)}) . In both double mutants, filamentation was repressed at 42 degrees C, but it was induced again by addition of cAMP in strain NC6707 and introduction of pHA7 containing wild-type crp in TR3318 . These results indicate that lateral wall synthesis in the E . coli cell cycle is triggered by the cAMP-cAMP receptor protein complex. J Bacteriol, 1989 May, 171(5), 2689 - 96 Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature; Strauch KL et al.; The degP gene, required for proteolysis in the cell envelope of Escherichia coli, maps at approximately 3.5 min on the chromosome . Null mutations in degP result in temperature-sensitive growth . In certain genetic backgrounds, expression of abnormal periplasmic or inner membrane proteins (protein fusions or proteins with internal deletions) enhances the temperature-sensitive phenotype . Such growth defects were used as a selection for cloning the degP gene into Mud4042 and pACYC184 plasmid vectors, and a restriction map was determined . Analysis of deletion and insertion mutations on one of these plasmids showed that the degP gene is approximately 1.5 kilobases in size . The plasmid-encoded DegP protein had an apparent molecular weight of 50,000, as determined by maxicell analysis . Protein fusions between DegP and alkaline phosphatase had high alkaline phosphatase enzymatic activity, indicating that DegP is a periplasmic or membrane protein. Pathol Res Pract, 1989 May, 184(5), 489 - 93 Role of endotoxin on hypoglycaemia in acute hepatic failure . Relationship between hypoglycaemia and hepatic injury following endotoxaemia in diabetic and nondiabetic rats; Shibayama Y; To clarify the relation of endotoxaemia to hypoglycaemia and consequent death in acute hepatic failure, the interrelationship between the degree of hepatic injury, blood glucose level and mortality following endotoxaemia were examined in streptozotocin-induced diabetic and nondiabetic rats . Endotoxin hepatotoxicity was not enhanced in diabetic state . Blood glucose level was markedly reduced after endotoxin administration in the nondiabetic rats, and the reduction of blood glucose level closely correlated with the degree of hepatic injury (serum transaminase activities), namely, the more severe the hepatic injury, the lower the blood glucose level . Hypoglycaemia due to endotoxaemia occurred also in the diabetic rats when the hepatic injury was severe, but the reduction of blood glucose level was slight when it was mild . The mortality for endotoxaemia in diabetic rats was significantly lower than that in the nondiabetic rats . These experimental data suggest that endotoxaemia may play a role in the development of hypoglycaemia in acute hepatic failure, and that the diabetic state may lower the mortality for endotoxaemia. FEMS Microbiol Lett, 1989 May, 50(1-2), 15 - 9 Nucleotide sequence of the Escherichia coli entE gene; Staab JF et al.; The Escherichia coli entE gene encodes a polypeptide necessary in the latter stages of biosynthesis of the siderophore enterobactin . The entE gene and adjacent DNA were sequenced . The predicted EntE polypeptide consists of 536 amino acids and has a Mr of 58,299 and a net charge of -7.33 . Genetic evidence combined with this and previous sequencing data indicate that the genes entCEB(G)A are transcribed as unit from a promoter upstream of entC. Circ Shock, 1989 May, 28(1), 69 - 77 Protective effects of ONO 3708, a new thromboxane A2 receptor antagonist, during experimental endotoxin shock; Taneyama C et al.; The effects of ONO 3708, a new thromboxane A2 (TXA2) receptor antagonist, on platelet aggregation in human plasma, the survival rate of rats subjected to lethal endotoxin shock, and the pathophysiological consequences of endotoxin shock in anesthetized dogs were investigated . ONO 3708 inhibited dose dependently human platelet aggregation induced by 2.5 microM of STA2, analogue of TXA2 . Treatment with ONO 3708, 1 mg/100 g i.v., significantly improved the survival rate of rats in endotoxin shock from 38 to 72% at 24 hr and from 27 to 61% at 48 hr . ONO 3708 significantly attenuated endotoxin-induced thrombocytopenia, but not leukopenia . In anesthetized dogs, endotoxin-induced pulmonary hypertension was completely prevented, and increased airway pressure was significantly attenuated by ONO 3708 . These results suggest that ONO 3708, the antagonist of TXA2 receptor, has beneficial effects during endotoxin shock, at least in part by inhibiting platelet aggregation. Radiat Res, 1989 May, 118(2), 257 - 68 Immunochemical quantitation of thymine glycol in oxidized and X-irradiated DNA; Hubbard K et al.; The present study demonstrates the usefulness of immunochemical assays for quantitating modified bases in oxidized and X-irradiated DNA . Escherichia coli, phi X174 RF I, PM2, and M13 DNA containing thymine glycols introduced by OsO4 oxidation were used as antigens in a direct enzyme-linked immunosorbent assay (ELISA) . The number of thymine glycols per DNA molecule was determined by reactivity with antithymine glycol antibody standardized either to the acetol fragment assay or to the number of Escherichia coli endonuclease III-sensitive sites . The number of thymine glycols was also determined in phi X174 RF I DNA X-irradiated in either phosphate or Tris buffer under air . Using a direct ELISA with phi X174 RF I DNA irradiated in a phosphate buffer solution, the anti-thymine glycol antibody detected damage at the level of 40 Gy . The immunochemical assay was sensitive, specific, quantitative, and independent of DNA structure. Gene Anal Tech, 1989 May-Jun, 6(3), 47 - 56 Expression and characterization of a protein encoded by the human c-myc exon 1 in Escherichia coli; Psallidopoulos MC et al.; Our previously reported data from DNA sequence studies of the c-myc locus show that the human c-myc exon 1 has an open reading frame capable of encoding a protein of 188 amino acid residues . To confirm the presence of the open reading frame, we constructed a recombinant vector (pMCP60) that contains a segment of the lambda cII translational initiation region, a portion of the N-terminus of the v-mos gene, and 639 base pairs of the first exon of the human c-myc gene . pMCP60 expresses a 38 kilodalton tripartate protein (cII-mos-myc), which was purified by high-pressure liquid chromatography . The presence of myc exon 1 sequences in the cII-mos-myc fusion protein was confirmed by partial amino acid sequence analysis . These experiments further establish that the first exon of the human c-myc gene contains an open reading frame capable of expressing a protein in Escherichia coli. Proc Natl Acad Sci U S A, 1989 May, 86(9), 3085 - 8 Proofreading by the epsilon subunit of Escherichia coli DNA polymerase III increases the fidelity of calf thymus DNA polymerase alpha; Perrino FW et al.; Addition of the 3'----5' proofreading exonuclease, epsilon subunit of Escherichia coli DNA polymerase III, to DNA polymerase alpha from calf thymus has been studied . Alone, calf thymus DNA polymerase alpha terminates in vitro DNA synthesis upon insertion of noncomplementary nucleotides . Upon addition of the epsilon subunit, DNA polymerase alpha elongates the newly synthesized DNA as a result of hydrolysis of the 3'-terminal mispair . The fidelity of DNA polymerase alpha in vitro is increased 7-fold by addition of the exonuclease . The functional interaction between DNA polymerase alpha and the epsilon subunit is independent of any detectable physical association . This suggests that a mechanism for proofreading could exist in mammalian cells involving sequential catalysis by DNA polymerase alpha excision of errors by a separate 3'----5' exonuclease, and further elongation onto correctly base-paired 3' termini by DNA polymerase alpha. Proc Natl Acad Sci U S A, 1989 May, 86(9), 3065 - 9 Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration; Fujiwara T et al.; After retroviral infection of a permissive cell, the viral RNA is reverse-transcribed to make a DNA copy of the viral genome . Integration of this DNA copy into the host genome is a necessary step for efficient viral replication . We have developed a cell-free system for integration of exogenous mini-retroviral DNA . The termini of this linear mini-Moloney murine leukemia virus (MoMLV) DNA are designed to mimic the ends of authentic unintegrated MoMLV DNA . The viral proteins required for integration can be provided either as a cytoplasmic extract of MoMLV-infected NIH 3T3 cells or as disrupted MoMLV particles . Phage lambda DNA serves as the target for integration . Genetic markers present on the mini-MoMLV DNA enable integration events to be detected, and the recombinants recovered, by selection in Escherichia coli . Integration, which occurs at heterogeneous locations in the target DNA, is absolutely dependent on the presence of a source of viral proteins and a divalent cation in the reaction mixture . The fidelity of the integration reaction was confirmed by sequencing the junctions between the integrated MoMLV DNA and adjacent lambda DNA sequence . In each case, as expected for authentic MoMLV DNA integration, a 4-base-pair duplication of target DNA sequence flanked the integrated MoMLV DNA. Eur J Biochem, 1989 May 1, 181(2), 485 - 91 Cross-reconstitution of the F0F1-ATP synthases of chloroplasts and Escherichia coli with special emphasis on subunit delta; Engelbrecht S et al.; F0F1-ATP synthases catalyse ATP formation from ADP and Pi by using the free energy supplied by the transmembrane electrochemical potential of the proton . The delta subunit of F1 plays an important role at the interface between the channel portion F0 and the catalytic portion F1 . In chloroplasts it can plug the protonic conductance of CF0 and in Escherichia coli it is required for binding of EF1 to EF0 . We wanted to know whether or not delta of one species was effective between F0 and F1 of the other species and vice versa . To this end the respective coupling membrane (thylakoids, everted vesicles from E . coli) was (partially) depleted of F1 and purified F1, F1(-delta), and delta were added in various combinations to the F1-depleted membranes . The efficiency or reconstitution was measured in thylakoids via the rate of phenazinemethosulfate-mediated cyclic photophosphorylation and in E . coli everted vesicles via the degree of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching . Addition of CF1 to partially CF1-depleted thylakoid vesicles restored photophosphorylation to the highest extent . CF1(-delta)+chloroplast delta, EF1, EF1(-delta)+E . coli delta were also effective but to lesser extent . CF1(-delta)+E . coli delta and EF1(-delta)+chloroplast delta restored photophosphorylation to a small but still significant extent . With F1-depleted everted vesicles prepared by repeated EDTA treatment of E . coli membranes, addition of CF1, CF1 (-delta)+chloroplast delta and CF1(-delta)+E . coli delta gave approximately half the extent of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching as compared to EF1 or EF1(-delta)+E . coli delta by energization of the vesicles with NADH, while Ef1(-delta)+chloroplast delta was ineffective . All 'mixed' combinations were probably reconstitutively active only by plugging the protonic leak through the exposed F0 (structural reconstitution) rather than by catalytic activity . Nevertheless, the cross-reconstitution is stunning in view of the weak sequence similarity between chloroplast delta and E . coli delta . It favors a role of delta as a conformational transducer rather than as a proton conductor between F0 and F1. Chest, 1989 May, 95(5), 1114 - 20 Multiple organ damage caused by tumor necrosis factor and prevented by prior neutrophil depletion; Mallick AA et al.; The effect of TNF on nonpulmonary multiple organ damage (MOD) was studied . Since polymorphonuclear leukocytes (PMN) are thought to play an important role in septic or TNF-induced MOD, we investigated both neutrophil sufficient (PMN+) and neutropenic (PMN-) guinea pigs . Sepsis was induced by Escherichia coli administration (2 x 10(9)/kg) or recombinant human TNF (1.4 x 10(6) U/kg) was infused into PMN+ and PMN- guinea pigs . During necropsy, the PMN+/TNF and PMN+/E coli animals exhibited marked damage in the adrenal glands, kidneys and liver as evidenced by hemorrhage, congestion, and PMN sequestration on histopathologic examination . There was also increased tissue albumin accumulation in the adrenal glands, kidneys, spleen, heart, and liver as demonstrated by 125I-labeled albumin determinations . In contrast, the PMN-/TNF group did not reveal histopathologic damage in any organ system and there was no abnormal organ accumulation of 125I-albumin . However, in PMN-/E coli animals, marked histopathologic damage in the adrenal glands and liver was evident . Furthermore, there were marked accumulations of 125I-albumin in the adrenals, heart, kidneys, liver, and spleen . Moreover, the PMN-/E coli guinea pigs had a much greater accumulation (p less than 0.01) of 125I-albumin in the kidneys than any other group including the PMN+/E coli group . Thus, nonpulmonary MOD in guinea pigs is caused by TNF administration and can be prevented by PMN depletion . However, while E coli administration also caused marked nonpulmonary MOD in neutrophil sufficient guinea pigs, equivalent or greater damage was produced in neutropenic animals . This suggests that while TNF-induced MOD may be primarily mediated by PMN, E coli-induced MOD seems to be mediated by more than PMN. Drug Des Deliv, 1989 May, 4(3), 217 - 25 The effect of C-terminal processing on the activity of human interferon-gamma; Arakawa T et al.; Homogeneous recombinant human interferon-gamma (IFN-gamma) obtained from Escherichia coli (E . coli) was treated with a protease-containing fraction prepared from mechanically lysed E . coli cells . Polyacrylamide gel electrophoresis of the resulting product revealed two major components of molecular weight less than that of intact IFN-gamma . These were purified by ion exchange chromatography in the presence of 7 M urea and shown to have intact IFN-gamma N-terminal sequences, suggesting that they resulted via C-terminal cleavages of IFN-gamma . Amino acid analysis indicated that 4 C-terminal residues of IFN-gamma were lacking in one, and 15 in the other . The species lacking 4 C-terminal residues had activities virtually indistinguishable from those of IFN-gamma in antiviral and growth inhibitory assays using Encepharomyocarditis-treated HeLa or T98G cells and in a macrophage activation assay using macrophage-like U937 cells . The species lacking 15 C-terminal residues had markedly decreased activities in each of these assays, and had decreased binding affinity for IFN-gamma cell surface receptors . These observations define the C-terminal residues important for IFN-gamma's biological activity--information which should be useful in designing analogs of IFN-gamma with enhanced or altered biological activities. Kekkaku, 1989 May, 64(5), 351 - 60 {Molecular cloning and expression of Mycobacterium tuberculosis strain Aoyama B peptide antigen genes in Escherichia coli--a gene encoding 15 kD antigen (AT 01)}; Tanaka T; A genomic library of Mycobacterium tuberculosis strain Aoyama B in Escherichia coli K-12 was constructed by cloning Sau 3A I cleaved M . tuberculosis Aoyama B chromosomal DNA into pUC18, pUC181 or pUC182 . Clones reacting with anti-PPDs-rabbit-serum were screened by immunoblotting among 3 x 10(4) clones . Seven clones were selected; designating pAT 01, pAT 101, pAT 102, pAT 103, pAT 104, pAT 105 and pAT 201 . On Western blotting, they were shown to produce 15 kD (pAT 01, pAT 101, pAT 105), 18 kD (pAT 103) and 60 kD (pAT 102, pAT 201) peptide antigens . Restriction endonuclease map of each of the above clones was composed, and putative coding frames of anti-PPDs-rabbit-serum reactive peptides were deduced by analysing deletion derivatives . Nucleotide sequence of pAT 01, encoding for 15 kD peptide antigen was analyzed, and a Hinf I-Hinf I fragment in pAT 01 was subcloned into pUC 18 lambda CPL 1 to determine the direction of its reading frame . The origin of promoter that drives cloned mycobacterial genes in E . coli was discussed. Plasmid, 1989 May, 21(3), 226 - 37 Replication of an RK2 miniplasmid derivative in vitro by a DNA/membrane complex extracted from Escherichia coli: involvement of the dnaA but not dnaK host proteins and association of these and plasmid-encoded proteins with the inner membrane; Kostyal DA et al.; A DNA/membrane complex extracted from a miniplasmid derivative of the broad host range plasmid RK2 cultured in Escherichia coli capable of synthesizing new plasmid supercoiled DNA in vitro was treated with antibodies that were made against or reacted with the dnaA and dnaK host-encoded proteins, respectively . Anti-dnaA protein antibody inhibited total plasmid DNA synthesis significantly and the synthesis of supercoil plasmid DNA almost completely . In contrast, anti-dnaK protein antibody and nonimmune serum had little or no effect on total plasmid DNA synthesis . Both proteins were found to be present in the inner but not outer membrane fraction of E . coli . A variety of miniplasmid-encoded proteins which had previously been found in the DNA/membrane complex have also been localized to the inner but not outer membrane fraction . These include an essential initiation protein of 32 kDa (and an overlapping protein of 43 kDa coded for by the same gene), as well as a 30-kDa protein that may be linked to incompatibility functions . Various extraction methods were used to distinguish between the associated and the integral nature of the plasmid-encoded proteins . The results demonstrated that the essential replication proteins (32 and 43 kDa) as well as the 30-kDa protein was tightly bound to the inner membrane . Computer analysis of the amino acid sequence of the 32 (and 43)-kDa protein revealed a hydrophobic region that is only half that normally required to span the membrane . Other interactions are discussed with respect to attaching this protein to the membrane. Mol Gen Genet, 1989 May, 217(1), 20 - 5 Mutagenesis by aflatoxin in M13 DNA: base-substitution mechanisms and the origin of strand bias; Sahasrabudhe S et al.; The two goals of the experiments described here are: (a) to examine whether there is a strand bias in mutagenic processing of bulky lesions in M13 replicative form (RF) DNA, and (b) to examine the mutational mechanisms of metabolically activated aflatoxin . For these experiments, two types of nicked heteroduplex M13 RF DNA molecules (+WT/-am1 and +am1/-WT) in which either the minus (-) or the plus (+) strand carried a gene 1 amber nonsense codon, were constructed . Heteroduplex DNAs were modified in vitro with aflatoxin B1 activated by hamster liver S9 enzymes, and transfected into SOS(UV)-induced Escherichia coli (Supo/uvrA-/mucAB+) . Forward mutations in the lacZ alpha-complementing gene segment were scored and sequenced . Results indicated that aflatoxin-induced mutation frequencies in the +WT/-am1 heteroduplex were significantly greater than those in the +am1/-WT heteroduplex, suggesting more efficient mutagenic processing of lesions in the plus strand . These results permit specific suggestions for improved mutation detection in the extensively used M13 forward mutagenesis system . Sequence analysis of point mutations from the +WT/-am1 experiments showed that most substitutions were targeted to plus-strand guanines . Both G-to-A transitions and G-to-T transversions were induced with equal efficiency . Since activated aflatoxin B1 is known to react almost exclusively with DNA guanines at the N7 position, these results suggest that bulky lesions at guanine N7 position may have the properties of mis-instructional as well as non-instructional lesions. Mol Gen Genet, 1989 May, 217(1), 118 - 25 Cloning and molecular characterisation of the amdR controlled gatA gene of Aspergillus nidulans; Richardson IB et al.; The gamma-amino-n-butyrate transaminase gene (gatA) of Aspergillus nidulans is one of several genes under positive control by the regulatory gene amdR (also called intA) . The gatA gene has been cloned from a cosmid library by complementation of a gatA mutation . The sequence of a 2.6 kb genomic fragment containing gatA has been determined . An open reading frame of 1497 bp within this sequence is interrupted by three putative introns and predicts a protein of 55 kDa . Northern analysis confirms control of gatA RNA levels by amdR and also indicates that gatA is not strongly regulated by areA-mediated nitrogen metabolite repression . A . nidulans transformants containing multiple copies of a plasmid carrying an 88 bp fragment from the 5' untranscribed region of gatA grew poorly on substrates whose utilisation is dependent on genes controlled by amdR . This indicated titration of limiting amounts of the amdR gene product by this 88 bp fragment . Comparison of this sequence with the 5' region of the coregulated gene, amdS, reveals probable sites of action for the amdR protein. EMBO J, 1989 May, 8(5), 1559 - 65 A 14 bp promoter element directs the testis specificity of the Drosophila beta 2 tubulin gene; Michiels F et al.; To analyze the regulation of gene expression during male germ cell development, we investigated the testis-specific expression of the Drosophila beta 2 tubulin gene . Germ line transformation experiments with the